Thomas’ Hematopoietic Cell Transplantation Stem Cell Transplantation Fourth Edition
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
Dedication This book is dedicated to our patients and their families whose courage and trust enabled them to choose a difficult, dangerous, and sometimes unproven therapy which offered the only chance in their fight against a fatal disease. Today, many thousands of patients are alive after a successful hematopoietic cell transplantation which would have been impossible without the fortitude of our patients, who took risks and thereby paved the way for the increasing application of this therapy. The progress in the area of clinical transplantation of hematopoietic cells would not have been possible without the dedicated, tireless work of the nurses and the supporting staff in the many transplant units around the world.
Thomas’
Hematopoietic Cell Transplantation Stem Cell Transplantation
Edited by
Frederick R. Appelbaum,
MD
Member and Director, Clinical Research Division Fred Hutchinson Cancer Research Center Professor and Head, Division of Medical Oncology University of Washington School of Medicine Seattle, Washington
Stephen J. Forman,
MD
Director, Department of Hematology and Hematopoietic Cell Transplantation Staff Physician, Department of Medical Oncology and Therapeutics Research City of Hope Comprehensive Cancer Center Duarte, California
Robert S. Negrin,
MD
Professor of Medicine Chief, Division of Blood and Marrow Transplantation Stanford University Stanford, California
Karl G. Blume,
MD
Emeritus Professor of Medicine Division of Blood and Marrow Transplantation Stanford University Stanford, California
Fourth Edition
A John Wiley & Sons, Ltd., Publication
This edition first published 1994, © 1994, 1999, 2004 by Blackwell Publishing Ltd Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical and Medical business to form WileyBlackwell. Registered office: John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wileyblackwell The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. The contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting a specific method, diagnosis, or treatment by physicians for any particular patient. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. Readers should consult with a specialist where appropriate. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. Library of Congress Cataloging-in-Publication Data Thomas’ hematopoietic cell transplantation / edited by Frederick R. Appelbaum . . . [et al.]. – 4th ed. p. ; cm. Includes bibliographical references and index. 1. Hematopoietic stem cells–Transplantation. I. Appelbaum, Frederick R. II. Thomas, E. Donnall. III. Title: Hematopoietic cell transplantation. [DNLM: 1. Hematopoietic Stem Cell Transplantation. 2. Transplantation Immunology. WH 380 T457 2008] RD123.5.B652 2008 617.4′4 – dc22 2008030325 ISBN: 978-1-4051-5348-5 A catalogue record for this book is available from the British Library. Set in 8.75 on 11 pt Times by SNP Best-set Typesetter Ltd., Hong Kong Printed & bound in Singapore by Fabulous Printers Pte Ltd 1
2009
Contents
Contributors, ix Preface to the First Edition, xvii Preface to the Second Edition, xviii Preface to the Third Edition, xix Preface to the Fourth Edition, xx Thomas HCT Fourth Edition, xxi List of Abbreviations, xxiii
9 Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation, 102 Edwin M. Horwitz 10 Genetic Manipulation of Hematopoietic Stem Cells, 116 Grant D. Trobridge & Hans-Peter Kiem Section 2b Immunology
SECTION 1 History and Use of Hematopoietic Cell Transplantation 1 A History of Allogeneic Hematopoietic Cell Transplantation, 3 E. Donnall Thomas 2 The History of Autologous Hematopoietic Cell Transplantation, 8 James O. Armitage 3 Uses and Growth of Hematopoietic Cell Transplantation, 15 Mary M. Horowitz SECTION 2 Scientific Basis for Hematopoietic Cell Transplantation Section 2a Hematopoiesis and Stem Cell Biology
4 Generation of Definitive Engraftable Hematopoietic Stem Cells from Human Embryonic Stem Cells, 27 Laurence Dahéron & David T. Scadden 5 Biology of Hematopoietic Stem and Progenitor Cells, 36 Susan Prohaska & Irving Weissman 6 Molecular Biology of Stem Cell Renewal, 64 Peter M. Lansdorp 7 Cellular Biology of Hematopoiesis, 72 Catherine M. Flynn & Catherine M. Verfaillie 8 Expansion of Hematopoietic Stem Cells, 88 Colleen Delaney & Irwin Bernstein
11 Overview of Hematopoietic Cell Transplantation Immunology, 131 Paul J. Martin 12 Histocompatibility, 145 Eric Mickelson & Effie W. Petersdorf 13 Natural Killer Cells and Allogeneic Hematopoietic Cell Transplantation, 163 Michael R. Verneris & Jeffrey S. Miller 14 Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect, 176 Robert Korngold & Thea M. Friedman 15 Mechanisms of Tolerance, 188 Megan Sykes 16 The Pathophysiology of Graft-Versus-Host Disease, 208 James L.M. Ferrara & Joseph H. Antin 17 Immune Reconstitution Following Hematopoietic Cell Transplantation, 222 Robertson Parkman & Kenneth I. Weinberg 18 The Human Graft-versus-Tumor Response – and How to Exploit It, 232 Edus H. Warren 19 Dendritic Cells in Hematopoietic Cell Transplantation, 248 Miriam Merad, Matthew P. Collin & Edgar G. Engleman 20 The Experimental Basis for Hematopoietic Cell Transplantation for Autoimmune Diseases, 264 Judith A. Shizuru Section 2c Technical Aspects
21 Pharmacologic Basis for High-dose Chemotherapy, 289 James H. Doroshow & Timothy W. Synold v
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Contents
22 High-dose Preparatory Regimens, 316 William I. Bensinger 23 Radiotherapeutic Principles of Hematopoietic Cell Transplantation, 333 Jeffrey Y.C. Wong & Timothy Schultheiss 24 Radioimmunotherapy and Hematopoietic Cell Transplantation, 351 Damian J. Green & Oliver W. Press 25 Documentation of Engraftment and Characterization of Chimerism Following Hematopoietic Cell Transplantation, 365 Paul J. Martin 26 The Detection and Significance of Minimal Residual Disease, 376 Jerald P. Radich & Marilyn L. Slovak 27 Pathology of Hematopoietic Cell Transplantation, 390 Howard M. Shulman, Robert C. Hackman & George E. Sale 28 Biostatistical Methods in Hematopoietic Cell Transplantation, 406 Joyce C. Niland & Paul Frankel 29 Outcomes Research in Hematopoietic Cell Transplantation, 428 Stephanie J. Lee
SECTION 3 Patient-oriented Issues in Hematopoietic Cell Transplantation 30 The Evaluation and Counseling of Candidates for Hematopoietic Cell Transplantation, 445 Karl G. Blume & Robert A. Krance 31 Nursing Role in Hematopoietic Cell Transplantation, 461 Rosemary C. Ford & Mihkaila M. Wickline 32 Ethical Issues in Hematopoietic Cell Transplantation, 478 David S. Snyder 33 Psychosocial Issues in Hematopoietic Cell Transplantation, 488 Richard P. McQuellon & Michael Andrykowski 34 Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients, 502 Karen L. Syrjala & Samantha Burns Artherholt 35 Sexuality Following Hematopoietic Cell Transplantation: An Important Health-related Quality of Life Issue, 515 D. Kathryn Tierney 36 Hematopoietic Cell Transplantation: The Patient’s Perspective, 526 Susan K. Stewart
SECTION 4 Sources of Hematopoietic Cells for Hematopoietic Cell Transplantation 37 Hematopoietic Cell Procurement, Processing, and Transplantation: Standards, Accreditation, and Regulation, 535 Phyllis I. Warkentin & Elizabeth J. Shpall 38 Bone Marrow and Peripheral Blood Cell Donors and Donor Registries, 544 Dennis L. Confer, John P. Miller & Jeffrey W. Chell 39 Cord Blood Hematopoietic Cell Transplantation, 559 Hal E. Broxmeyer & Franklin O. Smith 40 In Utero Transplantation, 577 Alan W. Flake & Esmail D. Zanjani 41 Mobilization of Autologous Peripheral Blood Hematopoietic Cells for Cellular Therapy, 590 Thomas C. Shea & John F. DiPersio 42 Removal of Tumor Cells from the Hematopoietic Graft, 605 John G. Gribben 43 Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation, 618 Norbert Schmitz 44 Cryopreservation of Hematopoietic Cells, 631 Scott D. Rowley 45 Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation, 645 Jürgen Finke & Roland Mertelsmann 46 Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors, 657 Claudio Anasetti, Franco Aversa & Andrea Velardi 47 Hematopoietic Cell Transplantation from Unrelated Donors, 675 Effie W. Petersdorf 48 Donor Selection for Hematopoietic Cell Transplantation, 692 Ann E. Woolfrey SECTION 5 Hematopoietic Cell Transplantation for Acquired Diseases 49 Hematopoietic Cell Transplantation for Aplastic Anemia, 707 George E. Georges & Rainer Storb 50 Hematopoietic Cell Transplantation for Paroxysmal Nocturnal Hemoglobinuria, 727 Robert P. Witherspoon 51 Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia, 734 Jerald P. Radich & Ravi Bhatia
Contents
52 Hematopoietic Cell Transplantation for Juvenile Myelomonocytic Leukemia, 751 Charlotte M. Niemeyer & Franco Locatelli 53 Hematopoietic Cell Transplantation for Adult Acute Myeloid Leukemia, 761 Frederick R. Appelbaum 54 Hematopoietic Cell Transplantation for Childhood Acute Myeloid Leukemia, 775 Julie-An M. Talano, James T. Casper & David A. Margolis 55 Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults, 791 Stephen J. Forman 56 Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Children, 806 Parinda A. Mehta & Stella M. Davies 57 Hematopoietic Cell Transplantation for Myelodysplastic Syndrome and Myeloproliferative Disorders, 827 H. Joachim Deeg 58 Hematopoietic Cell Transplantation for Multiple Myeloma, 845 Muzaffar H. Qazilbash & Sergio A. Giralt 59 Hematopoietic Cell Transplantation for Hodgkin’s Disease, 860 Philip J. Bierman & Auayporn Nademanee 60 Non-Hodgkin’s Lymphoma, 878 Laura J. Johnston & Sandra J. Horning 61 Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia, 897 David B. Miklos 62 Autologous Hematopoietic Cell Transplantation for Systemic Light Chain (AL-) Amyloidosis, 914 Raymond L. Comenzo & Morie A. Gertz 63 Hematopoietic Cell Transplantation for Breast Cancer, 931 Yago Nieto & Elizabeth J. Shpall 64 Hematopoietic Cell Transplantation in Germ Cell Tumors, 948 Christie J. Moore, Brandon Hayes-Lattin & Craig R. Nichols 65 Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors, 958 Richard W. Childs & Ramaprasad Srinivasan 66 Hematopoietic Cell Transplantation for Neuroblastoma, 970 Jason Law & Katherine K. Matthay 67 Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors, 985 David M. Loeb & Allen R. Chen 68 Hematopoietic Cell Transplantation for Patients with Human Immunodeficiency Virus Infection, 1001 John A. Zaia, Amrita Krishnan, & John J. Rossi 69 Hematopoietic Cell Transplantation for Autoimmune Diseases, 1014 Richard A. Nash
vii
70 Hematopoietic Cell Transplantation for Rare Hematologic Malignancies, 1030 Vinod Pullarkat & Stephen J. Forman 71 Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies, 1043 Brenda M. Sandmaier & Rainer Storb 72 Management of Relapse after Hematopoietic Cell Transplantation, 1059 Ginna G. Laport & Robert S. Negrin
SECTION 6 Hematopoietic Cell Transplantation for Inherited Diseases 73 Hematopoietic Cell Transplantation for Thalassemia, 1079 Guido Lucarelli & Javid Gaziev 74 Hematopoietic Cell Transplantation for Sickle Cell Disease, 1090 Mark C. Walters 75 Hematopoietic Cell Transplantation for Immunodeficiency Diseases, 1105 Trudy N. Small, Wilhelm Friedrich, & Richard J. O’Reilly 76 Hematopoietic Cell Transplantation for Osteopetrosis, 1125 Peter F. Coccia 77 Hematopoietic Cell Transplantation for Storage Diseases, 1136 Charles Peters 78 Hematopoietic Cell Transplantation for Macrophage and Granulocyte Disorders, 1163 Rajni Agarwal 79 Hematopoietic Cell Transplantation for Fanconi’s Anemia, 1178 John E. Wagner, Margaret L. MacMillan, & Arleen D. Auerbach SECTION 7 Complications of Hematopoietic Cell Transplantation 80 Mechanisms and Treatment of Graft Failure, 1203 Robert Lowsky & Hans Messner 81 Blood Group Incompatibilities and Hemolytic Complications of Hematopoietic Cell Transplantation, 1219 Margaret R. O’Donnell 82 Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation, 1226 Jeffrey McCullough
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83 Vascular Access and Complications, 1244 I. Benjamin Paz 84 Pharmacologic Prevention of Acute Graft-Versus-Host Disease, 1257 Nelson J. Chao & Keith M. Sullivan 85 T-Cell Depletion to Prevent Graft-versus-Host Disease, 1275 Robert J. Soiffer 86 Manifestations and Treatment of Acute Graft-Versus-Host Disease, 1287 Corey Cutler & Joseph H. Antin 87 Chronic Graft-versus-Host Disease: Clinical Manifestations and Therapy, 1304 Steven Z. Pavletic & Georgia B. Vogelsang 88 Bacterial Infections, 1325 Helen L. Leather & John R. Wingard 89 Fungal Infections after Hematopoietic Cell Transplantation, 1346 Janice (Wes) M.Y. Brown 90 Cytomegalovirus Infection, 1367 John A. Zaia 91 Herpes Simplex Virus Infections, 1382 James I. Ito 92 Varicella-zoster Virus Infections, 1388 Dora Y. Ho & Ann M. Arvin 93 Epstein–Barr Virus Infection, 1410 Wen-son Hsieh & Richard F. Ambinder 94 Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation, 1419 Michael Boeckh 95 Gastrointestinal and Hepatic Complications, 1434 Simone I. Strasser & George B. McDonald 96 Lung Injury Following Hematopoietic Cell Transplantation, 1456 Kenneth R. Cooke & Gregory A. Yanik 97 Kidney and Bladder Complications of Hematopoietic Cell Transplantation, 1473 Sangeeta Hingorani 98 Endocrine Complications Following Hematopoietic Cell Transplantation, 1487 Fouad R. Kandeel
99 Common Potential Drug Interactions Following Hematopoietic Cell Transplantation, 1523 Anne Poon & Lowan Ly 100 Critical Care of the Hematopoietic Cell Transplant Recipient, 1539 Gundeep S. Dhillon & Norman W. Rizk 101 Nutrition Support of the Hematopoietic Cell Transplant Recipient, 1551 Polly Lenssen & Saundra Aker 102 Pain Management, 1570 Noelle V. Frey & Jonathan R. Gavrin 103 Oral Complications of Hematopoietic Cell Transplantation, 1589 Mark M. Schubert & Douglas E. Peterson 104 Growth and Development after Hematopoietic Cell Transplantation, 1608 Jean E. Sanders 105 Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation, 1620 Mary E.D. Flowers & H. Joachim Deeg 106 Secondary Malignancies after Hematopoietic Cell Transplantation, 1638 Smita Bhatia & Ravi Bhatia 107 Neurologic Complications of Hematopoietic Cell Transplantation, 1653 Harry Openshaw 108 Vaccination of Allogeneic and Autologous Hematopoietic Cell Recipients, 1664 Trudy N. Small
SECTION 8 Looking Forward 109 Hematopoietic Cell Transplantation in the Future, 1673 Ernest Beutler Colorplates, facing page 514 Robert C. Hackman Index, 1677
Companion CD-ROM A companion CD-ROM is included with the book, containing: • The complete text • A database of figures from the book for downloading • A Search feature The CD-ROM is suitable for PC and Mac computers.
Contributor list
Rajni Agarwal, MD Assistant Professor of Pediatrics, Division of Stem Cell Transplantation, Department of Pediatrics, Stanford University School of Medicine, Stanford, California Saundra Aker, RD, CD Consultant, Nutrition and Patient Food Services, Seattle Cancer Care Alliance, Seattle, Washington Richard F. Ambinder, MD, PhD Professor in the Departments of Oncology, Pathology, Pharmacology and Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland
Samantha Burns Artherholt, PhD Research Fellow, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington Ann M. Arvin, MD Professor of Pediatrics, Microbiology and Immunology, Department of Pediatrics, Infectious Disease Division, Stanford University School of Medicine, Stanford, California Arleen D. Auerbach, PhD Director of the Laboratory of Human Genetics and Hematology, The Rockefeller University New York, New York
Claudio Anasetti, MD Chair, Department of Blood and Marrow Transplantation, Moffitt Cancer Center Professor of Medicine and Oncology, University of South Florida, Tampa, Florida
Franco Aversa, MD Head, Hematopoietic Stem Cell Transplant Unit Professor, Department of Clinical and Experimental Medicine Sections of Hematology and Immunology Perugia University, School of Medicine, Perugia, Italy
Michael A. Andrykowski, PhD Department of Behavioural Science, University of Kentucky College of Medicine, Seattle, Washington
William I. Bensinger, MD Professor of Medicine, University of Washington Fred Hutchinson Cancer Research Center, Seattle, Washington
Joseph H. Antin, MD Professor of Medicine, Harvard Medical School, Dana Farber Cancer Institute, Boston, MA
Irwin Bernstein, MD Member, Fred Hutchinson Cancer Research Center; and Professor of Medicine, University of Washington School of Medicine, Seattle, Washington
Frederick R. Appelbaum, MD Member and Director, Clinical Research Division, Fred Hutchinson Cancer Research Center, Professor and Head, Division of Medical Oncology, University of Washington School of Medicine, Seattle, Washington James O. Armitage, MD The Joe Shapiro Professor of Medicine Section of Oncology/ Hematology, University of Nebraska, Medical Center, Nebraska Medical Center, Omaha, Nebraska
Ernest Beutler, MD Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California Ravi Bhatia, MD Director, Stem Cell Biology Program, Division of Hematology and Bone Marrow Transplantation, City of Hope National Medical Center, Duarte, California ix
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Contributor list
Smita Bhatia, MD, MPH Staff Physician; Director, Epidemiology and Outcomes Research, Division of Pediatric Hematology/Oncology and Bone Marrow Transplantation, City of Hope National Medical Center, Duarte, California Philip J. Bierman, MD Associate Professor of Medicine, Department of Internal Medicine, Section of Oncology and Department Hematology, University of Nebraska Medical Center, Omaha, Nebraska Karl G. Blume, MD, FACP Emeritus Professor of Medicine, Division of Bone Marrow Transplantation, Department of Medicine, Stanford University, School of Medicine, Stanford, California Michael Boeckh, MD Assistant Member, Program in Infectious Diseases Fred Hutchinson Cancer Research Center, Assistant Professor, University of Washington School of Medicine, Seattle, Washington Janice M. Y. Brown, MD Associate Professor Divisions of Blood and Marrow Transplantation and Infectious Diseases, Stanford University School of Medicine, Stanford, California Hal E. Broxmeyer, PhD Chairman and Mary Margaret Walther Professor of Microbiology and Immunology, Professor of Medicine Scientific Director, the Walther Oncology Center Indiana University School of Medicine Cancer Research Institute, Indianapolis, Indiana James A. Casper, MD Professor of Pediatrics, Section of Pediatric HematologyOncology-Transplantation Department of Pediatrics Medical College of Wisconsin Children’s Hospital of Wisconsin, Milwaukee, Wisconsin Nelson J. Chao, MD Professor of Medicine and Imunology, Duke University Medical Center, Durham, North Carolina Jeffrey W. Chell, MD Chief Executive Officer, National Marrow Donor Program, Minneapolis, Minnesota Allen R. Chen, MD, PhD, MHS Associate Professor, Oncology and Pediatrics, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Pediatrics, Baltimore, Maryland
Richard Childs, MD Senior Investigator, National Heart, Lung and Blood Institute, National Institutes of Health, Bethseda, Maryland Peter F. Coccia, MD Ittner Professor of Pediatrics and Chief Section Pediatric Hematology/Oncology; and Director, Pediatric Bone Marrow Transplantation Program; and Vice-Chairperson, Department of Pediatrics, University of Nebraska Medical Center, Omaha, Nebraska Matthew Collin, MD, PhD Department of Gene and Cell Medicine, Mount Sinai Medical School, New York, New York Raymond L. Comenzo, MD Associate Attending, Hematology Service Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York Dennis L. Confer, MD Clinical Professor of Medicine, University of Minnesota; and Chief Medical Officer, National Marrow Donor Program, Minneapolis, Minnesota Kenneth Robert Cooke, MD Associate Professor of Pediatrics, Division of Hematology/ Oncology, Blood and Marrow Transplant Program, Case Western Reserve School of Medicine, Rainbow Babies and Children’s Hospital, Cleveland, Ohio Corey S. Cutler, MD MPH FRCPC Assistant Professor of Medicine, Harvard Medical School, Dana Farber Cancer Institute, Boston, Massachusetts Laurence Daheron, PhD Massachusetts General Hospital, Center for Regenerative Medicine, Boston, Massachusetts Stella Davies, MB, BS, PhD, MRCP Jacob G. Schmirdlapp Endowed Chair and Professor of Pediatrics; and Director, Blood and Marrow Transplantation Program, Division of Hematology/Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio H. Joachim Deeg, MD Member, FHCRC Professor of Medicine, University of Washington School of Medicine, Seattle, Washington Colleen Delaney, MD, MSc Assistant Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Assistant Professor, Department of Pediatrics, University of Washington, School of Medicine, Seattle, Washington
Contributor list
Gundeep S. Dhillon, MD, MPH Assistant Professor of Medicine, Stanford University School of Medicine, Stanford, California John Dipersio, MD, PhD Chief, Division of Oncology, Deputy Director, Siteman Cancer Research, Washington University School of Medicine, St Louis, Missouri
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Stephen J. Forman, MD Director, Department of Hematology and Hematopoietic Cell Transplantation, Staff Physician, Department of Medical Oncology and Therapeutics Research, City of Hope Comprehensive Cancer Center, Duarte, California Paul Frankel, PhD Statistician, City of Hope National Medical Center, Duarte, California
James H. Doroshow, MD, FACP Chairman, Department of Medical Oncology and Therapeutics Research, Associate Director for Clinical Investigation, City of Hope Comprehensive Cancer Care Center, Duarte, California
Noelle V. Frey, MD Instructor of Medicine Division of Hematology-Oncology Abramson Cancer Center University of Pennsylvania, Philadelphia, Pennsylvania
Edgar G. Engleman, MD Professor of Pathology and Medicine, Stanford University, School of Medicine, Stanford Blood Center, Stanford, California
Thea M. Friedman, PhD Director of Research Laboratory Services, The Cancer Center at Hackensack Medical University, Hackensack, New Jersey
James L. M. Ferrara, MD Professor of Medicine and Pediatrics; and Director, Blood and Marrow Transplantation Program, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan
Wilhelm Frierich, MD Professor of Pediatrics, Department of Pediatrics, University Children’s Hospital Ulm, University of Ulm, Ulm, Germany
Jürgen Finke, MD Head, Division of Allogeneic Stem Cell Transplantation, Dept Hematology and Oncology, Medizinische Klinik Univesity, Medical Center, Frieberg, Germany Alan W. Flake, MD Professor of Surgery and Obstetrics and Gynecology, University of Pennsylvania; and Ruth and Tristram Colket Professor of Pediatric Surgery, Director, Institute for Surgical Science, Children’s Hospital of Philadelphia Abramson Research Center, Philadelphia, Pennsylvania Mary E. D. Flowers, MD Assistant Member, Fred Hutchinson Cancer Research Center; and Assistant Professor, University of Washington School of Medicine, Seattle, Washington Catherine Flynn, MB BCH BAO Faculty of Medicine, Stem Cell Institute Leuven, Catholic University Leuven, Belgium Rosemary C. Ford, RN, BSN, OCN Nurse Manager, Transplant Clinic, Seattle Cancer Care Alliance, Seattle, Washington
Jonathan R. Gavrin, MD Director, Symptom Management and Palliative Care Interim Director, Pain Management Services, Physician Co-chair HUP Ethics Committee Department of Anesthesiology and Critical Care Department of Medicine Hospital of the University of Pennsylvania Javid Gaziev, MD Senior Hematologist, Mediterranean Institute of Hematology International Centre for Transplantation in Thalassemia and Sickle Cell Anaemia, Rome, Italy George Earl Georges, MD Associate Member, Fred Hutchinson Cancer Center Associate Professor of Medicine, University of Washington Seattle, Washington Morie Gertz, MD Professor of Medicine, Mayo Medical School; and Chair, Division of Hematology, Mayo Clinic, Rochester, Minnesota Sergio A. Giralt, MD Professor of Medicine, Department of Stem Cell Transplantation and Cellular Therapy, University of Texas, MD Anderson Cancer Center, Houston, Texas Damian J. Green, MD Research Associate, Fred Hutchinson Cancer Research Center and The University of Washington, Seattle, Washington
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Contributor list
John G. Gribben MD, DSc Professor of Experimental Cancer Medicine, St. Bartholomew’s Hospital, Barts and The London NHS Trust, London, UK Robert C. Hackman, MD Member and Director of Pathology, Fred Hutchinson Cancer Research Center, Seattle, Washington Brandon Hayes-Lattin, MD Associate Professor of Medicine, Division of Hematology/ Medical Oncology Director, Adolescent and Young Adult Oncology Program, Oregon Health and Science University Portland, Oregon
Fouad R. Kandeel, MD, PhD Director, Department of Diabetes, Endocrinology and Metabolism, City of Hope National Medical Center, Associate Clinical Professor, UCLA School of Medicine, Duarte, California Hans Peter Kiem, MD Member, Fred Hutchinson Cancer Research Center Professor of Medicine, University of Washington, Seattle, Washington Robert Korngold, PhD Chief, Division of Research, The Cancer Center Hackensack University Medical Center, Hackensack, New Jersey
Sangeeta Ram Hingorani, MD, MPH Assistant Professor, Pediatrics University of Washington Children’s Hospital and Regional Medical Center, Division of Nephrology, Seattle, Washington
Robert A. Krance, MD Professor of Pediatric and Medicine, Baylor College of Medicine; and Director of Pediatric Stem Cell Transplantation Program, Texas Children’s Hospital, Houston, Texas
Dora Y. Ho, MD Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California
Peter M. Lansdorp, MD, PhD Senior Scientist, Terry Fox Laboratory, BC Cancer Research Centre; Professor, Division of Hematology, University of British Columbia, Vancouver, BC
Sandra J. Horning, MD Professor of Medicine, Stanford University Medical Center, Stanford Cancer Center, Stanford, California
Ginna G. Laport, MD Assistant Professor of Medicine, Stanford University Medical Center, Division of Blood and Marrow Transplantation, Stanford, California
Mary M. Horowitz, MD, MS Chief Scientific Director, Center for Blood and Marrow Transplant Registry (CIBMTR), Medical College of Wisconsin, Milwaukee, Wisconsin Edwin M. Horwitz, MD, MS Children’s Hospital of Philadelphia, Abramson Research Center, Philadelphia, Pennsylvania Wen-son Hsieh, MD Assistant Professor in the Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland James I. Ito, MD Director, Department of Infectious Diseases, City of Hope National Medical Center, Duarte, California Laura J. Johnston, MD Assistant Professor of Medicine, Stanford University Medical Center, Division of Bone Marrow Transplantation, Stanford, California
Helen L. Leather, B. Pharm, BCPS Clinical Pharmacy Specialist BMT/Leukemia, Shands at the University of Florida; and Clinical Associate Professor, College of Pharmacy, University of Florida, Gainesville, Florida Stephanie J. Lee, MD, MPH Associate Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Polly Lenssen, MD Manager, Clinical Nutrition Children’s Hospital and Regional Medical Center, Seattle, Washington David M. Loeb, MD, PhD Assistant Professor, Oncology and Pediatrics, Director, Musculoskeletal Tumor Program, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University Robert Lowsky, MD Assistant Professor of Medicine, Stanford University Medical Center, Stanford, California
Contributor list
Guido Lucarelli, MD Director, Mediteranean Institute of Hematology, Policlinic of the University of Roma Tor Vergata, Roma, Italy Lowan L. Ly, Pharm D Clinical Pharmacist, Hematopoietic Stem Cell Transplant, University of Washington Medical Center/Seattle Cancer Care Alliance, Seattle, Washington David A. Margolis, MD Associate Professor, Medical College of Wisconsin, Milwaukee, Wisconsin Paul J. Martin, MD Member, Fred Hutchinson Cancer Research Center, Professor of Medicine, University of Washington, School of Medicine, Seattle, Washington Kathryn K. Matthay, MD Professor of Pediatrics, University of California School of Medicine, Chief, Pediatric Hematology-Oncology Department UCSF, San Francisco, CA Jeffrey McCullough, MD Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota George B. McDonald, MD Professor of Medicine, University of Washington; and Head Gastroenterology/Hepatology Section, Fred Hutchinson Cancer Research Center, Seattle, Washington Margaret L. MacMillan, MD Assistant Professor of Pediatrics, Division of Hematology/ Oncology and Blood and Marrow Transplantation, University of Minnesota Medical School, Minneapolis, Minnesota Richard P. McQuellon, PhD Comprehensive Cancer Center of Wake Forrest University, Bowman Gray School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina Parinda A. Mehta, MD Director in the Blood Disease Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio Miraim Merad, MD Department of Gene and Cell Medicine, Mount Sinai Medical School New York, New York Roland Mertelsmann, MD, PhD Medical Director, University Medical Center, Department Medicine I Hematology/Oncology, Frieberg, Germany
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Hans Messner, MD, PhD Director of Bone Marrow Transplant Program, Princess Margaret Hospital, and Senior Scientist, Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Canada Eric Mickelson, BSc Associate Staff Scientist, Fred Hutchinson Cancer Research Center, Seattle, Washington David B. Miklos, MD, PhD Assistant Professor of Medicine, Division of Blood and Marrow Transplantation Department of Medicine, Stanford University, Stanford, California Jeffrey S. Miller, MD Professor of Medicine, Division of Hematology, Oncology & Transplantation, University of Minnesota, Minneapolis, Minnesota Christie J. Moore, DO Providence Cancer Center Division of Medical Oncology Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Portland, Oregon Auayporn P. Nademanee, MD Associate Clinical Director, Division of Hematology and Bone Marrow Transplantation, Department of Medicine, Stanford University School of Medicine, Stanford, California Richard A. Nash, MD Member, Program in Transplantation Biology Clinical Research Division Fred Hutchinson Cancer Research Center; and Associate Professor, University of Washington, Seattle, Washington Robert S. Negrin, MD Professor of Medicine and Chief, Division of Blood and Marrow Transplantation, Stanford University, Stanford, CA Craig R. Nichols, MD Director, Oregon Testicular Cancer Program, Division of Medical Oncology, Robert W. Franz Cancer Research Center Earle A. Chiles Research Institute, Portland, Oregon Lewis Nick, RN, BSN, MT(ASCP) Technical Director, Accreditation Program, Foundation for the Accreditation of Cellular Therapy, University of Nebraska Medical Center, Omaha, Nebraska Charlotte Niemeyer, MD Professor of Pediatrics, Medicical Director, Division of Hematology and Oncology, University Children’s Hospital Frieberg, Germany
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Contributor list
Yago Nieto, MD, PhD Department of Stem Cell Transplantation and Cellular Therapy, the University of Texas, M. D. Anderson Cancer Center, Houston, Texas
Anne Poon, Pharm D Clinical Pharmacist, Hematopoietic Stem Cell Transplant University of Washington Medical Center/Seattle Cancer Care Alliance, Seattle, Washington
Joyce C. Niland, PhD Chair, Division of Information Sciences Edward and Estelle Alexander Chaired Professor, Beckman Research Center Associate Director for Informtation Sciences, City of Hope Cancer Center, Duarte, California
Oliver W. Press, MD, PhD Member, Fred Hutchinson Cancer Research Center, Penney E. Petersen Chair for Lymphoma, Research, Professor of Medicine and Biological Structure, University of Washington, Seattle, Washington
Margaret R. O’Donnell, MD, FRCPC Associate Clinical Director Division of Hematology/ Hematopoietic Cell Transplantation, City of Hope, Duarte, California
Susan Prohaska, PhD Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford, California
Richard O’Reilly, MD Chairman, Department of Pediatrics, Chief, Marrow Transplantation Service, Memorial Sloan-Kettering Center, New York, New York Harry Openshaw, MD City of Hope National Medical Center, Duarte, California Robertson Parkman, MD Professor of Pediatrics, Children’s Hospital of Los Angeles Division of Research Immunology and Bone Marrow Transplantation, Keck School of Medicine, University of Southern California, Children’s Hospital of Los Angeles, Los Angeles, California Steven Z. Pavletic, MD Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland I. Ben Paz, MD Associate Professor, Division of Surgery, Director, Department of General Oncologic Surgery, City of Hope, Duarte, California Charles Peters, MD Professor of Pediatrics, University of Missouri – Kansas City, School of Medicine, The Children’s Mercy Hospital, Kansas City, Missouri Effie W. Petersdorf, MD Member, Fred Hutchinson Cancer Research Center, Professor of Medicine, University of Washington School of Medicine, Seattle, WA Douglas E. Peterson, DMD, PhD Professor and Interim Head, University of Connecticut Health Center, Department of Oral Health and Diagnostic Sciences Farmington, Connecticut
Vinod Pullarkat, MB, BS, MRCP Assistant Professor Division of Hematology and Hematopoietic Cell Transplantation, City of Hope Medical Center, Duarte, California Muzaffar H. Qazilbash, MD Associate Professor of Medicine, Department of Stem Cell Transplantation and Cellular Therapy, M.D. Anderson Cancer Center, Houston, Texas Jerald P. Radich, MD Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington Norman Rizk, MD Guggenhime Professor of Medicine, Stanford University School of Medicine, Stanford, California Scott D. Rowley, MD, FACP Chief, Adult Blood and Marrow Transplant Program Hackensack University Medical Center, Hackensack, NJ; Clinical Associate Professor of Medicine, University of New Jersey School of Medicine, Newark, NJ George E. Sale, MD Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Jean E. Sanders, MD Full Member, Clinical Research Division Director, Pediatric Hematopoietic Stem Cell Transplantation, Fred Hutchinson Cancer Research Center, Professor of Pediatrics, University of Washington, Seattle, Washington Brenda Sandmaier, MD Member, Fred Hutchinson Cancer Center, Professor of Medicine, University of Washington, Seattle, Washington
Contributor list
Dave Scadden, MD Gerald and Darlene Jordan Professor of Medicine, CoChair, Department of Stem Cell and Regenerative Biology, Co-Director, Harvard Stem Cell Institute, Harvard University Director, MGH Center for Regenerative Medicine Chief, Hematologic Malignancies, MGH Cancer Center Massachusetts General Hospital, Boston, Massachusetts Norbert Schmitz, MD Director, Department of Hematology and Stem Cell Transplantation, Lohmiehlenstr. Germany
David Snyder, MD Chair, City of Hope Bioethics Committee Associate Director, Division of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, Calitornia Robert J. Soiffer, MD Dana Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA Rampprasad Srinivasan, MD, PhD Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Mark M. Schubert, DDS, MSD Professor of Oral Medicine School of Dentistry, University of Washington; and Director of Oral Medicine, Seattle Cancer Care Alliance and Fred Hutchinson Cancer Research Center, Seattle, Washington
Susan K. Stewart Blood & Marrow Transplant Information Network (BMT InfoNet), Highland Park, Illinois
Timothy Schulteiss, PhD Department Director and Professor, Division of Radiation Oncology, City of Hope Cancer Center, Duarte, California
Simone I. Strasser, MBBS, MD, FRACP A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia
Thomas Shea, MD University of North Carolina at Chapel Hill, School of Medicine, Division of Hematology/Oncology, Chapel Hill, North Carolina Judith A. Shizuru, PhD, MD Associate Professor of Medicine, Division of Blood and Marrow Transplantation, Department of Medicine, Stanford University, School of Medicine, Stanford, California E. J. Shpall, MD Professor of Medicine, Baylor College of Medicine, Houston, Texas Howard M. Shulman, MD Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Marilyn L. Slovak, PhD, FACMG Director, Department of Cytogenics, City of Hope, Duarte, California Trudy N. Small, MD Associate Attending, Department of Pediatrics Bone Marrow Transplantation Service, Memorial Sloan-Kettering Cancer Center, New York, New York Franklin O. Smith, MD Director, Division of Hematology/Oncology Professor of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
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Rainer Storb, MD Head & Member, Transplantation Biology Program, Fred Hutchinson Cancer Research Center, Professor of Medicine University of Washington School of Medicine, Seattle, Washington Simone I. Strasser, MBBS, MD, FRACP Clinical Associate Professor, Senior Staff Specialist, A.W. Morrow Gastroenterology and Liver Center, Royal Prince Alfred Hospital, Australia Keith M. Sullivan, MD James B Wingaarden Professor of Medicine, Director, LongTerm Follow-up and Information Research Division of Cellular Therapy, Department of Medicine, Durham, North Carolina Megan Sykes, MD Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts Timothy W. Synold, Pharm.D. Associate Professor, Department of Clinical and Molecular Pharmacology, Co-Director, Analytical Pharmacology Core Facility, City of Hope Comprehensive Cancer Center, Duarte, California Karen Syrjala, PhD Director of Biobehavioral Sciences and Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
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Contributor list
Julie Talano, MD Assistant Professor of Pediatrics, Department of Pediatric Hematology and Oncology, Medical College of Wisconsin, MaccFund Research Center, Milwaukee, Wisconsin E. Donnall Thomas, MD Member, Fred Hutchinson Cancer Research Center, Professor Emeritus, University of Washington School of Medicine, Nobel Laureate Medicine/Physiology, 1990, Seattle, WA D. Kathryn Tierney, RN, PhD Oncology Clinical Nurse Specialist, Division of Blood and Marrow Transplantation, Stanford University Medical Center, Stanford, California Grant D. Trobridge, PhD Fred Hutchinson Cancer Research Center, Seattle, WA Andrea Velardi, MD Professor, Department of Clinical and Experimental Medicine, Sections of Hematology and Clinical Immunology Perugia University, School of Medicine, Perugia, Italy Catherine M. Verfaillie, MD Professor of Medicine, Director, Stem Cell Institute, Director, Stamcelinstituut, Leuven, Belgium Michael R. Verneris, MD Assistant Professor Department of Pediatrics – BMT University of Minnesota, Minneapolis, Minnesota Georgia B. Vogelsang, MD The Johns Hopkins University, Sidney Kimmel Cancer Center, Baltimore, Maryland John E. Wagner, MD Professor of Pediatrics, Director, Division of Hematology/ Oncology and Blood and Marrow Transplantation CoDirector, Center for Translational Medicine, University of Minnesota Medical School, Minneapolis, Minnesota Mark C. Walters, MD Blood and Marrow Transplantation Program, Children’s Hospital & Research Center, Oakland, CA and the Dept. of Pediatrics, University of California, San Francisco School of Medicine, San Francisco, California Phyllis I. Warkentin, MD Professor of Pathology and Pediatrics, University of Nebraska Medical Center, Department of Pediatrics, Omaha, Nebraska
Edus H. Warren, III, MD, PhD Associate Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Kenneth I. Weinberg, MD Department of Pediatrics, Division of Pediatric Stem Cell Transplantation, Anne T. and Robert M. Bass Professor in Pediatric Cancer and Blood Diseases, Stanford University, Stanford, California Irving Weissman, MD Director, Stanford Institute for Stem Cell Biology and Regenerative Medicine Director, Director, Ludwig Center at Stanford, University of Stanford, California Mihkaila Wickline, MN, RN, AOCN HSCT Clinical Nurse Specialist, Seattle Cancer Care Alliance, Seattle, Washington John R. Wingard, MD Price Eminent Scholar and Professor of Medicine, Director, Bone Marrow Transplant Program, Division of Hematology/ Oncology, Deputy Director of the University of Florida, Gainesville, Florida Robert R. Witherspoon, MD Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Professor of Medicine, University of Washington, Medical Director, Transplant Clinic, Seattle Cancer Care Alliance, Seattle, Washington Jeffrey Y. C. Wong, MD Professor & Chair, Division of Radiation, Oncology, City of Hope Cancer Center, Duarte, California Ann E. Woolfrey, MD Director, Unrelated Donor Program, Associate Member, Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington Gregory A. Yanik, MD Clinical Associate Professor of Pediatrics and Communicable Diseases, Division of Hematology/Oncology, Blood and Marrow Transplantation Program, University of Michigan Cancer Center, Ann Arbor, Michigan John A. Zaia, MD Chair, Division of Virology, Beckman Research Institute of City of Hope, Duarte, California Esmail D. Zanjani, PhD Professor, Department of Medicine, University of NevadaReno, Reno, Nevada
Preface to the First Edition
The widespread application of bone marrow transplantation (BMT) to the treatment of a steadily increasing number of life-threatening hematological, oncological, hereditary, and immunological disorders is the culmination of more than four decades of research by many investigators. Early attempts in the 1950s to transplant living cells from one individual to another were carried out in the face of considerable skepticism. It was generally accepted as axiomatic that the immunological barrier to “foreign tissue” could never be overcome. The horrors of Nagasaki and Hiroshima spurred interest in studies of the lethal effects of irradiation. It was discovered that mice given total body irradiation in doses lethal to the marrow could be protected from death by shielding the spleen or by an infusion of marrow, and that the marrow of such animals contained living cells of donor origin. These observations suggested that patients with leukemia might be given a lethal exposure of total body irradiation, which would destroy the malignant cells along with remaining normal marrow. The exposure would also destroy the immune system, making it possible to protect against lethality by a transplant of normal marrow cells. The theory was correct, but results were disappointing. Because the procedure was both unproved and dangerous, only those patients who had no other options were considered. Except for a few patients with an identical twin donor, there were no survivors beyond a few months. Understanding of the human leukocyte antigen (HLA) system was not yet available, and little was known about the complication we now call graft-versus-host disease (GVHD). Thus, after a brief period of enthusiasm, most investigators abandoned this seemingly hopeless pursuit. Fortunately, work in animal models continued. Studies in inbred rodents defined the genetics of the major histocompatibility system and the fundamental rules of transplantation biology. Immunosuppressive drugs were developed to limit the severity of the immune reactions between donor and host. Demonstration of successful marrow transplants in the canine model using littermates matched for the major histocompatibility complex set the stage for successful transplantation of marrow between human siblings. Thus, it is clear that a long series of experimental studies in animals ultimately made human marrow transplantation possible. By the late 1960s, much was known about the HLA system, more effective antibiotics were available, and platelet transfusions were becoming routine. Thus began the modern era of human BMT. THe past 25 years have witnessed an almost exponential growth in the number of transplants being performed and the number of diseases being considered for BMT. Initially, most grafts employed marrow from an HLAidentical sibling. Autologous marrow, long known to be effective in animal systems, is now being used with increasing frequency following intensive cancer chemotherapy. Hematopoietic progenitor cells from the peripheral blood are now being used for BMT, either alone or to supplement marrow. As a result of increasing national and international cooperation, large panels of volunteer marrow donors of known HLA type are becoming available to patients whose own marrow cannot be used or who do not have a family donor.
Currently, thousands of transplants are being performed each year world-wide. With the demonstration that marrow could be transplanted and that the cure rate would be substantial, the logical step was taken to treat patients early during the course of their respective disease (i.e. in leukemia when the burden of malignant cells was relatively low and when the patient was in excellent clinical condition). With improved patient selection, development of improved tissue typing methods, availability of potent antimicrobial agents, advances in supportive care, and improved prevention of GVHD, the results of BMT have continued to improve. Marrow transplantation is now being applied to a long list of diseases with a wide range of results depending on the disease, the type of transplant, and the stage of the disease. For some of the diseases, BMT has already proven to be the most effective therapy (e.g., some leukemias and severe aplastic anemia), whereas for others it is the only available curative treatment (e.g., thalassemia). In very rare genetic disorders, one successful BMT may establish the success of the treatment. For other more common disorders, controlled trials are necessary to define the proper role of allogeneic or autologous BMT, or therapy not involving BMT. Only through rigorous study and long-term follow-up can novel approaches be confirmed as effective (or ineffective). For those working in the field of marrow transplantation, a source of intellectual satisfaction has been the interdisciplinary nature of the studies. A view of the wide-ranging disciplines involved can be gleaned by reading the chapter titles for this book. A successful BMT program is always a team effort. There must be cooperation between blood banks, referring physicians, radiation oncologists, immunologists, and physicians from many subspecialties. A dedicated support staff of technicians, data managers, and, above all, nurses, is crucial. The nursing team in particular is responsible for the day-to-day care of patients. Nurses not only provide the bedside management of complex protocol studies, but also bear the burden of emotional support through the difficult hospital period. They are the most readily available source of information for the patients and families day and night. Without a strong nursing team, the entire BMT program is jeopardized. Most important are the patients who come to the transplant center with the courage to accept days, weeks, and sometimes months of discomfort in the hope of surviving a fatal disease. We must ensure that we acknowledge and respect the dignity and individuality of each patient, that we provide adequate information for informed decision making and then include patients and families in the decision process. The greatest reward for clinical investigators is to see patients reintegrated into their personal, social, and professional lives, free of their disease and its complications. Stephen J. Forman Karl G. Blume E. Donnall Thomas Summer, 1993
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Preface to the Second Edition
During the five-year period since the first edition of this book was published, an enormous amount of new scientific information has been generated through experimental research and through clinical trials. This growth in knowledge is reflected by the doubling in the volume of this book and a 50 percent increase in the number of new chapters. The selection of a new title Hematopoietic Cell Transplantation became necessary because bone marrow is no longer the major source of allogeneic and autologous hematopoietic cells. Hematopoietic stem cells from peripheral blood collections and from umbilical cord blood are rapidly gaining clinical importance for hematologic and immunologic reconstitution following myeloablative, high-dose therapies for malignant and non-malignant, hereditary or acquired life-threatening disease. It would be impossible to identify a single scientific observation as the leading contribution to the progress made during the past five years. Exciting new data have been reported in all areas of preclinical and clinical research: New histocompatibility antigens for optimal donor selection have been described, novel concepts have been developed to eradicate the underlying malignancies, engineering of allogeneic and autologous grfts has been further developed, previously unavailable drugs for post-transplant immunosuppression and for the prevention or therapy of infectious complications have been tested. The indication list
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for high-dose therapy and hematopoietic cell transplantation has been extended and the long-term evaluation and management of transplant recipients has been further defined. Still, the editors would caution the young investigator that “breakthroughs” do not occur. Each step towards a cure has to be earned through thoughtfully designed and carefully conducted pre-clinical experiments and clinical trials. Single institution studies require confirmation through prospective randomized trials, each needing several years before a new concept can be fully accepted as a proven advance. The editors hope that the new edition will serve as a resource for physicians and many other members of health care teams at transplant centers and for new and established investigators in the area of hematopoietic cell transplantation and related fields of research; in brief, for everyone who wishes to learn about this exciting field which is now at the beginning of its fifth decade of development as a curative treatment modality for disorders with an otherwise poor prognosis. E. Donnall Thomas Karl G. Blume Stephen J. Forman Summer, 1998
Preface to the Third Edition
Ten years after the first and five years following the publication of the second edition of this textbook, the editors and the publisher, Blackwell Publishing, present now the third edition. Because of the continued impressive growth of knowledge in the field of hematopoietic cell transplantation, a new and completely revised book was needed to document and critically review the scientific progress made during the past five years. The editors wish to emphasize several changes in the third edition compared to the second edition. First, the title of the book has been changed to honor the pioneering work of E. Donnall Thomas, the 1990 Nobel Laureate in Physiology or Medicine (see Tribute). Second, the reader will find ten new chapters which deal with new experimental or clinical research observations while four prior chapters have been deleted. Topics for the new chapters include stem cell expansion, the theory and application of non-myeloablative regimens followed by allogeneic hematopoietic cell transplantation, the experimental basis for transplantation for autoimmune diseases, long-term complications and
others. Third, we have again tried to avoid overlap and contradiction between chapters, however, following academic principles, we have allowed the expression of opposing views and opinions in certain areas, for example, on the topic of plasticity of embryonic and adult stem cells. The editors are indebted to the publishing house which again has produced a high quality product in a timely fashion for the international community of investigators at all professional levels and disciplines in the field of hematopoietic cell transplantation. Finally, this book would not have been possible without the dedicated work of our assistants Sara E. Clark, Stephen J. Loy and Kristine A. Logan. Karl G. Blume Stephen J. Forman Frederick R. Appelbaum Spring 2003
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Preface to the Fourth Edition
Fifteen years after the first, and five years following the third edition of this textbook, the editors and the publisher, Blackwell Publishing, are proud to present the fourth edition of Thomas’ Hematopoietic Cell Transplantation. The impetus for the development of this fourth edition came from the rapid expansion in our understanding of the biologic basis of hematopoietic cell transplantation coupled with its rapidly expanding clinical application. When compared to the last edition, readers will find fourteen entirely new chapters. These new chapters focus on the scientific basis of transplantation (including chapters on embryonic stem cells, mesenchymal stem cells, NK cell biology, and dendritic cell biology), on emerging applications of hematopoietic cell transplantation (including chapters on hematopoietic cell transplantation for chronic lymphocytic leukemia and other less common indications), and on complications of transplantation (including chapters on graft failure, vascular access and complications, interstitial pneumonia, nephro-urologic complications, endocrine complications and common potential drug interactions). While new chapters have been added, others have been dropped or their materials combined in an effort to avoid redundancies. The remaining chapters have been revised and updated to reflect the current state of the science.
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For the first time, a CD-ROM accompanies this textbook. The CDROM contains the entire text of the book in easily searchable form, as well as all figures in a convenient downloadable format. We are hopeful that our readers will find this addition useful. The editors greatly appreciate the assistance of Blackwell Publishing, which has been extremely helpful in the process of creating this volume and in producing a finished product of the highest quality. Also, this book would not have been possible without the dedicated work of our assistants, Kimberly Laramie, Sara E. Clark-Fuentes, and especially, Tanya Chiatovich. Finally, this edition honors the memory of Dr. Ernest Beutler whose laboratory and clinical contributions helped begin the field of transplantation as a curative therapy for patients suffering from leukemia. He was a colleague and mentor for many people who have made significant contributions to the field of hematopoietic cell transplantation. Frederick R. Appelbaum, MD Stephen J. Forman, MD Robert S. Negrin, MD Karl G. Blume, MD
Tribute
Thomas HCT Fourth Edition
Dr. E. Donnall Thomas and King Carl Gustaf of Sweden. Dr. Thomas and Dr. Joseph Murray (not shown in the picture) shared the Nobel Prize for Physiology or Medicine, 1990, recognizing the field of organ transplantation. Dr. Thomas was honored for endeavors in experimental and clinical bone marrow transplantation. Their prizes emphasize the importance of patient-related research.
Our textbook title honors Dr. E. Donnall Thomas, the single individual most responsible for creating the subject of this book. Don was born on March 15, 1920, the son of a solo general practitioner in a small Texas village. He recalls as a child accompanying his father to his small office and to patients’ homes. As Don has mentioned, between he and his father, they span the period from horse and buggy house calls to our modern high-tech medicine. He received his B.A. and M.D. from the University of Texas and that is where he met his wife Dottie. Besides raising three children and eight grandchildren together, Dottie has been Don’s partner in every aspect of his professional life, from working in the laboratory, to editing manuscripts and administering grants. Anyone who has been lucky enough to work with Don knows that if he is the father of marrow transplantation, then Dottie is, without question, the mother.
Don graduated from Harvard Medical School in 1947 and completed his internship and residency at Peter Brent Brigham Hospital in Boston. It was while in medical school that Don first became interested in normal and malignant hematopoiesis. During those years, Sydney Farber was initiating his first studies of the use of antifolates to treat children with acute leukemia and Don witnessed the very first patient to achieve a remission with this approach. He was exposed to the pioneering work of Allan Erslev and his search for erythropoietin. Most importantly, he learned of Leon Jacobsen and his studies showing that shielding the spleen protected mice from the otherwise lethal effects of total body irradiation. As data emerged that a similar irradiation protection effect could be achieved by transferring bone marrow from a non-irradiated to an irradiated mouse, Don became convinced of the clinical potential of marrow transplantation.
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In 1955, Don moved to Cooperstown, New York and the Imogene Basset Hospital, a Columbia University affiliate, where he began working on marrow transplantation both in the canine model and in humans with Dr. Joseph Farrebee. In 1957, Don published the first report in human patients showing that complete remissions of leukemia could be achieved using total body irradiation followed by infusion of marrow from an identical twin. At that time, there was little understanding of the principles of human histocompatibility and so attempts to expand these studies to patients without identical twins were uniformly unsuccessful. These failures were the stimulus for a long series of experiments conducted by Don in the canine model showing it was possible to expose dogs to supralethal doses of irradiation and rescue them by reinfusing their own marrow, that the marrow could be cryopreserved, and that large doses of peripheral blood could substitute for marrow. But attempts at allogeneic transplantation in this outbred species continued to fail because of graft-versus-host disease or graft rejection. In 1963, Don moved to Seattle and the University of Washington to become the first head of the Division of Oncology. There he developed techniques for rudimentary histocompatibility typing in the dog and by the mid-1960s showed that by selecting matched donors and using methotrexate post-transplant, it was possible to successfully transplant marrow between matched littermates in almost every case. At the same time, based on the work of Dausset, Payne, Amos and others, the understanding of human histocompatibility also dramatically increased and so, in the late 1960s, Don made the decision to return to the subject of allogeneic transplantation in humans. He began to assemble a team of physicians, nurses and support personnel (many of whom are still part of the current Seattle Transplant Program) and obtained a program project grant from the National Cancer Institute. In November 1968, Dr. Robert Good and his colleagues carried out the first marrow transplant from a matched sibling for an infant with immunodeficiency and in March 1969, Don performed the first matched sibling donor transplant for a patient with leukemia. The Seattle Transplant Program was originally housed at the Seattle Public Health Hospital but in 1972, when the hospital was faced with closure by the federal government, the Program moved to Providence Hospital. In 1975, Don and his team moved to the newly created Fred Hutchinson Cancer Research Center, a move that provided Don with increased space, resources and scientific collaborations. That same year, he and his colleagues published their classic New England Journal of Medicine paper summarizing the field of allogeneic transplantation and particularly the early Seattle experience. These results demonstrated not
only the feasibility of the procedure, but also that there was a plateau on the survival curves following transplantation, suggesting that some of these patients were cured with this novel technique. Don continued to lead the Clinical Research Division of the Center and its transplant program until his partial retirement in 1989. Yet he continues to work writing manuscripts, delivering lectures and participating in research discussions at the Center. Appropriately, Don has received almost every possible award for his work, including the American Society of Hematology’s Henry M. Stratton Award, the General Motor’s Kettering Prize, the American Society of Oncology’s Karnofsky Award, the Presidential Medal of Science, and of course, the 1990 Nobel Prize in Medicine which he shared with Joseph Murray. With each award, Don has always emphasized how much his work was a team effort. He invariably mentions the contributions of Rainer Storb, Dean Buckner, Reg Clift, Paul Neiman, Alex Fefer, and Bob Epstein, who helped form the original Seattle Transplant Team, and of Ted Graham, who moved with Don from Cooperstown to help in the animal research. Don never fails to credit the nursing and support staff who played such a critical role in these efforts, and he always acknowledges the patients and their families who have been true partners in his work. Although Don is most noted for his pioneering scientific achievements, for those of us who have been fortunate enough to work with him, Don is equally admired for the way in which he achieved his success. He has always been focused, hardworking and uncompromising in his laboratory and clinical research. He can be a demanding critic and holds others accountable for their actions, yet at the same time he has always been generous with his ideas, loyal to his employees and quick to deflect praise to his coworkers. Although he has dedicated an enormous amount of himself to a life in medical science, he has managed to maintain a great deal of balance. He has traveled widely as a visiting lecturer, donated time to professional organizations and supported local and international charities. He and Dottie are avid and expert hunters and fishers and have a close and loving extended family. It is difficult to think of many other fields of modern medicine that are so much the result of a single man’s work. Don’s vision and dedication have changed the lives of literally hundreds of thousands of patients. By naming this textbook for him, we are joining those patients, and the many nurses, physicians and staff privileged to work in the field he created, in thanking Don both for what he has accomplished and for the way he has done it.
List of Abbreviations
AABB AAV ABMTR ABW ACC ACEI ACIF ACIP ACP ACTH ACV AD ADA ADCC ADL ADP ADR AEL AF AFO AFP AGM AGU AH AHCT AHRQ AIBW AICD AIDS AIHA AITD AITL AKI ALC ALCL ALD ALG ALI ALL ALT AMKL AML AMM
= American Association of Blood Banks = Adeno-Associated Virus = Autologous Blood and Marrow Transplant Registry – North America = Actual Body Weight = Acute Chest Syndrome = Angiotensin Converting Enzyme Inhibitor = Anticomplement Immunofluorescence = Advisory Committee on Immunization Practices = Advanced Care Planning = Adrenocorticotropic Hormone = Acyclovir = Autoimmune Disease = Adenosine Deaminase = Antibody Dependent Cell Mediated Cytotoxicity = Activities of Daily Living = Adenosine Diphosphate = Adverse Drug Reaction = Acute Erythroleukemia = Amniotic Fluid = Air Flow Obstruction = α-Feto Protein = Aorta-Gonad-Mesonephros (Region) = Aspartylglucosaminuria = Ancestral Haplotype = Autologous Hematopoietic Cell Transplantation = Agency for Health Care Research and Quality = Adjusted Ideal Body Weight = Activation Induced Cell Death = Acquired Immune Deficiency Syndrome = Auto-Immune Hemolytic Anemia = Autoimmune Thyroid Disease = Angioimmunoblastic T-Cell Lymphoma = Acute Kidney Injury = Absolute Lymphocyte Count = Anaplastic Large Cell Lymphoma = Adrenoleukodystrophy = Antilymphocyte Globulin = Acute Lung Injury = Acute Lymphoblastic Leukemia = Alanine Aminotransferase = Acute Megakaryoblastic Leukemia = Acute Myeloid Leukemia = Agnogenic Myeloid Metaplasia
AMN ANA ANC ANRC AP APC APL ARAC ARB ARDS ARF ARL ARSA ASBMT ASHI AST ATG ATL ATP ATRA ATTL AUC AVN AYA AZT B2M BACT BAL BAVC BCAA BCMA BCNU BCG BCR BCR BCRP BDNF BDP BEAC BEAM
= Adrenomyeloneuropathy = Anti-Nuclear Antibody = Absolute Neutrophil Count = Anthony Nolan Research Center = Alkaline Phosphatase = Antigen Presenting Cell = Acute Promyelocytic Leukemia = Cytosine Arabinoside = Angiotensin Receptor Blocker = Adult Respiratory Distress Syndrome = Acute Renal Failure = AIDS Related Lymphoma = Arylsulfatase A = American Society for Blood and Marrow Transplantation = American Society for Histocompatibility and Immunogenetics = Aspartate Aminotransferase = Antithymocyte Globulin = Adult T-Cell Leukemia = Autoimmune Thrombocytopenia = All-Transretinoic Acid = Acute T-Cell Leukemia/Lymphoma = Area under the Curve = Avascular Necrosis = Adolescent and Young Adult = Zidovudine = Beta-2-Microglobulin = BCNU, Cytosine-Arabinoside, Cyclophosphamide, Thioguanine = Bronchoalveolar Lavage = BCNU, Amsacrine, Etoposide, Cytosine-Arabinoside = Branched Chain Amino Acid = B Cell Maturation Antigen = 1,3-Bis(2-Chloroethyl)-1-Nitrosourea (Carmustine) = Bacillus Calmette-Guerin = B Cell Receptor = Breakpoint Cluster Region = Breast Cancer Resistance Protein = Brain Derived Neurotropic Factor = Beclomethasine-17, 21-Dipropionate = BCNU, Etoposide, ARA-C, Cyclophosphamide = BCNU, Etoposide, Cytosine Arabinoside, Melphalan
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List of Abbreviations
BED BEE BEP BES BFU-E BL-CFC BLS BM BMC BMD BMDW BMI BMP BMSC BMT BMTSS BNLI BO BOOP BOS BSA BSE BSI BU BUN
= = = = = = = = = = = = = = = = = = = = = = = = =
CA11 CAFC CALGB CA-MRSA CAR CARES CBC CBER CBSC CBV CC CCI CCI CCNU CCR CCSG CD CDAD CDC CDC CDK CDRH CEH CFU-B CFU-BLAST CFU-F CFU-GEMM
= Carbonic Anhydrase Isoenzyme 11 = Cobblestone Area Forming Colony = Cancer and Acute Leukemia Group B = Community Acquired MRSA = Chimeric Antigen Receptor = Cancer Rehabilitation Evaluation System = Cord Blood Cell = Center for Biologics Evaluation and Research = Cord Blood Stem Cell = Cyclophosphamide, BCNU, Etoposide = Complete Chimerism = Charlson Comorbidity Index = Corrected Count Increment = 1-(2-Chloroethyl)-3-Cyclohexyl-1-Nitrosurea = Complete Cytogenetic Response = Children’s Cancer Study Group = Cluster Designation = Clostridium Difficile Associated Diarrhea = Complement Dependent Cytotoxicity = Center for Disease Control = Cyclin/Cyclin Dependent Kinase = Center for Devices and Radiological Health = Conserved Extended Haplotype = Colony Forming Units-B-Lymphocyte = Blast Forming Colony Units = Colony Forming Unit – Fibrolast = Colony Forming Unit – Granulocyte/Erythroid/ Macrophage/Megakaryocytic = Colony Forming Unit – Granulocyte Macrophage = Colony Forming Unit – Megakaryocytic = Colony Forming Unit – Mixed = Colony Forming Unit – Spleen = Colony Forming Unit-T-Lymphocyte = Chronic Granulomatous Disease = Comparative Genomic Hybridization = Current Good Tissue Practice
CFU-GM CFU-MEG CFU-Mix CFU-S CFU-T CGD CGH CGTP
Biologically Effective Dose Basic Energy Expenditure Bleomycin, Etoposide, Cisplatin BMT Events Scale Burst Forming Units – Erythroid Blast-Colony-Forming Cell Bare Lymphocyte Syndrome Bone Marrow Bone Marrow Cell Bone Mineral Density Bone Marrow Donors Worldwide Body Mass Index Bone Morphogenic Protein Bone Marrow Stem Cell Bone Marrow Transplantation Bone Marrow Transplant Survivor Study British National Lymphoma Investigation Bronchiolitis Obliterans Bronchiolitis Obliterans Organizing Pneumonia Bronchiolitis Obliterans Syndrome Body Surface Area Bovine Spongiform Encephalopathy Brief Symptom Inventory Busulfan (Myleran) Blood Urea Nitrogen
CMV-IP CNI CNL CNS CNS CNV COG COHNMC Con A CONSORT COX-2 CP CPR CR CREG CRH CSF CSP CSSCD CT CTC CTL CTLP CTN CTZ CVB CVC CVS CY CYP
= Coronary Heart Disease = Congestive Heart Failure = Cyclophosphamide, Hydroxydaunomycin, Vincristin, Prednisone = Chediak-Higashi Syndrome = Confidence Interval = Center for International Blood and Marrow Transplant Research = Chlorine Channel Pump 7 = Chronic Idiopathic Myelofibrosis = Combined Immunodeficiency Syndrome = Chemical Inducers of Dimerization = Critical Illness Polyneuropathy = Ciprofloxacin = Creutzfeldt-Jakob Disease = Chronic Kidney Disease = Cutaneous Lymphocyte Antigen = Common Lymphocyte Progenitor = Clinical and Laboratory Standards Institute = Chronic Myelogenous Leukemia = Chronic Myelomonocytic Leukemia = Common Myeloid Progenitor = Cytomegalovirus = CMV-Antibody Enriched Intravenous Immunoglobulin = Cytomegalovirus-Associated Interstitial Pneumonia = Calcineurin Inhibitor = Chronic Neutrophilic Leukemia = Central Nervous System = Clinical Nurse Specialist = Copy Number Variation = Children’s Oncology Group = City of Hope National Medical Center = Concanavilin A = Consolidated Standards of Reporting Trials = Cyclo-oxygenase-2 (inhibitor) = Chronic Phase = Cardio-Pulmonary Resuscitation = Complete Remission = Cross-Reactive Group = Corticotropin Releasing Hormone = Colony Stimulating Factor = Cyclosporine = Cooperative Study of Sickle Cell Disease = Computerized Tomography = Cyclophosphamide, Thiotepa, Carboplatin = Cytotoxic T-Lymphocyte = Cytotoxic T-Lymphocyte Precursor = Clinical Transplant Network = Chemotactic Trigger Zone = Cyclophosphamide, Etoposide, BCNU = Central Venous Catheter = Chorionic Villous Sampling = Cyclophosphamide = Cytochrome P
DAD DAG DAH DAT DC DCC
= Diffuse Alveolar Damage = Diacylglycerol = Diffuse Alveolar Hemorrhage = Direct Agglutinin Test = Dendritic Cells = Data Coordinating Center
CHD CHF CHOP CHS CI CIBMTR CICN7 CIMF CID CID CIPN CIPRO CJD CKD CLA CLP CLSI CML CMML CMP CMV CMV-IG
List of Abbreviations
DEB DEXA DEXA DFA DFCI DFO DFP DFS DHAP DHFR DHTR DIC DISF DKA DLA DLCL DLCO DLI DLT DM DMSO DNA DNAX DNR DPC DPT DRG DRS DSMB DSRCT DST DTH DTPA
= Diepoxybutane = Dual Energy X-ray Absorptiometry = Dual Energy X-ray Absorptiometry = Direct Fluorescent Antibody (Test) = Dana-Farber Cancer Institute = Desferrioxamine = Deferiprone = Disease-Free Survival = Decadron, High dose Cytarabine, Cisplatinum = Dihydrofolate Reductase = Delayed Hemolytic Transfusion Reaction = Disseminated Intravascular Coagulation = Derogatis Interview of Sexual Functioning = Diabetic Ketoacidosis = Dog Leukocyte Antigen = Diffuse Large Cell Lymphoma = Diffusion Capacity = Donor Lymphocyte Infusion = Dose Limiting Toxicity = Diabetes Mellitus = Dimethylsulfoxide = Deoxyribonucleic Acid = DNA Accessory Molecule = Do Not Resuscitate = Days Post Conception = Diphtheria/Pertussis/Tetanus = Dorsal Root Ganglion = Disability Rating Scale = Data and Safety Monitoring Board = Desmoplastic Small Round Cell Tumor = Donor Specific Transfusion = Delayed Type Hypersensitivity = Diethylenetriaminepentaacetic Acid
EACA EAE EB EBMT
= Epsilon Amino Caproic Acid = Experimental Autoimmune Encephalomyelitis = Embryoid Body = European Group for Blood and Marrow Transplantation = Epstein-Barr (Virus) Nuclear Antigen = Epstein-Barr Virus = Epstein-Barr Virus Associated Lymphoproliferative Disorder = Extracorporal Adsorption Therapy = Electrocardiogram = Extracellular Matrix = Eastern Cooperative Oncology Group = Extra Corporal Phototherapy = Early Death = Erectile Dysfunction = Etoposide, Dexamethasone, Cytosine Arabinoside, Cisplatin = Expanded Disability Status Scale = European Federation for Immunogenetics = Event-Free Survival = Enzyme-Linked Immunosorbent Assay = Eutectic Mixture of Local Anesthetic = European Neuroblastoma Study Group = European Organization for Research and Treatment of Cancer = Erythropoietin = Endoplasmic Reticulum
EBNA EBV EBV-LDP ECAT ECG ECM ECOG ECP ED ED EDAP EDSS EFI EFS ELISA EMLA ENSG EORTC EPO ER
xxv
ERCP ERT ESA ESBL ESC ESFT ESRD ET ETP EWOG
= Endoscopic Retrograde Cholangiopancreatography = Enzyme Replacement Therapy = Erythropoiesis Stimulating Agent = Extended-Spectrum beta-Lactamase = Embryonic Stem Cell = Ewing Sarcoma Family of Tumors = End Stage Renal Disease = Essential Thrombocythemia = Early Thymic Progenitor = European Working Group
FA FAB FACS FACT
FLC FLIC FLIPI FLU FLV FMF FNA FSCL FSH 5-FU FUO FVC
= Fanconi Anemia = French American British Classification = Fluorescence Activated Cell Sorter = Foundation for the Accreditation of Cellular Therapy = Foundation for the Accreditation of Hematopoietic Cell Therapy = Fluorescent Antibody to Membrane Antigen = Fas Ligand = Facilitator Cell = Famciclovir = Food and Drug Administration = Forced Expiratory Ventilation in 1 Second = Fresh Frozen Plasma = Fred Hutchinson Cancer Research Center = Familial Hemophagocytic Lymphohistiocytosis = Fluorescence in-situ Hybridization = Tacrolimus = Tacrolimus (FK) Binding Protein = Follicular Lymphoma = Face, Legs, Activity, Chest X-ray, Consolability (Pain Assessment Tool) = Free Light Chain = Functional Living Index – Cancer = Follicular Lymphoma International Prognostic Index = Fludarabine = Friend Murine Leukemia Virus = Flow Microfluorometry = Fine Needle Aspiration = Follicular Small Cleaved Cell Lymphoma = Follicle Stimulating Hormone = 5-Fluorouracil = Fever of Unknown Origin = Forced Volume Capacity
GAD GAD65 GAG GALC GALT GALV GAP GAVE G-CSF GCV GDP GELA GEP GF GFJ GFP
= Glutamic Acid Decarboxylase = Glutamic Acid Decarboxylase 65 = Glycosaminoglycan = Galactoceribrosidase = Gut Associated Lymphoid Tissue = Gibbon Ape Leukemia Virus = GTP-ase Activating Protein = Gastric Antral Vascular Ectasia = Granulocyte-Colony Stimulating Factor = Ganciclovir = Guanosine Diphosphate = Groupe d’Etude des Lymphomes d’Adulte = Gene Expression Profiling = Graft Failure = Grapefruit Juice = Green Fluorescent Protein
FAHCT FAMA FasL FC FCV FDA FEV1 FFP FHCRC FHL FISH FK506 FKBP FL FLACC
xxvi
List of Abbreviations
GITMO GLD GLR GLSG GMBP GM-CSF GMP GMP GnRH GOG G-6-PD GRE GRH GSH GSSG GST GTP GTP GU GvA GVHD GVLE
= Glomerular Filtration Rate = Growth Hormone = Growth Hormone Releasing Hormone = German Hodgkin’s Lymphoma Study Group = Gastro-Intestinal (Tract) = Gruppo Italiano Malattie Ematologiche Maligne dell’ Adulto = Gruppo Italiano Trapianti di Midolio Osseo = Globoid Cell Leukodystrophy = Glomerular Filtration Rate = German Lymphoma Study Group = Guinea Pig Myelin Basic Protein = Granulocyte/Macrophage-Colony Stimulating Factor = Good Manufacturing Practices = Granulocyte Macrophage Progenitor = Gonadotropin Releasing Hormone = Gynecologic Oncology Group = Glucose-6-Phosphate Dehydrogenase = Glucocorticoid Response Elements = Gonadotropin-Releasing Hormone = Glutathione (Reduced) = Glutathione (Oxidized) = Glutathione-S-Transferase = Good Tissue Practice = Guanosine Triphosphate = Genitourinary = Graft-versus-Autoimmunity = Graft-versus-Host Disease = Graft-versus-Leukemia Effect
4-HC HA HAART HAMA HBV HC HC HCFA HCG HCoV HCT HCV HD HD-AC HDC HDC HD-CY HDIT HDR HDT HES HES HHV6 HIPAA HIV HL HLA HLH HMPV HPC HPLC HPP
= 4-Hydroperoxycyclophosphamide = Hemagglutinins = Highly Active Anti-Retroviral Therapy = Human Antimouse Antibody = Hepatitis B Virus = Hematopoietic Cell = Hemorrhagic Cystitis = Health Care Financing Administration = Human Chorion Gonadotropin = Human Coronavirus = Hematopoietic Cell Transplantation = Hepatitis C Virus = Hodgkin Disease = High Dose ARAC = High Dose Chemotherapy = High Dose Conditioning = High Dose Cyclophosphamide = High Dose Immunosuppressive Therapy = Hypersensitivity Delayed Reaction = High Dose Therapy = Hydroxyethyl Starch = Hypereosinophilic Syndrome = Human Herpes Virus 6 = Health Insurance Portability and Accountability Act = Human Immunodeficiency Virus = Hodgkin Lymphoma = Human Leukocyte Antigen = Hemophagocytic Lymphohistiocytosis = Human Metapneumovirus = Hematopoietic Progenitor Cells = High-Pressure Liquid Chromatography = High Proliferative Potential
GFR GH GHRH GHSG GI GIGEMA
HPRT HPFH HPV HRPBC HRQOL HRSA HRT HSA HSC HSP HSV HSV-tk HTC HTLP HTLV HU HUVEC HUS HVEM HVG HVR
= Hypoxanthine-Guanine Phosphoribosyltransferase = Hereditary Persistence of Fetal Hemoglobin = Human Papillomavirus = High Risk Primary Breast Cancer = Health Related Quality of Life = Health Resources and Services Administration = Hormone Replacement Therapy = Heat Stable Antigen = Hematopoietic Stem Cell = Heat Shock Protein = Herpes Simplex Virus = Herpes Simplex Virus Thymidine Kinase = Homozygous Typing Cells = Helper T-Lymphocyte Precursor = Human T-cell Lymphotropic Virus = Hydroxyurea = Human Umbilical Cord Endothelial Cells = Hemolytic Uremic Syndrome = Herpesvirus Entry Mediator = Host-versus-Graft (Reaction) = Hyper-Variable Region
IA IAA IBD IBMTR IBW ICA ICAM ICH ICL 670 ICOS ICU iDC IDDM IDE IDSA IE IEF IFAR IFI IFN IFRT IG IGF-1 IHC IIP IL IL-1 IM IMPDH IMUST
= Invasive Aspergillus (Infection) = Insulin Autoantibody = Inflammatory Bowel Disease = International Bone Marrow Transplant Registry = Ideal Body Weight = Islet Cell Antibody = Intracellular Adhesion Molecule = International Conference on Harmonization = Deferasirox = Inducible Costimulator (Protein) = Intensive Care Unit = Immature Dendritic Cells = Insulin Dependent Diabetes Mellitus = Investigational Device Exemption = Infectious Diseases Society of America = Immediate Early (Antigen Expression) = Isoelectric Focusing = International Fanconi Anemia Registry = Invasive Fungal Infection = Interferon = Involved Field Radiotherapy = Immunoglobulin = Insulinlike Growth Factor-1 = International Conference on Harmonization = Idiopathic Interstitial Pneumonia = Interleukin = Interleukin-1 = Infectious Mononucleosis = Inosine Monophosphate Dehydrogenase = International Marrow Unrelated Search and Transplant = Investigational New Drug = International Neuroblastoma Research Group = International Neuroblastoma Staging System = Intraoperative Radiation Therapy = Interstitial Pneumonia = Intraperitoneal = Inositol-l, 4, 5-Triphosphate = Inherited Paternal Antigen = Invasive Pulmonary Aspergillosis
IND INRG INSS IORT IP IP IP3 IPA IPA
List of Abbreviations
IPI IPS IPS IPSS IRB IS IS ISCT ISO ISS IST IT ITD ITIM ITR IU-HCT IV IVF IVIG
= = = = = = = = = = = = = = = = = = =
JACIE
= Joint Accreditation Committee of ISCT-Europe and EBMT = Joint Commission of Health Care Organizations = Juvenile Idiopathic Arthritis = Juvenile Myelomonocytic Leukemia
JAHCO JIA JMML
International Prognostic Index Idiopathic Pneumonia Syndrome Interstitial Pneumonia Syndrome International Prognostic Scoring System Institutional Review Board Immunosuppressive Immune Synapse International Society for Cellular Therapy Isohemagglutinin International Staging System Immunosuppressive Therapy Intrinsic Timetable (Model) Internal Tandem Duplication Immunoreceptor Tyrosine Binding Inhibition Motif Inverted Terminal Repeats Intrauterine-Hematopoietic Cell Transplantation Intravenous In Vitro Fertilization Intravenous Immunoglobulin
KLH KPC KPS
= Keratoconjunctivitis Sicca = Killer-Cell Inhibitory Receptor or Killer Ig-like Receptor = Keyhole Limpet Hemocyanin = Klebsiella Pneumoniae Carbapenemase = Karnofsky Performance Score
LA LAD LAK LAMP LAT LC LCH LCL LCR LCT LD LDA LDH LET LFA LFR LFS LH LHRH LIF LIN LMP LMWH LOH L-PAM LPC LPD LPHD LPR
= Latex Agglutination = Leukocyte Adhesion Deficiency = Lymphokine Activated Killer (Cells) = Loop Mediated Isothermal Amplification = Latency Associated Factor = Langerhans Cell = Langerhans Cell Histiocytosis = Lymphoblastic Cell Lines = Locus Control Region = Long Chain Triglyceride = Linkage Disequilibrium = Limiting Dilution Assay = Lactate Dehydrogenase = Linear Energy Transfer = Leukocyte Function-Associated Antigen = Limited Field Radiation = Leukemia-free Survival = Luteinizing Hormone = Luteinizing Hormone Releasing Hormone = Leukemia Inhibitory Factor = Lineage = Latency Membrane Protein = Low Molecular Weight Heparin = Loss of Heterozygosity = L-Phenylalanine Mustard = Leukemic Progenitor Cell = Lymphoproliferative Disease = Lymphocyte Predominant Hodgkin Disease = Lipoprotein Receptor-Related Protein
KCS KiR
xxvii
LPS LTA LTC-IC LTR LUTS LV LVEF LY
= = = = = = = =
MAB MACS MALT MAOI MAPC MAPC MAPK MBC MBL MBP MCA MCD MCL MCL MCP MCR M-CSF MCT mDC MDR MDS MEAC MEC MEG-CSA MEG-GPA MEL MEP mESC MF MG MGDF MGF MGUS
= Monoclonal Antibody = Multipotent Adult Stem Cell = Mucosa Associated Lymphoid Tissue = Monoamine Oxidase Inhibitor = Mesenchyme Associated Progenitor Cell = Multipotent Adult Progenitor Cell = Mitogen Activated Protein Kinase = Metastatic Breast Cancer = Metallo-beta-Lactamase = Myelin Basic Protein = Middle Cerebral Artery = Minimal Change Disease = Mantle Cell Lymphoma = Mast Cell Leukemia = Monocyte Chemoattractant Protein = Major Cytogenetic Response = Macrophage-Colony Stimulating Factor = Median Chain Triglyceride = Mature Dendritic Cells = Multi Drug Resistance = Myelodysplastic Syndrome = Minimum Effective Analgesic Concentration = Medullary Epithelial Cell = Megakaryocyte-Colony Stimulating Activity = Megakaryocyte-Growth Promoting Activity = Melphalan = Megakaryocytic Erythrocyte Progenitor = Mouse Embryonic Stem Cell = Mycosis Fungoides = Myasthenia Gravis = Megakaryocyte Growth and Development Factor = Mast Cell Growth Factor = Monoclonal Gammopathy of Undetermined Significance = Minor Histocompatibility Antigen = Modified Health Assessment Questionnaire = Major Histocompatibility Complex = Metaiodobenzylguanidine = Minor Histocompatibility Antigens = Medical Internal Radiation Dose = Mucolipidosis = Mixed Leukocyte Culture = Metachromatic Leukodystrophy = Myeloid-Lymphoid-Initiating Cell = Mixed Lineage Leukemia = Mixed Leukocyte Reaction = Maroteaux-Lamy Syndrome = Moloney Leukemia Virus = Multiple Myeloma = Mitomycin C = Mycophenolate Mofetil = Matrix Metalloprotease = Measles/Mumps/Rubella
mHA MHAQ MHC MIBG miHA MIRD ML MLC MLD ML-IC MLL MLR MLS MLV MM MMC MMF MMP MMR
Lipopolysaccharide Latency Associated Transcripts Long Term Culture Initiating Cell Long Terminal Repeat Lower Urinary Tract Syndrome Lenti Virus Left Ventricular Ejection Fraction Life Years
xxviii
List of Abbreviations
MMR MN MODS MOG MOPP MP MP MPA MPB MPO MPP MPS MPSV MPT MPV MR MRC MRD MRI m-RNA MRP MRS MRSA mRSS MS MSC MSC MSCH MSD MSFC MSI MSKCC MS-LCH MSOF MTD MTOR MTX MUGA
= Mannose Macrophage Receptor = Membranous Nephropathy = Multiple Organ Dysfunction Syndrome = Myelin Oligodendrocyte Glycoprotein = Mustargen, Vincristine, Prednisone, Procarbazine = Melphalan Prednisone = Methylprednisolone = Mycophenolic Acid = Mobilized Peripheral Blood (Cells) = Myeloperoxidase = Multipotent Progenitor = Mucopolysaccharidosis = Myeloproliferative Sarcoma Virus = Melphalan, Prednisone, Thalidomide = Metapneumovirus = Molecular Remission = Medical Research Council (United Kingdom) = Minimal Residual Disease = Magnetic Resonance Imaging = Messenger RNA = Multidrug Resistance-Associated Protein = Magnetic Resonance Spectroscopy = Methicillin Resistant Staphylococcus Aureus = Modified Rodman Skin Score = Multiple Sclerosis = Mesenchymal Stem Cell = Mesenchymal Stromal Cells = Mouse Spinal Cord Homogenate = Multiple Sulfatase Deficiency = Multiple Sclerosis Functional Composite = Microsatellite Instability = Memorial Sloan Kettering Cancer Center = Multisystem Langerhans Cell Histiocytosis = Multi-System Organ Failure = Maximum Tolerated Dose = Mammalian Target of Rapamycin = Methotrexate = Multiple Gated Acquisition (Scan)
NAB NANT NAPI NASBA NAT NB NCCN NCI NCIC NCM nCR NCR NEO Nf1 NF-AT NGFR NGVL NHL NIH NIMA NIPA NK NK-T
= Natural Antibody = New Approaches to Neuroblastoma Therapy = North American Pulsed Field Type 1 = Nucleic Acid Sequence-Based Amplification = Nucleic Acid Amplification Test = Neuroblastoma = National Comprehensive Cancer Network = National Cancer Institute = National Cancer Institute of Canada = Nurse Case Manager = Near Complete Response = Natural Cytotoxicity Receptor = Neomycin = Neurofibromatosis type 1 = Nuclear Factor of Activated T Cells = Nerve Growth Factor Receptor = National Gene Vector Laboratory = Non-Hodgkin Lymphoma = National Institutes of Health = Non-Inherited Maternal Antigen = Non-Inherited Paternal Antigen = Natural Killer (Cells) = NK-T cells
NO NOD NPC NRH NRM NS NS NSAID NYBC NZW
= Non-Myeloablative = National Marrow Donor Program = Non-Myeloablative Hematopoietic Cell Transplantation = Nitric Oxide = Non-Obese Diabetic (Mouse) = Non-Protein Calories = Nodular Regenerative Hyperplasia = Non-Relapse Mortality = Nephrotic Syndrome = Natural Suppressor (Cells) = Non-Steroidal Anti-Inflammatory Drug = New York Blood Center = New Zealand White (Mouse)
OCTGT ODN OLD OMIM ONJ ONS OPA OPG OPM OR OS OSTM1
= = = = = = = = = = = =
PACT PAIgG PAIS PB PBHC PBHCT PBMC PBP PBPC PBS PCA PCNSL PCR PD PD PDC PDQ PDT PEC PET PFA PFS PFT PGD PGF PGK Ph PH PHA PHN PHQ PHS PIC PICC
= Psychological Assessment for Transplantation Scale = Platelet Associated Immunoglobulin = Psychological Adjustment to Illness Scale = Peripheral Blood = Peripheral Blood Hematopoietic Cells = Peripheral Blood Hematopoietic Cell Transplantation = Peripheral Blood Mononuclear Cells = Penicillin-Binding Protein = Peripheral Blood Progenitor Cells = Primer Binding Site = Patient Controlled Analgesia = Primary Central Nervous System Lymphoma = Polymerase Chain Reaction = Programmed Death = Protective Dose = Plasmacytoid Dendritic Cell = Physician Data Query = Photodynamic Therapy = Cisplatin, Etoposide, Cyclophosphamide = Positron Emission Tomography = Phosphonoformic Acid (Foscarnet) = Progression-Free Survival = Pulmonary Function Test = Preimplantation Genetic Diagnosis = Poor Graft Function = Phosphoglycerokinase = Philadelphia (Chromosome) = Pulmonary Hypertension = Phytohemagglutinin A = Post-Herpetic Neuralgia = Perceived Health Questionnaire = Public Health Service = Pre-Integration Complex = Peripherally Inserted Central Venous Catheter
NM NMDP NMHCT
Office of Cellular, Tissue and Gene Therapies Oligodeoxynucleotide Obstructive Lung Disease Online Mendelian Inheritance in Man Osteonecrosis of the Jaw Oncology Nursing Society Office of Patient Advocacy Osteoprotegerin Oropharyngeal Mucositis Odds Ratio Overall Survival Osteopetrosis Membrane Protein 1
List of Abbreviations
PIG-A PIH PIQ PIXY 321 P-gp PK PKC PLL PLP PLPHA PLS PLT PMC PML PMR PMRD PN PNET PNH POG POMS PORN PORT POV PR PRA PRCA PRO PRP PSA PSE PTA PTCL PTH PTLD PTT PTX PUVA PV PVP PVR
= Phosphatidyl Inositol Glycan-A = Prolactin Inhibiting Hormone = Performance IQ = Fusion Protein: IL-3/GM-CSF = P-Glycoprotein = Pharmacokinetic = Protein Kinase C = Prolymphocytic Leukemia = Proteolipid Protein = Post-Lumbar Puncture Headache = Psychosocial Level System = Platelet = Persistent Mixed Chimerism = Progressive Multifocal Leukoencephalopathy = Progressive Muscle Relaxation = Partially Matched Related Donor = Parenteral Nutrition = Primitive Neuroectodermal Tumor = Paroxysmal Nocturnal Hemoglobinuria = Pediatric Oncology Group = Profile of Mood Status = Progressive Outer Retinal Necrosis = Patient Outcomes Assessment Research Team = Premature Ovarian Failure = Partial Remission or Partial Response = Panel Reactive Antibody = Pure Red Cell Aplasia = Patient Reported Outcome = Polyribosylphosphate = Prostate Specific Antigen = Prednisone = Peripheral Tissue Antigen = Peripheral T Cell Lymphoma = Parathyroid Hormone = Post-Transplant Lymphoproliferative Disorder = Partial Thromboplastin Time = Pentoxifylline = Psoralen-Ultraviolet A Irradiation (Treatment) = Polycythemia Vera = Polyvinylpyrrolidone = Polio Virus Receptor
QALY QLQ QOL QTWIST
= = = =
RA RA RAC RAEB RAEB-T
= Refractory Anemia = Rheumatoid Arthritis = Recombinant DNA Advisory Committee = Refractory Anemia with Excess Blasts = Refractory Anemia with Excess Blasts in Transformation = Radioimmunotherapy = Receptor Activator of NF-Kb Ligand = Refractory Anemia with Ringed Sideroblasts = Red Blood Cell = Relative Biological Effectiveness = Renal Cell Cancer = Ratio of Cost and Charges = Replication-Competent Lentivirus = Refractory Cytopenia with Multilineage Dysplasia
RAIT RANKL RARS RBC RBE RCC RCC RCL RCMD
Quality-Adjusted Life Years Quality of Life Questionnaire Quality of Life Quality Time Without Symptoms of Toxicity
xxix
RCT RDA REE RFS RHC RIA RIC RIFLE RIR RISC RIT RFLP RLD RNA ROI ROS RPM RQ RR RRT RRT RSCH RSV RT RT3U RTOG
= Randomized Controlled Trial = Recommended Daily Allowance = Resting Energy Expenditure = Relapse-Free Survival = Residual Host Cells = Radioimmune Assay = Reduced Intensity Conditioning = Risk, Injury, Failure, Loss, Endstage = Reduced Intensity Regimen = RNAi Induced Silencing Complex = Radioimmunotherapy = Restriction Frequent Length Polymorphism = Restrictive Lung Disease = Ribonucleic Acid = Region of Interest = Reactive Oxygen Species = Rapamycin (Sirolimus) = Respiratory Quotient = Relative Risk = Renal Replacement Therapy = Regimen-Related Toxicity = Rat Spinal Cord Homogenate = Respiratory Syncytial Virus = Reverse Transcriptase = Resin T3 Uptake = Radiation Therapy Oncology Group
SAA SAE SAP SAS-SR SBA SCC SCD SCF SCID SCID-hu
= Severe Aplastic Anemia = Severe Adverse Event = Serum Amyloid Protein P = Social Adjustment Scale – Self Report = Soy Bean Agglutinin = Squamous Cell Carcinoma = Sickle Cell Disease = Stem Cell Factor = Severe Combined Immunodeficiency Syndrome = Severe Combined Immunodeficiency Syndrome/ Human (Mouse) = Symptoms Check List = Stem Cell Therapeutics Outcomes Database = Standard Deviation = Standard Dose Chemotherapy = Stroma Cell Derived Factor = Shwachman Diamond Syndrome = Standard Error = Sleep, Energy, Appetite Scale = Surveillance Epidemiology and End Result = Symptom Experience Report = Serological Identification of Antigens by Recombinant Expression Cloning = Spleen Focus-Forming Virus = Societe Francaise de Greffe de Moelle = French Society of Pediatric Oncology = Sex Hormone Binding Globulin = Stanford Hospital and Clinics = Sonic Hedgehog = Sickness Impact Profile = Systemic Inflammatory Response Syndrome = Signaling Lymphocyte Activation Molecule = Systemic Lupus Erythematosus = SLE Disease Activity Index = Small Lymphocytic Leukemia
SCL SCTOD SD SDC SDF SDS SE SEAS SEER SER SEREX SFFV SFGM SFOP SHBG SHC Shh SIP SIRS SLAM SLE SLEDAI SLL
xxx
List of Abbreviations
SM SMP SNP SNRI SOP SOS SPA SPECT sPLA2 SRBC SRC SS SSC SSOP SSP SSRI STAMP-I STAMP-V SUMC SVC SWOG
= Systemic Mastocytosis = Sympathetically Mediated Pain = Single Nucleotide Polymorphism = Serotonin-Norepinephrine Reuptake Inhibitor = Standard Operating Procedure = Sinusoidal Obstruction Syndrome = Staphylococcal Protein A = Single Photon Emission Computed Tomography = Secretory Phospholipase A2 = Sheep Red Blood Cell = SCID Repopulating Cells = Sézary Syndrome = Systemic Sclerosis = Sequence Specific Oligonucleotide Probes = Sequence Specific Primer = Selective Serotonin Reuptake Inhibitor = BCNU-Cisplatinum-Cyclophosphamide = Thiotepa-Carboplatin-Cyclophosphamide = Stanford University Medical Center = Superior Vena Cava = Southwest Oncology Group
α-TIF T1DM T3 T4 TAA TAI TAM TAP TBI TC TCA TCD TCD TCIRG1 TCR TDM TdT TERS TERT TFO TGF-β TGN T-GVHD TH TIL TIP TIPS TK TLC TLI TLR TM TMA TMC TMD TMP-SMX
= α-Transinducing Factor = Type 1 Diabetes Mellitus = Triiodothyronine = Thyroxine = Tumor Associated Antigen = Thoraco Abdominal Irradiation = Transplant-Associated Microangiopathy = Transporter-Associated Antigen Processing = Total Body Irradiation = Cytotoxic T cells = Tricyclic Antidepressant = T Cell Depletion = Transcranial Doppler (Ultra-sonography) = T Cell Immune Regulator 1 = T Cell Receptor = Therapeutic Drug Monitoring = Terminal Deoxynucleotidyl Transferase = Transplant Evaluation Rating Scale = Telomerase Reverse Transcriptase = Triple Helix Forming Oligodeoxynucleotide = Transforming Growth Factor Beta = Transgolgi Network = Transfusion Associated Graft-versus-Host Disease = Helper T Lymphocytes = Tumor Infiltrating Lymphocytes = Paclitaxel, Ifosfamide, Cisplatin = Transjugular Intrahepatic Portosystemic Shunt = Thymidine Kinase = Total Lung Capacity = Total Lymphoid Irradiation = Toll Like Receptor = Thrombotic Microangiopathy = Thrombotic Microangiopathy = Transient Mixed Chimerism = Temporomandibular Dysfunction = Trimethoprim-Sulfamethoxazole
TNC TNF TNF-α TNFR TPA TPMT TPN TPO TPO TRAIL TRALJ TREC TREG TRH TRIM TRM TSH TSP TTP TTR
= Total Nucleated Cells = Tumor Necrosis Factor = Tumor Necrosis Factor α = Tumor Necrosis Factor Receptor = Tissue Plasminogen Activator = Thiopurine S-Methyltransferase = Total Parenteral Nutrition = Thrombopoietin = Thyroid Peroxidase = TNF-Related Apoptosis-Inducing Ligand = Transfusion-Related Acute Lung Injury = T Cell Receptor Excision Circles = Regulatory T Cell = Thyrotropin-Releasing Hormone = Transfusion-Related Immune Modulation = Treatment Related Mortality = Thyroid Stimulating Hormone = Tropical Spastic Paraparesis = Thrombotic Thrombocytopenic Purpura = Transthyretin
UAMS UCB UCB UGD URD URI UV
= = = = = = =
VAD VATS VCAM VEGF VGPR VIP VLA VLCFA VNTR VOD VP-16 VRE VSV VSV-G VWF VWMD VZIG VZV
= Vincristine, Adriamycin, Decadron = Video-Assisted Thoracoscopic Surgery = Vascular Cell Adhesion Molecule = Vascular Endothelial Growth Factor = Very Good Partial Remission = Etoposide, Ifosfamide, Cisplatin = Very Late Activation Antigen = Very Long Chain Fatty Acid = Variable Number of Tandem Repeat = Venocclusive Disease = Etoposide = Vancomycin Resistant Enterococcus = Vesicular Stomatitis Virus = Vesicular Stomatitis Rhabdovirus = von Willebrand Factor = Vanishing White Matter Disease = Varicella Zoster Immune Globulin = Varicella Zoster Virus
WAS WBC WBM WBRT WHI WMDA WNV
= Wiskott-Aldrich Syndrome = White Blood Cell = Whole Bone Marrow = Whole Brain Radiotherapy = Women’s Health Initiative = World Marrow Donor Association = West Nile Virus
X-AND
X-Linked Adrenoleukodystrophy
University of Arkansas Medical Science Center Umbilical Cord Blood Unrelated Cord Blood Uridine 5’ Diphosphate Glucoronosyl Transferase Unrelated Donor Upper Respiratory Infection Ultraviolet (Light)
Plate 5.1 Chimeric analysis of yolk sac blood islands with fluorescent embryonic stem cell clones. Tetrachimeric mice were generated by the injection of four distinctly colored single cells into developing blastocysts. The resulting embryos were then analyzed for color combinations observed in the yolk sac, and revealed endothelial and hematopoietic cells of different colors in all individual blood islands, indicating that they developed from more than one cell. (a) A chimeric embryo at the early neural plate stage (EB). A white rectangle indicates an immature blood island. The arrow indicates the direction of tissue extension. al, allantois; am, amnion; ch, chorion; ec, embryonic ectoderm; me, intraembryonic mesoderm; ve, visceral endoderm. (b) Experimental scheme indicating the possible derivations of blood islands and the percentage of each type observed. (c) A type IV chimeric blood island with EGFP, ECFP, and mRFP1 endothelial cells, and ECFP, EGFP, and nonfluorescent hematopoietic cells. (Adapted from [172].)
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
Progression to acute myelogenous leukemia (AML) or myeloid blast crisis, CML LT-HSC
ST-HSC
MPP
CLP
Progenitors (GMP)
CMP Blood cells
Self-renewal (regulated)
EVENTS ACCUMULATED: 1. Bcr-Abl or Aml-1–Eto, or jun-b, etc. 2. Antisenescence (e.g. tert) 3. Antiapoptosis 1 3. Antiapoptosis 2 5. Evasion of immune cells (1–4 events) 6. Activation/overexpression self-renewal genes
Self-renewal (Unregulaed)
blasts
blasts
blasts Leukemic Stem Cell
Plate 5.2 Comparison of stem cell self-renewal during normal hematopoiesis and leukemic transformation. During normal hematopoiesis, the signaling pathways that regulate self-renewal are tightly controlled, allowing proper differentiation to each of the mature blood cell lineages (top). Accumulation of mutations in the self-renewing hematopoietic stem cells (HSCs) over time until dysregulated self-renewal ability is attained, and leukemia propagation is initiated. Dysregulation of self-renewal mechanisms in transformed cancer stem cells leads to uncontrolled self-renewal and the production of leukemic blast cells. The leukemic population capable of sustaining disease is often distinct from the blast population, and may reside in the stem cell or progenitor cell compartment. Importantly, if the transformation event occurs in a progenitor cell, it must endow the progenitor cell with the self-renewal, because the progenitors would otherwise differentiate. See text for abbreviations.
5‘ 3‘
W W‘ C
-
W C C‘
+
a1A2 b 1b 2 c 1c 2
a
-
A
c A a
D a A
b B
c C
3‘ 5‘
+
b B
A1a2 B1B2 C1C2
C c
S asymmetric division
maintenance
A1a2 b 1B2 C1c 2
a1A2 B1b 2 c 1C2
S symmetric division
S self-renewal
stem cell niche
Cell Counts
Plate 6.1 The “silent sister” hypothesis [32]. Top panel: According to this hypothesis the two sister chromatids in metaphase chromosomes carry distinct epigenetic marks (indicated by + and − signs) at centromeric DNA and at certain “stem cell” genes (shown here is a single gene expressed as “A” or silent as “a”). Epigenetic differences at centromeres between sister chromatids are proposed to enable chromatid-specific segregation of chromosomes during mitosis. Such nonrandom partition of chromatids is proposed to regulate the expression of certain genes present on those chromatids in the daughter cells following mitosis. Bottom panel: Selective attachment of microtubules (MT) coming from the “mother” centrosome [96] (indicated by the red dot close to the stem cell niche) to sister chromatids containing the Watson (W) template strand (here defined as the 3′ to 5′ template strand indicated by the solid red line) during a polar, asymmetric stem cell division. Shown are three metaphase chromosomes (e.g. out of the 46 chromosomes in a human cell). The preference for one of the chromatids could result from a gradient of MT guiding proteins (red) that regulate preferential MT binding to the kinetochore at the sister chromatid containing the Watson template strand. Selective partition of sister chromatids results in one stem cell (S) with two active copies of “self-renewal” genes (the two parental copies of these genes are indicated by 1 and 2) ready to be transcribed (A, B, and C) and one cell committed to differentiate (D) in which expression of the two copies of the “stem cell” genes is suppressed (a, b, and c). Note that an occasional sister chromatid exchange event between a centromere and a relevant “stem cell” gene (illustrated as a2 in cell S and A2 in cell D) is not incompatible with the “silent sister” hypothesis. In stem cells (S) expression of silenced “stem cell” genes is predicted to be restored by default (bottom panel, right-hand side), and aberrant expression of a single “stem cell” gene in a differentiating cell (A2 in cell D) is not predicted to prevent differentiation altogether. However, inappropriate expression of multiple “self-renewal” genes (perhaps in combination with inappropriate suppression of tumor suppressor genes) in differentiating cells could result in abnormal cell proliferation. Such defects could result from high levels of sister chromatid exchange, defects in cell polarity, failure to establish or recognize epigenetic marks at centromeres or other factors involved in the proposed strand-specific chromatid segregation pathways. (c) Random segregation of sister chromatids restores expression of “silent” stem cell genes and results in self-renewal of stem cells. Without a polar distribution of guiding proteins (even red color), both daughter cells inherit a random mixture of active and inactive “stem cell” genes, which is predicted to restore the expression of the “stem cell” genes in both daughter cells if both cells are in the proper microenvironment (the stem cell “niche”). Epigenetic differences between sister chromatids are restored following expression during replication. Note that only one out of eight possible expression patterns for the three genes in the two daughter cells following random sister chromatid segregation is shown.
MSCs
Plate 9.1 A panel of data demonstrating the defining characteristics of mesenchymal stromal cells (MSCs): adherence, immunophenotype, and in vitro differentiation. Top left: Photomicrograph of undifferentiated MSCs showing the characteristic spindle shape and adherent properties of the cells (original magnification ×40). Top right: Flow cytometry histograms demonstrating the typical expression pattern of surface antigens (—) and isotype control (- - -), as indicated. Bottom: Immunocytochemical staining demonstrating the differentiation of MSCs into osteoblasts (Alizarin red stain), adipocytes (oil red O stain), and chondroblasts (Alcian blue stain). (Reproduced from [43], with permission.)
Osteoblasts
CD105
CD45
CD34
CD73
CD11b
CD19
CD90
CD3
HLA-DR
Log Fluoresence Intensity
Adipocytes
Chondroblasts
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Plate 14.1 Evaluation of murine graft-versus-host disease (GVHD) lingual tissue for apoptosis, T-cell infiltration, and cytokeratin 15 expression. (a) Light microscopy of hematoxylin and eosin-stained tissue from GVHD at day 11 after bone marrow transplantation reveals lymphocytic infiltration and apparent apoptosis in the rete-like prominences (RLPs) (arrow). (b) Apoptosis restricted to RLPs was confirmed by TUNEL immunohistochemistry (arrows). (c) Transmission electron microscopy demonstrates RLP infiltrated by a lymphocyte (left arrow) in apposition to an epithelial cell undergoing mitotic division (right arrow). (d,e) Immunohistochemistry for CD4 at day 4 (d) and day 7 (e) after bone marrow transplantation with progressive infiltration of RLPs by CD4+ T cells. Note the focus where CD4+ T cells ((e); arrow) surround a centrally located nonreactive cell ((e) inset). (f,g) K15 expression in sections adjacent to (d) and (e), respectively, reveals focal loss of K15 immunoreactivity ((f); arrow). Note the cell strongly positive for K15 and surrounded by nonreactive cells ((g); arrow and inset), a pattern reciprocal to the inset in (e) and suggestive of satellitosis. (Adapted from [98], with permission.)
Plate 21.1 Theoretical models of drug resistance. In the conventional model of drug resistance (a) a subpopulation of cells (yellow) have pre-existing genetic variations that confer multidrug resistance (MDR). Following chemotherapy, the resistant cells survive and proliferate, forming a recurrent tumour that is composed of the drug-resistant clone. In the cancer stem-cell model (b), tumors have a small population of tumor stem cells (red) and their differentiated progeny (blue). Following chemotherapy, the differentiated cells are killed, but the stem cells survive. In the “acquired resistance” stem-cell model (c), the tumor stem cells (red) survive therapy, and differentiated cells are killed. Mutations in the surviving stem cells (yellow) and their progeny (purple) may also display the drug-resistant phenotype. In the “intrinsic resistance” model (d), both the stem cells (yellow) and the differentiated cells (purple) are inherently drug resistant. (From [34], with permission.)
Plate 23.1 Dose distribution through T5–6 showing 6, 8, 10, 12, and 13.2 Gy isodose lines. The prescription dose was 12 Gy, with 50% transmission lung blocks.
Plate 23.2 Color wash demonstrating the typical dose distribution of a patient treated with targeted total body irradiation using tomotherapy. The target structure is skeletal bone. A, anterior; L, left; R, right. (Reproduced from [4], with permission from the American Society for Blood and Marrow Transplantation.)
Plate 23.3 Color wash demonstrating dose distribution of 30 Gy to the site of a chloroma in the maxillary sinus and high neck, combined with 12 Gy total marrow irradiation, in a patient with acute myeloid leukemia.
(a)
(b)
Plate 25.1 Assessment of chimerism in sorted granulocytes (a) and leukemic blasts (b) by dual-color fluorescence in situ hybridization (FISH). Hybridization was carried out with a biotin-labeled Y chromosome-specific DNA probe and a digoxigenin-labeled X chromosome-specific DNA probe. Hybridization was detected with Texas Red-conjugated avidin and fluorescein-conjugated antibody against digoxigenin. Cells were counterstained with 4′,6-diamidino-2phenylindole API. Male cells contain a single red fluorescent spot and a single green fluorescent spot, while female cells contain two green fluorescent spots and no red fluorescent spots. In this case, granulocytes were derived from a male donor, and leukemic blasts were derived from the female recipient.
14 14
der(4) der ?
4
(b)
(a)
(c)
(d)
(e)
Targeted scanning with CD19 monoclonal antibody CD19
+
lymphoblast
der(9)
9
22 BCR/ABL (f)
(g)
CD10 positive
BCR–ABL positive
(i)
(h) CD10 negative
BCR–ABL negative
Plate 26.1 Plasma cell-specific fluorescence in situ hybridization (FISH) analysis. (a–e) Cytospin slides are made from the bone marrow aliquot and stained with May-Grünwald stain. After the cells have been mapped to the slide, the slide is destained and hybridized with the selected DNA probe. For details, see [5]. (a) A plasma cell that has been mapped. This same cell is seen in panel (b) showing a variant immunoglobulin H (IGH) pattern using a dual-color 5′IGH/3′IGH break apart probe. Once an IGH gene rearrangement is found in a patient with multiple myeloma, the same slide is rehybridized to determine its partner chromosome in the IGH translocation. (c) A fusion signal is detected with the FGFR3 (fibroblast growth factor receptor-3)/IGH probe indicating the derivative chromosome 4 of the t(4;14) or der(4)t(4;14). The second fusion that would have indicated the der(14)t(4;14) is not present in this patient, the result of loss of the derivative 14 chromosome. In another slide from this patient (d and e), monosomy 13 is shown in the neoplastic plasma cells, but the segmented neutrophil adjacent to the plasma cells shows both chromosome 13 signals (one each in each of the segments of the nucleus). The chromosome 13 probe set used was composed of D13S319 probe (red), which maps to 13q14.3, and the LAMP1(lysosomal-associated membrane protein1)/13q34 reference probe labeled in green. Sequential immunohistochemistry (IHC)/FISH is shown in (f–i). (f) A CD19+ lymphoblast that is BCR/ABL1 fusion gene positive (g), indicating residual disease and not a hematogone. Similarly, (h) and (i) show the cell that is CD10+ is positive for the BCR/ABL1 fusion gene, and the CD− cell is negative for the BCR/ABL1 fusion gene. The BCR/ ABL1 probe used for the sequential IHC/FISH studies was the single fusion probe spiked with 9q34/argininosuccinate synthetase gene probe labeled in Spectrum aqua. All probes were obtained from Abbott Molecular Inc. (Des Plaines, IL).
Plate 26.2 Flow cytometry assays for detection of minimal residual disease. Leukemia samples can be labeled with multiple fluorescent antibodies, and cells can be distinguished by the size of the cell and the types of antibody that bind the cell surface. Normal hematopoiesis has very stereotypical cell antigen expression linked to differentiation; leukemia cells are distinguished by inappropriate expression patterns of these antibodies. In the example, a residual acute monocytic leukemia is analyzed with multiple antigens. In the bottom left panel, the sample shows an inappropriate expression of CD56 and CD4 consistent with residual disease. In this example, these cells made up 0.9% of the total nucleated cells. (Courtesy of Dr Brent Wood, University of Washington.)
Plate 26.3 Quantitative polymerase chain reaction (PCR). The cartoon shows the principle behind the two most popular quantitative approaches: the Taqman (ABI) and the LightCycler (Roche). In the Taqman (I) reaction, the probe 5′ reporter dye and a 3′ quencher molecule. When the probe is intact, the quencher blocks laser excitation of the reporter. During PCR, the 5′ exonuclease activity of Taq polymerase releases the reporter, which, free from the quencher, is excited by the laser detection system. In the LightCycler system (II), annealing of juxtaposition probes during PCR synthesis allows for the transference of resonance energy that can be stimulated by the laser and detected. In both systems, a standard curve is generated that plots dilutions of the standards versus the PCR cycle number at which a threshold signal is obtained (middle panel). The starting amount of target in unknown samples is thus detected by a backcalculation of cycle threshold in the unknown (Ct) against the standard curve. (Courtesy of Dr John Goldman.)
Plate 27.1 Fatal Mylotarg-associated sinusoidal obstruction syndrome. The obliterated central venule is surrounded by focal sinusoidal fibrosis. (Trichrome stain)
Plate 27.2 Markedly hypocellular marrow without evidence of engraftment, day 7. The interstitial fluid accumulation, fat necrosis, and iron deposition are consistent with chemoirradiation damage.
Plate 27.3 Post-transplant Epstein–Barr virus-associated lymphoma infiltrating the intestinal mucosa in a boy treated for graft-versus-host disease (GVHD) with cyclosporine and a monoclonal antibody to T lymphocytes. Although the GVHD improved, large bizarre cells with atypical nuclei characteristic of a high-grade post-transplant lymphoproliferative disorder rapidly formed tumor nodules in liver, spleen, lymph nodes, and mesentery.
(a)
(b)
Plate 27.4 (a) Unsuspected Epstein–Barr virus post-transplant lymphoproliferative disorder. After the first hematopoietic cell transplantation (HCT), the patient developed severe liver graft-versus-host disease. Donor lymphocyte infusion was followed by loss of engraftment. A second HCT was carried out following conditioning with antithymocyte globulin, Campath, and steroids. Jaundice developed 35 days later. At low power, the portal space contains a prominent infiltrate that extends outward into the periportal zone. (b) In the same case, higher magnification shows the large atypical immunoblastic cells that were positive for CD20 and EBER staining.
Plate 27.5 Skin biopsy from the recipient of identical twin marrow who had received a single 15 mg/kg dose of dimethylbusulfan 15 days previously. The epidermal separation, keratinocyte atypia, and scanty lymphoid dermal infiltrate can also be seen in graft-versus-host disease.
Plate 27.7 Severe mucositis of the lips and tongue with pseudomembranous exudate 20 days after hematopoietic cell transplantation following conditioning with total body irradiation and chemotherapy. Similar changes may be present with severe acute graft-versus-host disease or secondary infection such as with a herpes virus. Plate 27.6 Acute glossitis 1 day post hematopoietic cell transplantation after a conditioning regime of 60 mg/kg of cyclophosphamide followed by 12 Gy of fractionated total body irradiation. There is early mucosal damage with severe erythema and focal atrophy.
Plate 27.8 Effect of high-dose therapy on colonic mucosa, day 7. This biopsy specimen was taken following therapy with busulfan, cyclophosphamide, and total body irradiation (12 Gy). There has been obliteration of almost all epithelial cells, leaving cystic crypt remnants. (Hematoxylin and eosin with alcian blue counterstain)
Plate 27.9 Recovery from the effects of high-dose therapy on colonic mucosa, day 16. This repeat biopsy from the same patient as in Plate 27.8 indicates that regeneration is occurring, although some cystic crypts remain. (Hematoxylin and eosin with alcian blue counterstain)
Plate 27.10 Hepatic sinusoidal obstructive syndrome (SOS) 95 days post hematopoietic cell transplantation for malignancy. The trichrome stain shows the blue outline of an hepatic venule with marked luminal narrowing (arrow shows lumen diameter) caused by subendothelial trapping of red cells. The hepatocytes of acinar zone 3 are severely disrupted. When the clinical diagnosis of hepatic SOS is uncertain, it may be confirmed by a transvenous liver biopsy with measurement of an elevated gradient between wedged and free hepatic venous pressures.
Plate 27.13 Fatal hepatic sinusoidal obstructive syndrome (SOS) at autopsy 12 days post bone marrow transplantation for leukemia. At this low magnification, the hematoxylin and eosin-stained section demonstrates striking congestion, hemorrhage, and hepatocyte disruption in zone 3 (centrilobular) of the liver acinus toward the right. The portal space and surrounding acinar zone 1 on the left are well preserved. These early severe hepatic SOS lesions at times can be identified with the hematoxylin and eosin stain after tissue fixation with B5 or methyl Carnoy’s. However, stains for connective tissue are more effective.
Plate 27.11 Early hepatic sinusoidal obstructive syndrome (SOS) showing perivenular staining for procoagulants. Immunohistochemical staining for factor VIII (von Willebrand) demonstrates postsinusoidal obstruction corresponding to the endothelium-lined pores through which the sinusoids drain into the venules. Immunostaining for fibrin localizes in the same regions. These observations provide some of the rationale for using anticoagulation and antithrombotic therapy for hepatic SOS; however, these therapies are ineffective.
Plate 27.14 CD31 immunostaining of liver with fatal sinusoidal obstructive syndrome. There has been partial to complete loss of sinusoidal endothelial staining of the hepatic cords in zone 3 (arrows) which surround the subtotally occluded venule lacking an endothelium (arrowhead).
Plate 27.12 Late hepatic sinusoidal obstructive syndrome, day 63. The trichrome stain demonstrates fibrotic obliteration of the venous lumen with extensive fibrosis in the surrounding sinusoids. The resulting vascular obstruction produced intractable ascites. Immunohistochemical stains at this stage reveal extensive deposition of collagen, while fibrin and other blood procoagulants are no longer identifiable.
Plate 27.15 Hepatic sinusoidal obstructive syndrome (SOS) and coexistent graft-versus-host disease (GVHD), day 26. On this trichrome stain, the two most common liver problems after hematopoietic cell transplantation can be differentiated by the zone of the liver acinus they affect. The expanded portal space in the lower right demonstrates changes from GVHD, including fibrosis, proliferation of atypical cholangioles along the limiting plate, and destruction of small interlobular bile ducts. The SOS lesion along the left margin shows striking collections of embolized hepatocytes beneath the endothelial basement membrane.
Plate 27.16 Fatal sinusoidal obstructive syndrome. (a) Hematoxylin and eosin staining demonstrates a large perivenular zone of hemorrhagic necrosis with destruction of hepatocyte cords surrounding the damaged venule (V); the areas around the portal spaces (PS) are spared. (b) Trichrome staining demonstrates the subtotal occlusion of the venular lumen by collagen and extracellular matrix. (c) Immunostain Mac 387 for macrophage/ monocytes demonstrates a zone of cells at the interface of the viable and necrotic hepatocytes. (d) Smooth muscle actin immunostain demonstrates marked expansion of hepatic stellate cell processes within the damaged sinusoids surrounding the venule.
Plate 27.18 Acute skin graft-versus-host disease, day 28 post mismatched allogeneic hematopoietic cell transplantation. The epidermis demonstrates a prominent lichenoid reaction involving the tip of the rete ridge in the center of the specimen, a location of epithelial stem cells within the epidermis.
Plate 27.17 Hepatic phlebosclerosis, day 96. Eccentric thickening of the venular wall without striking luminal narrowing was associated with an early liver toxicity syndrome. The patient had received a 5-day infusion of highdose arabinoside-C followed by cyclophosphamide and total body irradiation. Liver enzyme and bilirubin elevations developed prior to the infusion of marrow. Changes of phlebosclerosis were widespread, but there were no identifiable intravenular hepatic sinusoidal obstructive syndrome lesions at autopsy. (Trichrome stain)
Plate 27.19 Early skin graft-versus-host disease involving the parafollicular bulge region of the pilar unit. Note the small numbers of mononuclear inflammatory cells which infiltrate the parafollicular bulge area (*) near the arrector pilorum muscle in association with apoptotic bodies.
Plate 27.20 Gastric biopsy with graft-versus-host disease (GVHD), day 35. Several studies have shown that of all upper endoscopic biopsy sites, the stomach most frequently shows histologic changes of GVHD. In the area shown here, large numbers of lymphocytes infiltrate the lamina propria and the basilar portions of the gastric crypts. There is apoptosis of crypt epithelial cells and frank early crypt destruction. Inflammation and apoptosis of this intensity are not required for the diagnosis of gastric GVHD. More subtle yet still diagnostic alterations are illustrated in Plates 27.27 and 27.28.
Plate 27.21 Erythema and desquamation before treatment for acute graftversus-host disease, day 27 (see also Plate 27.24).
Plate 27.22 Maculopapular rash of skin graft-versus-host disease without a notable decrease in inflammation, 1 week after institution of PUVA therapy (see also Plate 27.25).
Plate 27.23 Painful red-to-violaceous maculopapular rash involving the sole of the foot consistent with acute graft-versus-host disease in a 2-year-old girl 11 days after human leukocyte antigen-nonidentical hematopoietic cell transplantation.
Plate 27.25 Marked improvement in skin graft-versus-host disease following 6 weeks of PUVA treatment (same patient as in Plate 27.22).
Plate 27.24 Virtually normal skin with focal depigmentation after treatment for acute graft-versus-host disease, day 74 (same patient as in Plate 27.21).
Plate 27.26 Endoscopic appearance of acute graft-versus-host disease in the stomach, day 32. The pyloric channel is in the center of the picture. The mucosa of the gastric antrum is edematous, reddened, and friable.
Plate 27.27 Gastric graft-versus-host disease in which the severe mucosal erythema, edema, and erosion seen endoscopically on the left are more striking than the focal, mild, epithelial cell apoptosis (arrow) in the histologic section on the right. Although a lymphocytic infiltrate is absent, apoptosis in multiple crypts is consistent with graft-versus-host activity. Inflammation may have been partially controlled by immunosuppressive therapy. (Reproduced from [42], with permission.)
Plate 27.28 Gastric graft-versus-host disease (GVHD) in which the antral mucosa is endoscopically normal aside from edema (left). With air insufflation through the endoscope, the antrum did not distend and there was no motor activity. Biopsy of such a bland area can be diagnostic. In this case, as shown in the photomicrograph on the right, there was epithelial cell apoptosis (arrow) and moderate lymphocytic infiltration diagnostic for GVHD. (Reproduced from [42], with permission.)
Plate 27.29 Gastric graft-versus-host disease. Within the edematous antral mucosa, numerous lymphocytes infiltrate the glands. Apoptosis is widespread, along with segmental disruption of the glands.
Plate 27.30 Rectal biopsy with graft-versus-host disease, day 53. Three mucosal crypts display varying degrees of damage. The crypt on the right shows many apoptotic cells. More extensive cell damage is present in the crypt on the lower left. The upper crypt has lost all enterocytes. Note infiltrating lymphocytes in the lower left crypt associated with nuclear debris characteristic of apoptosis.
Plate 27.31 Surgical resection of severe longstanding intestinal graft-versushost disease (GVHD), day 207. Scarred, ulcerated mucosa alternates with dilated areas of mucosal regeneration. The ulcerated portions demonstrate complete loss of epithelium and severe submucosal fibrosis. Very few patients have responded to intestinal resection for localized GVHD.
Plate 27.34 Fatal pseudomembranous colitis. An acute abdomen developed 15 days post transplant. The degenerative colonic crypts were covered by a cap of mucin admixed with sloughed cellular debris containing large colonies of bacteria consistent with Clostridium spp.
Plate 27.32 Severe graft-versus-host disease (GVHD) of ileum and cecum, day 80. The mucosal lining and folds are replaced by a friable, beefy red, diffusely ulcerated lamina propria. The numerous exposed capillaries ooze blood and serum, a major cause of morbidity from intestinal GVHD. The endothelial cells and fibroblasts within the lamina propria are often infected with cytomegalovirus, as was true in this patient.
Plate 27.35 Endoscopic appearance of cytomegalovirus esophagitis, day 61. A large shallow ulcer with serpiginous, reddened borders is seen in the distal esophagus. The ulcer base (arrows) has a yellow, reticulated appearance.
Plate 27.33 Collagen deposition in the colon with longstanding refractory extensive chronic graft-versus-host disease. The thickened bowel has extensive deposition of collagen in the submucosa, serosa, and lamina propria of the mucosa.
Plate 27.36 Esophageal biopsy showing cytomegalovirus (CMV) infection, day 61. Beneath the epithelium are two cytomegalic cells (large arrows), each containing a single, large, oval intranuclear inclusion surrounded by a clear halo (Cowdry type A). A third cell (small arrow) is probably also infected but is morphologically nondiagnostic. It would presumably be positive by immunocytochemistry and in situ hybridization techniques. Studies of cell morphology as well as viral DNA and antigen expression indicate that the esophageal squamous epithelium is never infected with CMV.
Plate 27.37 Autologous gastric graft-versus-host disease (GVHD) or lymphocytic gastritis. The focal mononuclear inflammatory cell infiltration of the lamina propria and mucosal crypt associated with crypt epithelial cell apoptosis is histologically indistinguishable from GVHD in an allograft recipient.
Plate 27.38 Hepatic graft-versus-host disease. Periodic acid–Schiff-stained section highlights a damaged interlobular bile duct that is infiltrated by lymphocytes. The nuclei vary in size and are irregularly arranged. The syncytium-like segment reflects nuclear loss.
Plate 27.39 Acute hepatitic onset of graft-versus-host disease at 10 months post transplant. Three months after tapering off immunosuppression, the aminotransferases rose to 2000 U, and alkaline phosphatase was three times normal, with normal bilirubin. This biopsy (1) was done 26 days from the onset and shows an extensive lobular hepatitis with intense lymphocytic inflammation of sinusoids and portal spaces, and hepatocellular necrosis.
Plate 27.40 This biopsy (2) was done 1 month later when the total bilirubin was 30 mg/dL. (a,b) Lymphoplasmacytic portal infiltrate which obscures the damaged interlobular bile ducts. (c) Immunostaining with cytokeratin 7 demonstrates that damaged bile ducts are still present. (d) Perivenular (zone 3) cholestasis. Hepatocytes show ballooning, and there is focal dropout associated with a patchy lymphocytic infiltrate. The patient made a full recovery after immunosuppressive treatment for graft-versus-host disease was initiated.
Plate 27.42 Lichen planus-like chronic graft-versus-host disease of the forearm of a 24-year-old African-American male 220 days after bone marrow transplantation of human leukocyte antigen-identical marrow (see also Plates 27.44 and 27.45). Plate 27.41 Hepatic graft-versus-host disease with severe cholangitis lentalike cholangiolar hepatocellular cholestasis. The interlobular bile duct is no longer recognizable. A ductular reaction with large bile-filled cholangioles lies along the periphery of the enlarged irregular portal space. The adjacent hepatocyte cords display marked unrest, disarray, and cytoplasmic ballooning.
Plate 27.43 Periungual erythema and onychodystrophy 100 days after allogeneic bone marrow transplantation in a patient with ocular sicca and oral lesions of chronic graft-versus-host disease.
Plate 27.44 Lichen planus-like chronic graft-versus-host disease before treatment, day 220 (see also Plates 27.42 and 27.45).
Plate 27.45 Same patient shown in Plates 27.42 and 27.44 after treatment, day 770.
Plate 27.46 Poikiloderma in late chronic graft-versus-host disease in a 2year-old girl, day 450. Thinning of the epidermis and dermis is present, along with telangiectasia and reticulated pigmentation.
Plate 27.47 Localized late phase of chronic graft-versus-host disease resembling morphea with atrophic plaque formation, induration, and peripheral hyperpigmentation, day 684.
Plate 27.48 Generalized scleroderma with atrophy, sclerodactyly, and joint contracture, day 540 after human leukocyte antigen-identical bone marrow transplantation.
Plate 27.50 Late chronic graft-versus-host disease (GVHD) of skin, day 959. As this patient’s GVHD progressed, the epidermis became atrophic, with straightening of the dermal-epidermal junction. All dermal appendages were destroyed. The reticular dermal collagen became increasingly sclerotic. A panniculitis seen at the base of the specimen resulted in fibrosis of the subcutaneous fat and large vessels. This histologic picture corresponds to the dense hidebound and sclerodermatous changes of late chronic GVHD.
Plate 27.49 Oral labial biopsy with early chronic graft-versus-host disease (GVHD), day 92. The minor salivary gland exhibits a periductal mononuclear inflammatory infiltrate involving the duct epithelium, in which there is focal epithelial apoptosis. There is inflammation of adjacent acini. This process leads to fibrosis with acinar atrophy in lobules drained by the affected ducts. The squamous surface epithelium (not present here) showed grade II lesions similar to those previously illustrated in skin (see Plate 27.18). The lip biopsy is valuable in the diagnosis and staging of chronic GVHD because it frequently mirrors destructive inflammatory changes in the lacrimal, tracheal, and esophageal glands, as well as bile ducts.
Plate 27.51 Atrophy and fibrosis of the thymus associated with chronic graft-versus-host disease (GVHD) in a 13-year-old, day 350. The patient had not received immunosuppressive drugs, so the destruction was presumably the result of chronic GVHD activity.
Plate 27.52 Poikiloderma, day 719. The patient was treated vigorously for extensive chronic graft-versus-host disease with corticosteroids plus several other immunosuppressive agents. Although he had extensive dyspigmentation, telangiectasia, and atrophy of the skin, there were no contractures or scleroderma. Histologically, the epidermis is atrophic, with loss of rete ridges. The fibrotic papillary dermis contains many telangiectatic vessels, and there is intracellular melanin pigment. The deep dermal sweat glands remain despite earlier inflammatory involvement, and the reticular dermal collagen is normal.
Plate 27.54 Polymyositis in deltoid muscle biopsy associated with chronic graft-versus-host disease, day 1129. There is extensive endomysial chronic inflammation associated with myocyte destruction. The condition responded to immunosuppressive therapy.
Plate 27.55 Coronary arteriopathy associated with chronic graft-versus-host disease in a 15-year-old who died of myocardial infarction preceded by severe anginal symptoms. The lumen and muscular wall of all the major coronary arteries were subtotally to completely obliterated by a cellular infiltrate of lymphoid and monocytoid cells admixed with extracellular matrix. Plate 27.53 Lichenoid early chronic graft-versus-host disease (GVHD) of the skin, day 233. Biopsy of a papulosquamous lesion from the posterior neck demonstrates the characteristic features of the lichen planus-like type of chronic cutaneous GVHD. The thickened epidermis displays hyper- and parakeratosis, hypergranulosis, and acanthosis. The extensive destruction along the dermal-epidermal junction results in sawtooth-like changes of the rete ridges, mimicking lichen planus. The papillary dermis has considerable perivascular inflammation. The lichenoid reaction also involves the hair follicles and eccrine glands seen deep in the reticular dermis. At this stage, treatment can prevent progression to fibrosis (see also Plate 27.50).
Plate 27.56 Two Toxoplasma tachyzoites are present near the ciliated surface of a bronchial epithelial cell. Diagnosis of Toxoplasma pneumonia was established in less than 2 hours after bronchoalveolar lavage. (Wright–Giemsa stain of cytospin preparation)
Plate 27.57 Respiratory syncytial virus pneumonia in a lung biopsy, day 48. The presence of dark purple cytoplasmic inclusions in multinucleated epithelial cells (arrows) suggests the diagnosis but may be found with other viruses such as parainfluenza. During severe community outbreaks, infections may spread to hospitalized bone marrow recipients.
Plate 27.58 Varicella-zoster virus (VZV) hepatitis in a percutaneous needle biopsy, day 79. On the right, a periodic acid–Schiff stain demonstrates a pale necrotic area (*) bordered by cells containing nuclear inclusions (large arrows) visible by hematoxylin and eosin staining on the lower left. Immunofluorescence confirmed the presence of VZV (upper left), and intravenous acyclovir was begun within hours of the biopsy.
Plate 27.60 Fusarium hyphae invade the dermis in pretransplant skin biopsy.
Plate 27.59 Overwhelming adenovirus hepatitis at autopsy 92 days after allogeneic hematopoietic cell transplantation for acute myeloid leukemia. Dark basophilic nuclear inclusions produce “smudge cells” (large, lower arrows) with blurred nuclear margins. Occasional cells with a halo around the inclusion (small, upper arrow) suggestive of cytomegalovirus (CMV) infection are sometimes seen when adenovirus infection is severe. In this case, immunohistology studies for CMV were negative.
Plate 27.61 Pulmonary sinusoidal obstructive syndrome in a lung biopsy from a patient with pulmonary hypertension 48 days post second allogeneic bone marrow transplantation for acute lymphoblastic leukemia. Loose intimal fibrosis partially occludes the interlobular vein. This complication probably results from chemotherapy and may be more frequent than previously suspected. (Verhoeff–Van Gieson elastin stain)
Plate 27.62 Bronchiolitis with evolving subepithelial fibrosis and lymphohistiocytic infiltration. There is marked epithelial damage. In this patient with chronic graft-versus-host disease (GVHD) and obstructive pulmonary function abnormalities, the alterations are consistent with pulmonary GVHD. Similar changes could be seen in respiratory virus infection, which was absent in this biopsy. The ongoing inflammation and subtotal constrictive obliteration of this airway suggest that immunosuppressive therapy may be beneficial.
Plate 27.63 In a patient with a history similar to the preceding case, the bronchiolitis obliterans of chronic pulmonary graft-versus-host disease has progressed. This bronchiole is completely occluded by granulation tissue, and scattered mononuclear inflammatory cells are present. Without successful intervention with immunosuppressive therapy, this airway will finally be replaced by probably permanent scar tissue. The arrows indicate residual smooth muscle. The elastin fibers are black with this Verhoeff–Van Gieson stain. (a)
(c)
(bi)
(bii)
(biii)
(biv)
(d)
Plate 39.1 Proliferative capacity of human cord blood hematopoietic progenitor and stem cells. (a) Colony formation by multipotential (CFU-GEMM) (lower-left) and granulocyte–macrophage (CFUGM) (upper-right) progenitor cells from cord blood cells thawed after 21 years in a frozen cryopreserved state. (b) Colony formation from a high proliferative potential (i and ii) and a smaller proliferative potential (iii and iv) progenitor cell arising from a single high-CD34-expressing single cell sorted into a single microtiter plate (i and iii: magnification × 20; ii and iv: magnification × 80). (Reproduced from [102] with permission.) (c) A secondary umbilical cord blood CFU-GEMM colony derived from replating of a single CFU-GEMM colony onto a secondary plate, a measure of self-renewal of CFUGEMM. See [104] for more information. (d) Immunohistochemical staining of CD34+ cord blood cells that engrafted NOD/SCID mouse bone marrow using human Ki67, a marker of cell proliferation. The conclusion is that cord blood progenitors and stem cells have extensive proliferative capacity. See [238] for more information.
1.0
Probability of survival
0.8
0.6
0.4 Early match, CP (n= 16) Late match, CP (n= 40) Late mismatch, CP (n= 23) Early mismatch, CP (n= 81) Late match, beyond CP (n= 13) Late mismatch, beyond CP (n= 33)
0.2
0.0 0
2
4 6 8 Years after transplant
10
12
Plate 47.1 Impact of disease phase and human leukocyte antigen matching for chronic myeloid leukemia. CP, chronic phase. (Reproduced from [2].)
Plate 62.1 Renal biopsy showing amyloidosis. These images show three views of a renal biopsy: with periodic-acid Schiff (PAS) staining showing amorphous material in glomeruli (top), Congo red staining showing apple-green birefringence in polarized light (middle) and electron microscopy showing nonbranched linear fibrils about 10 nm in length. (See also Chapter 62.)
Plate 70.1 (a, b) Hands of a patient with Sézary syndrome show fissuring, cracking, and nail destruction. (c, d) One year after allogeneic hematopoietic cell transplantation (HCT), there is regression of skin involvement and normalization of the nails. (Reproduced from [94], with permission.)
Plate 79.1 Progression of dysplasia to refractory anemia with excess blasts in a single patient with Fanconi’s anemia over a 9-month period. (a) Marrow aspirate on February 21, 1996 demonstrating pancytopenia and early dysplastic changes in neutrophils. (b) Marrow aspirate on July 3, 1996 demonstrating progressive dysplasia and 3% blasts. (c) Marrow aspirate on September 24, 1996 demonstrating progressive dysplasia and 12% blasts. (d) Marrow biopsy on September 24, 1996 demonstrating severe aplasia. (See Chapter 79.)
Plate 79.2 Above: G-banded karyotypes from an abnormal clone in the marrow of a patient with Fanconi’s anemia. This is a hyperdiploid metaphase cell with 48 chromosomes including two extra derivative chromosomes (designated by arrows); one derivative chromosome is composed of two copies of the long arm of chromosome 1, and the other contains two copies of a small portion of the long arm of chromosome 3. This cell contains a total of four copies of portions of chromosomes 1 and 3. Below: A karyotype of the same abnormal clone as above, as characterized by fluorescent in situ hybridization (FISH) using a multicolor paint probe (Vysis spectral vision M-FISH). MFISH confirmed that the two derivative chromosomes detected by G-banding represented extra copies of portions of chromosomes 1 and 3. (Courtesy of Betsy Hirsch, PhD, Director of the Cytogenetics Laboratory of the University of Minnesota Medical Center, Fairview, Minneapolis, MN.) (See Chapter 79.)
Plate 79.3 Model of the Fanconi’s anemia (FA) pathway. The model shows the sequential assembly of the FA core complex in the cytoplasm and nucleus. DNA replication or DNA damage activates the complex, either directly or indirectly, leading to the activation of FANCD2 by monoubiquination and its targeting to nuclear foci. The association of FANCD2 with BRCA1, FANCD1/BRCA2, FANCN/PALB2, and FANCI, leads to as yet incompletely defined downstream events, such as DNA repair or the activation of DNA synthesis (S) checkpoints. In this model, the core complex is the sensor for DNA damage and participates by activating FANCD2 through ubiquination. FANCD2 and its associates facilitate repair. (Reproduced from [65], with permission.) (See Chapter 79.)
Plate 79.4 Hepatic adenoma. Pathologic specimen obtained at autopsy from a patient with Fanconi’s anemia with multiple adenomata (arrow in upper panel pointing to an adenoma) not revealed by ultrasound prior to hematopoietic cell transplantation. The lower specimen shows area of massive intrahepatic hemorrhage (arrowed) in one previously unidentified adenoma.
Plate 79.5 Before and after blastomere biopsy of the six to eight-cell embryo to diagnose Fanconi’s anemia and assess human leukocyte antigen type. (Electron micrograph provided by Mark Hughes, M.D., Genesis Genetics Institute, Detroit, Michigan.)
Plate 87.1 Poikiloderma, a diagnostic manifestation of chronic graft-versushost disease, which includes both atrophic and pigmentary changes. Note the combination of reticulated pattern, epidermal atrophy (cigarette-paper wrinkling of the skin surface), erythema, and brown pigment forming the borders of skin-colored or depigmented skin.
Plate 87.2 Lichen planus-like eruption involving the palmar surfaces, a diagnostic manifestation of chronic graft-versus-host disease. Note the erythematous/violaceous flat-topped papules or plaques with or without surface reticulations or a silvery or shiny appearance on direct light.
Plate 87.4 Alternative manifestation of deep sclerosis common in areas of excess subcutaneous tissue such as the abdomen, upper arms, and buttocks. Tissue is not hidebound but demonstrates prominent rippling, dimpling or a cellulite-like appearance.
Plate 87.5 Morphea-like superficial sclerotic skin changes, a diagnostic feature of chronic graft-versus-host disease. Note the localized, patchy areas of movable smooth or shiny skin with a leathery-like consistency, often with dyspigmentation. In this picture, there is a fibrotic, hypopigmented area in the center of the plaque with a slightly hyperpigmented border.
Plate 87.3 Diffuse deep sclerotic features involving the lower extremities, a diagnostic feature of chronic graft-versus-host disease. Note the nonmovable/ hidebound subcutaneous tissues, erosions and ulcerations, loss of hair follicles, and toenail dystrophy.
(a)
(b)
Plate 87.6 Lichen sclerosis-like lesion, a diagnostic manifestation of chronic graft-versus-host disease. Note the discrete to coalescent gray to white movable papules or plaques, with a shiny appearance and leathery consistency (a) or cigarette paper-like textural changes (b).
Plate 87.7 Nail dystrophy involving multiple digits is a distinctive feature of chronic graft-versus-host disease (GVHD), although it is insufficient alone to establish the diagnosis of chronic GVHD. Note the longitudinal ridging, splitting and brittle nail features, as well as periungual erythema.
Plate 87.8 New onset of patchy alopecia after recovery from chemoradiotherapy is another distinctive manifestation of chronic graftversus-host disease (but is insufficient alone to establish the diagnosis). Patchy alopecia may involve loss of scalp or body hair.
Plate 87.9 Lichenoid lesions of oral chronic GVHD showing crythema, white striae, plaque, and ulcer formation, day 394. (See also Chapter 23, p. 294; Chapter 50, p. 647; Chapter 67, pp. 920, 922, 923.)
Plate 87.11 Numerous vesicle-like mucoceles (a distinct manifestation of chronic graft-versus-host disease [GVHD]) are seen along the center of the soft palate. Patchy white lichenoid hyperkeratosis (diagnostic of chronic GVHD) and interspersed moderate erythematous changes are also evident across the soft palate. Additionally, note the foamy mucus saliva consistent with GVHD salivary gland dysfunction.
Plate 87.10 Decreased oral range of motion and microstomia in a patient with sclerotic features of chronic graft-versus-host disease. This is a diagnostic manifestation. Note the erythema, edema, and atrophy of the vermillion lip and labial mucosa.
Plate 87.12 Keratoconjunctivitis sicca with blepharitis. The eyelid margins are thickened, edematous, and erythematous. Also note plugging of the meibomian gland orifices (along the eyelid margin) and significant conjunctival hyperemia/injection. A punctal plug can be seen at the lower left margin of the photo.
Plate 87.13 Chronic graft-versus-host disease (GVHD) involving the vulvovaginal tissues. Note lichen planus-like manifestations, which are diagnostic of chronic GVHD. Also seen are erythema and agglutination of the labial tissues.
Plate 87.14 Chronic GVHD of liver, day 166. This biopsy shows a prominent plasmacytic infiltrate in the portal area and a paucity of interlobular bile ducts. The histological dichotomy between acute and chronic GVHD is less clear in liver than in skin. However, increased portal inflammation and loss of bile ducts are more common in chronic GVHD. Despite the frequency of chronic hepatic GVHD, cirrhosis and liver failure are rare and may reflect superinfection with hepatitis C virus. (See also Chapter 23, p. 292.)
Plate 87.15 Bronchiolitis obliterans in a lung biopsy, day 396. The patient had chronic GVHD with severe obstructive pulmonary disease. Similar small airway inflammation with fibrosis is seen in lung allograft rejection. (See also Chapter 23, p. 297; Chapter 50, p. 647.)
(a)
Plate 87.16 Edema in the extremities can be bilateral or, as shown, unilateral. Edema may be an early sign of evolving subcutaneous sclerosis and fasciitis.
(b)
Plate 87.17 (a,b) Manifestations suggestive of fascial involvement by chronic graft-versus-host disease (a diagnostic feature) can include rippling or the groove sign.
Plate 92.1 Modeling varicella zoster virus (VZV) pathogenesis in human tissue xenografts in vivo. Human tissue xenografts are maintained in mice with severe combined immunodeficiency. VZV infection of cells within their tissue microenvironments can be evaluated in mice with skin, T-cell (thymus/liver) or dorsal root ganglia xenografts.
Chronic lymophocytic bronchiolitis
Obstructive bronchiolitis obliterans (OLD)
Acute pneumonitis (IPS)
OLD b
c
Chronic interstitial pneumonitis
Restrictive interstitial fibrosis (RLD)
RLD
a
d Phase one
e Phase two
Phase three
Plate 96.1. A triphasic model of both obstructive lung disease (OLD) and restrictive lung disease (RLD) following allogeneic stem cell transplantation (SCT). Immune-mediated injury to the lung after allogeneic SCT can be conceptualized in three phases. In phase one, acute lung injury develops as a consequence of an allogeneic immune response, and results in the sequential influx of lymphocytes, macrophages, and neutrophils into an inflamed pulmonary parenchyma (a). Persistence of an inflammatory signal in the setting of dysregulated repair mechanisms promotes the transition from acute to chronic injury in phase two (b and d). As chronic inflammation proceeds to phase three, lung fibroblasts increase dramatically in number and contribute to the enhanced deposition of collagen and granulation tissue in and around bronchial structures, ultimately resulting in complete obliteration of the small airways and fixed OLD (c). If, by contrast, the principal target of early damage is the alveolar epithelium, fibroblast proliferation and intraseptal collagen deposition ultimately results in interstitial fibrosis and RLD (e).
(a)
(b)
Plate 103.1 Severe oral mucositis occurring 8 days after hematopoietic cell transplantation following conditioning with VP-16, cyclophosphamide, and total body irradiation (World Health Organization mucositis score of 4). Bleeding ulcerations are noted on the anterior ventral area of the tongue (a) in this patient, with simultaneous pseudomembranous fibrin exudate-covered ulcers evident on the lower labial mucosa (b).
Plate 103.2 Oral chronic graft-versus-host disease in a patient 1 year post hematopoietic cell transplantation, with lichenoid hyperkeratotic changes (stria-, papule-, and plaque-like changes) associated with generalized atrophic and erythematous mucosal changes involving the right buccal mucosa. Small initial pseudomembranous fibrin exudate-covered ulcers are indicated by arrows.
Plate 103.3 Severe oral chronic graft-versus-host disease showing distinct hyperkeratotic lichenoid striae and a pseudomembranous fibrin exudatecovered ulceration with intense surrounding inflammation in a patient approximately 18 months post hematopoietic cell transplantation.
Plate 103.4 A 45-year-old male with a history of multiple myeloma who had been treated with intravenous zoledronate for 2 years prior to having two lower right molars extracted. He presented with three areas of bisphosphonate-associated osteonecrosis of the lower right mandible 6 months following the extractions. Lesions were nonpainful and stable. Arrows indicate areas of bone exposure.
Plate 103.5 Bisphosphonate-associated osteonecrosis of the jaw involving the lower right lingual ridge in a patient with multiple myeloma treated with intravenous zoledronate monthly for 20 months. The patient’s second bicuspid and first molar had been extracted 3 months earlier due to what was thought to be severe peridontal disease; the primary extraction site healed, but an extensive area of osteonecrosis developed along the mandibular lingual torus that was present lingual to the original extraction site.
Section 1 History and Use of Hematopoietic Cell Transplantation
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
1
E. Donnall Thomas
A History of Allogeneic Hematopoietic Cell Transplantation
In the broad sweep of history, it seems strange that our knowledge of the functions of the bone marrow is less than two centuries old, and the knowledge of transplantation of bone marrow occupies little more than half a century. History, as defined in this chapter, will be restricted to those findings which led to human bone marrow transplantation. It will not deal with solid organ transplantation, extensive studies in mice or advances in immunology and immunosuppressive drugs except as related to clinical marrow transplantation. Attempts to employ marrow for therapeutic purposes began in 1939 and 1940 [1,2]. However, infusions of a few milliliters of marrow were not attempts at transplantation, and no useful results were seen. During World War II, some very interesting experiments were carried out by Rekers and colleagues in the then classified laboratories of the Atomic Energy Commission at Rochester, New York. When these studies were published in 1950 [3], they were found to be carefully conducted trials of infusion of bone marrow from normal dogs into dogs exposed to 350 R. The investigators found no significant effect on pancytopenia or on survival. They were not aware that at least twice this exposure is necessary to provide sufficient immunosuppression to achieve engraftment. Thus, they failed in their attempts to achieve marrow recovery by intravenous marrow infusion.
The beginning The seminal studies, first published in 1949, were those of Jacobson and colleagues, who found that a mouse would survive otherwise lethal irradiation if the spleen were exteriorized and protected from the irradiation [4]. They found that an intraperitoneal injection of cells from the spleen (a hematopoietic organ in the mouse) would achieve the same effect. Lorenz et al. [5] extended these observations by demonstrating a similar protective effect by an infusion of bone marrow cells. At first, it was hypothesized that this “irradiation protection” effect was due to some kind of hormone or growth factor contained in the infusion. Most of these early studies involved measuring death at 30 days after lethal irradiation. Infusions of preparations from a donor of different H2 type were effective when survival was measured at 30 days. Such a result favored a humoral mechanism since it would not be expected if cells were involved. However, in 1954, Barnes and Loutit reported that mice protected with syngeneic marrow survived beyond
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
100 days. Mice given allogeneic marrow (A into CBA) survived at 30 days (“academic survivors”) but died thereafter of a “secondary disease” [6]. Another argument in favor of a hormonal effect was that “cell-free” extracts contained the protective effect. The investigators did not yet appreciate the very small number of cells required for transplantation in the syngeneic situation. Barnes and Loutit [7] sounded a warning against the hormonal hypothesis in which they noted that the “cellular hypothesis” had not been excluded as an explanation of the irradiationprotective effect. The demise of the humoral hypothesis became inevitable in 1955 when Main and Prehn published an article in the Journal of the National Cancer Institute [8]. They studied mice given lethal irradiation and marrow from an H2-incompatible (BALB/cAnN into DBA/2JN) strain. Subsequently, the surviving recipient mice given a skin graft from the donor strain accepted the skin graft for the rest of their life. Trentin was able to show that the tolerance of the graft was specific for the marrow donor strain [9]. Survival of the graft could only be explained by the persistence of cells of the donor strain leading to “tolerance.” Proof that these animals protected against lethal irradiation by marrow infusion were true radiation chimeras came from several sources. In 1956, Nowell et al. described the growth and continued function of rat marrow cells in X-irradiated mice [10]. Most convincingly, Ford et al. showed that the marrow of lethally irradiated mice given infusions of syngeneic marrow marked by the T6 chromosome was made up of cells of the cytogenetic type of the donor [11].
Early clinical studies At this point, it became evident that marrow transplantation might be of use not only in irradiation protection, but also in therapeutic application to marrow aplasia, leukemia, and other diseases of the marrow and lymphoid system. In 1956, Barnes et al. described the treatment of mice with leukemia by lethal irradiation and marrow transplantation [12]. In Cooperstown, NY, Thomas and Ferrebee and their colleagues had already begun comparable studies in terminal patients with hematological malignancies. In 1957, they reported six patients treated with irradiation and intravenous infusion of marrow from a normal individual [13]. Only one patient showed a transient marrow graft. These failures were to be duplicated by many investigators, so that in 1970 Bortin was able to compile a list of approximately 200 attempts at allogeneic marrow grafting, all of which had failed [14]. In 1959, Thomas et al. [15] reported an identical twin with terminal leukemia who was given 850 R (748 rad) total body irradiation from
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Chapter 1
Fig. 1.1 A symposium on bone marrow transplantation in Paris in 1971. From left to right: George Mathé, Dirk van Bekkum, George Santos, Don Thomas, Charles Congdon, Delta Uphoff.
opposing cobalt-60 sources and an intravenous infusion of marrow from the healthy twin (a syngeneic transplant). This dose of irradiation would be expected to produce prolonged pancytopenia and death [16]. However, the patient showed prompt recovery and disappearance of the leukemia for 4 months. This study demonstrated that lethal irradiation followed by compatible marrow could have an antileukemic effect even in advanced leukemia. Most importantly, it showed that compatible marrow infused intravenously could restore marrow function in human beings after lethal irradiation. Also in 1959, Mathé et al. reported the infusion of marrow into patients exposed to potentially lethal irradiation in a reactor accident [17]. Subsequent analysis raised doubt about whether there had been a therapeutic effect [18]. Nevertheless, this experience gave a great boost to interest in marrow transplantation and attracted many investigators to the field. One of the driving spirits of the early studies was Charles Congdon of the Oak Ridge National Laboratory, TN. In 1957, he organized an informal series of conferences at Oak Ridge. The early results of these conferences were published in abstract form in small leaflets called Fundamental and Clinical Aspects of Radiation Protection and Recovery. From 1964 to 1970, the abstracts appeared under the title Experimental Hematology. These small booklets may be difficult to find but are well worth the effort. They list the authors and investigations of that early time: there was life before Medline. In 1971, these meetings were organized into the International Society for Experimental Hematology, which publishes the modern journal Experimental Hematology (Fig. 1.1).
Advances from animal studies Thus, it was soon recognized that marrow grafting in man would be very difficult. Many investigators therefore turned to animal studies for definition and resolution of the problems. Much early information came from the work of Brent, Billingham, Medawar, and their colleagues [19]. They studied the induction of tolerance in newborn mice. In the course of these studies, they described the syndrome of “runt disease” in newborn mice given allogeneic cells. The principles they established were to prove the same as for “secondary disease” in irradiated mice and, eventually, for graft-versus-host disease (GVHD) in man. Although there had been great concern about the possibility of runt disease in the early studies of human marrow transplantation [13], it was apparent that this would be a problem only with a successful graft.
In 1959, Billingham and Brent published a landmark study of runt disease in newborn mice [20], and in 1966, Billingham described in detail the biology of graft-versus-host reactions [21]. These authors noted the following: • Syngeneic cells did not result in runt disease. • Persistence of allogeneic cells in the recipient was necessary for the development of runt disease. • The severity and incidence of runt disease were determined by antigenic differences between donor and host. • Tolerance could occur in the absence of runt disease. • The severity of runt disease was enhanced by cells already sensitized by exposure to host strain cells. The principles they elaborated were applicable to all studies of hematopoietic cell transplantation (HCT). The mouse has been used widely in studies of transplantation biology. Numerous studies in the murine system are cited in the book by van Bekkum and de Vries [22]. The availability of inbred strains has permitted extensive studies of genetic factors in transplantation. Uphoff [23] showed that the severity of the secondary disease after irradiation and marrow grafting was controlled by genetic factors. Uphoff [24] and Lochte and colleagues [25] showed that methotrexate (MTX) could prevent or ameliorate the secondary disease now known as GVHD. The dog has been frequently used in transplantation and surgical studies. Dogs are outbred animals comparable to humans. They come in large families, permitting a study of inherited factors. Studies in dogs first demonstrated the utility of autologous marrow grafts [26,27]. Marrow from one group of dogs was collected and set aside. The marrow donor or control animals were then given lethal or supralethal irradiation. The control animals invariably died whereas those treated with their own marrow promptly recovered. Obviously, this type of study could not be done with human beings. Successful cryopreservation of marrow had been accomplished by freezing in glycerol [28] using the technique of Polge and Smith. A careful step-wise removal of the glycerol before infusion of the cells was essential. Cavins et al. showed that canine marrow could be cryopreserved by freezing in dimethylsulfoxide (DMSO), thawed and injected intravenously without removal of the DMSO [29]. This technique was to become standard for marrow transplantation. Goodman and Hodgson had shown that mice could be protected against lethal irradiation by the infusion of blood cells [30]. Cavins et al. showed that autologous canine buffy coat cells from the peripheral blood could be
A History of Allogeneic Hematopoietic Cell Transplantation
collected, cryopreserved in DMSO, and infused after lethal irradiation with subsequent recovery of marrow function [31]. The practical application of peripheral blood cells for transplantation had to await better methods of centrifugation for separation of the buffy coat cells and techniques for mobilizing hematopoietic cells into the blood. The dog was most informative in extensive studies of allogeneic marrow grafting in an outbred species. Ferrebee et al. [32] reported the first allogeneic marrow graft in an irradiated dog. Four dogs were given 400 R on each of 3 days. One control and two dogs given marrow from unrelated dogs died of the acute irradiation syndrome. The male dog given marrow from a female littermate engrafted, and the leukocyte nuclei showed female drumsticks characteristic of the donor. Thomas et al. carried out a series of studies of irradiation and allogeneic marrow grafting in the dog [33,34]. Allogeneic graft recipients showed all of the problems that were soon to be recognized in human patients. The problems were graft failure, graft rejection, GVHD and/or death from opportunistic infections. A few animals did not develop these problems and became long-term survivors with marrow cells of donor type. Presumably these survivors fortuitously had donors of sufficient histocompatibility to permit long-term survival. In 1960, methods for selecting compatible donors were unknown. Early studies of marrow grafting after irradiation were carried out with non-human primates. Autologous marrow grafts were effective in protecting monkeys against lethal irradiation [35]. After allogeneic marrow grafting, they developed early severe secondary disease [35] that was somewhat reduced by treatment with antilymphocyte serum [36]. Rhesus monkeys given blood transfusions before irradiation rarely accepted an allogeneic graft [37]. In the dog, prior transfusions from histocompatible donors also prevented successful engraftment [38]. Unfortunately, marrow-grafting studies in non-human primates were not very useful for application to human patients. The non-human primates were expensive and difficult to work with. Therefore, the number of subjects studied was small. They were also not available in families. In addition, survival studies were difficult because of infection and parasitism in wild-caught animals [39]. In 1960, Medawar and Burnett received the Nobel Prize for their discovery “of acquired immunological tolerance.” When the prize was announced, my wife Dottie and I were attending a transplantation meeting in Switzerland. The joy and celebration knew no bounds. We were not only honoring the winners, but also acknowledging that transplantation biology had become a recognized field of scientific endeavor.
Advances in knowledge of histocompatibility Techniques for defining tissue antigens in humans were crucial to the development of HCT. In 1954, Miescher and Fauconnet recognized antibodies induced by transfusion or pregnancy that reacted with antigens on white blood cells [40]. In 1958, Dausset and van Rood and their colleagues recognized that human leukocyte antigens (HLAs) followed the genetic principles of inheritance [41,42]. There followed numerous studies designed to elucidate the role of these antigens in transplantation. HLA proved, however, to be of minimal value in the prediction of the outcome of skin grafts [43,44]. Epstein et al. [45] developed typing sera for dog leukocyte antigens (DLAs). Storb and colleagues studied dogs given lethal irradiation and a marrow graft from a littermate [46,47]. They showed that those marrow grafts between mismatched littermates always failed. Grafts between DLA-matched donors and recipients showed much improved survival. The administration of a short course of MTX for immunosuppression after grafting improved the long-term survival of matched recipients to 90% or better [48]. Thus, studies in dogs illustrated the importance of
5
DLA matching. At about the same time, Terasaki showed that the outcome of kidney grafts between siblings was highly dependent on HLA matching [49]. Taken together, these studies showed that matching for leukocyte antigens would be essential in human marrow grafting.
Renewed clinical studies After all the disappointments of clinical marrow transplants in the late 1950s and early 1960s, there was general pessimism about the field, and many of the early investigators had moved on to other studies. Nevertheless, improvements in transfusion medicine and the treatment of infections, and especially an improved understanding of the importance of HLA typing, encouraged a renewed attack on the clinical application of marrow grafts. The first good news came from studies of children with an immunological deficiency. Because of their disease, these children could not reject a foreign graft, and therefore immunosuppression before grafting should not be necessary. In November 1968, Gatti et al. performed the first successful allogeneic graft in a patient with severe combined immunodeficiency [50]. Two similar successes were reported immediately thereafter [51,52]. All three patients were alive and well 25 years later [53]. In late 1967, the Seattle marrow transplantation team received a grant to support clinical marrow transplantation. The year 1968 was spent in assembling a team of nurses, technicians, dietitians, etc. who were to be dedicated to patients undergoing intensive therapy and marrow transplantation with a special emphasis on post-transplant clinical care. The first transplant by this team was carried out in March 1969 [54]. The patient was a 46-year-old man with the blastic crisis of chronic myelogenous leukemia. An initial typing showed the patient’s sister to be an HLA match. After an irradiation exposure of a calculated midline dose of 954 rad, marrow was infused intravenously. Engraftment by donor cells was evident in 13 days. The patient developed mild GVHD controlled by MTX. Subsequently, he developed fever and died after 56 days. Autopsy showed cytomegalovirus pneumonia, but there was no evidence of leukemia or GVHD. This patient showed successful engraftment and control of GVHD but illustrated the problem of opportunistic infection. Thus began the Seattle marrow transplant team’s long series of patients given marrow grafts from HLA-matched siblings. In 1972, Thomas et al. reported the first experience with allografting for severe aplastic anemia [55]. The first four patients were referred for marrow grafting after a failure of conventional therapy with steroids and multiple transfusions. Since these patients did not involve the problem of eradication of a malignant disease, they were prepared with immunosuppression by four large doses of cyclophosphamide following the regimen of Santos et al. [56]. All four grafts were initially successful. One patient died of GVHD, and one died of graft rejection. Two patients were long-term survivors. In 1975, the Seattle team published a landmark Medical Progress review in the New England Journal of Medicine that was to become a highly cited article [57]. That article reviewed the rationale and experimental background for marrow transplantation. It emphasized the importance of histocompatibility and the possibility of using unrelated donors. It described the preparation of the patient, the technique of marrow transplantation, and the importance of supportive care. It also described the use of HLA-matched siblings for 37 patients with aplastic anemia and 73 with leukemia who had reached an advanced stage of their disease before transplantation. Death from recurrent disease, opportunistic infection, and GVHD were analysed. Most importantly, the article described a number of survivors. In 1977, the Seattle team reported 100 patients with end-stage acute leukemia treated by chemotherapy, total body irradiation and allogeneic
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marrow transplantation [58]. Thirteen of these patients became longterm survivors. Also, by 1977, the time of follow-up of transplanted patients was long enough to show a change in the shape of the survival curve indicating a plateau, which suggested that some patients were cured of their disease [59]. The survival of some patients transplanted in a terminal stage of their disease raised the possibility of transplantation earlier in the course of the disease. Transplantation before the development of drug resistance and before the complications of advanced disease might be expected to improve survival. In 1979, two reports described marrow grafts for patients with acute myeloid leukemia transplanted in first remission [60,61]. As expected, survival was greatly improved, and approximately 50% of the patients became long-term survivors. Mathé had coined the term “adoptive immunotherapy” to indicate the possibility that lymphoid elements in the engrafted marrow might react against malignant cells in the patient to aid in eradication of the remaining malignant cells [62]. Initial attempts to evaluate the possibility that GVHD might include graft-versus-leukemia effects were unrewarding [57]. However, when enough cases had been accumulated, Weiden et al. were able to demonstrate that the more severe the GVHD, the less likely was the recurrence of leukemia [63]. In the 1970s, a major concern was the limitation of allogeneic grafting to HLA-matched sibling pairs. Obviously, only about one-fourth of the patients would have a suitable marrow donor. Because of the complexity of the HLA system, finding a matched unrelated donor seemed a formidable task. In 1979, Hansen et al. performed the first successful marrow graft from an unrelated donor for a patient with leukemia [64].
A young patient with refractory acute leukemia did not have a matched sibling. Quite by chance, it was observed that one of the hematology technicians had an HLA type that matched the patient. The graft was successful without GVHD until the leukemia recurred 2 years later. This patient’s experience stimulated the formation of the National Marrow Donor Program.
History is now The use of donor peripheral blood leukocytes or cord blood cells for HCT has become a routine part of transplantation. The use of donor lymphocytes to cure recurrent leukemia and the use of non-myeloablative preparative regimens are but a few of the advances that are current subjects of research. Molecular biological techniques have greatly improved the accuracy of tissue typing. We are just beginning to appreciate the importance of the microenvironment, the cytokines, and the multitude of genes regulating cell division and senescence. Each advance with new agents, such as imatinib mesylate, forces us to redefine the role of HCT in therapy of disease. Thus, at the end of the 20th century, grafting of hematopoietic cells had become widely employed for research and as a form of therapy for patients with a variety of diseases. Many investigators have been attracted to the field, and many institutions have established HCT programs. Progress in the clinical application of HCT and in new avenues of research is the subject of the remaining chapters of this book and is described by the investigators who carried out much of the work.
References 1. Osgood EE, Riddle MC, Mathews TJ. Aplastic anaemia treated with daily transfusions and intravenous marrow. Ann Intern Med 1939; 13: 357– 67. 2. Morrison M, Samwick AA. Intramedullary (sternal) transfusion of human bone marrow. JAMA 1940; 115: 1708–11. 3. Rekers PE, Coulter MP, Warren S. Effect of transplantation of bone marrow into irradiated animals. Arch Surg 1950; 60: 635–67. 4. Jacobson LO, Marks EK, Robson MJ, Gaston EO, Zirkle RE. Effect of spleen protection on mortality following X-irradiation. J Lab Clin Med 1949; 34: 1538–43. 5. Lorenz E, Uphoff D, Reid TR, Shelton E. Modification of irradiation injury in mice and guinea pigs by bone marrow injections. J Natl Cancer Inst 1951; 12: 197–201. 6. Barnes DWH, Loutit JF. What is the recovery factor in spleen [Letter]? Nucleonics 1954; 12: 68–71. 7. Barnes DWH, Loutit JF. Spleen protection: The cellular hypothesis. In Bacq ZM, editor. Radiobiology symposium. London: Butterworth; 1955. 8. Main JM, Prehn RT. Successful skin homografts after the administration of high dosage X radiation and homologous bone marrow. J Natl Cancer Inst 1955; 15: 1023–9. 9. Trentin JJ. Mortality and skin transplantability in X-irradiated mice receiving isologous or heterologous bone marrow. Proc Soc Exp Biol Med 1956; 92: 688–93. 10. Nowell PC, Cole LJ, Habermeyer JG, Roan PL. Growth and continued function of rat marrow cells in X-radiated mice. Cancer Res 1956; 16: 258– 61.
11. Ford CE, Hamerton JL, Barnes DWH, Loutit JF. Cytological identification of radiation-chimaeras. Nature 1956; 177: 452–4. 12. Barnes DWH, Corp MJ, Loutit JF, Neal FE. Treatment of murine leukaemia with X-rays and homologous bone marrow. Preliminary communication. Br Med J 1956; 2: 626–7. 13. Thomas ED, Lochte HL, Jr., Lu WC, Ferrebee JW. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med 1957; 257: 491–6. 14. Bortin MM. A compendium of reported human bone marrow transplants. Transplantation 1970; 9: 571–87. 15. Thomas ED, Lochte HL, Jr., Cannon JH, Sahler OD, Ferrebee JW. Supralethal whole body irradiation and isologous marrow transplantation in man. J Clin Invest 1959; 38: 1709–16. 16. Cronkite EP, Bond VP. Radiation injury in man. Oxford, UK: C.C. Thomas/Blackwell Scientific Publications; 1960. 17. Mathé G, Jammet H, Pendic B et al. Transfusions et greffes de moelle osseuse homologue chez des humains irradiés a haute dose accidentellement. Rev Franc Etudes Clin et Biol 1959; IV: 226–38. 18. Andrews GA. Criticality accidents in Vinca, Yugoslavia, and Oak Ridge Tennessee. Am J Roentgenol Radium Ther Nucl Med 1965; 93: 56–74. 19. Billingham RE, Brent L, Medawar PB. “Actively acquired tolerance” of foreign cells. Nature 1953; 172: 603–6. 20. Billingham RE, Brent L. Quantitative studies on tissue transplantation immunity. IV. Induction of tolerance in newborn mice and studies on the phenomenon of runt disease. Philos Trans R Soc Lond B Biol Sci 1959; 242: 477.
21. Billingham RE. The biology of graft-versus-host reactions. In: The Harvey lectures. New York: Academic Press; 1966. pp. 21–78. 22. van Bekkum DW, de Vries MJ. Radiation chimaeras. London: Logos Press; 1967. 23. Uphoff DE. Genetic factors influencing irradiation protection by bone marrow. I. The F1 hybrid effect. J Natl Cancer Inst 1957; 19: 123–5. 24. Uphoff DE. Alteration of homograft reaction by A-methopterin in lethally irradiated mice treated with homologous marrow. Proc Soc Exp Biol Med 1958; 99: 651–3. 25. Lochte HL, Jr., Levy AS, Guenther D, Thomas ED, Ferrebee JW. Prevention of delayed foreign marrow reaction in lethally irradiated mice by early administration of methotrexate. Nature 1962; 196: 1110–11. 26. Alpen EL, Baum SJ. Modification of X-radiation lethality by autologous marrow infusion in dogs. Blood 1958; 13: 1168–75. 27. Mannick JA, Lochte HL, Jr., Ashley CA, Thomas ED, Ferrebee JW. Autografts of bone marrow in dogs after lethal total-body radiation. Blood 1960; 15: 255–66. 28. Barnes DWH, Loutit JF. The radiation recovery factor: Preservation by the Polge–Smith– Parkes technique. J Natl Cancer Inst 1955; 15: 901–5. 29. Cavins JA, Kasakura S, Thomas ED, Ferrebee JW. Recovery of lethally irradiated dogs following infusion of autologous marrow stored at low temperature in dimethyl-sulphoxide. Blood 1962; 20: 730–4. 30. Goodman JW, Hodgson GS. Evidence for stem cells in the peripheral blood of mice. Blood 1962; 19: 702–14.
A History of Allogeneic Hematopoietic Cell Transplantation
31. Cavins JA, Scheer SC, Thomas ED, Ferrebee JW. The recovery of lethally irradiated dogs given infusions of autologous leukocytes preserved at −80°C. Blood 1964; 23: 38–43. 32. Ferrebee JW, Lochte HL, Jr., Jaretzki A, III, Sahler OD, Thomas ED. Successful marrow homograft in the dog after radiation. Surgery 1958; 43: 516– 20. 33. Thomas ED, Ashley CA, Lochte HL, Jr., Jaretzki A, III, Sahler OD, Ferrebee JW. Homografts of bone marrow in dogs after lethal total-body radiation. Blood 1959; 14: 720–36. 34. Thomas ED, Collins JA, Herman EC, Jr., Ferrebee JW. Marrow transplants in lethally irradiated dogs given methotrexate. Blood 1962; 19: 217–28. 35. Crouch BG, van Putten LM, van Bekkum DW, de Vries MJ. Treatment of total-body X-irradiated monkeys with autologous and homologous bone marrow. J Natl Cancer Inst 1961; 27: 53–65. 36. Merritt CB, Darrow CC, II, Vaal L, Rogentine GN, Jr. Bone marrow transplantation in rhesus monkeys following irradiation. Modification of acute graftversus-host disease with antilymphocyte serum. Transplantation 1972; 14: 9–20. 37. van Putten LM, van Bekkum DW, de Vries MJ, Balner H. The effect of preceding blood transfusions on the fate of homologous bone marrow grafts in lethally irradiated monkeys. Blood 1967; 30: 749–57. 38. Storb R, Epstein RB, Rudolph RH, Thomas ED. The effect of prior transfusion on marrow grafts between histocompatible canine siblings. J Immunol 1970; 105: 627–33. 39. van der Waay D, Zimmerman WMTh. Problems in the sanitation of monkeys for whole-body irradiation experiments. Proceedings of the International Symposium on Bone Marrow Therapy and Chemical Protection in Irradiated Primates 1962. 1962. pp. 231–40. 40. Miescher PP, Fauconnet M. Mise en évidence de différents groupes leucocytaires chez l’homme. Schweiz Med Wochenschr 1954; 84: 597–9. 41. Dausset J. Iso-leuco-anticorps. Acta Haematol 1958; 20: 156–66.
42. van Rood JJ, Eernisse JG, van Leeuwen A. Leukocyte antibodies in sera from pregnant women. Nature 1958; 181: 1735–6. 43. Rapaport FT, Lawrence HS, Thomas L et al. Crossreactions to skin homografts in man. J Clin Invest 1962; 41: 2166–72. 44. Dausset J, Rapaport FT, Legrand L et al. Studies on transplantation antigens (HL-A) by means of skin grafts from 90 children onto their fathers [French]. Nouv Rev Fr Hematol 1969; 9: 215–29. 45. Epstein RB, Storb R, Ragde H, Thomas ED. Cytotoxic typing antisera for marrow grafting in littermate dogs. Transplantation 1968; 6: 45–58. 46. Storb R, Epstein RB, Bryant J, Radge H, Thomas ED. Marrow grafts by combined marrow and leukocyte infusions in unrelated dogs selected by histocompatibility typing. Transplantation 1968; 6: 587–93. 47. Storb R, Rudolph RH, Thomas ED. Marrow grafts between canine siblings matched by serotyping and mixed leukocyte culture. J Clin Invest 1971; 50: 1272–5. 48. Storb R, Epstein RB, Graham TC, Thomas ED. Methotrexate regimens for control of graft-versushost disease in dogs with allogeneic marrow grafts. Transplantation 1970; 9: 240–6. 49. Singal DP, Mickey MR, Terasaki PI. Serotyping for homotransplantation: XXIII analysis of kidney transplants from parental versus sibling donors. Transplantation 1969; 7: 246–58. 50. Gatti RA, Meuwissen HJ, Allen HD, Hong R, Good RA. Immunological reconstitution of sexlinked lymphopenic immunological deficiency. Lancet 1968; 2: 1366–9. 51. Bach FH, Albertini RJ, Joo P, Anderson JL, Bortin MM. Bone-marrow transplantation in a patient with the Wiskott–Aldrich syndrome. Lancet 1968; 2: 1364–6. 52. deKoning J, van Bekkum DW, Dicke KA, Dooren LJ, Rádl J, Van Rood JJ. Transplantation of bonemarrow cells and fetal thymus in an infant with lymphopenic immunological deficiency. Lancet 1969; 1: 1223–7.
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53. Bortin MM, Bach FH, van Bekkum DW, Good RA, van Rood JJ. 25th anniversary of the first successful allogeneic bone marrow transplants. Bone Marrow Transplant 1994; 14: 211–12. 54. Buckner CD, Epstein RB, Rudolph RH, Clift RA, Storb R, Thomas ED. Allogeneic marrow engraftment following whole body irradiation in a patient with leukemia. Blood 1970; 35: 741–50. 55. Thomas ED, Storb R, Fefer A et al. Aplastic anaemia treated by marrow transplantation. Lancet 1972; 1: 284–9. 56. Santos GW, Sensenbrenner LL, Burke PJ et al. Marrow transplantation in man following cyclophosphamide. Transplant Proc 1971; 3: 400–4. 57. Thomas ED, Storb R, Clift RA et al. Bone-marrow transplantation. I and II. N Engl J Med 1975; 292: 832–43, 895–902. 58. Thomas ED, Buckner CD, Banaji M et al. One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 1977; 49: 511–33. 59. Thomas ED, Flournoy N, Buckner CD et al. Cure of leukemia by marrow transplantation. Leuk Res 1977; 1: 67–70. 60. Thomas ED, Buckner CD, Clift RA et al. Marrow transplantation for acute nonlymphoblastic leukemia in first remission. N Engl J Med 1979; 301: 597–9. 61. Blume KG, Beutler E. Allogeneic bone marrow transplantation for acute leukemia [Letter]. JAMA 1979; 241: 1686. 62. Mathé G, Amiel JL, Schwarzenberg L, Catton A, Schneider M. Adoptive immunotherapy of acute leukemia: Experimental and clinical results. Cancer Res 1965; 25: 1525–31. 63. Weiden PL, Flournoy N, Thomas ED et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 1979; 300: 1068–73. 64. Hansen JA, Clift RA, Thomas ED, Buckner CD, Storb R, Giblett ER. Transplantation of marrow from an unrelated donor to a patient with acute leukemia. N Engl J Med 1980; 303: 565–7.
2
James O. Armitage
The History of Autologous Hematopoietic Cell Transplantation
Introduction The procedure known variously as autologous bone marrow transplantation, autologous peripheral blood cell transplantation, autologous stem cell transplantation, and autologous hematopoietic cell transplantation involves the intravenous infusion of a patient’s own hematopoietic stem cells to rescue the patient from severe bone marrow injury. Usually, the bone marrow injury has been caused by high-dose chemotherapy and/or radiotherapy as part of a treatment for cancer. Usually, but not always, the patient’s hematopoietic stem cells have been cryopreserved and thawed before reinfusion. Autologous hematopoietic cell transplantation tests the hypothesis that the resistance of cancer cells to chemotherapy and/or radiotherapy can be relative rather than absolute, and that the resistance might be overcome by dose escalation. The importance of dose in cancer therapy has been known for some time [1]. While it has been clear that arbitrary dose reductions can reduce the chance of a good outcome with potentially curative cancer chemotherapy, autologous hematopoietic cell transplantation tests the hypothesis that dose escalation to a point that would cause irreversible myelotoxicity (but tolerable because of replacing myeloid function with the infused autologous hematopoietic stem cells) might be able to cure otherwise incurable patients. This hypothesis is illustrated in Fig. 2.1. Both chemotherapeutic agents and total body radiotherapy injure organs other than the bone marrow. Thus, for effective autologous hematopoietic cell transplantation, drugs whose initial life-threatening toxicity is myeloid injury need to be chosen, and radiation doses must be kept below that which would cause lethal pulmonary, gastrointestinal or central nervous system toxicity.
Early attempts at autologous hematopoietic cell transplantation The use of bone marrow to treat cancer has been considered by healthcare providers for a very long time. The first reports of the use of bone marrow as a treatment for cancer appeared in the 1890s [2]. The theoretical basis for the use of hematopoietic stem cells to rescue patients from irreversible myeloid injury was provided by canine studies [3,4] showing that dogs could survive otherwise lethal total body radiotherapy after the infusion of their own autologous hematopoietic cells. A report
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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of the first attempts at autologous hematopoietic cell transplantation in humans appeared in 1958 [5] and reported two patients, one suffering from teratocarcinoma and one from renal cell carcinoma, who received wide-field “intensive” radiotherapy followed by the infusion of glycerolpreserved hematopoietic cells derived from the bone marrow. A subsequent report by the same investigators [6] described four patients managed in a similar way who seemed to have hematopoietic recovery after infusion of the autologous bone marrow cells. McFarland et al. in 1959 described five patients with non-Hodgkin’s lymphoma or Hodgkin’s disease who received high doses of mechlorethamine followed by the infusion of noncryopreserved autologous bone marrow cells [7]. Three of the five patients had hematopoietic recovery, but there was only transient improvement in clinical status in a few. Clifford et al., working in Kenya in 1961, reported three patients with lymphoma or anaplastic nasopharyngeal carcinoma who received highdose mechlorethamine followed by an infusion of glycerol-preserved bone marrow cells [8]. All three patients demonstrated tumor regression and clinical improvement in addition to hematopoietic recovery. In 1962, Pegg et al. described 29 patients with cancer who received intensive, large-volume radiotherapy or chemotherapy with one of several alkylating agents followed by autologous bone marrow cell infusion. Most patients showed hematopoietic recovery, and a few had significant tumor regression and clinical improvement [9]. In 1962, Kurnick described 82 patients (presumably including some from his previous reports) who received high-dose chemotherapy and/or radiotherapy for a variety of malignancies including germ-cell tumors, lymphomas, leukemias, carcinomas, and sarcomas. The report expanded his previous observations and described hematopoietic recovery in most patients with, apparently, modest clinical benefit [10]. In many of these early reports, the intensity of chemotherapy would not necessarily have caused extremely prolonged marrow suppression, and it is difficult to be certain of the contribution of the reinfused hematopoietic cells. In the case of cryopreserved cells, it is not certain that viable cells were reinfused. The first patients apparently cured of otherwise incurable malignancy using high-dose therapy and autologous hematopoietic cell transplantation were reported by investigators from the National Cancer Institute in 1978. The authors reported 22 patients with resistant malignant lymphoma who were treated with high-dose chemotherapy utilizing carmustine, cytarabine, cyclophosphamide, and thioguanine [11]. Twelve patients received an infusion of their own cryopreserved autologous bone marrow, and 10 patients served as controls without reinfused cryopreserved marrow. Recovery of peripheral blood counts occurred with a median of 13 days in the patients who received autologous
The History of Autologous Hematopoietic Cell Transplantation
Increasing dose
3
Death secondary to other organ toxicity 2
1
Death secondary to marrow toxicity
Dose level necessary to cure three hypothetical patients
Fig. 2.1 Potential for cure with autologous hematopoietic stem-cell transplantation. The patient represented by column 1 could be cured with doses that would not cause permanent marrow injury. The patient represented by column 2 could be uniquely cured by doses requiring autotransplantation. The patient represented by column 3 would die of nonhematologic toxicity with the doses necessary to cure the cancer.
hematopoietic cells, and at a median of 23 days in those patients who did not receive marrow infusion. In a separate report, the same authors described 14 patients with Burkitt’s lymphoma resistant to conventional therapy who received the same high-dose chemotherapy regimen and autologous hematopoietic cells when these were available. Eight of the 14 patients received autologous hematopoietic cell transplantation; three of those were apparently cured, whereas none of the six patients who did not receive autologous hematopoietic cell transplantation had a long-term, disease-free survival [12]. The success in these patients ushered in the modern era of autologous hematopoietic cell transplantation and was made possible by the reproducible freezing and thawing of autologous hematopoietic cells using dimethylsulfoxide, resulting in viable and transplantable stem cells after thawing. This was initially reported by Cavins et al. [13] in dogs, and the work by Appelbaum et al. at the National Cancer Institute [12] demonstrated that the same approach worked in humans. Although almost all autologous hematopoietic cell transplantations are carried out with cryopreserved cells, some investigators have utilized noncryopreserved hematopoietic progenitor cells for this procedure. For this to be practical, the treatment has to be completed very rapidly, and the rescue product has been reinfused in most studies within 48 hours. Early studies in the United States and Italy showed the feasibility of this approach [14–16]. Carella subsequently reported 10 patients with Hodgkin’s disease and non-Hodgkin’s lymphoma who received highdose therapy with carmustine or vinblastine, cyclophosphamide, and carmustine. Seven of 10 patients with resistant, refractory disease achieved remissions, and hematopoietic recovery occurred in nine of the 10 patients [17]. A subsequent report by the same authors described 13 patients with Hodgkin’s disease and showed similar results, with all but one of the patients having hematopoietic recovery [18]. However, despite occasional other reports, this approach has been rarely used, with almost all patients receiving cryopreserved products to re-establish hematopoiesis.
Autologous hematopoietic cell transplantation utilizing peripheral blood-derived cells The existence of circulating hematopoietic stem cells capable of reestablishing hematopoiesis was established in mice in 1962 [19] and subsequently in other species [20,21]. Initial attempts to utilize circulat-
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ing autologous hematopoietic stem cells for autologous transplantation in humans failed [22,23], probably related to the infusion of an inadequate number of hematopoietic stem cells. Although an adequate number of hematopoietic stem cells for transplantation can be achieved utilizing multiple apheresis procedures [24], the practical application of this treatment required methods to increase the number of circulating hematopoietic stem cells. This increase in circulating hematopoietic stem cells can be achieved utilizing endotoxin [25] and recovery from chemotherapy [26,27]. However, it required the availability of hematopoietic growth factors, as well as the demonstration that their administration could dramatically increase the number of circulating hematopoietic progenitor cells and decrease the number of apheresis procedures required for an adequate dose [28] to make this approach the standard method for autologous transplantation. The hematopoietic progenitor cells collected after the administration of hematopoietic growth factors altered the kinetics of recovery of hematopoiesis. Both granulocyte colony-stimulating factor (G-CSF) [29] and granulocyte–macrophage colony-stimulating factor (GM-CSF) [30] were found to expand the number of circulating hematopoietic progenitor cells. Sheridan et al. [31] showed that hematopoietic stem cells collected after the administration of G-CSF not only shortened neutrophil recovery time, but also dramatically shortened platelet recovery. This does not appear to be the case using autologous peripheral blood stem cells collected after “priming” with GM-CSF [24,32]. Other hematopoietic growth factors, including erythropoietin [33], for mobilizing circulating progenitor cells have been used, but once again platelet recovery was delayed. However, whatever method of mobilization of circulating hematopoietic progenitor cells is utilized, extensive preceding therapy decreases the ease of collection of an adequate number of hematopoietic progenitor cells and delays recovery of hematopoiesis [34,35]. Peripheral blood-derived hematopoietic progenitor cells and bone marrow-derived hematopoietic progenitor cells have been compared in two randomized trials [36,37]. Both studies showed that autologous peripheral blood hematopoietic progenitor cells were associated with more rapid engraftment and had a similar treatment outcome. The relative ease of collecting peripheral blood-derived hematopoietic progenitor cells has made this the most frequent approach utilized for autologous hematopoietic cell transplantation in the world today.
In vitro treatment of autologous hematopoietic blood or marrow collections The risk of reinfusing cancer cells during autologous hematopoietic cell transplantation has been understood to be a risk for decades. The presence of tumor cells in rescue products has been shown using immunohistochemistry [38], bone marrow culture [39], and the polymerase chain reaction [40,41]. Some studies have found a poorer outcome in patients in whom residual disease in the graft could be identified [41,42], whereas others have questioned the significance of this finding [40]. The description of patients with early pulmonary recurrence after autologous bone marrow infusion in patients with lymphoma suggested that reinfusion of tumor cells may have been the cause [43,44]. Schultz et al., however, used a mathematical model to suggest that, in leukemia, reinfusion of tumor cells was unlikely to be the cause of relapse after autologous hematopoietic cell transplantation [45]. Brenner et al., using gene-marking techniques, showed that in some patients who relapsed after autologous hematopoietic cell transplantation for acute myeloid leukemia, the tumor cells at relapse were derived from the reinfusion product [46]. Attempts at removing tumor cells have used multiple approaches. Nadler et al. first reported the use of antibody treatment of autologous hematopoietic cells in an attempt to eliminate tumor cells [47]. De Fabritiis et al. showed that clonogenic Burkitt’s lymphoma cells could
Chapter 2
be eliminated from human bone marrow using a combination of monoclonal antibodies, compliment, and 4-hydroperoxycyclophosphamide [48]. Hagenbeek and Martens suggested that cryopreservation of the hematopoietic cells was more toxic to leukemic blasts than normal progenitor cells, and that freezing might reduce the risk of reinfusing of clonogenic tumor cells [49]. Attempts to reduce the risk of reinfusion of tumor cells has also been addressed by attempts to concentrate CD34positive hematopoietic stem cells and “leave behind” the tumor cells [50,51]. The use of antibodies directed against some tumor cells administered systemically at the time of peripheral blood cell collection might also reduce the number of tumor cells being collected.
100 Percent surviving disease-free
10
80
60 Sensitive relapse
40
Resistant relapse
20 No remission
0
Diseases treated by autologous hematopoietic cell transplantation Non-Hodgkin’s lymphoma The first malignancy successfully treated by high-dose therapy and autologous hematopoietic cell transplantation was non-Hodgkin’s lymphoma [11] in the late 1970s. In the early 1980s, as techniques of cryopreservation of hematopoietic cells improved, numerous singlecenter publications appeared demonstrating the curative potential of high-dose therapy and autologous hematopoietic cell transplantation in patients who had failed standard chemotherapy for aggressive nonHodgkin’s lymphoma [52–57]. Several of those centers pooled data and reported on the results on autologous hematopoietic cell transplantation in 100 patients with relapsed or refractory aggressive non-Hodgkin’s lymphoma [58]. Observations from that paper have affected the way in which autologous hematopoietic cell transplantation has been applied to patients with lymphoma and other diseases. Patients who had once achieved a complete remission, relapsed, and responded again to salvage chemotherapy before undergoing autologous hematopoietic cell transplantation had a higher complete remission rate following the autotransplant and were more likely to remain in remission than those who had relapsed from complete remission but were no longer chemotherapy sensitive. Both groups had a better result than patients who were primarily refractory to treatment and never achieved a complete remission (Fig. 2.2). These observations were tested in a phase II trial of 50 patients with aggressive non-Hodgkin’s lymphoma who relapsed from complete remission. All patients received dexamethasone, high-dose cytarabine, and cisplatin to test for chemotherapy sensitivity. The actuarial 2-year event-free survival for patients who were chemotherapy sensitive and underwent autotransplantation was 40% [59], confirming the retrospective study and forming the basis for a prospective trial comparing autotransplantation with standard-dose chemotherapy [60]. In that subsequent trial, 215 patients were treated and 109 responded to standard-dose salvage chemotherapy. They were then randomly assigned to continuing standard-dose chemotherapy followed by radiation or autologous hematopoietic cell transplantation. At 5 years, the event-free survival was 46% in the group undergoing transplantation and 12% in the standard therapy group – a difference that was highly significant. In addition, overall survival significantly favored the transplant (53% versus 32%), despite the fact that patients from the standard chemotherapy arm were able to be transplanted at a later time. This study established high-dose therapy and autologous hematopoietic cell transplantation as the standard treatment for patients with relapsed aggressive non-Hodgkin’s lymphoma. The history of the use of high-dose therapy and autologous hematopoietic cell transplantation in other types of non-Hodgkin’s lymphoma has not led to the same consensus as for the common aggressive nonHodgkin’s lymphomas. A number of single centers had reported encour-
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Fig. 2.2 Results of a retrospective study of 100 patients with diffuse, aggressive lymphoma showing that patients with chemotherapy-sensitive relapse do better with autologous transplantation than those who are chemotherapy resistant. Both groups have a better outcome than patients never achieving an initial complete remission [58].
aging results with the use of high-dose therapy and autologous hematopoietic cell transplantation in patients with relapsed follicular lymphoma [61–64]. One prospective trial has been completed and showed an advantage in disease-free and overall survival to patients undergoing hematopoietic stem cell transplantation [65]. However, the application of autologous hematopoietic cell transplantation in patients with follicular lymphoma has been much more controversial, and the development of a consensus has been much slower. Patients with Burkitt’s lymphoma were the first to be treated successfully with high-dose therapy and autologous hematopoietic cell transplantation [11]. The advent of high-dose chemotherapy regimens not requiring hematopoietic stem cell support has been so successful in curing patients with Burkitt’s lymphoma that this treatment is rarely utilized today. Several early reports of autologous hematopoietic cell transplantation in the treatment of lymphoblastic lymphoma [66,67] led to an international randomized trial [68]. However, the results did not reach statistical significance, and the use of autotransplantation in lymphoblastic lymphoma has remained controversial. Hodgkin’s disease High-dose therapy and autologous hematopoietic cell transplantation as a successful treatment for patients with relapsed Hodgkin’s disease was first reported in the 1980s [18,69–72]. By the early 1990s. Hodgkin’s disease had become one of the most frequent diagnoses for which autologous transplantation was applied. Subsequently, the British National Lymphoma Investigation carried out a randomized trial of high-dose therapy and autologous hematopoietic cell transplantation for patients with relapsed or refractory Hodgkin’s disease, the control arm being the same drugs used in the high-dose regimen but at lower doses [73]. Another randomized trial carried out by the German Hodgkin’s Lymphoma Study Group compared autologous hematopoietic stem cell transplantation with further conventional therapy in patients with chemotherapy-sensitive relapsed Hodgkin’s disease [74]. Both studies showed an improvement in failure-free survival but no difference in overall survival. Multiple myeloma Although currently one of the diseases most frequently treated using high-dose therapy and autologous transplantation, the fact that visible
The History of Autologous Hematopoietic Cell Transplantation
tumor cells were being reintroduced with the stem cell infusion probably reduced early enthusiasm for this treatment. Multiple myeloma has been treated with alkylating agents for several decades. McElwain and Powles showed in the early 1980s that high doses of melphalan could produce responses in patients refractory to the drug given orally at standard doses [75], but with prolonged and severe myelosuppression. In 1988, Barlogie et al. reproduced the high response rate with high-dose melphalan and showed that the intravenous infusion of autologous hematopoietic stem cells could reduce the duration of myelosuppression [76]. Subsequent studies tested the value of ex vivo treatment of the rescue product to attempt to eliminate tumor cells [77,78], and peripheral blood-derived hematopoietic cells [79,80]. The combination of blood- and marrow-derived hematopoietic cells [81] and the use of CD34-positive enriched hematopoietic stem cells [82] were both tested without any clear advantage being seen to either approach. These studies reflected the increasing interest in autotransplantation for multiple myeloma. However, the widespread acceptance of this treatment followed the publication of the first randomized trial demonstrating the superiority of high-dose therapy and autologous transplantation over standard chemotherapy [83].
Acute leukemia Perhaps surprisingly, autologous hematopoietic cell transplantation has been extensively pursued in the treatment of patients with acute leukemia. This was originally based on the lack of matched, allogeneic donors for most patients, the apparent responsiveness of the leukemia to very high doses of therapy, and the hope that small numbers of reinfused leukemia cells in patients in remission, or the infusion of no or very low numbers of cells after ex vivo treatment of the reinfusion product, might lead to an improved treatment outcome. This treatment was particularly popular in Europe. The first report of autologous hematopoietic stem cell transplantation in acute myeloid leukemia appeared in the late 1970s [84]. Treatment carried out for patients in relapse yielded remission in most, but was usually followed by rapid relapse. The use of ex vivo marrow treatment of the autograft to reduce leukemia cell reinfusion [85,86] and the treatment of patients in remission improved the duration of remission. Similar or somewhat less good results were seen with early attempts of autologous hematopoietic cell transplantation in acute lymphoblastic leukemia [87]. The early prospective trials of autologous hematopoietic cell transplantation in acute myeloid leukemia [88] and acute lymphoid leukemia [89] showed a higher relapse rate with autologous transplantation compared with allogeneic transplantation, but no clear advantage over intensive treatments not involving hematopoietic cell transplantation.
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remissions but most patients relapsing [94,95]. No prospective randomized trials have been completed.
Carcinomas and sarcomas Probably the carcinoma most frequently treated using high-dose therapy and autologous hematopoietic cell transplantation has been breast cancer. The rationale was a dose response in metastatic breast cancer [96] and the ability to collect autologous cells apparently free of tumor. Early attempts with high-dose therapy and autologous hematopoietic cell transplantation in breast cancer yielded primarily partial responses [97]. There was some evidence of a higher response rate with high-dose alkylating agents as opposed to other cytotoxic agents [98]. A number of high-dose combination chemotherapy regimens were developed in phase II trials, and several randomized trials were subsequently carried out [99]. In the United States, a high level of antipathy developed between physicians using high-dose therapy and autologous hematopoietic cell transplantation, and insurance companies that were reticent to fund this procedure. The borderline or negative results of most randomized trials and the revelation of fraud in one positive trial [100,101] dramatically reduced the frequency of utilization of this treatment approach. High-dose therapy and autologous hematopoietic cell transplantation has been studied in numerous carcinomas and sarcomas, generally with discouraging results. These studies include the treatment of patients with malignant glioma [102,103], malignant melanoma [104–106], neuroblastoma [107], ovarian cancer [108], lung cancer [109–112], and germcell tumors [113]. Of these diseases, only the treatment of germ-cell tumors has continued as a standard therapy [114].
Nonmalignant disorders Allogeneic bone marrow transplantation has been known for some time to have the potential to cure autoimmune disease [115]. It was much less clear that autologous transplantation could be beneficial since cells of the original immune system were being reinfused [116]. Two patients who underwent autologous bone marrow transplantation for lymphoma seemed to show an improvement in their markers for an autoimmune disorder (myasthenia gravis in one case and systemic lupus in another) [117,118]. These results led to attempts at autologous transplantation aimed at treating autoimmune diseases. The first studies focused on rheumatoid arthritis [119–121]. Studies in this and other autoimmune diseases continue.
Conclusion Chronic leukemia Autologous hematopoietic cell transplantation has been utilized to treat patients with chronic myeloid and with chronic lymphocytic leukemia. The rationale for autotransplantation in chronic myeloid leukemia was the existence of surviving normal hematopoietic stem cells [90] and the occasional durable remissions seen in patients who received toxic doses of busulfan [91]. Treatment of patients with chronic myeloid leukemia that had transformed to the acute phase using autologous transplantation of cells stored during the chronic phase of the disease yielded remissions but rapid relapse [92]. Treatment of patients in chronic phase occasionally produced durable remissions [93]. Autologous hematopoietic cell transplantation in chronic lymphocytic leukemia has also been tested. However, early results showed frequent
The history of autologous hematopoietic cell transplantation is a clear example of how clinical advances reflect advances in basic science and come as the result of innovative, but sometimes painstaking, clinical studies. Autologous hematopoietic cell transplantation was developed to test the hypothesis that dose escalation could affect cure in patients who had otherwise incurable malignancies. This turned out to be true for groups of patients with lymphoma, and the treatment continues to be widely utilized today. Studies in most patients with carcinoma and sarcoma showed minimal benefit and, with the exception of germ-cell tumors and certain pediatric malignancies, the treatment is rarely utilized. Although not curative, prolonged survival in patients with multiple myeloma has made the treatment widely utilized, and the results of autologous hematopoietic cell transplantation in nonmalignant disease have been sufficiently interesting to lead to ongoing studies.
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Chapter 2
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18. Carella AM, Santini G, Santoro A et al. Massive chemotherapy with non-frozen autologous bone marrow transplantation in 13 cases of refractory Hodgkin’s disease. Eur J Cancer Clin Oncol 1985; 21: 607–13. 19. Goodman JW, Hodgson GS. Evidence for stem cells in the peripheral blood of mice. Blood 1962; 19: 702–14. 20. Storb R, Graham TC, Epstein RB, Sale GE, Thomas ED. Demonstration of hemopoietic stem cells in the peripheral blood of baboons by cross circulation. Blood 1977; 50: 537–42. 21. Appelbaum FR. Hemopoietic reconstitution following autologous bone marrow and peripheral blood mononuclear cell infusions. Exp Hematol 1979; 7 Suppl 5: 7–11. 22. Hershko C, Gale RP, Ho WG, Cline MJ. Cure of aplastic anaemia in paroxysmal nocturnal haemoglobinuria by marrow transfusion from identical twin: Failure of peripheral-leucocyte transfusion to correct marrow aplasia. Lancet 1979; 1: 945–7. 23. Abrams RA, Glaubiger D, Appelbaum FR, Deisseroth AB. Result of attempted hematopoietic reconstitution using isologous, peripheral blood mononuclear cells: A case report. Blood 1980; 56: 516–20. 24. Kessinger A, Armitage JO, Landmark JD, Weisenburger DD. Reconstitution of human hematopoietic function with autologous cryopreserved circulating stem cells. Exp Hematol 1986; 14: 192–6. 25. Cline MJ, Golde DW. Mobilization of hematopoietic stem cells (CFU-C) into the peripheral blood of man by endotoxin. Exp Hematol 1977; 5: 186– 90. 26. Richman CM, Weiner RS, Yankee RA. Increase in circulating stem cells following chemotherapy in man. Blood 1976; 47: 1031–9. 27. Abrams RA, McCormack K, Bowles C, Deisseroth AB. Cyclophosphamide treatment expands the circulating hematopoietic stem cell pool in dogs. J Clin Invest 1981; 67: 1392–9. 28. Gianni AM, Siena S, Bregni M et al. Granulocytemacrophage colony-stimulating factor to harvest circulating haemopoietic stem cells for autotransplantation. Lancet 1989; 2: 580–5. 29. Duhrsen U, Villeval JL, Boyd J, Kannourakis G, Morstyn G, Metcalf D. Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients. Blood 1988; 72: 2074–81. 30. Socinski MA, Cannistra SA, Elias A, Antman KH, Schnipper L, Griffin JD. Granulocytemacrophage colony stimulating factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet 1988; 1: 1194–8. 31. Sheridan WP, Begley CG, Juttner CA et al. Effect of peripheral-blood progenitor cells mobilised by filgrastim (G- CSF) on platelet recovery after high-dose chemotherapy [see comments]. Lancet 1992; 339: 640–4. 32. Haas R, Ho AD, Bredthauer U et al. Successful autologous transplantation of blood stem cells mobilized with recombinant human granulocytemacrophage colony-stimulating factor. Exp Hematol 1990; 18: 94–8. 33. Kessinger A, Bishop MR, Jackson JD et al. Erythropoietin for mobilization of circulating progeni-
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The History of Autologous Hematopoietic Cell Transplantation 47. Nadler LM, Takvorian T, Botnick L et al. Anti-B1 monoclonal antibody and complement treatment in autologous bone-marrow transplantation for relapsed B-cell non-Hodgkin’s lymphoma. Lancet 1984; 2: 427–31. 48. De Fabritiis P, Bregni M, Lipton J et al. Elimination of clonogenic Burkitt’s lymphoma cells from human bone marrow using 4-hydroperoxycyclophosphamide in combination with monoclonal antibodies and complement. Blood 1985; 65: 1064–70. 49. Hagenbeek A, Martens AC. Cryopreservation of autologous marrow grafts in acute leukemia: Survival of in vivo clonogenic leukemic cells and normal hemopoietic stem cells. Leukemia 1989; 3: 535–7. 50. Gorin NC, Lopez M, Laporte JP et al. Preparation and successful engraftment of purified CD34+ bone marrow progenitor cells in patients with nonHodgkin’s lymphoma. Blood 1995; 85: 1647–54. 51. Berenson RJ, Bensinger WI, Hill RS et al. Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma. Blood 1991; 77: 1717–22. 52. Philip T, Biron P, Herve P et al. Massive BACT chemotherapy with autologous bone marrow transplantation in 17 cases of non-Hodgkin’s malignant lymphoma with a very bad prognosis. Eur J Cancer Clin Oncol 1983; 19: 1371–9. 53. Gorin NC, Najman A, Douay L et al. Autologous bone marrow transplantation in the treatment of poor prognosis non-Hodgkin’s lymphomas. Eur J Cancer Clin Oncol 1984; 20: 217–25. 54. Appelbaum FR, Sullivan KM, Thomas ED et al. Experimental hematology today. New York: Springer-Verlag; 1985. 55. Verdonck LF, Dekker AW, van Kempen ML et al. Intensive cytotoxic therapy followed by autologous bone marrow transplantation for nonHodgkin’s lymphoma of high-grade malignancy. Blood 1985; 65: 984–9. 56. Armitage JO, Jagannath S, Spitzer G et al. High dose therapy and autologous marrow transplantation as salvage treatment for patients with diffuse large cell lymphoma. Eur J Cancer Clin Oncol 1986; 22: 871–7. 57. Armitage JO, Gingrich RD, Klassen LW et al. Trial of high-dose cytarabine, cyclophosphamide, total-body irradiation, and autologous marrow transplantation for refractory lymphoma. Cancer Treat Rep 1986; 70: 871–5. 58. Philip T, Armitage JO, Spitzer G et al. High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediate-grade or high-grade nonHodgkin’s lymphoma. N Engl J Med 1987; 316: 1493–8. 59. Philip T, Chauvin F, Armitage J et al. Parma international protocol: Pilot study of DHAP followed by involved-field radiotherapy and BEAC with autologous bone marrow transplantation. Blood 1991; 77: 1587–92. 60. Philip T, Guglielmi C, Hagenbeek A et al. Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkin’s lymphoma [see comments]. N Engl J Med 1995; 333: 1540–5. 61. Liang R, Chen F, Lee CK et al. Autologous bone marrow transplantation for primary nasal T/NK cell lymphoma. Bone Marrow Transplant 1997; 19: 91–3.
62. Colombat P, Binet C, Linassier C et al. High dose chemotherapy with autologous marrow transplantation in follicular lymphomas. Leuk Lymphoma 1992; 7 Suppl: 3–6. 63. Rohatiner AZ, Johnson PW, Price CG et al. Myeloablative therapy with autologous bone marrow transplantation as consolidation therapy for recurrent follicular lymphoma. J Clin Oncol 1994; 12: 1177–84. 64. Schouten HC, Bierman PJ, Vaughan WP et al. Autologous bone marrow transplantation in follicular non-Hodgkin’s lymphoma before and after histologic transformation. Blood 1989; 74: 2579– 84. 65. Schouten HC, Qian W, Kvaloy S et al. High-dose therapy improves progression-free survival and survival in relapsed follicular non-Hodgkin’s lymphoma: Results from the randomized European CUP trial. J Clin Oncol 2003; 21: 3918– 27. 66. Milpied N, Ifrah N, Kuentz M et al. Bone marrow transplantation for adult poor prognosis lymphoblastic lymphoma in first complete remission. Br J Haematol 1989; 73: 82–7. 67. Santini G, Coser P, Chisesi T et al. Autologous bone marrow transplantation for advanced stage adult lymphoblastic lymphoma in first complete remission. A pilot study of the non-Hodgkin’s Lymphoma Co-operative Study Group (NHLCSG). Bone Marrow Transplant 1989; 4: 399–404. 68. Sweetenham JW, Santini G, Qian W et al. High-dose therapy and autologous stem-cell transplantation versus conventional-dose consolidation/maintenance therapy as postremission therapy for adult patients with lymphoblastic lymphoma: Results of a randomized trial of the European Group for Blood and Marrow Transplantation and the United Kingdom Lymphoma Group. J Clin Oncol 2001; 19: 2927– 36. 69. Jagannath S, Dicke KA, Armitage JO et al. Highdose cyclophosphamide, carmustine, and etoposide and autologous bone marrow transplantation for relapsed Hodgkin’s disease. Ann Intern Med 1986; 104: 163–8. 70. Philip T, Dumont J, Teillet F et al. High dose chemotherapy and autologous bone marrow transplantation in refractory Hodgkin’s disease. Br J Cancer 1986; 53: 737–42. 71. Carella AM, Congiu AM, Gaozza E et al. High-dose chemotherapy with autologous bone marrow transplantation in 50 advanced resistant Hodgkin’s disease patients: An Italian study group report. J Clin Oncol 1988; 6: 1411–6. 72. Phillips GL, Wolff SN, Herzig RH et al. Treatment of progressive Hodgkin’s disease with intensive chemoradiotherapy and autologous bone marrow transplantation. Blood 1989; 73: 2086–92. 73. Linch DC, Winfield D, Goldstone AH et al. Dose intensification with autologous bone-marrow transplantation in relapsed and resistant Hodgkin’s disease: Results of a BNLI randomised trial. Lancet 1993; 341: 1051–4. 74. Schmitz N, Pfistner B, Sextro M et al. Aggressive conventional chemotherapy compared with highdose chemotherapy with autologous haemopoietic stem-cell transplantation for relapsed chemosensitive Hodgkin’s disease: A randomised trial. Lancet 2002; 359: 2065–71.
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75. McElwain TJ, Powles RL. High-dose intravenous melphalan for plasma-cell leukaemia and myeloma. Lancet 1983; 2: 822–4. 76. Barlogie B, Alexanian R, Smallwood L et al. Prognostic factors with high-dose melphalan for refractory multiple myeloma. Blood 1988; 72: 2015–19. 77. Anderson KC, Andersen J, Soiffer R et al. Monoclonal antibody-purged bone marrow transplantation therapy for multiple myeloma. Blood 1993; 82: 2568–76. 78. Reece DE, Barnett MJ, Connors JM et al. Treatment of multiple myeloma with intensive chemotherapy followed by autologous BMT using marrow purged with 4-hydroperoxycyclophosphamide. Bone Marrow Transplant 1993; 11: 139–46. 79. Dimopoulos MA, Alexanian R, Przepiorka D et al. Thiotepa, busulfan, and cyclophosphamide: A new preparative regimen for autologous marrow or blood stem cell transplantation in high-risk multiple myeloma. Blood 1993; 82: 2324–8. 80. Fermand JP, Chevret S, Ravaud P et al. High-dose chemoradiotherapy and autologous blood stem cell transplantation in multiple myeloma: Results of a phase II trial involving 63 patients. Blood 1993; 82: 2005–9. 81. Jagannath S, Vesole DH, Glenn L, Crowley J, Barlogie B. Low-risk intensive therapy for multiple myeloma with combined autologous bone marrow and blood stem cell support. Blood 1992; 80: 1666–72. 82. Schiller G, Vescio R, Freytes C et al. Transplantation of CD34+ peripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma. Blood 1995; 86: 390–7. 83. Attal M, Harousseau JL, Stoppa AM et al. A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Engl J Med 1996; 335: 91–7. 84. Gorin NC, Najman A, Duhamel G. Autologous bone-marrow transplantation in acute myelocytic leukaemia. Lancet 1977; 1: 1050. 85. Gorin NC, Douay L, Laporte JP et al. Autologous bone marrow transplantation using marrow incubated with Asta Z 7557 in adult acute leukemia. Blood 1986; 67: 1367–76. 86. Yeager AM, Kaizer H, Santos GW et al. Autologous bone marrow transplantation in patients with acute nonlymphocytic leukemia, using ex vivo marrow treatment with 4-hydroperoxycyclophosphamide. N Engl J Med 1986; 315: 141– 7. 87. Carella AM, Martinengo M, Santini G et al. Autologous bone marrow transplantation for acute leukemia in remission. The Genoa experience. Haematologica 1988; 73: 119–24. 88. Zittoun RA, Mandelli F, Willemze R et al. Autologous or allogeneic bone marrow transplantation compared with intensive chemotherapy in acute myelogenous leukemia. European Organization for Research and Treatment of Cancer (EORTC) and the Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto (GIMEMA) Leukemia Cooperative Groups. N Engl J Med 1995; 332: 217–23. 89. Kersey JH, Weisdorf D, Nesbit ME et al. Comparison of autologous and allogeneic bone marrow transplantation for treatment of high-risk
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refractory acute lymphoblastic leukemia. N Engl J Med 1987; 317: 461–7. Coulombel L, Kalousek DK, Eaves CJ, Gupta CM, Eaves AC. Long-term marrow culture reveals chromosomally normal hematopoietic progenitor cells in patients with Philadelphia chromosome-positive chronic myelogenous leukemia. N Engl J Med 1983; 308: 1493–8. Finney R, McDonald GA, Baikie AG, Douglas AS. Chronic granulocytic leukaemia with Ph 1 negative cells in bone marrow and a ten year remission after busulphan hypoplasia. Br J Haematol 1972; 23: 283–8. Buckner CD, Stewart P, Clift RA et al. Treatment of blastic transformation of chronic granulocytic leukemia by chemotherapy, total body irradiation and infusion of cryopreserved autologous marrow. Exp Hematol 1978; 6: 96–109. Brito-Babapulle F, Apperley JF, Rassool F, Guo AP, Dowding C, Goldman JM. Complete remission after autografting for chronic myeloid leukaemia. Leuk Res 1987; 11: 1115–17. Rabinowe SN, Soiffer RJ, Gribben JG et al. Autologous and allogeneic bone marrow transplantation for poor prognosis patients with B-cell chronic lymphocytic leukemia. Blood 1993; 82: 1366–76. Khouri IF, Keating MJ, Vriesendorp HM et al. Autologous and allogeneic bone marrow transplantation for chronic lymphocytic leukemia: Preliminary results. J Clin Oncol 1994; 12: 748–58. Hryniuk W, Bush H. The importance of dose intensity in chemotherapy of metastatic breast cancer. J Clin Oncol 1984; 2: 1281–8. Tannir N, Spitzer G, Schell F, Legha S, Zander A, Blumenschein G. Phase II study of high-dose amsacrine (AMSA) and autologous bone marrow transplantation in patients with refractory metastatic breast cancer. Cancer Treat Rep 1983; 67: 599–600. Antman KH. Dose-intensive therapy in breast cancer. In: Armitage JO, Antman KH, editors. High-dose cancer therapy – pharmacology, hematopoietins, stem cells. Baltimore, MD: Williams & Wilkins; 1992. pp. 701–718. Rodenhuis S, Richel DJ, van der Wall E et al. Randomised trial of high-dose chemotherapy and haemopoietic progenitor-cell support in operable breast cancer with extensive axillary lymph-node involvement. Lancet 1998; 352: 515–21. Weiss RB, Gill GG, Hudis CA. An on-site audit of the South African trial of high-dose chemo-
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Mary M. Horowitz
Uses and Growth of Hematopoietic Cell Transplantation
Introduction The first successful transplantations of allogeneic hematopoietic cells were performed in 1968 in three children with congenital immune deficiency diseases [1–4]. In each instance, hematopoietic cells were collected from the bone marrow of sibling donors who were genotypically identical or closely matched to the recipient for human leukocyte antigens (HLAs). Since then, more than 800,000 patients have received hematopoietic cell transplantations (HCTs) to treat life-threatening malignant and nonmalignant diseases. Current estimates of annual number of HCTs are 55,000–60,000, worldwide (Fig. 3.1). Reasons for widespread use include proven and potential efficacy in many diseases, better understanding of the appropriate timing of transplantation and patient selection, greater availability of donors, greater ease of hematopoietic progenitor cell collection, and improved transplantation strategies and supportive care, leading to less transplantation-related morbidity and mortality and an increased ability to perform the procedure in older and sicker patients.
Changing indications for HCT HCT has efficacy in many diseases (Table 3.1). In some, transplantation corrects congenital or acquired defects in blood cell production and/or immune function. In others, it restores hematopoiesis after high-dose (myeloablative) cytotoxic therapy for malignancy and/or provides potent anticancer adoptive immunotherapy. In the 1970s, more than half of the diseases for which HCT was performed were nonmalignant disorders: 40% were for aplastic anemia and 15% for immune deficiencies. Fewer than half were for cancers, and these were mostly for advanced acute leukemia. In the 1970s, Thomas and colleagues showed convincingly that some patients with refractory acute leukemia could achieve longterm leukemia-free survival with high-dose therapy and HLA-identical sibling marrow transplantation (see Chapter 1) [5,6]. Better outcome was subsequently demonstrated in patients transplanted in first or second remission [7–9]. Syngeneic (identical twin) and allogeneic HCTs were shown to produce cytogenetic remissions and long-term leukemia-free survival in chronic myeloid leukemia (CML) in the late 1970s and early 1980s [10–12].
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
Use of allogeneic HCT to treat leukemia increased dramatically in the 1980s. By 1985, about 75% of allogeneic transplantations were for leukemia, with approximately equal numbers for CML, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL); more than 90% were from HLA-identical sibling donors. Introduction of alternative targeted therapies for CML in the early 2000s led to decreased use of HCT, with current guidelines reserving HCT for patients who fail to respond to nontransplant therapy or who lose their response [13]. However, leukemia treatment still accounts for about 65% of allogeneic HCT procedures (Fig. 3.2) (Center for International Blood and Marrow Transplant Research [CIBMTR] Statistical Center, unpublished data). Experimental and clinical evidence for a dose–response effect of drugs used in lymphoma therapy led to trials of autologous HCT to allow dose intensification in non-Hodgkin’s lymphoma in the middle 1980s (see Chapter 2) [14–16]. Results were promising, and there was rapid acceptance of autologous transplantation as salvage therapy in persons failing conventional chemotherapy for lymphoma. This was followed by increasing use of autologous transplantation as consolidation of primary therapy in patients with high-risk disease. There is now more frequent use of allogeneic transplantation in lymphoma to harness immunemediated graft-versus-lymphoma effects, especially in patients whose disease recurs after autologous transplantation, and in patients with follicular histology who are rarely cured by autologous transplantation [17]. Lymphoma accounted for 12% of allogeneic HCTs in 2006 (CIBMTR Statistical Center, unpublished data). The rationale of dose intensification led to the application of autologous transplants to many other hematologic and nonhematologic cancers over the past 10 years. One striking development was a dramatic increase in their use for breast cancer in the early 1990s. Breast cancer accounted for 16% of autologous transplants done in 1989–90 and 40% in 1994–95 [18]. However, results of randomized clinical trials in early and advanced breast cancer were disappointing (reviewed in [19–21]). In 1999, the use of HCT for breast cancer declined dramatically, and in 2006 breast cancer accounted for fewer than 5% of autologous transplants in North America. Treatment of solid tumors still accounts for about 10% of autologous transplants (Fig. 3.2). In 1996, results of a randomized trial comparing high-dose therapy and autologous HCT with conventional therapy for multiple myeloma were published; this study found a significant survival benefit with autologous transplantation [22]. Autologous HCT for myeloma increased dramatically after this report. Subsequent data suggested improved results with sequential autologous transplantations or autologous transplantation followed by allogeneic transplantation [23,24]. Multiple myeloma is now the single most common indication for HCT, account-
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Chapter 3
ing for 45% of autologous transplants but fewer than 5% of allogeneic transplants (Fig. 3.2). Thirty percent of HCTs for myeloma in 2006 involved a planned sequential autologous–autologous or autologous– allogeneic transplant approach (CIBMTR Statistical Center, unpublished data). The most common indications for allogeneic and autologous HCT in North America in 2006 are shown in Fig. 3.2. About 70% of allogeneic HCTs are for leukemia or preleukemia: 34% for AML, 16% for ALL, 6% CML, 10% for myelodysplastic or myeloproliferative syndromes, and less than 5% for other leukemias. About 15% are for other cancers, including non-Hodgkin’s lymphoma (11%), multiple myeloma (2N DNA content) are less efficient at reconstitution, and are similarly less efficient at homing directly to the bone marrow rather than the spleen or liver [69,70,72]. Within the 2N DNA pool of LT HSCs, the G0 cells transplant much more readily than those in G1 [70]. Following transplantation, a much higher fraction of LT-HSCs remain in cell cycle in the radioprotected host, and this period can extend for at least 4–5 months after transplantation [40,68,69,73]. Given the poor transplantability of cycling HSCs, it is conceivable that protocols that test cells for “stemness” by serial transplantation could be confounded by these lingering effects of mobilization and radiation on cell-cycle status and reconstitution ability [68,69,73]. Therefore, experiments that are designed to measure the life span of expanding and
38
Chapter 5 Kinetics of mobilization of LT-HSC following Cy/G-CSF
Cell-cycle status of LT-HSC in blood 298/307 0 40 80 120 100 200
1,000,000
Total HSC
100,000
10,000 Bone marrow Spleen Blood
1000
100 –1
0
1
2
3
4
5
6
7
8
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self-renewing HSCs by transplantation should be carried out in ways that remove the induced cell-cycle phenomenon from consideration. Although in the steady state only a small fraction of LT-HSCs is in the cell cycle, this ratio can be dramatically altered by a number of circumstances. For example, upon transplantation into irradiated hosts, probably every HSC that lands in a hematopoietic microenvironment undergoes, at least initially, symmetrical self-renewing cell divisions to expand the HSC pool. In addition, the techniques used to mobilize HSCs into the blood stream usually operate by first causing all HSCs to enter the cell cycle nonrandomly [72,74,75]. As shown in Fig. 5.2, treatment of a mouse with cyclophosphamide (which should kill no more than 4–8% of LT-HSCs, but should have a dramatic effect on both MPPs and oligolineage progenitors [see below] because they are largely in cell cycle), followed by granulocyte-colony-stimulating factor (G-CSF) treatment, results in every HSC entering the cell cycle, which leads to a 12–15-fold increase in the steady state number of LT-HSCs [74]. Even bleeding of mice, up to 25% or 50% of their blood volume two or three times in a week, results in a measurable increase in the fraction of HSCs in the cell cycle [76]. Replacement of RBCs lost due to bleeding suppressed the expansion, whereas replacement of fluid volume or white blood cells did not, indicating that HSCs are selectively responsive to decreases in RBC levels. Thus, positive and negative feedbacks on the regulation of HSC cell division must exist, but currently their nature and mechanisms are unknown. HSCs must go through many self-renewing cell divisions over the life of an animal, and it is reasonable to wonder if they are susceptible to a natural limit on the number of cell divisions they can undergo, often called the “Hayflick limit” [77], and whether HSCs have a special mechanism for maintaining the ends of their chromosomes, called telomeres, to avoid programmed cell senescence resulting from critical telomere shortening [78,79] (see Chapter 6). Both mouse and human HSCs have telomerase activity, but telomere length declines in the progeny of HSCs that have undergone a large number of cell divisions, in the context of normal blood development and in response to radiation and transplant recovery [80]. Therefore, the relatively high telomerase activity in HSCs [81] is not sufficient to completely prevent telomere shortening through several cell division cycles. The role of telomerase is complex. In the absence of a functional telomerase-reverse transcriptase (TERT) component of the telomerase complex, the progeny of dividing HSCs lose telomeres much more rapidly, confirming that it functions to maintain telomere length [27,80]. However, transgenic mouse strains that overexpress the TERT component at high levels in HSCs do not reduce the length of their telomeres, even following successive induced cell cycles by transplantation, and there is also evidence that TERT has a protective effect independent of its enzymatic function [82]. Nevertheless, these HSCs still lose trans-
5.2% S/G2/M
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Fig. 5.2 The total number of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow, spleen and blood of mice on successive days of cyclophosphamide/granulocyte colony-stimulating factor (CY/G-CSF) treatment. Total bone marrow hematopoietic stem cells (HSCs) were calculated by assuming that the femurs and tibias (less the epiphyses) contained 15% of all bone marrow in the mouse. Total HSC level in the blood was calculated by assuming that the total blood volume was 1.8 mL. Cell-cycle status of LT-HSCs in the blood was determined by Hoechst DNA staining [72].
plantation activity after the fourth or fifth serial HSC transplant generation [27]. This loss of transplantability with HSCs that retain telomeres points to other phenomena limiting the transplantation of these cells, including perhaps the persistent entry into the cell cycle, programmed cell life spans independent of telomere length, or random factors associated with transplantation and/or cell division [80,83]. Programmed cell death or senescence of HSCs need not occur only as a result of extensive numbers of HSC cell divisions. There are several genetic loci that regulate, in mice, both the frequency of HSCs and the frequency of HSCs that are in the cell cycle [84–86]. One genetic program that likely regulates HSC numbers is that leading to programmed cell death inhibitable by the antiapoptotic protein BCL-2 (and by extension, its antiapoptotic gene relatives such as BCL-x). In mice that have enforced high-level expression of human BCL-2 in HSCs, the steady-state levels of HSCs are increased four- to fivefold, and the competitive repopulation activity of these HSCs, cotransplanted with CD45 congenic HSCs into the same irradiated host, is high [87,88]. Therefore, it is likely that programmed cell death is one regulator of HSC numbers, and it is yet to be determined what stimuli to HSCs enact the programmed death pathway inhibitable by BCL-2. There are several genes whose expression is required to initiate hematopoiesis, hematopoiesis and angiogenesis, or angiogenesis resulting in failed hematopoiesis; these include AML-1/RUNX-1 [89], SCL-TAL [90], FLK1 [91,92]. It is unclear whether these genes act directly within HSCs, within their immediate precursors to generate HSCs or on other cell populations that have a role in the survival or stimulation of HSCs, or whether, as in the case of a developing vasculature, they regulate the movement of HSCs or their precursors from one part of the body to another [93]. Reverse transcription polymerase chain reaction (RT-PCR) analysis of highly purified HSCs, or the use of highly purified HSC messenger RNA probes to interrogate extensive mouse complementary DNA libraries has revealed that at least some of these genes are expressed intrinsically in HSCs [55,94–98]. It is important to point out that many factors could be playing a part in the movement of pre-HSCs or HSCs during embryonic and fetal development. Factors affecting the release of cells from a particular microenvironment, their movement through tissues or the blood stream, the recognition of endothelium in tissues, and their homing to stem cell niches could easily be read out as genes affecting HSCs without directly affecting either their development or their self-renewal capacity (see below). Genetic pathways for self-renewal of HSCs Self-renewal is the single distinguishing characteristic between LTHSCs and the rest of the multipotent and oligopotent progenitors in the hematopoietic system (see Chapter 6). While LT-HSCs can be expanded
Biology of Hematopoietic Stem and Progenitor Cells
in a variety of situations in vivo, the attempt to expand rigorously purified mouse or human HSCs in vitro with conventional cytokines such as steel factor (SLF), thrombopoietin (TPO), interleukin-11 (IL-11), IL-6, IL-3, and Flt3L, alone or in combination in serum-containing medium, has never resulted in more than a minor expansion in HSCs by phenotype, and little or no expansion by function [99–105]. Many investigators were led astray by the expansion of cells expressing CD34, but a broad variety of non-HSCs are also CD34+, and these cells were the result of proliferation of HSCs and progenitors coupled with cell differentiation. The introduction of the antiapoptosis protein BCL-2 into HSCs in mice allowed HSCs to survive serum-free culture conditions, so the response of LT-HSCs to factors in the absence of differentiating factors present in serum, such as monocyte/macrophage-CSF (M-CSF), G-CSF, granulocyte–macrophage CSF (GM-CSF), and others, could be studied [87,88]. Any combination of these cytokines and others when added to these BCL-2-containing HSCs led to a massive burst of proliferation and progenitor expansion by most HSCs, but no detectable HSC expansion [87,88]. These HSCs could respond to three factors as single factors in vitro: IL3, SLF, and TPO [88]. HSCs responding to IL-3 give large bursts of proliferation with concomitant maturation along the myeloid and mast cell lineages, without self-renewal of HSCs. One might have hoped that the HSC response to SLF alone might have been self-renewing divisions, as mutations in the SLF gene or its receptor, c-kit, lead to fetal or early neonatal lethality, usually because of profound anemia [106–111] (reviewed in [112]). However, the response of single highly purified Bcl-2-expressing LT-HSCs to SLF in serum-free medium was proliferation without detectable self-renewal, but with maturation through both CLP and CMP pathways [88]. Stimulation with TPO led to mild proliferation, and dedicated differentiation along the megakaryocytic lineage. Interestingly, RT-PCR analysis of messenger RNA from HSCs and highly purified myeloid progenitors revealed that HSCs might have the highest expression of the TPO receptor c-mpl of any of these cell populations [26,94,113]. Recent reports support a complex role for TPO in HSC biology. Neither TPO nor c-mpl is necessary for fetal HSC development, but HSCs in adult TPO- or c-mpl-deficient mice are cycling more and have impaired reconstitution ability compared with wild-type HSCs [114]. Further, TPO is produced by osteoblasts in the bone marrow, and blocking tpo antibody induces release from the niche and increased proliferation [115]. Taken together, these data suggest that TPO is important for HSC proliferation and self-renewal after transplantation, but also promotes quiescence by promoting retention in the niche with increased integrin expression and preventing cycling and subsequent HSC exhaustion. Some early experiments indicated that other factors might be involved in HSC expansion. For example, activation of the Notch-1 receptor, mimicked by retroviral transduction with the intracellular activated form of Notch-1, occasionally led to the generation of mouse cell lines with many of the characteristics of HSCs, although this response was neither general nor robust [116,117]. Provision of HoxB4 as a retroviral insert to semipurified mouse hematopoietic progenitors led to vigorous proliferation and at least some retention of hematopoietic multilineage and long-term reconstituting activity in vitro and in vivo [118–120], although a rigorous demonstration that this outcome was caused by expansion of HSCs alone, rather than provision of other signals that in addition enable their engraftment has not yet been established [119–121]. The Wnt/Fzd/Dsh/GSK-3β/β-catenin pathway is also implicated in HSC self-renewal (reviewed in [122,123]). Wnt, a highly hydrophobic protein, binds to surface frizzled (Fzd), a 7TM protein, in combination with a low-density lipoprotein-associated receptor, usually LRP5. Binding of Wnt to Fzd leads to activation of disheveled (Dsh). The action of Dsh prevents GSK-3 (glycogen synthase kinase) β-phosphory-
39
lation of β-catenin, and also allows β-catenin to dissociate from the complex and translocate to the nucleus, where it becomes available for binding to Lef/TCF factors and activation of transcription [122,123]. The addition of partially or fully purified Wnt3A to highly purified LT-HSCs in serum-free medium, with or without SLF, results in clonal proliferation of HSCs. In vitro, these HSCs go through a significant expansion, and up to 50% of the expanded progeny are cells of the LTHSC phenotype [124,125]. Transfection of nondegradable β-catenin lacking the N-terminal phosphorylation sites was reported to drive LTHSCs into massive and prolonged expanding cell divisions, with expansion of LT-HSCs both by phenotype and by function upon transplantation [124]. Inhibitors of Wnt signaling even blocked SLF and other cytokine-mediated proliferation of LT-HSCs [124]. When LTHSCs are transfected with a reporter of LEF/TCF + β-catenin activity in the nucleus and then transplanted into lethally irradiated hosts, LTHSCs reading out the reporter are retained in vivo long term, whereas myeloid progenitors derived from these LT-HSCs do not express the reporter [124]. Taken together, these results indicated that the Wnt/Fzd/ β-catenin pathway is likely to be an important pathway utilized by LTHSCs in self-renewing cell division. When activated by Wnt 3A, LTHSCs upregulate expression of both Notch-1 and HoxB4, genes also implicated in the self-renewal of HSCs. However, the regulated deletion of the β-catenin gene in transplanted bone marrow failed to affect the engraftment of hematopoiesis, or the retransplantation capacity of this marrow [126]. Contradictory results were recently reported with a different gene promoter encoding the cre recombinase required for βcatenin deletion [127]. The bmi-1 gene is a member of the polycomb family, which has a role in silencing gene expression by site-specific deacetylation of histones associated with those genes [128]. BMI-1 is expressed in high levels in mouse and human LT-HSCs, and mouse-made mutants for the BMI-1 gene have a profound defect in the transplantation and selfrenewal of LT-HSCs [97,128]. The two transcripts of the p16 locus, P16INK4A and P19ARF, are overexpressed selectively in the hematopoietic tissues in the BMI-mutant mice, thus implicating a role of BMI1 in the downregulation of genes whose expression is contrary to self-renewal of HSCs [129]. These could include decreased viability, enforced differentiation, or direct regulation or inhibition of genes involved in the self-renewing pathways such as those of the Wnt/Fzd/βcatenin and Notch pathways [116,117,122,124]. BMI-1 mutants do not show differences in expression in either HoxA9 or HoxB4 (Clarke et al., unpublished data), genes that also have some role in HSC functions [121,130]. JunB negatively regulates mouse LT-HSCs, and in the absence of JunB the HSC pool expands; in the expanded pool, p16ink4a and p19arf are expressed at lower levels than wild-type HSC, and Bcl-2 and Bcl-xl are increased in expression in JunB knockout compared with wild-type HSCs [131]. A number of genes have recently been reported to play a role in HSC self-renewal including junb, sox17, cited2, Mcl1, Tel/Etv6, Gfi1, Pten, and Stat5. Chapter 6 covers self-renewal in more detail, but taken together these results indicate that the control of self-renewal is a complex, multipathway process, much of which is probably controlled by the HSC niche. Migration of HSCs Hematopoietic capacity is known to be present in the adult mouse at highest levels in bone marrow, at about one-tenth that level in spleen, and at one hundredth or less of the level of bone marrow in blood [132]. In those early experiments, the ability of marrow or tissue transplants to retain erythropoiesis could not be assigned directly to actions of HSCs. There was no clear explanation as to why hematopoietic activity might
40
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be present in organs other than the bone marrow, and certainly not in the blood. It was only when clinical HCT groups described the phenomenon of hematopoietic activity increasing in the blood of cytotoxic drug-treated individuals that the possibility of HSC mobilization into the blood was considered [133,134]. Empirical protocols using cyclophosphamide plus cytokines such as G-CSF, GM-CSF, SLF alone or in various combinations with G-CSF, IL-1, and IL-3, led to efficient mechanisms to mobilize hematopoietic cells into the blood stream [135–139]. While at the early stages, this activity was measured in terms of in vitro tests for hematopoietic CFCs, but CFC assays alone are not specific for HSC versus oligolineage progenitor activities [37,58]. However, there could be no doubt that HSCs were involved when MPB transplants were used in hosts conditioned with high-dose chemotherapy, resulting in both early and sustained hematopoiesis. When cyclophosphamide and G-CSF are used in the mouse model, wherein one can analyze directly the daily changes in marrow, spleen, and blood, it is clear that nearly every HSC and MPP enters the cell cycle rapidly [74]. These reach a peak in the marrow, at which time the cells appear rapidly in blood and spleen (Fig. 5.2). At all times, the HSCs appearing in the blood have a 2N amount of DNA, indicating that they are not proliferating in the blood; in fact, the fold increase in their number would have ruled out a blood-only expansion of the HSC pool [74]. Bromodeoxyuridine incorporation into their chromosomal DNA during the expansion phase of HSC in the “mobilized” bone marrow results in the labeling of virtually every HSC, and, interestingly, just 1 or 2 days later the 2N HSCs found in the blood are all labeled with bromodeoxyuridine [74]. Thus, mobilization appears to result as a consequence of marrow HSC proliferation, and accumulation of emigrant HSCs in extramedullary sites such as the spleen, liver, and blood. The proliferation that precedes mobilization has been described above, and it is notable that the cytokines (e.g. G-CSF) that show high efficiency in the mobilization process do not have receptors on native HSCs [26,113,140]. In the early phases of mobilization, there is a rise in cells of the myelomonocytic series, and it has been shown that the elaboration locally by myeloid cells of matrix metalloproteinases such as MMP-9 can serve to cleave cell–cell interaction molecules known to be active between HSCs and marrow stroma [141]. Similarly, elaboration of the chemokine IL-8 occurs, and infusion of IL-8 alone can cause a significant and rapid mobilization of HSCs [142,143]. The actual nature of the molecules involved in HSC release is not clear, although it is known that HSCs express integrin α4β1, which allows them to attach to hematopoietic stromal cell vascular cell adhe-
Fig. 5.3 Rapid clearance of mobilized hematopoietic progenitor cells from the blood stream. Clearance from the blood of eGFP+ progenitors and PKH-26+ red blood cells (RBCs), and predicted progenitor and RBC frequencies, respectively. In vivo homing to different organs of mobilized and consecutively transplanted hematopoietic progenitor and stem cells 3 hours after injection. (From [72] with permission.)
sion molecule [144–148]. Also, HSCs express the c-kit receptor that allows binding to, at least, cell surface SLF on stroma. Although HSCs have a number of other members of the adhesion and integrin family members, the role of each of these in cell proliferation, detachment from stroma, local emigration into sinusoids, movement through the blood stream, margination on distant blood vessels, transendothelial migration at these vessel sites, and localization to microniches that support LTHSCs are still unclear. In the adult mouse, homing of infused HSCs appears to involve both cell surface integrin α4β1 and cell surface CXCR4, a receptor for the chemokine stromal cell-derived factor-1 (SDF-1) [149–151]. Of all known chemokines, adult mouse HSCs migrate in vitro only in response to SDF-1, and this is chemotactic movement [151]. However released, co-infusion of labeled HSCs and labeled RBCs from mobilized blood into the blood of same-stage mobilized animals results in the rapid emigration of HSCs out of the blood, with retention of RBCs in the vessels [72]. In fact, most HSCs are gone by 1–5 minutes, and they do not reappear in the blood stream within the next several hours (Fig. 5.3). These HSCs nonrandomly home to hematopoietic sites in mobilized animals: bone marrow, spleen, and liver, all tissues with sinusoids lined by macrophages. This observation fits with previous studies on the rapid egress of infused lymphocytes out of the blood stream into tissues via recognition of particular vascular addressins for which they have cognate homing receptors [152,153], as well as the rapid egress from the blood stream of other nucleated cells such as monocytes [154]. There are similar HSC, MPP, and myeloid progenitor fluxes in normal mice. To maintain the approximate 100 HSCs found in the blood of mice, given a residence time in the blood of 5 minutes or less, would require fluxes of well over tens of thousands of HSCs and MPPs per day. Recent data suggest that HSCs and progenitors circulate through the blood and reside in the tissues for up to 36 hours before returning to the blood via the lymphatics [155]. It is not yet clear whether these cells are passing through the blood in one pass or whether they form a specialized pool of HSCs and MPPs yet have a higher probability of re-entering the blood than cells resident in deep marrow niches. If HSCs found in the blood are also derived from dividing HSCs, nearly one out of every two daughter cells from a self-renewing HSC division would have to enter the blood stream. Consistent with this emigration rate, infusion of purified LT-HSCs into untreated immunodeficient mice (to avoid immune rejection of cells with antigenic markers) led to a dose–response engraftment of marrow sites of between 0 and 250 HSCs, while up to 5000 HSCs engrafted no
41
% granulocyte chimerism
Biology of Hematopoietic Stem and Progenitor Cells
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Fig. 5.4 Hematopoietic stem cell (HSC) niches can be transiently saturated in unconditioned animals [157]. (a) RAG2−/−γc−/− (CD45.2) mice were transplanted with 10, 50, 250, 500 or 1000 HSCs from green fluorescent protein (GFP) transgenic mice (shaded bars). Eight weeks after transplantation, mice were given a second transplant of 4000 non-GFP HSCs from CD45.1 donors (open bars). Peripheral blood granulocyte chimerism was analyzed at 16 weeks after the first transplantation. The dashed line indicates theoretical chimerism if endogenous HSCs were displaced from bone marrow niches. (b) Additive donor HSC engraftment following repetitive transplantation. Unconditioned CD45.2 RAG2−/−γc−/− mice were transplanted with saturating doses of GFP+ CD45.1 HSCs (250–1000) and retransplanted with 4000 GFP− CD45.1 HSCs 8 weeks after the first transplantation. Bone marrow was harvested and donor HSCs were quantified as Lin−c-kit+Sca-1+Slamf1+CD34−CD45.1+ cells that are either GFP+ (first transplant) or GFP− (second transplant).
more niches than 50 cells. This plateau was about 0.5% of marrow sites. However, 50 cells on day 1 and another 50 cells on day 2 gave about 0.5% of each population (Fig. 5.4) [156]. Thus, about 0.5% of the marrow niches for HSC appear to be available at any moment, and these appear to be occupied by the next interval. Preclearing of HSC with a cytoreductive anti-c-kit antibody alone in immunodeficient mice leads to up to 80% engraftment with purified HSCs given in large numbers at the point that the monoclonal antibody reaches its nadir concentration in the blood [157]. The migration of HSCs into hematopoietic tissues involves a step of crossing macrophage-lined sinusoids. We have recently found that, during the mobilization process, HSCs and progenitors upregulate the expression of the integrin-associated protein CD47 [158]. One function of CD47 is as a ligand for the signal regulatory protein-α macrophage receptor [159]. In the absence of CD47, macrophage-bound cells are phagocytosed, whereas cells expressing high levels of CD47 signal the macrophage through the receptor to block phagocytosis. We have found that leukemias transfected with mouse CD47 at levels approximating those found on mobilized HSCs are protected from phagocytosis in vitro and engraft in vivo, whereas the same leukemias lacking these levels of CD47 are phagocytosed in vitro and fail to engraft in vivo [158]. Further, HSCs from CD47 knockout mice fail to engraft wild-type congenic mice [158]. Taken together, these experiments provide evidence that, during
migration, itinerant HSCs express high levels of CD47 to gain entry to hematopoietic sites. This interpretation of the fate of recently self-renewed and also resting HSCs calls for a deeper examination of hematopoietic niches in the marrow; niches that commit recently entering HSCs into the myeloid or lymphoid pathways need not be adjacent to the HSC niche if the daughter cells of HSCs are constantly migrating. In addition, the existence of a large flux of recirculating HSCs likely provides for the continuous replacement of empty HSC niches with functional HSCs; in the absence of such a pool of migrating HSCs, the death of an HSC could mean the permanent loss of that functional niche. This finding also has relevance when one considers claims of plasticity of stem cells in one or more tissues [5]. If tens of thousands of HSCs are fluxing through tissues every day, finding HSC activity in those tissues might only result from these itinerant HSCs, rather than from transdifferentiation of local tissue-specific stem cells into hematopoietic fates [5]. Ontogeny and aging of HSCs It is tacitly assumed that HSCs derive from the embryonic mesoderm. In mammals, the first site of definable blood cells appears to be the yolk sac blood islands, in mice at about 7.5 dpc [160,161]. Although the terminology used for development from the time of conception is E
42
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Fig. 5.5 The ontogeny of hematopoietic progenitors. The sites of and duration of hematopoiesis in the fetus and neonate are shown. (Adapted from [178], with permission.)
(embryonic day) 1–21 (for mouse), in fact the transition from the embryo to fetus, at organogenesis, occurs at about 8–9 days post conception or coitus (dpc), so we will hereafter use the “dpc” abbreviation. At dpc 7–8, mouse yolk sacs contain cells capable of responding in vitro to produce hematopoietic CFCs [160,161]. The yolk sac connects to the developing embryo/fetus [162] at about dpc 8.5 later via the development of the vitelline vessels, which feed into the developing fetal liver. At very nearly that dpc 8–8.5 time interval, hematopoietic cells appear in and around the dorsal aorta of the developing fetus (reviewed in [163]). Transplantation of dpc 8–9 yolk sac blood island cells into the yolk sac cavity of dpc 8 allogeneic hosts results in mice that have a lifelong presence of donor-derived day 10 CFU-spleen (CFU-S) in marrow as well as thymocytes [93]. At about dpc 10–11, hematopoiesis begins in the fetal liver in the mouse, and continues there throughout fetal life (Fig. 5.5). Where do the first HSCs develop [164]? At dpc 3.5, the blastocyst implants into the uterus, and its inner cell mass appears to be a population of pluripotent cells without defined commitments [162]. Between dpc 3.5 and dpc 5.5, gastrulation occurs with the formation of the three germ layers [162]. It has been a mystery whether the developing postgastrulation mesoderm cells emigrate separately to yolk sac and embryo to give rise to independent origins of hematopoiesis, or whether only one of these sites is the initial site of commitment to hematopoiesis, the later appearance of hematopoiesis at the other site resulting from migration [93,163,165,166]. The idea of separate origins for extraembryonic (yolk sac) hematopoiesis and intraembryonic hematopoiesis was supported by the fact that yolk sac erythropoiesis resulted in the production of embryonic and fetal hemoglobins, while lifelong hematopoiesis resulted in the production of mainly adult hemoglobin [165,167]. These were called, respectively, primitive and definitive hematopoiesis [167]. Until recently, no experiment directly assessed whether these two kinds of hematopoiesis have distinct or common origins at the cellular level, To address this question, Nishikawa’s group used an inducible Runx1/ aml1 reporter system to label extraembryonic HSCs [168]. When embryos were induced at dpc 7.5, the time that HSCs first appear in the yolk sac blood islands [93,169], labeled hematopoietic cells were observed in the fetus and adult. As circulation was not established at this time point, these data suggest that the labeled HSCs that persist and
give rise to ongoing hematopoiesis must have derived from yolk sac HSCs and migrated into the embryo once circulation was established [168]. Because the allantois at dpc 9 has some runx1+ cells, it is another possible site of HSC generation in these experiments if the tamoxifen inducer has a half-life in the embryo of greater than a day. There is a close association of hematopoiesis and angiogenesis in both the developing blood islands and the developing dorsal aorta, and there is good evidence that mutant mice lacking the cell surface receptors for angiogenic peptides fail to develop either hematopoietic or angiogenic cells [91,170]. While these experiments suggest that a bipotent precursor called the hemangioblast exists that can give rise to both angiogenic stem cells and HSCs, only a small number of hemangioblast cells capable of producing both outcomes in vitro can be prospectively isolated from the pre-yolk sac embryo [171]. Rather than there being a hemangioblast precursor, it is possible that mutant mice that cannot make blood vessels properly cannot make blood because primitive stem cells travel via the blood, or that blood vessels make factors important for hematopoiesis. A recent set of experiments supports the hypothesis that the majority of embryo blood cells derive from distinct progenitors for hematopoiesis and endothelium [172]. Tetrachimeric mice were generated by the injection of four distinctly colored single cells into developing blastocysts. Analysis of the color combinations observed in the yolk sac of the resulting embryos revealed endothelial and hematopoietic cells of different colors in all individual blood islands, indicating that they developed from more than one cell (Plate 5.1). If both hematopoietic and endothelial cells derived from a common hemangioblast progenitor, each blood island would be monochromatic in these mice. However, single-color blood islands were not observed. Instead, the majority of blood islands contained blood and endothelial cells of multiple but not necessarily the same colors, indicating that multiple progenitors of each type came together to form each blood island [172]. About 15–20% of the blood islands examined could have derived from hemangioblasts; when we lineage-marked cells with the Flk1.cre/floxed Lacz-GFP (green fluorescent protein) constructs in the same ES cells, about 20% of blood cells were GFP+, showing an activated flk1 locus, but 80% were lacz+GFP−. Taken together, these data support about 20% of early blood cells deriving from a possible hemangioblast progenitor, and about 80% being derived from a hematopoietic progenitor that has not gone through a hemagioblast pathway. Similar conclusions were drawn by Shalaby et al. [91]. There is an interesting point coming out of ES cell biology that might be relevant to these issues. ES cells are derived from the inner cell mass of blastocysts, and likely represent pluripotent stem cells in the mouse at approximately dpc 3.5 age. They are maintained as pluripotent cells by the addition of leukemia inhibitory factor to their cultures, at least in mouse ES lines [173]. Removal of leukemia inhibitory factor from these pluripotent ES lines results in the onset of hematopoiesis, and during this development, at about 4 days, cells capable of in vitro hematopoiesis emerge, which express some markers shared with HSCs (Yamane, Domen, and Weissman, unpublished data). This interval, 3.5–4.0 days, correlates in developmental time to about dpc 7.5, when HSCs first appear in yolk sac blood islands [93,169]. Thus, these two systems appear in parallel to produce embryonic HSCs. The major site of the next (fetal) stage of hematopoiesis appears to be the fetal liver [161]. The numbers of HSCs in the fetal liver increase logarithmically from dpc 11–15. After dpc 15, fetal liver HSC numbers level off, but hematopoiesis continues to expand in the liver [53,174,175]. At dpc 16 hematopoiesis appears in the fetal spleen in mice, and at dpc 18 in the fetal bone marrow. The initiation of fetal liver hematopoiesis is blocked in mice with mutations in integrin α4β1 (very late antigen-4 [VLA-4]) or integrin α5β1
Biology of Hematopoietic Stem and Progenitor Cells
(VLA-5) [176,177]. Because integrins are involved in cell migration, it is reasonable to propose that immigration of these cells into or beyond the fetal liver might require steps that include the expression of α4β1, or induce the expression of integrin α5β1 at this stage of the liver. Integrin α4β1 is used by trafficking HSCs throughout life to enter sites of hematopoiesis [148], so the endowment of expression of integrin α4β1 permits fetal liver HSCs to be assayed by transplantation in fetuses or adults. It is important to note that neither yolk sac blood island HSCs nor ESderived HSCs in their native state can be transplanted into adult irradiated mice. It has been reported that the transition of yolk sac HSCs and ES-derived HSCs from that embryonic stage to the fetal stage can be facilitated by enforced expression by HoxB4, or by HoxB4 and cdx4 in these cells [118]. Therefore, there are important clues to how embryonic HSCs, presumably capable of primitive hematopoiesis, can develop into fetal HSCs capable of definitive hematopoiesis. During the various stages of fetal HSC function, it seemed possible that waves of HSCs emerge from tissues such as the fetal liver at specific time points to be accepted by and to engraft in secondary sites such as fetal spleen, fetal bone marrow, and lymphoid sites such as the thymus [93]. However, HSCs are found constitutively in the blood throughout fetal life, and it must be the preparedness of the developing organs such as the spleen, marrow, and thymus that results in the movement of hematopoiesis to those sites [178]. A detailed examination of HSCs in the developing fetal liver with respect to T-cell developmental potentials has revealed that changes are occurring at the level of HSCs which show that HSCs develop in a quantal but dynamic process [179]. Developing T cells gain their specificity and function in a coordinated series of molecular events, including selection of which class of T-cell receptor (TCR) genes are rearranged and selected, and to which distant sites the T cells will migrate. Within the thymus during fetal development, the first T cells to emerge use products of the TCR Vγ3 and TCR δ gene families, to be followed by use of the TCR Vγ4, then Vγ2 and Vγ5 and TCR αβ gene families. In mice, the TCR V genes at the γ locus that must rearrange to be expressed are, extending from the most proximal site of potential rearrangements, Vγ3, Vγ4, Vγ2, and then Vγ5. The first T cells to emerge in the thymus are Vγ3 cells, which colonize the skin only during fetal life [180]. While Vγ3 T-cell development is shutting down in the thymus, Vγ4 T-cell development arises, and these T cells will migrate during their fetal life to the presumptive female genital tract epithelia and the tongue epithelium of both sexes [179,180]. The development of Vγ4 T cells decreases at about day 16 or 17 of fetal life, to be overtaken by the almost simultaneous onset of that will inhabit the entire gastrointestinal tract, and αβ T cells that are largely restricted to lymphoid tissues. The kinetics of T-cell development can be studied with the use of fetal thymic organ cultures, although it should be noted that these are not physiologic cultures. When single clonogenic dpc 12 fetal liver HSCs are added to fetal thymic organ cultures, those thymuses produce Vγ3, then Vγ2, then Vγ5, and TCR αβ T cells [179]. When dpc 14 fetal liver HSCs are placed at clonal levels into the same stage fetal thymic organ culture, Vγ3 T cells do not emerge, but the rest of the repertoire is developed. Finally, dpc 12 fetal liver HSCs added directly into the adult thymus cannot produce the fetal program of Vγ3 T cells, but can produce the mature program of Vγ2, Vγ5, and αβ TCR cells [179]. Taken together, these studies indicate that, probably with each cell division, the fetal liver HSCs are changing (usually reducing) their developmental fates that would only be read out when their progeny entered the fetal thymus, and the change in these fates and the readout of these fates is dependent upon factors within HSCs and within the thymus microenvironment. After birth, and at least in young adult mice, the numbers of LT-HSCs, ST-HSCs, and MPPs are regulated at a relatively constant level.
43
HSCs in the mouse go through cell autonomous changes in number and developmental potential during aging. We first reported that there is a gradual loss of cells of the MPP phenotype, and an increase in both the number of LT-HSCs and the fraction of LT-HSCs that are in the cell cycle [83], most dramatically seen in the marrow of geriatric mice. With a refinement of markers for LT-HSC versus all other MPPs, we found that the number of LT-HSCs increased as reported, but that they retained a low level of cell-cycle activity; the increase was found primarily in an MPP fraction [96]. In old mice, myelopoiesis is retained or even enhanced, while lymphopoiesis in both T and B lineages falls. These fates are cell intrinsic; co-transplantation of 2-month-old and 2-year-old LT-HSCs into irradiated young mice led to increased output of LT-HSCs from the old versus young stem cells, and decreased output of CLP and T and B cells from the old HSCs compared with the young stem cells [96]. These same trends were seen in the small number of aged hosts transplanted with young and old HSCs. The cell intrinsic biologic properties of old versus young HSCs were mirrored by a microarray analysis of these highly purified cells: young HSCs had both myeloid and lymphoid transcripts, but old HSCs overexpressed myeloid and underexpressed lymphoid transcripts [96]. Of the top 32 myeloid transcripts overexpressed in old HSCs, 13 have been identified as proto-oncogene or translocation partners of myelogenous leukemia proto-oncogenes in humans. As myelogenous leukemias increase in incidence with age, it is reasonable to propose that initiating events may occur in HSCs, and if so, it is possible that genomic events such as translocation, inversion, etc, occur preferentially at highly transcribed loci. Does hematopoiesis only derive from HSCs, and do HSCs only give rise to blood? Several years ago, there were reports that bone marrow hematopoietic cells could give rise to brain cells, that brain stem cell populations could give rise to blood, that fat cells could give rise to neurons and mesenchymal fates, that muscle stem cells could give rise to hematopoietic and myogenic outcomes, and a host of others (reviewed in [181]). What was lacking in most of these experiments was the demonstration that the transplanted population was in fact a stem cell purified to homogeneity that could give rise at the clonal level to two different tissue types, and that the tissue types that had been generated were robustly characterized as functional mature cells of that tissue without cell fusion. Attempts to repeat the experiments showing transdifferentiation of CNS stem cells to hematopoietic tissues have thus far failed. A reexamination of the cells in muscle that give rise to blood show them to be CD45+ committed HSCs, separable from muscle progenitors that, on their own, give rise to muscle [182,183]. In some circumstances, regenerating muscle following an injury can incorporate cells whose markers are expressed in the regenerated muscle tissue, and the most common transplantable source of these cells is bone marrow [184,185]. While some claims have been made that these are derived from HSCs, in fact single purified HSCs, even over long intervals, cannot contribute to muscle or any other tissue, except a rare set of liver cells in irradiated hosts [5]. In another example, liver regeneration of hepatocytes bearing markers from injected purified HSCs, following multiple rounds of selection through liver toxicity, can occur [186], but recent evidence [187,188] indicates that these are mainly a result of rare cell fusion events between myelomonoctic progeny of the HSCs and some precursor of liver cells. Furthermore, the marrow or HSC or circulating precursor cells that enter undamaged or kainite-damaged brains are limited to microglia and CD45+ cells, as well as rare binucleate Purkinje cells derived by cell fusion [189]. We had previously proposed that cases of transdifferentiation of one tissue-specific stem cell to another tissuespecific stem cell might not really be transdifferentiation but differen-
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tiation from a more pluripotent precursor [1]. That possibility is still viable. The many claims that transdifferentiation could occur reflect a problem in stem cell biology as a young field. Most new investigators to the field did not appreciate the importance of purifying the populations to homogeneity, populations of cells of apparent phenotypic and functional homogeneity, and did not mark cells retrovirally or with other genetic markers to follow the fate of clonogenic precursors. The transition from discovery to accepted scientific fact It is important to note that, in order for a reported discovery to become accepted as scientific fact, the following several criteria need to be met: 1. The initial discovery must be published in fully peer-reviewed journals. 2. The experiment as published must be repeatable in many independent laboratories. 3. The phenomenon described should be so robust that other experimental methods must reveal it. 4. In the case of transplantation, the tissue regeneration must be therapeutic in quantity and quality. Based on these criteria, we do not believe there is sufficient evidence for any of the transplant claims of transdifferentiation. In addition, bone marrow is not the same as HSCs, but is a population of mature or maturing cells, and certainly includes at least two or three stem and progenitor populations: HSCs, mesenchymal stem cells (MSCs), and endothelial precursors, which might really be MSCs [18]. Thus, it is possible that the transdifferentiation ability or “plasticity” ascribed to HSCs to interpret formerly unexpected outcomes from cell transplantations, if not the result of cell fusion, is more likely derived from a class of previously unappreciated adult pluripotent cells, or perhaps the normal developmental fates of MSCs are not yet appreciated. It is conceivable that some of these very rare cell fusions could be part of a regenerative process, but there is no evidence today that such is the case. Thus, it is inappropriate for such initial claims to be considered true enough to be the basis of clinical trials or care protocols or public funding and policy decisions.
Lineage-committed hematopoietic progenitor cells Considerations for the definition/isolation of hematopoietic progenitors The developmental process from HSCs to mature cells must involve intermediates that have lost stem cell potentials but are not yet terminally differentiated. An important question and active area of research is the sequence in which lineage commitment occurs. Several findings have suggested the existence of oligopotent progenitors. First, the lack of lymphocyte in patients with severe combined immunodeficiency syndromes was taken as evidence for the existence of oligopotent, lymphoid-committed progenitor cells or lymphoid stem cells. However, the loss of lymphocytes caused by adenosine deaminase deficiency [190,191] or by mutations of the common cytokine receptor γ-chain (γc) [192,193] does not necessarily imply a common progenitor. Rather, several lymphoid cell types or their progenitors might be most susceptible to the induced alterations [194] or might be dependent on the same signal transduction mechanisms. This same caution needs to be applied to the interpretation of hematopoietic phenotypes of genetically altered mice, as mutants of Ikaros [195] or Notch-1 [196] that could either be essential factors for a common or multiple different progenitors [197]. Second, leukemia cells can be viewed as cells that are arrested at early developmental stages but have gained self-renewal capacity. The occur-
rence of leukemias that either coexpress antigens normally associated with myeloid or lymphoid cell types (mixed-lineage leukemias) or that harbor two leukemia populations of clonal origin (bilineal leukemias) could be taken as evidence for the existence of bipotent B-myeloid or T-myeloid progenitors [198–203] (reviewed in [204–206]). However, leukemia clones could be derived from HSCs, from MPPs or from restricted progenitors that, because of their altered gene expression profile, display cell surface gene products which are normally not present at these developmental stages. Third, the fact that single cells in bone marrow give rise to colonies that contain all myeloid but no lymphoid progeny in vitro (colonyforming unit-granulocyte/erythrocyte/macrophage/megakaryocyte [CFUGEMM]) or in vivo (mixed CFU-S) is suggestive for the existence of oligopotent myeloid progenitors. However, cells that give myeloid readout could also be multipotent but did not find the conditions to read out all possible progeny [48,207,208]. In light of these problems, several prerequisites need to be met to define restricted progenitors in a developmental hierarchy. First, in order to prospectively identify candidate populations, they need to be isolated to highest possible homogeneity/purity. Second, if a population shows oligopotent differentiation activity, it must be demonstrated that at least some single cells within this population give oligopotent readout, i.e. that this population is not a mixture of different monopotent progenitors. Third, it further needs to be shown that single oligopotent progenitors are not HSCs or MPPs. These prerequisites are difficult to achieve. At any given time point, progenitor cells might undergo intrinsic “stochastic” random commitment events, might not find suitable microenvironments to read out in all their possibilities or might even not read out at all [209,210]. However, by applying the above criteria, we and others were able to identify oligopotent developmental intermediates in mice and humans. These data strongly support the hypothesis that multipotent hematopoietic progenitors first lose their self-renewal ability and consecutively commit to either the lymphoid or the myeloid developmental pathway. Although not formally proven, based on frequency, cycling status, and in vitro and in vivo expansion potential of bone marrow lymphoid and myeloid committed progenitors, it seems possible that all hematopoiesis develops through either a common lymphoid or common myeloid developmental stage. CLP cells and lymphoid development Lymphoid cell development from HSCs is dependent on externally provided differentiation, growth, and survival factors. Of those, IL-7 might be most important. Its cognate high-affinity receptor is a complex composed of the IL-7 receptor-α (IL-7Rα) chain [211] and the γc chain [193,212]. Neutralizing antibodies to IL-7 or genetic ablation of either IL-7 or IL-7Rα inhibits both T- and B-cell development in vivo [213,214]. Targeted deletion of the γc gene leads to the loss of T and B cells and the additional loss of NK cells [215–217], presumably because of the impaired formation not only of the IL-7R, but also the IL-2R and IL-15R that might be nonredundant cytokines for NK cell development [218– 220]. Further, mice that lack Jak3, a signal transduction molecule associated with γc [221,222], display a phenotype similar to the γc-deficient mice [223–225]. Also, patients with genetic defects in Jak3 show similar defects in lymphoid development to patients with γc gene disruptions (X-linked severe combined immunodeficiency syndrome) [226,227]. Based on these data, IL-7 is recognized as a nonredundant cytokine for both T- and B-cell development that could act as a proliferation factor or a survival factor and/or could initiate lineage-specific developmental programs. IL-7R is expressed in both developing T and B cells, and in mature T cells [228]. In genetically modified mice that are either IL-7Rα−/−,
Biology of Hematopoietic Stem and Progenitor Cells
45
HSC
Population
MPP
HSC
Lin–KSCD150+ Flk2–Thylo(CD105lo CD34–FcγRlo)
MPP
Lin–KSCD150–Flk2+ Thylo/–(CD105lo, CD34–/+ FcγRlo)
GMLP GMLP
MEP
GMP ProT
Lin–KitintScaintIL7Rα+Flk2+
MCP
Lin–Kit+Sca–Ly6c–FcεRIα–CD27–T1/ST2+β7+
CMP
Lin–Kit+ScaloCD150+CD34+FcγR+
GMP
Lin–Kit+Sca–CD150+ Flk2–/+FcγRlo
MEP
Lin–Kit+Sca–CD150+ Flk2–FcγRlo
MkP
Lin–Kit+Sca–CD150+ Flk2–CD41+CD9+
BLP
MCP
MkP
Lin–Kit+ScaloFlk2++
CLP CLP
CMP
Phenotype
EP T cells
Mast cells
B cells
EP
Lin–Kit+Sca–CD150+ Flk2–CD105+
Dendritic cells Platelets
RBC
Myelomonocytic cells
Fig. 5.6 Schematic of hematopoietic development. Composite of current data from mice indicating intermediates in the hierarchy of hematopoietic differentiation. Hematopoietic cells (HSC), long-term reconstituting, self-renewing; MPP multipotent progenitors with limited or no self-renewal leading to transient but multilineage reconstitution; GMLP; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; BLP, B-lymphocyte progenitor; ProT, T-cell progenitor; GMP, granulocyte–macrophage progenitor; MEP, megakaryocyte–erythroid progenitor; MCP, mast cell progenitor; MkP, megakaryocyte progenitor; EP, erythroid progenitor.
γc−/− or IL-7−/−, T-cell progenitors express low levels of Bcl-2, an antiapoptotic protein [229]. However, incubation of T cells from IL-7−/− mice with recombinant IL-7 results in the upregulation of Bcl-2 [230], and enforced expression of Bcl-2 in either IL-7Rα−/− or γc−/− mice leads to significant rescue of αβ T-cell development [25,230,231]. Therefore, a critical role of IL-7 in developing and mature αβ T cells is the promotion of survival via expression of Bcl-2 or other antiapoptotic proteins as possibly Bcl-Xl [232]. However, enforced expression of Bcl-2 was not sufficient to rescue B-cell development and αβ T-cell development [25,87,230]. Here, IL-7R-mediated signals are necessary for the rearrangement of immunoglobulin heavy chain V segments via Pax-5 gene activation [233,234] and for V–J recombination of αβ TCR genes via Stat5 [235,236]. Therefore IL-7R signaling can either lead to the transmission of “trophic” survival signals in the T-cell lineage or “mechanistic” differentiation and proliferation signals in the B and αβ T-cell lineages, respectively. Thus, it was reasonable to search for oligopotent CLPs within the Lin−IL-7Rα+ fraction in adult mouse bone marrow. We identified a population of Lin−IL-7Rα+ Thy-1−Sca-1loc-kit lo cells that also express γc, indicating that this population possesses functional IL-7R (Fig. 5.6) [25]. In contrast, both LT-HSC and ST-HSC populations are IL-7Rα−. These CLPs possess rapid and potent T-, B-, and NK-cell-restricted differentiation activity in reconstitution assays. In both in vitro and in vivo assays, Lin−IL-7Rα+ Thy-1−Sca-1loc-kit lo cells completely lack myeloid differentiation activity [25]. Also no day 8 and day 12 CFU-S activity could be detected [25,37]. Injection of 1000 CLPs could generate 0.6– 1.1 × 107 CD3+ spleen T cells by 4–6 weeks, and 1.4 × 107 B220+ spleen B cells by 2 weeks in vivo. CLP-derived T- and B-cell generation peaks
7–10 days earlier than similar numbers of T and B cells derived from the same number of HSCs [25]. However, in contrast to HSC-derived cells, numbers of CLP-derived T and B cells begin to decline after 4–6 weeks, indicating that this population has no or limited self-renewal activity. Using a two-step assay, we demonstrated that CLPs contain clonogenic progenitors for both T and B cells. Single cells were cultured in methylcellulose in the presence of SLF, IL-7, and Flt3L to expand cell numbers, and a portion of the day 3 colonies were picked up and injected directly into the thymus. Those injected into the thymus differentiated into all stages of T-cell development and, in some cases, differentiated into both T- and B-lineage cells. The further cultured cells formed Blineage colonies composed of pro-B and pre-B cells [25]. Therefore, the defined CLPs match the criteria for the definition of oligopotent progenitor cells. We have subsequently shown that the purity of CLPs is further refined based on their expression of Flk2, whereas the Flk2 fraction of the previously defined CLPs contains B-committed cells [237]. Accordingly, CLPs exist downstream of Flk2+ ST-HSCs and MPPs in normal hematopoiesis [51,52,238]. Based on their frequency in bone marrow, their cycling status and their in vivo T-, NK-, and B-cell generation potential, CLPs theoretically could be a developmental intermediate for most if not all mature lymphoid cells. The commitment of CLPs to either the T-, B- or dendritic cell lineage may be determined simply by the microenvironment and signals that CLPs encounter. However, it is still unclear whether CLPs are an indispensable stage of T- and B-cell development. Coffman and Weissman identified the B220 marker that is present on all B-lineage cells [239], and early B-cell progenitors were found to be
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B220+, CD43+, CD24-BP1/6C3− [240,241]. Hardy and his colleagues further subdivided B-cell progenitors into fractions: pre-pro-B cells (fraction A0: AA4.1+CD4loB220−HSA−; A1: AA4.1+CD4+B220+HSA−; A2: AA4.1+CD4−B220+HSA−) and pro-B cells (fraction B: B220+CD43+HSA+6C3/BP-1−; C: B220+CD43+HSA+6C3/BP-1+) [242– 244] (reviewed in [245]). Some fraction A0 cells express c-kit and Sca1 and can give rise not only to B cell, but also to myeloid and T-cell progeny. Therefore, they might contain MPPs. The majority of fraction A1 cells are c-kit − and lack myeloid potential, but minor T-cell potential can be detected after intrathymic injection. Pro-B cells are B-lineage committed, rearrange DH–JH genes (fraction B) and undergo V–DJ recombination (fraction C). In view of their T- and B-cell readout capacity, the A0 or A1 fraction could contain CLPs, but because different markers were used for their isolation, a direct comparison of data from these populations is not possible. However, a majority of A0 and A1 fraction cells do not express IL-7Ra, while IL-7Ra is expressed on fraction A2 cells. B-lineage development has subsequently been studied in detail by a variety of groups using different combinations of markers that prevented direct comparison of the data [242,244,246–252]. Recently, we performed analyses to clarify these data by incorporating the majority of these described markers and others in our experiments. These studies allowed us to identify a committed B-lineage progenitor downstream of the CLP with the surface phenotype B220+CD43+AA4.1+Flk2+CD27+IL 7Ra+c-kitloCD24−CD19−CD11c−Ly6c−CD4− [253]. This population is distinguished from CLPs by the expression of B220 and comprises only approximately 5% of fraction A/pre-pro B cells, while the remaining cells are dendritic cell (DC) progenitors. Using the same markers described above, we also identified heterogeneity within the fraction B cells, which are thought to lie immediately downstream of the fraction A cells. One subset, which we term fraction B1, is highly proliferative, while the second subset, fraction B2, is largely quiescent and smaller in size. In contrast to the refined pre-pro B cells, fraction B1 cells do not respond strongly to IL-1, suggesting that the sensitivity to inflammatory signals within B-committed progenitors is restricted to pre-pro B cells and is lost immediately upon differentiation to their immediate downstream progeny. The earliest thymic progenitors (double-negative-1 cells) are CD4loCD8−CD44+CD25−c-kit+ cells [254–257] and therefore closely resemble the phenotype of HSCs. Most of these early thymic progenitors are T-cell committed (pro-T cells); however, some cells within this population are capable of differentiating into B, NK, and dendritic cells and, at a low frequency, into myeloid cells [257], but their clonal origin has not been established [256–258]. Whether the earliest thymic progenitor population contains a small number of recently homed CLPs that, within this microenvironment, preferentially differentiate into T cells, or whether CLPs commit to the T-cell lineage while still in bone marrow and successively home to the thymus [259], has been controversial. Allman et al. [260] suggest that thymopoiesis is maintained through early thymic progenitors that might develop from bone marrow progenitors more closely related to HSCs and not via a CLP-dependent pathway. This is based on findings that some cells (early thymic progenitors) within the double-negative-1 fraction do not express IL-7Rα at high levels, have some myeloid potential, give rise to T and B cells with kinetics that resemble those of ST-HSCs [52] more closely than those of CLPs, and were present at near-normal frequencies in Ikaros−/− mice while CLPs were not detectable by phenotype. While these data add important information on possible alternative T-cell developmental pathways, they do not assess directly how many early thymic progenitors or mature T cells are CLP-derived or whether most thymopoiesis is independent of CLPs. Both HSCs and CLPs might home to the thymus,
where T-cell commitment might immediately be initiated and thymus homing receptors might be lost. The issue of progenitor commitment to the T-cell lineage in bone marrow versus thymus is further complicated by the observation that, at least in experimental settings, extrathymic T-cell development can occur [261–263]. Recent studies comparing (Flk2+CD27+) MPPs and CLPs for their ability to seed the thymus and generate T cells indicate that CLPs are indeed the major source of normal thymic development (Serwold, Erhlich, and Weissman, submitted for publication). Both MPPs and CLPs injected intravenously give rise to thymocytes, but the MPPs do so with delayed kinetics reflective of their homing to the bone marrow and giving rise to CLPs that then go on to seed the thymus directly. Additionally, thymic myeloid cells were observed to derive from transplanted MPPs and not CLPs, suggesting that these cells are the progeny of myeloid progenitors that also enters the thymus. Further, when Flk2+ CLPs were plated in conditions capable of supporting differentiation into multiple lymphoid and myeloid lineages, single CLPs gave rise to T, B, NK, and DC lineages [264]. Approximately 50% of single CLP clones gave rise to both B- and T-lineage cells in this assay, and none gave rise to any macrophages or neutrophils. Taken together, these data confirm the role of CLPs as potent progenitors of both B and T lymphocytes in vivo and in vitro. Also of note, CLPs co-transplanted in addition to HSCs in autologous or allogeneic transplantation models can rescue mice from otherwise lethal cytomegalovirus (murine CMV) infections [265], providing additional evidence for the robust and functional T- and/or NKcell reconstitution capacity of CLPs in vivo. In humans, evidence for a T/NK bipotential precursor population in fetal thymus [266] and a terminal deoxynucleotidyl transferase (TdT)positive candidate lymphoid precursor population in CD34+ bone marrow cells have been reported [267]. However, clonal analysis was not carried out. A subpopulation of the CD34+TdT+ cells expresses the neutral endopeptidase CD10 [268]. Later, it was shown that fetal and adult bone marrow Lin−CD34+Thy-1−CD38+CD10+ cells contain clonal progenitors of B, NK, and dendritic cells [269]. As a population, these cells give rise to T cells in the SCID-hu thymus assay but could not generate myeloid progeny. In another report, it was shown that cord blood CD34+CD38−CD7+ cells contain clonal B, NK, and dendritic cell precursors; however, T-cell readout was not evaluated [242]. Therefore, both studies suggest but do not formally demonstrate the existence of CLPs in humans. IL-7 signaling might not be essential for normal B-cell development in humans because B cells can be generated without IL-7 in vitro [270], and disruption of the IL-7R in vivo causes T-cell but not consistently B-cell deficiencies [271,272] (reviewed in [268]). Human early lymphoid progenitors, in analogy to mouse CLPs, express IL-7Rα. Indeed, Lin−CD34+CD38+CD10+ cells can be subdivided into a CD10+IL-7Rα− and a CD10loIL-7Rα+ fraction, with the latter being highly enriched in clonal B-cell progenitors [273]. It has also been shown that pro-B cells proliferate and differentiate in culture with IL7 and bone marrow stromal cells, whereas pre-B cells are no longer responsive to IL-7 [274]. It is important to determine whether CD10loIL-7Rα+ cells or CD10+IL-7Rα− cells contain both T- and B-cell progenitors and whether thymus seeding cells share some of these phenotypes (for a review of human early thymocyte development, see [275]). CMP cells and myeloid development The identification of CLPs in the IL-7Rα-expressing fraction of mouse bone marrow which holds no myeloid differentiation activity [25] suggested that complementary progenitors common to all myeloid cells might exist in the IL-7Rα-negative cell fraction. In addition, we had noted that the Sca-1− subset of Thy-1loLin− cells contained myeloery-
Biology of Hematopoietic Stem and Progenitor Cells
throid progenitors [40]. Therefore, to exclude HSCs and CLPs, we searched for myeloid progenitors within the Lin−IL-7Rα−Sca-1−c-kit + bone marrow fraction. We identified three myeloid progenitor populations [26]: CMPs, FcgRloCD34+ (now known to be Sca-1lo not Sca-1− [276]), and their lineal descendants; megakaryocyte/erythrocyte progenitor cells (MEPs; FcgRloCD34−); and granulocyte/macrophage progenitor cells (GMPs; FcgRhiCD34+). These three myeloid progenitor subsets likely represent the major pathways for myeloid cell differentiation because they contain the vast majority of myeloid progenitor activity in steady-state bone marrow. CMPs give rise to all myeloid colonies in vitro, including CFU-mixed; GMPs give rise to CFU-granulocytes (CFU-G) and CFU-macrophage (CFU-M) as well as CFU-granulocyte–macrophage (CFU-GM); and MEPs give rise to burst-forming unit-erythroid (BFU-E), CFU-megakaryocyte (CFU-Meg) as well as CFU-megakaryocyte/erythroid (CFUMegE), each with high cloning efficiency. We demonstrated that CMPs as a population differentiate in vitro into cells with MEP and GMP phenotype and function [26]. Further, the majority of single CMPs generate both GM- and MegE-related progeny. Upon in vivo transfer, the three cell populations give short-term but not long-term readout corresponding to their in vitro activities, indicating that they have only limited if any self-renewal activity [37]. MEPs provide radioprotective cells for lethally irradiated mice housed under infection control conditions [37]. Lethally irradiated mice injected with MEPs, CMPs, ST-HSCs or MPPs show transient donor-derived hematopoiesis sufficient to sustain survival while reconstitution with residual host HSCs occurs [37,38,87]. In addition, the majority of day 8 CFU-S activity resides within the MEP population but is absent from GMPs [37], supporting previous findings that day 8–9 CFU-S are largely erythroid [40,277]. Our data also confirm previous findings that the majority of day 12 CFU-S activity resides not within lineage-committed progenitors but within the more primitive HSC populations with some activity in CMPs and MEPs [35,38,40]. Neither B- nor T-cell differentiation activity was detectable in either MEPs or GMPs. CMPs could not generate T cells; however, a small number of B-cell progeny were detectable in vitro and in vivo [26]. We also isolated a pure population of megakaryocyte progenitors in mice. These Lin−c-kit+Sca-1− CD34+CD9+CD41+ cells are highly efficient at producing megakaryocyte CFCs in vitro, micromegakaryocyte loci in vivo, and donor-derived platelet production in vivo, while they are devoid of erythrogenic activity [278]. The subsequent identification of additional markers has enabled higher-resolution delineation of myeloid developmental pathways. First, we and others found that the CMP population, like the CLP, was heterogenous for Flk2 expression [237,279] and the Flk2+ fraction contained the CMP activity and could give rise to GMPs, MEPs, and all myeloerythroid progeny including DCs. More recently, the addition of CD150 (Slamf1), CD105 (endoglin), and CD41 to the analysis revealed further heterogeneity within the myeloid progenitor populations [280]. These markers revealed that the previously defined CMPs could be further subdivided into CD150+ and CD150− fractions, and that MEPs can be divided into three subpopulations based on endoglin and CD150 expression. Detailed analyses with these markers led to the identification of isolatable pre-GM, pre-MegE, and pre-CFU-erythrocyte populations that give appropriate lineage restricted readouts in vitro and in vivo [280]. These data are consistent with another study that used a Gata1-GFP reporter to identify a population that refines the CMP phenotype or precedes it in the developmental scheme [276] (Fig. 5.6). Specifically, this population is Lin−Kit+ScaintFlk2−Gata1lo and gives rise in vivo to all myeloid lineages but very low lymphoid readout. Using a PU.1 reporter, this study also described a granulocyte–lymphocyte–macrophage progenitor that could give rise to lymphocytic and myeloid lineage cells but
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not platelets, similar to results from Adolfsson et al. which indicated that those MPPs that express the highest levels of Flk2 have lost platelet-forming ability [281]. This is also consistent with data suggesting that PU.1 represses MegE development [282]. Alternative developmental pathways There are several recent in vivo studies as well as in vitro data suggesting that myeloid versus lymphoid differentiation may not always be the first decision made during hematopoietic development, and either bipotent B-cell/myeloid or T-cell/myeloid progenitors, or both, might exist. In support of a B/macrophage progenitor, it was demonstrated that several B-cell lines can be modified to produce cells with features of macrophages by in vitro manipulations [283–285]. Importantly, transfection of v-raf into B-cell lines established from Eμ-myc transgenic mice could convert them into macrophages that maintained the identical rearrangement of an immunoglobulin H (IgH) gene [284]. This observation suggests that latent developmental potentials that are not accessible under normal conditions might be regained through genetic alteration. Indeed, we have shown that CLPs, which under normal conditions never generate myeloid readout, produced granulocytes and macrophages after they were genetically engineered to express the human IL-2 receptor β chain (IL-2Rβ) or the granulocyte/macrophage colony-CSF receptor and were stimulated with the cognate ligands [286]. This was blocked by enforced expression of Pax-5 [287]. Also in vitro, single fetal liver cells could be demonstrated to give rise to both B/myeloid and T/myeloid progeny [207,288,289]. In these experiments using a fetal thymic organ culture system in the presence of 60% oxygen for single cell readout, Kawamoto et al. [289] detected B/T/myeloid, B/myeloid, T/myeloid, but never T/B-bipotent progenitors from fetal liver cell populations. However, in these high oxygen concentration cultures, only approximately 5% of single Lin−Sca-1+c-kit + HSCs gave rise to multilineage (T/B/myeloid) outcomes, suggesting that this system is not reproducible enough to demonstrate all differentiation capacities of HSCs. Also, differentiation into T/myeloid or B/myeloid lineages might simply be because of the “random” commitment of HSCs in these culture conditions. Alternatively, T/B-committed bipotent progenitors might not be contained in the Lin−Sca-1+c-kit + population but only in the Lin−Sca-1loc-kit lo CLP fraction [25,290]. Recently, bipotent B/macrophage progenitors were also described at a very low frequency (~0.02%) in adult hematopoiesis in vitro [291], supporting the hypothesis that a B/macrophage developmental line might be conserved beyond fetal hematopoiesis. As described above, recent data also suggest that HSCs and progenitors circulate through the blood and reside in the tissues before returning to the blood via the lymphatics [155]. Further, these cells were shown to differentiate in the tissue directly into dendritic, myeloid, and B220+ cells. This result, taken together with studies that HSCs respond to reduced hematocrit [76], in vitro data providing evidence for direct differentiation to megakaryocyte development [88], and studies suggesting oligo- and bipotent progenitors, suggest that HSCs percolate through the tissues as a means of directly assessing the specific hematopoietic and immune demands. It is reasonable to hypothesize that these cells reflect the responsiveness of the hematopoietic system and indicate that steadystate hematopoiesis when demand for each lineage is constant is distinct from hematopoieis that occurs during infection or blood loss, when there is a specific need for an increase in certain lineages. Being able to shut down production of lineages not in demand would also be a means of conserving resources such that white blood cells are not generated at the expense of RBCs during anemia and, conversely, RBCs and platelets are not generated at the expense of innate or specific immune cells when they are in demand.
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Human myeloid progenitors As in the case of CLPs, multiple studies suggested the existence of human oligopotent myeloid progenitors [292–296]. Recently, we were able to identify human bone marrow and cord blood cell populations that were counterparts of mouse CMPs, GMPs, and MEPs [273]. They are negative for multiple mature lineage markers (including early lymphoid markers that might define human CLPs as CD7, CD10 or IL-7Rα), and all are CD34+CD38+. They are distinguished by the expression of CD45RA, an isoform of the CD45 cell surface tyrosine phosphatase that can negatively regulate at least some classes of cytokine receptor signaling [297], and IL-3Rα, a receptor that upon activation supports the proliferation and differentiation of primitive progenitors [88,298,299]. CD45RA−IL-3Ralo (CMPs), CD45RA+IL-3Rαlo (GMPs), and CD45RAIL-3Rα− (MEPs) cells show high myeloid cloning efficacy but low long-term (stromal hematopoietic) culture-initiating cell capacity, indicating that they do not self-renew. Although FcγRII–III (CD16/CD32) expression distinguishes mouse CMPs and GMPs, it was not detectable on any of the human myeloid progenitor populations, and all human clonal myeloid progenitors express CD34 while mouse MEPs are CD34− [26]. Interestingly, in mouse myeloid progenitors, IL-3Rα as well as CD45RA expression were both negative to low in MEPs, intermediate in CMPs, and positive in GMPs, without providing sufficient discrimination to clearly set a cut-off level between the different cellular fractions. As with the mouse myeloid progenitors, CMPs give rise to MEPs and GMPs in vitro, and a significant proportion of CMPs have clonal granulocyte/macrophage and megakaryocyte/erythrocyte readout potential. We did not test T-cell differentiation ability; however, no in vitro B- or NK-cell readout could be detected. However, some B cells might develop from large numbers of transplanted CMPs. As in the case of mouse CMPs, it is important to clarify whether the CMP population contains oligopotent B/myeloid progenitors, or monopotent B-cell progenitors occurred because of small numbers of contaminating B-cell progenitors in the large number of transplanted cells (see below).
Lineage commitment in fetal hematopoiesis While fetal liver HSCs show major similarities to adult HSCs, some phenotypic and functional differences exist [175,300–302]. Remarkably, fetal liver HSCs possess some cell fate potentials such as the generation of Vγ3+ and Vγ4+ T cells [179] and B-1a lymphocytes [262,303] that are not detectable in adult HSCs. Therefore, it was important to determine the phenotype and developmental capacities of fetal liver CLPs and CMPs. We recently identified fetal liver CLPs (Lin−IL-7Rα+ B220− /lo Sca-1loc-kit lo) [290], and the myeloid progenitors fetal liver CMPs (Lin−IL-7Rα−Sca-1−c-kit+AA4.1−FcgRloCD34+), fetal liver GMPs (Lin− IL-7Rα−Sca-1−c-kit +AA4.1−FcgRhiCD34+), and fetal liver MEPs (Lin−IL7Rα−Sca-1−c-kit+AA4.1−FcgRloCD34−) [304] that show high similarities to their adult counterparts. However, some significant differences in proliferative potential colony-forming activity, and lineage differentiation capacities exist. Of special note, fetal liver CLPs generate CD45+CD4+ CD3−LTb+ cells that are candidate initiators of lymph node and Peyer’s patch formation [305–308]. About 5% of these IL-7R+ fetal liver CLPs differentiated into macrophages as well as into B cells, at least in vitro. Interestingly, the fetal liver CLPs fail to express Pax-5, a suppressor of macrophage development [306] while adult marrow CLPs that lack myeloid potential are Pax-5+ [290]. Conversely, although fetal liver CMP and downstream fetal liver MEPs or fetal liver GMPs do not give rise to T cells, fetal liver CMPs possess some in vitro and in vivo B-cell potential. A close relation between B-cell and macrophage development for adult and fetal hematopoiesis was suggested before (see below). It can therefore be speculated that, during commitment to the
lymphoid or myeloid lineage in fetal liver hematopoiesis, the developmental capacity for macrophages and B cells might be the last to be lost in commitment to either the lymphoid or myeloid lineage. DC development DCs were initially defined as bone marrow-derived nonmacrophage leukocyte populations with high antigen presentation capacities and the ability to prime naïve T cells [309–311]. To date, with their numbers still growing, multiple DC subpopulations that differ in tissue distribution, surface marker expression, and functional capacities have been described in humans and mice (reviewed in [312,313] and Chapter 19). From an evolutionary point of view, DCs might belong to the myeloid as well as to the lymphoid lineage because they link the innate and adaptive immune responses. Based on current data, two opposing models of DC ontogeny can be suggested: either the mature DC subpopulation is determined at the level of an early hematopoietic progenitor or, alternatively, immature DCs retain the capacity to mature to distinct DC subpopulations in response to different environmental instructions. In mice, two major DC subpopulations are distinguishable by their expression of the CD8α chain [314]. It was reported that early intrathymic progenitors (CD4loCD44+CD25−c-kit +) and thymic pro-T cells (CD44+CD25+c-kit +) contain precursors capable to differentiate into CD8α+ DCs in vivo [258,315]. Therefore, and because the majority of thymic DCs and only a minority of secondary lymphoid organ DCs express CD8α+, the CD8α+ DCs had been considered to be related to the T-lymphoid lineage and were therefore termed “lymphoid DCs” in contrast to the CD8α− “myeloid DCs.” This model seemed to be supported, but not directly demonstrated, in mice deficient for the transcription factors RelB [316], PU.1 [317], and Ikaros [318] that lack only CD8α− DCs. However, other mutants, as in c-kit −/− , γc−/− [319], and Notch1−/− [320] mice, showed a developmental dissociation of CD8α+ DC and T cells. We and others therefore tested DC developmental potentials in adult and fetal lymphoid and myeloid committed progenitors. Surprisingly, CD8α+ and CD8α− DCs were produced both by highly purified CLPs and CMPs with similar efficacy on a per cell basis, therefore disproving the concept of CD8α+ “lymphoid”- and CD8α− “myeloid”-derived DCs in mice [290,320–322]. Further, beyond CLPs and CMPs, DC developmental potential is conserved in the lymphoid lineage in pro-T cells and in the myeloid lineage in GMPs, but it is lost once B-cell or megakaryocyte/erythrocyte commitment occurs [320,321]. In contrast to mice wherein DCs or their precursors are usually isolated from the lymphoid organs or bone marrow, human DCs and their precursors have usually been isolated from peripheral blood and only in rare cases from blood-forming organs, making direct comparisons difficult (reviewed in [313]). However, DCs could be generated from total CD34+ progenitor populations, lymphoid-restricted progenitor populations, and peripheral blood monocytes, suggesting that, in analogy to mice, DC developmental potential is conserved in myeloid- and lymphoid-restricted lineages [269,323–328]. Recently, an immediate DC precursor population that upon stimulation can produce high amounts of interferon-α, displays a set of distinct pattern recognition receptors, and can mature into DCs in vitro, was identified in humans [329–332] and later in mice [333–335]. This DC subpopulation has alternatively been referred to as plasmacytoid DCs, DC2 cells, and interferon-α-producing cells [312]. These cells can derive from both myeloid and lymphoid progenitors (reviewed in [336]) but are predominantly of myeloid origin [337]. Taken together, DCs are developing along a lymphoid and myeloid pathway in vitro and in vivo. By using simple assays as immunophenotyping, or functional readout as mixed lymphocyte reaction and cytokine
Biology of Hematopoietic Stem and Progenitor Cells
production, we did not detect differences in lymphoid precursor- and myeloid precursor-derived CD8α+ DC fraction DCs [321]. This is an unexpected finding, because it suggests developmental redundancy for DCs. Of note, DC developmental capacities in CLPs and pro-T cells correlate with their latent myeloid developmental potential that can be “rescued” by artificial introduction and activation of the IL-2β or GMCSF receptor [286,338]. It would therefore be important to characterize the signals that are involved to retain DC developmental capacities in the otherwise exclusively lymphoid- or myeloid-committed progenitor cells. Activation of the Flt3 tyrosine kinase could be one of them [237].
Gene expression profiles of HSCs Several powerful technologies have allowed the characterization of transcripts expressed in defined cells, populations of cells, tissues, and organs. These technical advances allow for reductionist approaches to understand the complexity of RNA transcripts found in these cells or tissues, and how the transcriptional profile changes upon differentiation, self-renewal or other functional events. One of the most powerful technologies is microarray analysis, wherein the expression profile of transcripts for a high fraction of all known genes in the species can be ascertained for purified cell populations. Several groups have performed microarray expression profiling of hematopoietic stem and progenitor cells from fetal, adult, and aged mice [55,56,94,97,98,238,280,339,340]. These studies have identified several genes that have been subsequently validated as important for HSC function, such as Bmi1 [97], or useful for HSC purification, such as Slamf1 [55,56]. Gene subtractions or other differential expression techniques have revealed a subset of genes that appear to be expressed in HSCs but not broadly elsewhere. In fact, some genes have been found that are expressed in HSCs and neurosphere cultures (highly enriched for CNS stem cells) [341], or HSCs and ES cells, or all three [140,342]. These gene expression arrays cannot detect important events that are essentially post-translational, for example the phosphorylation and dephosphorylation of proteins in signal transduction pathways, or the changes in subcellular location of proteins such as β-catenin under conditions when they are held within the degradation complex, or attached to the cytoplasmic face of cadherins, or allowed to be free in the cytoplasm and appear in the nucleus [122]. Most of the oligolineage progenitors downstream of mouse HSCs [25,26] and human HSCs [63,269,273,323] have been isolated to homogeneity, and in some instances a comparison of their gene expression profiles reveals important clues as to genes responsible for particular commitments to differentiated fates [140,342] (see below). It is reasonable to expect that the reductionist approach to cataloging genes in this way will reveal many new clues about stem cell behaviors, but it will require a similar cataloging of cellular proteins (proteomics) to begin to reveal cell protein modifications that lead one to identify important pathways in stem cell behavior.
Gene expression profile of stem and progenitor cells An important question is how the progeny of multipotent cells adopt one fate from a choice of several. Lineage commitment and subsequent differentiation of multipotent cells should involve the selective activation and silencing of particular gene expression programs. There are single or pairs of transcription factors that have “master” roles in hematopoiesis in altering the phenotype of hematopoietic cells in defined circumstances. Combinatorial control is operated at the level of two or more lineage-specific regulators. This transcriptional control might be more complicated, considering protein–protein interactions mounted by the
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formation of multiprotein hematopoietic-specific protein complexes [343], and changes in the expression levels of transcription factors [344]. Furthermore, as in the case of cytokine signals, roles in transcription factors are also sometimes redundant. Finally, single MPPs appear to coexpress transcription factors and cytokine receptors related to multiple lineages [345]. Promiscuous expression of multiple myeloid or lymphoid genes in hematopoietic branchpoints Prospectively purified LT-HSCs, oligopotent progenitors, and lineagecommitted progenitors have been used for a definitive sampling of the transcriptional profiles of cells at these particular stages of physiologic hematopoiesis. The expression patterns of lineage-affiliated genes in each stage of hematopoiesis are largely consistent with their known functions. Using semiquantitative RT-PCR assay targeted for each purified population, SCL (stem cell leukemia) [346,347] is expressed in HSCs and all myeloid progenitors. GATA-2 [348] and c-mpl are expressed in HSCs, CMPs, and MEPs, but not in GMPs [26,94,113]. Other MegErelated genes such as NF-E2 [347], GATA-1 [349,350], and the erythropoietin receptor are expressed in CMPs and MEPs but not in GMPs, and their expression levels are highest in MEPs [113,340]. Granulocyteaffiliated transcription factor C/EBPα [351] is expressed in HSCs, CMPs, and GMPs but not in MEPs, and its expression level is highest in GMPs [113,340]. All of these genes are expressed in CMPs, but not in CLPs or other late lymphoid progenitors, suggesting potential roles for each in myeloid-specific cell fate decisions. In contrast, the lymphoid-related transcription factor Aiolos [352] is not expressed in any myeloid progenitors, nor in HSCs. Ikaros, an Aiolos partner nuclear factor, thought initially to be a lymphoid-specific transcription factor [195,353], is expressed in HSCs and all stages of hematopoiesis [354]. Aiolos first appears at the CLP stage and is highest in T- and B-cell progenitors [26,113]. Lymphoid-related GATA-3 [355] and Pax-5 [356] are expressed at a low level in adult marrow CLPs but not in myeloid progenitors and fetal liver CLPs [26,290]. These data provide a clearer view of the regulation of gene expression in hematopoietic development. First, the expression of many myeloid and lymphoid genes is mutually exclusive in each pathway. Second, CMPs express both MegE- and GM-affiliated genes, and CLPs express both T- and B-lymphoid genes, although CMPs and CLPs have not committed to myeloid and lymphoid sublineages, respectively [26,113]. These data are consistent both with the priming hypothesis [357] and a model wherein chromatin is open prior to commitment, but the transcripts from that open chromatin are not critical for maturation [113]. The view of myeloid or lymphoid promiscuity has been significantly extended by using an oligonucleotide microarray analysis targeted for purified CMPs and CLPs. Genome-wide profiling using microarray methods revealed that CMPs and CLPs coexpress a vast majority of GM- and MegE-affiliated genes, and T-, B-, and NK-lymphoid genes, respectively [94,340]. Furthermore, coexpression of both GM- and MegE-related genes in CMPs and of both T- and B-lymphoid genes in CLPs have been formally demonstrated at the level of single cells [94]. By using RT-PCR analysis targeted for single cells, CMPs coexpress MegE-related β-globin, erythropoietin receptor, NF-E2 and GM-related myeloperoxidase, G-CSF receptor and PU.1, while CLPs coexpress Bcell-related l5 and/or Pax-5 and T-cell-related CD3d and/or GATA-3 [113]. These results indicate that the expression of lineage-related genes can precede commitment, and suggest that the “promiscuous” expression of multiple lineage-related genes may allow a flexibility of commitment at these oligopotent stages: for differentiation towards T- and B-cell lineages in CLPs, and towards GM and MegE lineages in CMPs.
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It is still unclear whether the primary mechanism of hematopoietic commitment could be operated first by opening chromatin at programmed sites, or whether the “priming” seen in promiscuous transcription patterns results from the presence of open chromatin and transcription factors that can form “sterile” transcripts. Nonetheless, in priming stages, multiple differentiation programs might collaborate or compete with each other until one becomes dominant. These programs should include cross-talk among transcription factors or transcriptional complexes, because transcription factor activity can be potentiated [358] or suppressed [359,360] by interaction with other transcription factors. Changes in levels of key transcription factors [344,361] may also be critical for the lineage decision. Cross-talk between different cytokine signals may also be important, as single progenitors coexpress multiple cytokine receptors. Further, fluctuations of gene expression of multiple differentiation programs in progenitors may lead to different outcomes depending on the microenvironment the cell inhabits. In this context, both intrinsic and extrinsic factors could play a part in lineage determination. However, data comparing HSCs from young and old mice show an intrinsic change in their differentiation potential with decreasing lymphoid potential with age, even when transplanted into a young mouse [96]. These data suggest that at least some portion of lineage determination is independent of the environment or that aging HSCs irreversibly lose responsiveness to signals provided by it. One way to view the hematopoietic system is to assume that, at or around the stage of yolk sac hematopoiesis, a pool of HSCs are created that are the only source of hematopoiesis for life. Each early HSC then is a clonogenic precursor of other HSCs, which self-renew, and their progeny, which do not. Over time and perhaps with a cell divisioncounting developmental clock, the clones begin to diversify in terms of responsive potential; these intrinsic epigenetic, and rarely genetic, changes prime the clones for their responsiveness to normal cues, such as diffusible or membrane-bound factors, or to pathologic cues, such as bacterial endotoxins. At any point, the clones that are present represent clonal competitions up to that point. In this way, intrinsic programs can select certain clones over others, but within an inbred colony of pretty clean mice, the clones chosen tend to be consistent, mouse-to-mouse. We later discuss changes that not only enable clonal selection, but are also proto-oncogenic in nature, so that the long process of preleukemic clonal progression is initiated and spread through the HSC clones.
Downregulation of genes irrelevant to committed lineages as a critical mechanism of lineage restriction According to the “priming” model, commitment could be dependent on two independent molecular events: upregulation of differentiation programs related to selected lineages, and the transcriptional abrogation of master control genes in the unselected lineages. A striking example is Pax-5, a key molecule for restriction to B-cell lineage [356]. In normal hematopoiesis, Pax-5 expression initiates at the CLP stage, and is upregulated in pro-B cells, but undetectable in pro-T cells or myeloid cells. Expression of Pax-5 in pro-B cells is important not to differentiate into other lineage cells, because pro-B cells from Pax-5-deficient mice (Pax5−/− pro-B cells) can differentiate into T cells, NK cells, granulocytes, macrophages, and osteoclasts as well as B cells [356]. Pax-5−/− pro-B cells rearrange the D–J locus of IgH genes and express other pro-B related genes. Thus, even after a set of B lineage-associated genes begins to be transcribed, differentiation programs for other lineages are still accessible. Conversely, enforced Pax-5 expression in myeloid progenitors suppresses myeloid development, presumably blocking signals from myelomonocytic cytokine receptors such as G-CSF receptor and GMCSF receptor [362].
Similarly, enforced expression of PU.1 in pro-T cells can block T-cell differentiation [363]. Ectopic GATA-1 expression in GMPs also inhibits differentiation into granulocyte/monocyte lineages, but induces transdifferentiation into megakaryocytes and erythrocytes [364]. A similar phenomenon is observed in ectopic cytokine receptors. Ectopic expression of IL-2Rβ or GM-CSF receptor in CLPs can deliver signals that redirect cells to myelomonocytic differentiation [286]. Further characterization of this latent myeloid potential revealed that CCAAT/enhancerbinding protein (CEBPα) is upregulated in response to the ectopic IL2 signal and leads to GM-CSF receptor expression and further myeloid differentiation [365]. This study also revealed that enforced expression of CEBPα in CLPs is itself sufficient to induce the myeloid outcome and, conversely, enforced Pax-5 expression blocks the myeloid differentiation induced from IL2Rβ. Similarly, Laiosa et al. showed that committed T cell progenitors could be induced to develop into macrophages or DCs by enforced expression of CEBPα or PU.1, respectively. These data suggest that committed T-cell progenitors are also susceptible to the lineage-instructive effects of myeloid transcription factors, but that T-lineage development occurs by the normal role of Notch signaling downregulating CEBPα and PU.1 in multilineage precursors. These data indicate that termination of lineage-related gene expression is equally important for completion of lineage commitment. First, in committed progenitors, abrogated differentiation programs for unselected lineages are not totally erased immediately after commitment, but could be reactivated by “instructive” signals mediated by ectopic cytokine receptors and transcription factors. For example, GMPs might be restricted to GM lineages simply because GATA-1 is downregulated, and CLPs are likely lymphoid restricted because they downregulate myeloid cytokine receptors. Second, programs for differentiation into committed lineages can be operated on condition that transcription factors for unselected lineages are downregulated, as Pax-5 and PU.1 inhibit myeloid and T-cell differentiation programs, respectively. Thus, a hierarchy of transcription factors and cytokine receptors organized at the epigenetic level is critical to maintain hematopoietic homeostasis. Changes in chromatin structure, allowing access for RNA polymerase to initiate transcription, are essential for genetic programs to be transcribed [366,367]. The activation of chromatin structure can occur prior to significant expression of genes [368]. It has been hypothesized that a wide-open chromatin structure is maintained in early hematopoietic progenitors, enabling multilineage-affiliated programs to be accessible [369]. This phenomenon of open chromatin in early hematopoietic progenitors could be the mechanism by which lineage priming occurs in stem or progenitor cells prior to their lineage determination [140,358,370,371]. Standard methods for studying chromatin structure require large numbers of cells, which has made their application to stem cell biology difficult, but we and others have made recent technical advances that have allowed chromatin immunoprecipitation to be performed on rare cells [372–374] so that we have been able to determine whether the epigenetic marks in control regions of lineage-directing genes modulate during hematopoiesis. Specifically, we analyzed histone modifications and DNA methylation patterns within the regulatory regions of erythroid (Gata1 and β-globin), myeloid (c-fms), and lymphoid lineage-affiliated genes (Gata3 and Ptcrα) in 50,000 prospectively purified stem and progenitor cells. By performing bisulfite sequencing analysis, we found that methylated H3K4 and AcH3 and unmethylated CpG dinucleotides localized at defined regulatory regions of these lineage-affiliated genes in HSC. These histone modifications are epigenetic markers of active transcription and either accumulated or were replaced by increased DNA methylation and H3K27 trimethylation in committed progenitors consistent with the pattern of gene expression. We also observed bivalent
Biology of Hematopoietic Stem and Progenitor Cells
domains at lymphoid-affiliated genes in HSC and downstream transitamplifying progenitors. Bivalent domains described by Bernstein et al. contain both activating and silencing histone modifications [375], and are associated with genes such as transcription factors that are expressed at low levels and affect developmental decisions. These data support the role of epigenetic modifications as being reflective of HSC multipotency and suggest that the remodeling of chromatin is an important mechanism of driving gene expression changes associated with developmental decisions.
Transplantation of HSCs in mouse and humans It is fair to say that the field of bone marrow transplantation, now more accurately called HCT, has provided one of the most striking medical advances in our lifetime and, similarly, the desire to understand cells involved in such transplants has opened the field of stem cell biology, first to HSC but then to other tissue-specific stem cells. It is important at the outset, however, to make sure that the scientific and clinical community uses the appropriate nomenclature for the type of transplant used as misconceptions as to the cell type transplanted lead to misconceptions about the scientific or clinical results obtained [14,15]. Thus, the transplantation of complex mixtures of hematopoietic cells in bone marrow or MPB is not the same as transplanting purified HSCs. Clinical HCTs have been used for a variety of purposes, but their primary use is the ability to regenerate the hematopoietic tissues of a cancer patient who has received otherwise lethal doses of a high-dose regimen of chemoradiotherapy [376,377]. Other uses have been the correction of inherited or acquired hematopoietic cell defects and dysplasias, and in animals the induction of transplantation tolerance to other cells, tissues or organs co-transplanted from the HSC donor, the hematopoietic cell donor, and also the reversal of autoimmune disease [19] (see Chapter 20). It was not clear whether purified HSCs could replace HCT, as it was not clear whether a rapid and sustained regeneration of a particular hematopoietic outcome could be accomplished with the primitive HSCs alone, without having oligolineage progenitors in the transplant. Several experiments have demonstrated in mice and in humans that HSCs are the principal cells that are functional in the transplant setting, and that
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by varying cell dose one could achieve both rapid and sustained regeneration of hematopoietic tissues [378]. Prospective isolation of HSCs with multiple markers allows the possibility that grafts would consist solely of HSCs and of no other contaminating cells. There are two important advantages, at least theoretically, to the transplantation of HSCs rather than HCT removal of cancer cells and removal of T cells. Autologous HCT from cancer patients can be accompanied by transplantation of the patient’s cancer cells; in some diseases, such as stage 4 breast cancer and many lymphomas, the HCT populations to be transplanted that are contaminated with cancer cells vary from 20% to 40% of all transplants [379–381]. In patients with multiple myeloma, contamination of the graft is the rule and not the exception [382]. Therefore, it was important to test whether positive selection of HSCs would effectively eliminate or purge cancer cells from the transplant. Deliberate spiking of MPB with cell lines representing human breast cancer, human non-Hodgkin’s lymphoma and human myeloma was performed, and four methods of HCT/HSC isolation were carried out to determine the full reduction of contaminating cancer cells. Two independent methods to identify breast cancer cells were used: microscopy to detect cells carrying breast cell cytokeratins, and a flow-based method. Cells representative of myeloma and B-cell lymphoma were detected by PCR specific for the immunoglobin heavy chain rearrangements in these B-lineage cells. All three techniques have a sensitivity of between 10−5 and 10−6. Over 105-fold reductions of contaminating cancer cells were accomplished with the multiparameter cell sorting of CD34+Thy+ cells from MPB, whereas techniques based on CD34 alone were at least two to three orders of magnitude less efficient (Fig. 5.7). The time to reach engraftment with purified HSCs (measured by absolute neutrophil count >500/mL and platelets >20,000/mL of blood [379,380,383]) (Fig. 5.8) is very similar to the time obtained when unmanipulated MPB is used as the HCT transplant, and similar to the dose–response observed for mouse HSCs in syngeneic transplants (Fig. 5.9) [378]. Even in acute myeloid leukemia (AML), HSCs can be separated from leukemic stem cells (LSCs) by surface markers [384]. Therefore, the stage is set to test whether the transplantation of cancer-depleted HSCs will lead to clinical benefits when compared with the transplantation of cancer-containing unmanipulated HCT grafts.
Comparison of fold depletion of cancer cells from mobilized peripheral blood CD34+ Thy1.1+ (flow) CD34+ (flow) CD34+ (paramagnetic beads) CD34+ (magnetic beads)
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Fig. 5.7 Comparison between CD34 only and CD34+Thy-1 isolation of human stem and progenitor cells in terms of fold depletion of human cancer cells. MPB (1.6 × 1010) was spiked with 1.2 × 108 cancer cells (T47D breast cancer, SU DHL 6 non-Hodgkin’s lymphoma cells, or RPMI-LAV myeloma cells) prior to cell sorting. The CD34+Thy-1 cells were flow sorted with a modified high-flow cytometer, as were the first CD34 flow sort test. The other devices were a commercial paramagnetic bead-coupled monoclonal passed through magnetized steel mesh, and the last a large magnetic bead device. Breast cancer cells were analyzed by immunofluorescence microscopy for cytokeratin, the non-Hodgkin’s lymphoma by Igh-BCL-2 real-time polymerase chain reaction (PCR), and the myeloma by CDR III real-time PCR. The data are represented as fold reduction of cancer cells from the sorted CD34 or CD34+Thy-1 cells. (Hanania, unpublished data.)
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50 Platelets >20,0000/μL ANC >500/μL
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Fig. 5.8 Transplantation of highly purified human hematopoietic stem cells in patients with metastatic breast cancer. Shown are the times to engraftment of neutrophils (absolute neutrophil count [ANC] > 500/mL] ) following transplantation with purified CD34+Thy1-1+ hematopoietic stem cells. (Reproduced from [379].)
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Fig. 5.9 Hematopoietic recovery in lethally irradiated C57BL/Ka mice syngeneically transplanted with different doses of purified hematopoietic stem cells (HSCs). Irradiated mice were injected with 100 (solid circles), 1000 (triangles), 5000 (solid triangles) or 10,000 (squares) HSCs. Shown are the recovery kinetics for white blood cell (WBC), and platelet (plts) counts. (From [378] with permission.)
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Allogeneic HCT transplants have been plagued from the beginning by the appearance of graft-versus-host disease as a result of T cells contained within the graft [385]. Mice receiving allogeneic HSCs have few or no contaminating T cells, and never develop graft-versus-host disease [378,386]. Dose–response comparisons of purified HSC transplants in syngeneic, in matched unrelated donor allogeneic, and in fully allogeneic circumstances have been carried out, and, for the most part, doses of HSCs sufficient to allow rapid and sustained engraftment can be achieved without contaminating T cells (Fig. 5.10) (see Chapter 20) [386]. However, the number of HSCs required for engraftment in the allogeneic setting can be 10 times higher than that required for syngeneic
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Fig. 5.10 Engraftment kinetics of allogeneic versus congeneic purified hematopoietic stem cells (HSCs). Comparison of stem cell dose required to achieve early hematopoietic recovery in syngeneic versus allogeneic irradiated mice. Syngeneic or allogeneic mice were transplanted with purified Thy-1loSca-1+ Lin−/loc-kit + (KTLS) cells and bled on sequential days post transplant. The kinetics of white blood cell (WBC) recovery were categorized into delayed, intermediate, and rapid recovery. (Adapted from [378] with permission.)
HSC engraftment (Fig. 5.10) [378]. One can achieve nearly equal levels of engraftment at the same HSC dose with syngeneic and allogeneic models if the allogeneic hosts are preconditioned with high-dose conditioning regimens that include either facilitator cells [387–389] from the donor, or monoclonal antibodies that eliminate NK- and T-cell populations [386,390]. High-dose conditioning alone results in donor B-celland myeloid-lineage conversion [386–390], but persistent donor and host T-cell chimerism requires elimination of the host residual T and NK cells with facilitator cells or antibodies. There are two kinds of facilitator cell found in mouse bone marrow and lymphoid tissues: classic CD8α+TCR+CD3+ T cells and CD8α+ TCR−
Biology of Hematopoietic Stem and Progenitor Cells
CD3− facilitator cells [387–389]. The immunocompetent CD8α+ TCR+ T cells have some capacity for graft-versus-host disease, so there is great interest in understanding the nature and function of CD8α+ TCR−CD3− facilitator cells [387]. There are two other kinds of cell that share this phenotype: CD8α+ immature DCs, and veto cells [387,391,392]. We have proposed that all three cell types are in fact a single cell class, and that it is only the assays that makes one believe that they are different lymphoid cell subsets [387]. This view is strengthened by the recent findings of Steinman and Nussenzweig [393] that CD8α+ DCs that do not have active or appropriate co-stimulation can in fact lead to the inactivation or disappearance of T cells (and, we propose, NK cells) that have receptors which recognize them. Thus, a cell that facilitates engraftment of allogeneic HSCs might be a cell that presents major histocompatibility complex/peptide antigens to reactive T and NK cells without delivering a co-stimulatory signal, or induces the cells to undergo activation-induced cell death. These same cells might also be responsible for the veto of active CD4 and CD8 T cells that recognize and respond to them. It has been known since the pioneering work of Main and Prehn that bone marrow chimeras are usually transplantation tolerant to cells, tissues or organs from the hematopoietic cell donor [359]. This method of inducing transplantation tolerance has been substantiated with pure HSC transplants [387,390], in either the high-dose or reduced-intensity setting, and whether the transplant is concurrent with the HSC transplant or occurs months later [387,390]. Hematopoietic chimeras in humans are also usually specifically transplant tolerant of donor tissues, and new trials for inducing tolerance with bone marrow cells in a reducedintensity but lymphoablative setting along with organ allografts from the bone marrow donor are underway [394]. Partial or complete replacement of an autoimmune diabetes-prone hematopoietic system in mice with purified HSCs from diabetes-resistant strains of mice in the prediabetic period completely precludes progression to diabetes (see Chapter 20), whereas the same treatment of already-diabetic hosts along with donor strain islet cell transplants cures the small numbers of mice undergoing this protocol [395]. This too has been accomplished in both high-dose and reduced-intensity conditioning settings. Additionally, in a mouse model of systemic lupus erythematosus, Smith-Berdan was able to achieve greatly increased overall survival by treating mice with established disease with reduced-intensity conditioning and transplants of major histocompatibility complex haplomismatched allogeneic HSCs [396]. Mice that received transplants exhibited stabilization or reversal of their lupus symptoms, including proteinuria, and elevated circulating immune complexes or autoantibodies compared with control mice. These results indicate that induction of durable mixed chimerism by transplantation of purified allogeneic HSCs after reducedintensity conditioning has the potential to reverse symptoms of established autoimmune disease.
Co-transplantation of HSCs and hematopoietic progenitors to protect against opportunistic infections The bane of clinical HCT in terms of infectious disease is the often lethal infections with Aspergillus fumigatus, CMV, and Pseudomonas [397]. Small numbers of each of these infectious organisms given to HSC transplanted mice can result in the morbidity and mortality that occurs in patients [397]. With the isolation of clonal CLPs, CMPs, and GMPs, cellular therapies for each of these disorders have been investigated. Co-transplantation of HSCs and CLPs can completely preclude the lethality of murine CMV infection in both syngeneic and allogeneic protocols [265]. Co-transplantation of HSCs and CMPs and GMPs can prevent the lethal consequences of Aspergillus infections if the Aspergil-
53
lus is given on day 9, 11 or 14 after HSC transplantation, and addition of G-CSF to these HSC/CMP/GMP transplant protocols precludes Aspergillus infections given just 3 days after transplantation [397]. Similar CMP plus GMP protocols are sufficient to eliminate Pseudomonas infections, and in both cases these treatments work in both the syngeneic and allogeneic settings.
Origin of leukemia: LSCs Poorly regulated or unregulated self-renewal is a property that all cancers have, and we call those cells within a cancer that have self-renewal capacity “cancer stem cells” [122]. Therefore, it is important for the reader to keep in mind that the major distinguishing property of normal and neoplastic stem cells is their capability of self-renewal, and thus it becomes important to understand whether the pathways that lead to self-renewal are novel in normal HSCs, or might be shared between different classes of tissue-specific multipotent stem cells, and might be acquired by cancer cell progenitors as they emerge as cancer stem cells [122]. Pten, a tumor suppressor that negatively regulates proliferation, is required to sustain long-term hematopoiesis and self-renewing LSCs [398]. To understand the mechanisms of leukemic transformation, it is important to understand the life history of leukemic progression (Plate 5.2). If one views leukemia development as a series of steps in malignant progression, malignant self-renewal may be obtained through several transformation steps that include a block in differentiation, the retention or acquisition of self-renewal, and the avoidance of programmed cell death by several independent cell-intrinsic pathways and by intrinsic and adaptive immune system immunosurveillance. A set of early stem/progenitor cells with extensive proliferation, or with reduced programmed cell death, that are not yet malignant may form “preleukemic” clones, and these clones may receive additional oncogenic events that induce differentiation blocks at a progenitor stage to become leukemia. Accumulation of mutations as a precondition for malignant transformation in human acute leukemias Molecular markers for leukemia, including oncogenic fusion proteins resulting from leukemia-specific chromosomal translocations, have disclosed the multistep nature of acute leukemia development. A typical case is chronic myelogenous leukemia (CML). CML-specific t(9;22) Philadelphia chromosome and its resultant Bcr–Abl fusion gene has been shown to be present in all myeloid cells, B cells, and rare T cells. Additional mutation(s) and/or epigenetic changes in patterns of gene expression are required for blastic transformation into acute leukemia. There are many other examples of human adult and childhood leukemias that support the early acquisition of predisposing mutations occurring in HSCs that require additional events to result in leukemic transformation. AML1/ETO chimeric protein that is the product of the t(8;21) translocated genomic region is continuously detectable in patients with t(8;21) AML who maintain long-term remission for more than 10 years. Although these patients are clinically “cured,” AML1/ETO remains detectable in HSCs (Thy-1+CD34+Lin−CD38), B cells, and erythroid and megakaryocyte CFCs [384]. In all of these cells, only HSCs self-renew, setting up clones that can acquire additional proto-oncogenic events. In childhood t(8;21) AML, AML1–ETO fusions are retrospectively detected in neonatal Guthrie spots [399], further suggesting that AML1–ETO fusion is at the level of HSCs (in some cases in utero). Acquisition of chromosomal translocations in utero is also demonstrated in infant t(4;11) and childhood t(12;21) acute leukemias. Clonotypic MLL–AF4 and TEL–AML1 genomic fusion sequences were demonstrated to be present in neonatal blood spots from patients who were
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diagnosed with acute lymphoblastic leukemia (ALL) [399–401] and were also detected in identical twins, whose genomic fusion sequence was identical [369]. In these cases, t(12;21) ALL developed at approximately 10 years of age, indicating that cells with TEL–AML1 persisted more than 10 years before leukemic transformation. Further, TEL– AML1+ ALL cells in twins sometimes possess an identical IgH rearrangement, implicating in utero transfer of preleukemic cells. In childhood ALL, preleukemic B- or T-cell progenitors appear to persist in the long term; “leukemic clones” possessing clonal TCR or IgH rearrangements are reported to persist for more than 3 years after achieving complete remission [402]; these might involve the only other blood cells that self-renew – memory T and B cells. These data strongly suggest that leukemic transformation commonly occurs by multistep processes. In mice and in humans, the only cell in the myeloid lineage that self-renews over long periods of time is the LT-HSC [1]. The preleukemic clone should exist in the LT-HSC to sustain multiple mutational events over time. Therefore, it appears that the accumulation of mutations occur as a precondition for leukemia development at the level of HSCs, or in progenitors that have gained the capacity of self-renewal.
Final leukemic transformation can occur at the level of myeloid progenitors Several lines of evidence show that final myeloid leukemic transformation can only occur at the level of progenitors that have acquired the property of self-renewal. In contrast, lymphoid leukemias are made up of neoplastic cells with clonally rearranged TCR or immunoglobulin genes. Whereas myeloid progenitors and myelomonocytic cells are not self-renewing, lymphocytes that contribute to the memory pool are selfrenewing. Thus, the final transformation to lymphatic neoplasms can occur in cells that already have regulated self-renewal capacity. The search for LSCs involves the one property shared by all malignancies – the ability to initiate and spread the disease. Transfer of human AML to immunodeficient mice was first demonstrated to be limited to CD34+CD38− or CD34+CD71−HLA-DR− cells, and was attributed to early progenitors such as HSCs [403]. We [396] and others [404] have shown that, in at least some AMLs, the LSCs lack CD90 expression. In our study of AML1/ETO AML, LSCs were in the MPP phenotype (CD34+CD38lo90− Lin−). Surprisingly, in the same patients, 1–40% of HSCs were positive for the t(8;21) translocation, but these HSCs in vitro gave rise to normal myeloerythroid colonies, not leukemic blasts [396]. As shown in Plate 5.2, these represent preleukemic clones; thus, AML1/ETO may be necessary but not sufficient for full leukemic transformation. A mouse model that expresses AML1/ETO driven by multidrug resistance protein-8 (MRP8) (GMP, granulocyte, and monocyte specific) promoter did not develop leukemia. After the injection of a DNA alkylating mutagen, N-ethyl-N-nitrosourea, however, almost 55% of mice develop AML-M2 [405]. Similarly, promyelocytic leukemia/retinoic receptor-α (PML/RARα) is a product of the t(15;17) translocation, and MRP8–PML/RARα transgenic mice develop acute promyelocytic leukemia-like disorders [406]. The frequency and rapidity of PML-RARα leukemias increases when they are crossed to MPR8–Bcl-2 mice [407]. In these mice, the proto-oncogenes were present in all cells of the GMP lineage, allowing rare subsequent events to be fixed. Inhibition of apoptotic pathways alone can allow leukemia development at progenitor stages. MRP8–Bcl-2 mice display increased numbers of mature monocytes, resembling chronic myelomonocytic leukemia, but do not develop acute leukemia. However, an additional introduction of Fas (lpr/lpr) mutations into these mice results in transformation into AML in about 15% of progeny, but only if leukemia emerges by age 8 weeks [408]. The transplantable GMP-stage leukemias are tert++, over-
express CD47, and have acquired the property of self-renewal, implicating a multistep process. Even mpr8–bcr–abl mice have increased frequencies of conversions to acute blastic leukemias if crossed to mpr8– bcl-2 strains. The general rules so far are that mouse AML LSCs induced as described are at the stage of the GMP, as is human CML myeloid blast crisis [409–412]. Most human AMLs are at the level of the MPP. No HSC-stage LSC has been found. LSC markers The identification and characterization of LSCs is crucial to the development of more effective treatment strategies. Ideal therapies will be those that target the LSCs while sparing normal HSCs, so identifying LSCspecific markers is an important strategy. To date we have identified several candidate LSC-specific markers, including CD96 and CD47 [158,413]. Fluorescence-activated cell sorter analysis indicates that, in many cases, CD96 is expressed on the majority of CD34+CD38− AML cells, whereas few, if any, normal HSCs (Lin−CD34+CD38−CD90+) expressed CD96 [413]. Further, in four of five samples, only CD96+ cells were able to propagate the leukemia in recipient immunocompromised mice, supporting its expression on LSCs and its usefulness as a potential therapeutic target. We identified CD47 in the mouse AML microarray screen, and have verified it as specifically upregulated in human AML. Subsequently, we have found it to be expressed in all mouse models of myeloid leukemia we have examined, and in human AML and blast crisis CML (manuscript submitted). Recent data suggests that expression of mouse CD47, which inhibits phagocytosis, is sufficient to allow the human myeloid leukemia cell line (MOLM-13) to engraft hematopoietic organs efficiently and develop into large tumors, whereas control MOLM-13 cells fail to engraft in immunocompromised Rag2−/−γc−/− cells [158]. Thus, CD47 does seem to play a role in the spread and/or life span of AML LSCs. These data also suggest that macrophagemediated killing may be a significant player in leukemia surveillance. There are increasing descriptions of mutations that occur at the posttranslational and epigenetic levels, and would not be detected by gene expression profiling. A study of 100 CML blood and marrow samples has demonstrated that GMPs from CML blast crisis have been shown to have increased levels of the active β-catenin in the nucleus compared with normal GMPs and to exhibit increased self-renewal in in vitro replating assays [412]. Dysregulation of microRNAs, small RNAs that bind to specific transcripts and either suppress their translation or target them for deletion, is another potential leukemogenic mechanism [414–417]. The important point here, as outlined in Plate 5.2, is that the elucidation of the phenotypes of normal HSCs and hematopoietic progenitors allows the classification of the various types of LSC, and the comparison of genes and gene transcripts between the normal and the neoplastic counterparts has revealed a plethora of new, and perhaps useful diagnostic and therapeutic, targets undetected when whole blood or marrow populations are analyzed.
Conclusion With the development of a general method to prospectively isolate hematopoietic stem and progenitor cells [1,40,45], it has become possible to construct increasingly detailed lineage pathways from LT-HSCs to mature blood cells. With the exception of memory T and B lymphocytes in the entire hematopoietic lineage, only LT-HSCs self-renew, and this self-renewal capacity is tightly regulated [1]. The genetic regulation of HSC self-renewal includes regulation of programmed cell death, regulation of cell-division frequency, and regulation of transition to
Biology of Hematopoietic Stem and Progenitor Cells
multipotent and to oligopotent progenitors. Gene expression analyses of prospectively isolated HSCs and progenitors are revealing insights into the mechanisms of self-renewal and those in lineage commitment. These same isolated HSCs and progenitors are increasingly useful in clinical HCT, whether to regenerate the myeloablated hematopoietic system in cancer patients, to condition allogeneic hosts for donor tissue or organ grafts, or even to replace an autoimmune-prone hematolymphoid system
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with a nonautoreactive donor population. Finally, the concept of selfrenewal as the critical regulated event limited to HSCs in normal hematopoiesis has now been extended to the hypothesis that within leukemias and cancers are relatively infrequent populations of LSCs that undergo poorly regulated self-renewal. Prospective isolation of LSCs should allow more direct approaches for immune and drug therapies for target molecules restricted to these malignant stem cells.
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337. Karsunky H, Merad M, Mende I, Manz MG, Engleman EG, Weissman IL. Developmental origin of interferon-alpha-producing dendritic cells from hematopoietic precursors. Exp Hematol 2005; 33: 173–81. 338. King AG, Kondo M, Scherer DC, Weissman IL. Lineage infidelity in myeloid cells with TCR gene rearrangement: a latent developmental potential of proT cells revealed by ectopic cytokine receptor signaling. Proc Natl Acad Sci U S A 2002; 99: 4508–13. 339. Rossi DJ, Bryder D, Seita J, Nussenzweig A, Hoeijmakers J, Weissman IL. Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age. Nature 2007; 447: 725–9. 340. Prohaska SS, Weissman I. Gene expression profiling of highly purified myeloid progenitor populations (submitted for publication). 341. Geschwind DH, Ou J, Easterday MC et al. A genetic analysis of neural progenitor differentiation. Neuron 2001; 29: 325–39. 342. Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, Lemischka IR. A stem cell molecular signature. Science 2002; 298: 601–4. 343. Sieweke MH, Graf T. A transcription factor party during blood cell differentiation. Curr Opin Genet Dev 1998; 8: 545–51. 344. DeKoter RP, Singh H. Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science 2000; 288: 1439– 41. 345. Hu M, Krause D, Greaves M et al. Multilineage gene expression precedes commitment in the hemopoietic system. Genes Dev 1997; 11: 774– 85. 346. Porcher C, Swat W, Rockwell K, Fujiwara Y, Alt FW, Orkin SH. The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 1996; 86: 47–57. 347. Shivdasani RA, Mayer EL, Orkin SH. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature 1995; 373: 432–4. 348. Tsai FY, Keller G, Kuo FC et al. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 1994; 371: 221–6. 349. Fujiwara Y, Browne CP, Cunniff K, Goff SC, Orkin SH. Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1. Proc Natl Acad Sci U S A 1996; 93: 12355–8. 350. Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin SH. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J 1997; 16: 3965–73. 351. Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ, Tenen DG. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proc Natl Acad Sci U S A 1997; 94: 569–74. 352. Morgan B, Sun L, Avitahl N et al. Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation. EMBO J 1997; 16: 2004–13. 353. Klug CA, Morrison SJ, Masek M, Hahm K, Smale ST, Weissman IL. Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin
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372. Attema JL, Papathanasiou P, Forsberg EC, Xu J, Smale ST, Weissman IL. Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis. Proc Natl Acad Sci U S A 2007; 104: 12371– 6. 373. Dahl JA, Collas P. Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells. Stem Cells 2007; 25: 1037–46. 374. O’Neill LP, VerMilyea MD, Turner BM. Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nat Genet 2006; 38: 835–41. 375. Bernstein BE, Mikkelsen TS, Xie X et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 2006; 125: 315–26. 376. Thomas ED. Bone marrow transplantation from bench to bedside. Ann N Y Acad Sci 1995; 770: 34–41. 377. Thomas ED. A history of haemopoietic cell transplantation. Br J Haematol 1999; 105: 330–9. 378. Uchida N, Tsukamoto A, He D, Friera AM, Scollay R, Weissman IL. High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts. J Clin Invest 1998; 101: 961–6. 379. Negrin RS, Atkinson K, Leemhuis T et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant 2000; 6: 262–71. 380. Vose JM, Bierman PJ, Lynch JC et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with recurrent indolent non-Hodgkin’s lymphoma. Biol Blood Marrow Transplant 2001; 7: 680–7. 381. Stadtmauer EA, O’Neill A, Goldstein LJ et al. Conventional-dose chemotherapy compared with high-dose chemotherapy plus autologous hematopoietic stem-cell transplantation for metastatic breast cancer. Philadelphia Bone Marrow Transplant Group. N Engl J Med 2000; 342: 1069–76. 382. Gazitt Y, Tian E, Barlogie B et al. Differential mobilization of myeloma cells and normal hematopoietic stem cells in multiple myeloma after treatment with cyclophosphamide and granulocyte-macrophage colony-stimulating factor. Blood 1996; 87: 805–11. 383. Michallet M, Philip T, Philip I et al. Transplantation with selected autologous peripheral blood CD34+Thy1+ hematopoietic stem cells (HSCs) in multiple myeloma: impact of HSC dose on engraftment, safety, and immune reconstitution. Exp Hematol 2000; 28: 858–70. 384. Miyamoto T, Weissman IL, Akashi K. AML1/ ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. Proc Natl Acad Sci U S A 2000; 97: 7521–6. 385. Ferrara JL, Levy R, Chao NJ. Pathophysiologic mechanisms of acute graft-vs.-host disease. Biol Blood Marrow Transplant 1999; 5: 347–56. 386. Shizuru JA, Jerabek L, Edwards CT, Weissman IL. Transplantation of purified hematopoietic stem cells: requirements for overcoming the barriers of allogeneic engraftment. Biol Blood Marrow Transplant 1996; 2: 3–14.
387. Gandy KL, Domen J, Aguila H, Weissman IL. CD8+TCR+ and CD8+TCR− cells in whole bone marrow facilitate the engraftment of hematopoietic stem cells across allogeneic barriers. Immunity 1999; 11: 579–90. 388. Kaufman CL, Colson YL, Wren SM, Watkins S, Simmons RL, Ildstad ST. Phenotypic characterization of a novel bone marrow-derived cell that facilitates engraftment of allogeneic bone marrow stem cells. Blood 1994; 84: 2436–46. 389. Martin PJ. Donor CD8 cells prevent allogeneic marrow graft rejection in mice: potential implications for marrow transplantation in humans. J Exp Med 1993; 178: 703–12. 390. Shizuru JA, Weissman IL, Kernoff R, Masek M, Scheffold YC. Purified hematopoietic stem cell grafts induce tolerance to alloantigens and can mediate positive and negative T cell selection. Proc Natl Acad Sci U S A 2000; 97: 9555–60. 391. Heeg K, Wagner H. Induction of peripheral tolerance to class I major histocompatibility complex (MHC) alloantigens in adult mice: transfused class I MHC-incompatible splenocytes veto clonal responses of antigen-reactive Lyt-2+ T cells. J Exp Med 1990; 172: 719–28. 392. Thomas JM, Verbanac KM, Carver FM et al. Veto cells in transplantation tolerance. Clin Transplant 1994; 8: 195–203. 393. Steinman RM, Nussenzweig MC. Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc Natl Acad Sci U S A 2002; 99: 351–8. 394. Millan MT, Shizuru JA, Hoffmann P et al. Mixed chimerism and immunosuppressive drug withdrawal after HLA-mismatched kidney and hematopoietic progenitor transplantation. Transplantation 2002; 73: 1386–91. 395. Beilhack GF, Scheffold YC, Weissman IL et al. Purified allogeneic hematopoietic stem cell transplantation blocks diabetes pathogenesis in NOD mice. Diabetes 2003; 52: 59–68. 396. Smith-Berdan S, Gille D, Weissman IL, Christensen JL. Reversal of autoimmune disease in lupus-prone New Zealand black/New Zealand white mice by nonmyeloablative transplantation of purified allogeneic hematopoietic stem cells. Blood 2007; 110: 1370–8. 397. BitMansour A, Burns SM, Traver D et al. Myeloid progenitors protect against invasive aspergillosis and Pseudomonas aeruginosa infection following hematopoietic stem cell transplantation. Blood 2002; 100: 4660–7. 398. Yilmaz OH, Valdez R, Theisen BK et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 2006; 441: 418–19. 399. Wiemels JL, Xiao Z, Buffler PA et al. In utero origin of t(8;21) AML1-ETO translocations in childhood acute myeloid leukemia. Blood 2002; 99: 3801–5. 400. Gale KB, Ford AM, Repp R et al. Backtracking leukemia to birth: identification of clonotypic gene fusion sequences in neonatal blood spots. Proc Natl Acad Sci U S A 1997; 94: 13950–4. 401. Hjalgrim LL, Madsen HO, Melbye M et al. Presence of clone-specific markers at birth in children with acute lymphoblastic leukaemia. Br J Cancer 2002; 87: 994–9. 402. Cave H, van der Werff ten Bosch J, Suciu S et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. Euro-
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6
Peter M. Lansdorp
Molecular Biology of Stem Cell Renewal
Several factors have led to intensive investigations of the hematopoietic system over the past few decades. The hematopoietic system represents the prototype of a self-renewing biologic system in that large numbers of blood cells have to be produced daily in order to compensate for the loss of relatively short-lived mature blood cells. The relative ease with which blood and marrow samples can be obtained and single-cell suspensions prepared has greatly facilitated in vitro and in vivo studies of hematopoietic progenitor and stem cells. Techniques to purify increasingly rare cells on the basis of cell surface antigens or functional properties have been refined and are available to most researchers. A large number of purified recombinant proteins and other molecules with activity on various hematopoietic cells have been discovered and have become available as well. In addition, a variety of in vitro and in vivo assays to measure functional properties of hematopoietic cells have been developed over the years, and some of these are now available as standardized tests. Together, these advances have allowed various studies with purified cells and molecules, some of which have revealed unexpected characteristics of stem cell “candidates.” From such studies, it has become clear that hematopoietic stem cells (HSCs) cannot be considered a homogeneous population of cells. Instead, HSCs represent a heterogeneous population of cells with functional properties that vary with replicative history, developmental stage, and age. This is illustrated by the pronounced developmental changes in the functional properties of purified “candidate” stem cell populations, the marked change in stem cell turnover in the first few years of life, and the age-related loss of telomeric DNA in HSCs. The age-related decline in stem cell renewal and replicative potential is clinically relevant as limited engraftment and failure of stem cell transplants could result from properties of HSCs that are predictable and, increasingly, measurable. In this chapter, some aspects related to the “self-renewal” of HSCs are reviewed. No attempt is made to cover the extensive literature in this general area. Specifically, the large number of studies aimed at dissecting the role of various transcription factors in stem cell properties and function are only briefly mentioned here. Instead, the focus is on the role of telomerase and the loss of telomere repeats in stem cells relative to their “self-renewal” in vitro and in vivo.
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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The stem cell hierarchy A recurrent theme in experimental hematology over the past 40 years has been the redefinition of what HSCs are, based on changes and improvements in assays. The trend has invariably been towards the identification of cells with a lower frequency than was previously reported or proposed [1]. The inevitable conclusion of this trend will no doubt be the identification of the fertilized egg as the “mother of all stem cells.” But what could be the explanation for the seemingly endless debate about HSC definition, HSC purification, and the apparent inability of an extensive field to agree on basic principles? The fact that the behavior of HSCs upon transplantation depends on many experimental variables, including the number and type of transplanted cells as well as host factors, has certainly contributed to the confusion. In animal models, only a limited number of blood- or bone marrow-derived HSCs are required to completely regenerate hematopoiesis in suitably conditioned recipients. Although the minimum number of HSCs required for sustained engraftment in human patients is not known, it has been shown that a single purified HSC in the mouse can reconstitute hematopoiesis [2]. This remarkable fact has supported the older definition of HSCs as “pluripotent cells with self-renewal potential” [3]. Implicit in the definition of “self-renewal” is that the two daughter cells, derived from the division of a single parental HSC, are identical to each other and indistinguishable from the parental cell. However, this is most likely an oversimplification as the behavior of HSCs is known to vary markedly with the developmental stage of the donor [4]. Thus, the source of HSCs is an important variable in transplant studies. Indeed, stem cell “candidates” purified from human [5] or murine [6] fetal liver versus adult bone marrow show remarkable functional differences that correlate with measurable molecular changes occurring with age [7] and upon transplantation [8]. Loss of telomere repeats in stem cells, developmental changes in HSC function, and the facultative, cell dosedependent symmetric versus asymmetric (self-renewal versus maintenance) cell divisions of HSCs [9] greatly complicate generalizations about the nature and function of HSCs. Nevertheless, the central dogma and current paradigm in experimental hematology has remained that perhaps the frequency of HSCs, but not HSCs themselves, are subject to significant change. In a way, it is understandable that experimental hematologists hate to part with simple notions about stem cells as “units” and “self-renewal” as a concept. After all, it is these notions that allow, in theory, an external control of HSC properties and the net “expansion” of HSCs in vitro. Although the inclusion of “relative” in the definition of “self-renewal”
Molecular Biology of Stem Cell Renewal
may seem annoying, the fact remains that hematopoietic tissues typically contain a spectrum of “stem” cells, each endowed with different replicative potential and functional properties. The relative frequency of various HSCs within the hierarchy is likely to vary with the age of the donor. The optimal clinical use of HSCs in transplantation settings will take not only the number and viability of HSCs in the transplant into account, but also their developmental stage and their estimated replicative potential.
Hematopoiesis: how many stem cell divisions? The realization that stem cells are subject to pronounced developmental changes has focused attention on the question, “How many times can stem cells divide?” Unfortunately, no techniques to address this important question are currently available, and possible answers range from fewer than 100 times [1] to over 5000 times [10,11]. The total number of blood cells required for the lifelong maintenance of normal levels of blood cells can be calculated to be in the order of 4.1016 cells (∼1012 cells per day × 365 days × 100 years). In theory, only 55 divisions of a single cell would satisfy this need (255 = 4.1016). Because many hundreds of different HSCs are contributing to hematopoiesis in a normal individual at any given time, an absolute replicative potential of fewer than 100 cell divisions per HSC could presumably easily accommodate a lifetime of blood cell production and allow for the known apoptosis of cytokinedeprived committed progenitor cells and the remarkable regenerative capacity of HSCs displayed in response to marrow injury and upon (serial) transplantation. An important related question is how many HSC clones are actually present in normal adults. Because genetic markers such as point mutations can occur at any given cell division, the presence of such a clonal marker does not a priori allow the conclusive identification of a single HSC clone. For example, in patients with paroxysmal nocturnal hemoglobinuria, the presence of a mutation in the PIG-A gene is often used to identify the abnormal HSC “clone” [12]. However, such a mutation could have occurred relatively early in development, for example before the major expansion of HSC numbers in early childhood [13]. As a result, many hundreds of adult HSC clones would be expected to show the mutation and “clonal” marker. Conversely, when a mutation takes place very late in the stem cell hierarchy, only committed progenitors could be affected, resulting in the transient appearance of a clonal marker in the peripheral blood. The transient presence of BCR–ABL fusion genes in the circulation of normal individuals could be an example of such a late occurrence [14]. Presumably the likelihood of PIG-A, BCR–ABL or other mutations will be directly related to the number of cells at risk: relatively few in early development and many in adult tissues. The notion that HSCs have a limited replicative potential is further supported by several observations. Both the in vivo regenerative capacity [15,16] and the in vitro expansion potential [4,5] of HSCs appear to be under developmental control (see also below). Most HSCs in adult tissue are quiescent cells in line with expectations for cells that divide very infrequently and have only a limited replicative potential that lack “self-renewal” properties in an absolute sense. The loss of telomere repeats in adult hematopoietic cells (including purified “candidate” HSCs) relative to fetal hematopoietic cells [7] also fits a model that postulates a finite and limited replicative potential of HSCs [1,17]. Recent longitudinal studies of telomere length in baboons provide further support for the idea that each cell division in HSC is accompanied by a measurable loss of telomere repeats [18]. In this study, it was found that the rate of telomere loss was greatly reduced after the first year of life in line with the rapid expansion of the HSC compartment in the first few years deduced from cross-sectional telomere length measurements in human leukocytes [13].
65
Development and organization of hematopoietic tissue The hematopoietic system has been subdivided into a hierarchy of three distinct populations [19,20]. In this model, the most mature cells are morphologically identifiable as belonging to a particular lineage and have very limited proliferative potential. The cells in this most mature compartment are derived from committed progenitor cells with a higher but finite proliferative potential. Committed progenitor cells are in turn produced by a population of multipotential HSCs with variable “selfrenewal” potential. Hematopoiesis in embryos is first observed in “blood islands” in the yolk sac [15]. Blood island precursor cells are mesenchymal cells derived from the primitive streak region of the early blastoderm. In the mouse, the yolk sac does not contain precursors of adult hematopoiesis, which can first be found in the aorta–gonad–mesonephros (AGM) region at day 10 of gestation [21]. From the AGM region, hematopoiesis appears to move to the fetal liver before reaching its final destination in the marrow. The concept of subsequent waves of progenitor cells seeding different tissues is compatible with various observations. However, the origin and number of cells seeding a particular hematopoietic niche still needs to be defined. Interestingly, some cells from yolk sac, fetal liver, and marrow are all capable of producing spleen colonies in irradiated adult recipients [15]. This observation, together with the questionable use of spleen colony assays to measure HSCs [22], is probably responsible for what appears to be an incomplete appreciation of the developmental changes in the biologic properties of primitive hematopoietic cells. Results showing that cells from early stages of development can function, to a large extent, in adult hosts and vice versa [23] illustrate a remarkable functional adaptive capacity or “plasticity” of hematopoietic cells at various stages of development. However, in the mouse, cells giving rise to spleen colonies upon transplantation into lethally irradiate recipients (colony-forming unit-spleen or CFU-S) from different tissues were found to be qualitatively different in that the number of CFU-S per individual spleen colony decreased during ontogeny [15]. In these experiments, yolk sac CFU-S could be transferred in vivo seven times, fetal liver CFU-S four to six times, and marrow CFU-S from young and adult mice up to three times [15]. These findings support a functional decline in stem cell self-renewal properties with each subsequent cell division and/or a decreasing and limited replicative potential of HSCs (see below). Several more recent observations provide further evidence of ontogeny-related differences between populations of primitive hematopoietic cells at various stages of development (reviewed in [4,24]). In general, fetal cells have a higher turnover rate and a higher replating potential than their functional counterparts from adult marrow. This observation is illustrated in Fig. 6.1 [5,25], which shows the production of CD34+ cells in culture from “candidate” HSCs with a CD34+CD45RAloCD71lo phenotype purified from, respectively, human bone marrow, cord blood, and fetal liver. Using identical growth factors and culture conditions, no significant production of CD34+ cells was observed in cultures initiated with adult bone marrow HSCs, whereas the number of CD34+ cells in cultures of fetal liver HSCs increased several thousand-fold. As the same number of cells was cultured under identical conditions, these observations point to intrinsic differences between the candidate HSCs themselves. Developmental switches have also been described in the immune system (reviewed in [26]). The switches from embryonic to fetal to adult hemoglobin in red cells reflect epigenetic changes in HSCs that could also affect their biologic properties upon transplantation. Because such epigenetic differences between HSCs at different developmental stages
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Fig. 6.1 Ontogeny-related differences in production of CD34+ cells in culture. Human candidate hematopoietic stem cells (HSCs) with a CD34+CD45RAloCD71lo phenotype were purified from bone marrow, umbilical cord blood and fetal liver, and cultured in serum-free culture medium supplemented with interleukin-6 (IL-6), IL-3, stem cell factor, and erythropoietin (Epo) as described in [25]. Cultures (1 mL each) were initiated with 104 sorted cells. At the indicated time interval, the total number of nucleated (䊐) and CD34+ (䊏) cells present in the cultures was calculated from the cell counts and the percentage of viable CD34+ cells measured by flow cytometry. All CD34+ cells from bone marrow cultures and fractions of the CD34+ cells from cord blood and fetal liver cultures were sorted and used for continuation of the cultures. Reproduced with permission from [5]. Copyright (1993), The Rockefeller University Press.
are likely, many assumptions about HSCs in adults need experimental verification. For example, it is currently not clear that adult HSCs have a similar potential to their fetal counterparts to produce lymphoid progenitors. In the mouse, the ability of HSCs to produce specific lymphocyte precursors is under developmental control [27], and the number of HSCs with full lymphoid potential in adults is expected to be limited. Similarly, the cells of the microenvironment are expected to show changes as a function of normal development and as a function of age. Uncertainties regarding the composition of the stem cell microenvironment and its function over time, combined with similar questions about the cells in the stem cell compartment, represent ongoing challenges in the basic science of HSCs and HSC transplantation.
Molecular control of stem cell fate Of fundamental interest to experimental hematologists and developmental biologists are the mechanisms that control the fate of HSCs. At any point in time, a “stem” cell has a choice to contribute to either the immediate future (by differentiating and producing committed progenitor cells) or the more distant requirements for mature cells (by undergoing a functional self-renewal division). It has been proposed that such decisions at the level of pluripotent HSCs can be depicted as stochastic processes that are intrinsic to the cells [20,28]. If this concept is correct, the distinction between HSCs with limited and extensive in vivo repopulation potential will likely remain elusive as the critical distinction between the cells, the number of actual self-renewal divisions executed in vivo, would be governed by chance. This possibility is certainly in agreement with the difficulties encountered by many investigators in purifying repopulating HSCs to homogeneity. However, as was outlined above, the situation is more complex as the self-renewal properties of HSCs are also subject to developmental control. Furthermore, it seems likely that the two daughter cells resulting from cell division of a single HSC may each differ in their functional properties. Such asymmetric cell divisions are key in animal development [29] and need to be taken into account in models of HSC biology and hematopoiesis [9,30]. The ability of a limited number of HSCs to reconstitute hematopoiesis in an irradiated recipient strongly suggests
that factors in the environment are also crucial in directing stem cell fate. Most likely, HSCs switch upon transplantation from symmetric “self-renewal” divisions (which increase the absolute number of stem cells, albeit at the expense of telomere loss) to asymmetric “maintenance” cell divisions as the bone marrow and its HSC “niches” become occupied with cells. Although the molecular details that govern this switch remain to be elucidated, it seems likely that the orientation of the mitotic spindle relative to the basal layer is involved [9,31]. One interesting possibility is that, following DNA replication but prior to mitosis, the two sister chromatids in the stem cells carry distinct epigenetic marks at the centromeric DNA as well as at the regulatory regions of specific genes [32]. The nonrandom segregation of sister chromatids could support asymmetric cell divisions and maintenance of stem cells, whereas the random segregation of sister chromatids could favor self-renewal and increased stem cell numbers (Plate 6.1).
Transcriptional control of stem cell function Despite intensive studies, the role of soluble factors involved in stem cell fate decisions is not clear. The dramatic clinical effect of cytokines such as erythropoietin, granulocyte colony-stimulating factor, stem cell factor, thrombopoietin, and others have underscored the important and critical role that cytokines play in the proliferation and differentiation of committed hematopoietic progenitor cells. However, attempts to modulate cell fate decisions in early hematopoietic cells using cytokines have invariably been unsuccessful [28,30,33,34]. Perhaps the primary role of cytokines and the microenvironment in HSC biology is to provide the essential but primarily permissive environment required for survival, proliferation, and differentiation. But if growth factors or factors in the microenvironment do not dictate stem cell fate decisions, what factors do? Important clues to this crucial question have been obtained by studies of the expression of homeobox genes in human hematopoietic cells [35]. It was found that expression of Hox genes is restricted to the most primitive hematopoietic progenitors and that the overexpression of HoxB4 in murine HSCs results in a remarkable expansion of cells with long-term lymphomyeloid repopulat-
Molecular Biology of Stem Cell Renewal
ing potential [36,37]. The involvement of Hox genes in stem cell biology is particularly striking because Hox genes are better known as key regulators of patterning along the body axis of developing embryos [38]. It is possible that Hox genes are part of the network that controls stem cell expansion during development. However, the precise mechanism by which overexpression of Hox (fusion) genes increases self-renewal is currently not understood. Another signaling pathway that is clearly very important for stem cell self-renewal is the WNT signaling pathway [39–41]. This evolutionary conserved signaling pathway spans all the way from the cell membrane to regulate transcription at several stages of B- and T-cell development, as well as HSC self-renewal. Unlike the Hox genes, for which the connection with extracellular regulation is not clear, WNT signaling provides a degree of extracellular control over the self-renewal of HSCs. How the WNT pathway regulates stem cell fate in relation to other regulatory factors, including HOX proteins and other transcription factors, remains to be established [42,43]. None of the current models of how stem cell fate is controlled has led to procedures that allow a manipulation of HSCs ex vivo in a way that is clinically meaningful. Given the known complexity of transcriptional regulation and the difficulty of assaying stem cell function, mentioned above, a detailed understanding of the factors that control HSC self-renewal is likely to remain elusive for the foreseeable future.
Telomere structure and function Without extremely reliable mechanisms to duplicate and segregate complete copies of genomes into daughter cells, life in any form could not exist. Although much progress has been made in deciphering the many factors and pathways involved in securing the fidelity of DNA replication and chromosome segregation [44], many questions in this general area remain. The ends of chromosomes are crucially important to maintain genome stability, and telomeres are of particular interest in relation to DNA damage responses and DNA repair pathways. The DNA component of telomeres in all vertebrates consists of (TTAGGG)n. Telomeres distinguish intact from broken chromosomes, stabilize chromosome ends, and provide protection against nuclear degradation and end-to-end fusion events [45]. Telomeres are furthermore involved in the positioning of chromosomes within the nucleus and are also important for chromosome segregation [46]. Almost 50 years ago, Hayflick suggested that most normal human cells are unable to divide indefinitely but are programmed for a given number of cell divisions [47]. In 1990, several papers described a loss of telomeres with replication and with age, and suggested that progressive telomere shortening could explain Hayflick’s original observation [48–50]. This model was confirmed by subsequent studies showing that transfer of the telomerase reverse transcriptase gene could prevent telomere erosion and resulted in immortalizing of the cells that Hayflick studied in most detail: normal diploid human fibroblasts [51,52]. Since then, many papers have appeared that are compatible with the notion that telomere shortening limits the number of times most normal diploid cells can divide (reviewed in [53]). Telomeric DNA is lost in human cells via several mechanisms that are related to DNA replication, remodeling, and repair. Causes of telomere loss include the “end replication problem” [54,55], the nucleolytic processing of 5′ template strands following DNA replication to create a 3′ single-strand overhang [56,57], and the failed repair of oxidative DNA damage to telomeric DNA [58–60]. The relative contribution of the different mechanisms of telomere shortening that have been proposed (reviewed in [61]) to the overall decline in telomere length with age is not known and most likely varies between cell types and individuals, and as a function of age.
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To compensate for the inevitable loss of telomere repeats with each cell division, most cells express telomerase. Telomerase is a ribonucleoprotein containing the reverse transcriptase telomerase protein (hTERT), the telomerase RNA template component (hTERC), and the dyskerin protein as essential elements [62]. In addition, a number of proteins have been described that are important for telomerase assembly (sub) nuclear localization [63] and stability (reviewed in [64]). Telomerase is capable of extending the 3′ ends of telomeres. Telomerase levels are typically high in immortal cells that maintain a constant telomere length, such as the stem cells of the germline in the testis and embryonic stem cells. For reasons that remain to be precisely defined, the telomerase activity that is readily detected in human candidate HSCs [65] (reviewed in [66]) appears to be incapable of maintaining telomere length in these cells. Nevertheless, existing telomerase levels in HSCs are functionally important, as is highlighted in patients with the disorder dyskeratosis congenita (DKC). One possibility is that only very short telomeres provide a suitable substrate for telomerase in somatic cells. Patients with the autosomal dominant form of DKC have one normal and one mutated copy of the telomerase RNA template gene [67]. As expected, such patients show a modest reduction in telomerase levels, yet they eventually typically suffer from marrow failure, immune deficiencies, pulmonary fibrosis or cancer and rarely live past the age of 50 [68–70]. These findings are in stark contrast to those in the mouse, where a complete lack of telomerase activity is tolerated for up to six generations [71]. An emerging consensus is that telomere shortening in long-lived mammals acts as a tumor suppressor mechanism that prevents an unlimited and life-threatening proliferation of organ-specific stem cells and lymphocytes [72]. Current data are compatible with a model in which telomerase levels in human HSCs are tightly regulated and sufficient to elongate only a limiting number of short telomeres. As a result, the telomere length in human HSCs declines with cell proliferation and with age, eventually triggering replicative senescence or apoptosis. In patients with inherited telomerase deficiencies, limitations in the proliferation of various stem cells imposed by telomere loss occur earlier in life than normal.
Telomeres in the hematopoietic system Since the important original observation that telomeres in adult blood leukocytes are significantly shorter compared with germline material (sperm) from the same donor [73], the decline in somatic telomeres has been documented in three ways. The original observation was confirmed [48,74], it was shown that telomeres in various tissues were shorter in older donors [49,50], and telomere shortening was documented during in vitro culture of human cells [49,75]. In 1994, it was reported that cells produced in culture by highly purified human “candidate” HSCs from fetal liver, cord blood, and adult bone marrow could be distinguished by reproducible differences in telomere length, and that these cells showed a measurable decline in telomere length upon culture in vitro (Fig. 6.2) [7]. In the years that followed these initial reports, a large number of papers have appeared that have greatly refined our understanding of telomere shortening in human nucleated blood cells (reviewed in [17]). Studies in this general area have been facilitated by the development of quantitative fluorescence in situ hybridization (FISH) techniques to measure the telomere length in suspension cells using flow cytometry (“flow FISH” [76, 77]). With this technique, it has been shown that the age-related decline in telomere length in lymphocytes is much more pronounced than in granulocytes, and that rapid telomere shortening early in life is followed by a much more gradual decline thereafter (Fig. 6.3) [13].
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Fig. 6.2 Loss of telomeric DNA in cells from cultures of purified human candidate hematopoietic stem cells (HSCs) with a CD34+CD45RA10 CD710 phenotype from fetal liver, cord blood, and bone marrow. The purified cells were cultured in serum-free medium supplemented with a mixture of cytokines to stimulate their proliferation and obtain sufficient numbers of cells for telomere restriction fragment (TRF) length measurements by Southern analysis. The mean ± SE of TRF measurements for two different donors of each tissue is shown for the cells produced in culture (䊐) as well as for the total nucleated cells from each donor before culture and cell purification ( ). Reproduced with permission from [7]. Copyright (1994), National Academy of Sciences, USA.
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If one assumes that the number of cell divisions between HSCs and granulocytes is relatively constant throughout life and that telomere shortening in HSCs is (i) primarily resulting from replication, and (ii) relatively constant with each cell division, the telomere length in granulocytes can be used as a surrogate marker for the average telomere length
Fig. 6.3 Age-related loss of telomere length in lymphocytes and granulocytes from peripheral blood of normal individuals measured by flow fluorescence in situ hybridization (FISH). The specific telomere fluorescence of lymphocytes and granulocytes was analyzed after gating on these cells based on light scatter properties and DNA fluorescence. Note the heterogeneity in telomere fluorescence values, the overall decline in telomere fluorescence with age in both cell types, and the higher rate of telomere attrition in lymphocytes. In this study, it was found that a bi-segmented line resulted in a significantly better fit with the data than the linear fit shown on the left. Inserts show the results of bi-segmented fit analysis for lymphocytes (top) and granulocytes (bottom). Arrows indicate the optimal intersection of calculated regression lines which for both cell types was before 2 years of age. Reproduced with permission from [13]. Copyright (1999), the Rockefeller University Press.
in the “average” HSC clone. While the telomere length in leukocytes shows an overall decline with age [13,78], the telomere length at any given age in humans is highly heterogeneous, as is illustrated in Fig. 6.3 below. This variation appears to be primarily genetically determined [13,79]. For example, monozygous twins of over 70 years of age were
Molecular Biology of Stem Cell Renewal
shown to have a very similar telomere length in both granulocytes and lymphocytes, whereas such values in dizygotic twins differed more but not as much as between unrelated individuals [13]. Using further refinements in the flow FISH method ([76]), it was recently shown that the rapid decline early in life is followed by a slow decline until the age of 50–60 years, after which the decline again accelerates [80]. The decline in both granulocytes and lymphocytes is nonlinear and fits a cubic curve. The pronounced decline in telomere length in specific T and natural killer cells could trigger apoptosis in these cells during a normal lifetime and compromise immune responses in the elderly. In contrast, granulocytes show a much more modest decline in telomere length with age, and critical telomere shortening in HSCs during normal hematopoiesis does not seem very plausible. More likely, the total production of blood cells from a single HSC is primarily determined by differentiation into committed progenitor cells and not by replicative senescence. Furthermore, the occasional loss of individual stem cells via telomere shortening is not expected to impact on overall hematopoiesis (or on the overall telomere length in granulocytes) as long as an excess of HSCs exists in the marrow. Indeed, studies in mice suggest that HSCs accumulate with age, be it that their functional properties in terms of replicative potential and production of B lymphocytes is diminished [81,82]. Nevertheless, the age-related loss of telomeres in granulocytes, the (modest) loss of telomeres following allogeneic transplantation [8,83], and the aplastic anemia that follows partial telomerase deficiency [84–86] all indicate that telomerase levels in normal HSCs are limiting and that the proliferation of HSCs is ultimately also limited by progressive telomere shortening. The number of mature “end” cells, such as granulocytes, that is produced by an individual HSC is expected to be highly variable as a result of the poorly understood regulation of symmetric versus asymmetric cell divisions. Even a very limited number of additional self-renewal divisions in a HSC will greatly increase cell output, and if endowed with a modest proliferative advantage, individual HSCs can produce a staggering number of cells. This phenomenon is illustrated in clonal proliferative disorders such as paroxysmal nocturnal hemoglobinuria and chronic myeloid leukemia. However, even in chronic myeloid leukemia, clonally expanded Philadelphia-positive stem cells eventually appear to encounter a telomere crisis [87]. Unfortunately, with a large number of cells to select from, the genetic instability triggered by the loss of functional telomeres appears to facilitate the emergence of subclones in blast crisis that typically show additional genetic abnormalities, more malignant properties, and higher levels of telomerase.
The telomere checkpoint Most human somatic cells, including HSCs, express limiting levels of telomerase, and as a result, telomere shortening effectively limits their proliferative potential. Most likely, telomere shortening evolved as a checkpoint function to suppress tumor growth in long-lived species. The function of this “telomere checkpoint” may help explain poorly understood aspects of stem cell biology, including stem cell “exhaustion” in aplastic anemia and other disorders. Cells may bypass the telomere checkpoint by expressing high levels of telomerase or by inactivating downstream signaling events, for example by loss of p53 function. Some cells, including subsets of B cells [88], appear to avoid the telomere checkpoints altogether, and this process could make these cells more vulnerable to tumor development. Loss of p53 function also inactivates the telomere checkpoint. This is expected to be a rare event as both copies of the normal p53 allele in a cell must typically be lost or mutated in order to continue proliferation in the presence of many short and dysfunctional telomeres [89]. Loss of p53 function allows survival of cells with dysfunctional telomeres. The resulting chromosome fusions result in chromosome breakage and, as a result,
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in the loss and amplification of genes [90]. This leaves us with a paradox: telomere shortening can act both as a tumor suppressor mechanism (by imposing limits to the replication of somatic cells) and as a tumor promoter mechanism (by favoring genetic instability once cells have reached their telomere-imposed limit in replicative potential). Whether tumor growth is suppressed or promoted by progressive telomere shortening may depend on the number of cells that encounter such a telomere crisis and the genetic changes acquired by such cells prior to this encounter.
Conclusion In this chapter, some molecular aspects related to stem cell renewal were discussed. Developmental changes in the functional properties of HSCs and loss of telomeric DNA in HSCs are important considerations in both laboratory studies and the clinical use of HSCs. It is clear that, despite much progress over the last several decades, we are still left with many questions. Some of the most urgent are: How many times can HSCs divide, and what is the actual replicative potential of HSCs at various stages of development? To what extent are factors in the microenvironment (i.e., cytokines) capable of modulating the self-renewal properties of HSCs? How does the microenvironment change during development and with age? What are the mechanisms controlling the turnover time of HSCs in steady state hematopoiesis and during hematopoietic regeneration? Are differences in the replicative history of HSCs reflected in their anatomic location, cytokine response, and mobilization properties? Hopefully, answers to these important questions will become available in the near future. Studies with cultured hematopoietic cells have shown that the formation of hematopoietic colonies, as well as the proliferation and survival of various hematopoietic cells, typically requires the presence of “hematopoietic growth factors,” many of which have been cloned over the last decade and produced for clinical trials (reviewed in Chapter 41). The availability of recombinant growth factors has led to frantic research efforts over the past two decades to achieve clinically useful manipulation of blood cell production in vitro. Two sets of observations that were discussed in this chapter cast doubt about the feasibility of some of the perceived applications of ex vivo “expansion” and should probably be taken into account when laboratory or clinical experiments involving limited numbers of HSCs are con-templated. First, there are the observations indicating that currently ill-defined, intrinsic (genetic) mechanisms with a developmental component control the fate of HSCs. Second, hematopoietic cells, including purified HSCs, appear to lose telomeric DNA with each cell division [7]. Recent studies indicate that inherited telomerase deficiencies can give rise to a large number of disorders including idiopathic pulmonary fibrosis [91], aplastic anemia [86,92], and dyskeratosis [93]. The expectation is that inherited defects in telomere maintenance pathways will prove to be involved in many more diseases. The studies of telomeres have focused attention on possible genetic limitations in the replicative potential of somatic cells, including HSCs, and such restrictions are compatible with most experimental evidence produced in clinical and laboratory studies. In view of these considerations, it seems prudent to use reasonably large numbers of HSCs for transplantation, especially from adult donors [83]. Although the number of HSCs in fetal liver or cord blood is small, such cells are likely to be superior in terms of replicative potential. This genetic superiority must be balanced against practical disadvantages including the small number of cells available for transplantation. The small size of fetal or neonatal stem cell grafts is expected to compromise clinical results, especially in the presence of histocompatibility differences between donor and recipient [94,95]. Possibly, this limitation could be addressed by more effective immunosuppression and conditioning regimens. In addition, the outcome of fetal liver and cord blood transplants could possibly be improved by increasing the number of cells in the transplant, for example by culture ex vivo.
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Chapter 6
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72. Maser RS, DePinho RA. Connecting chromosomes, crisis, and cancer. Science 2002; 297: 565– 9. 73. Cooke HJ, Smith BA. Variability at the telomeres of the human X/Y pseudoautosomal region. Cold Spring Harb Symp Quant Biol 1986; 51(Pt 1): 213–19. 74. Allshire RC, Gosden JR, Cross SH et al. Telomeric repeat from T. thermophila cross hybridizes with human telomeres. Nature 1988; 332: 656– 9. 75. Counter CM, Avilion AA, LeFeuvre CE et al. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. Embo J 1992; 11: 1921–9. 76. Baerlocher GM, Vulto I, de Jong G, Lansdorp PM. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat Protoc 2006; 1: 2365–76. 77. Rufer N, Dragowska W, Thornbury G, Roosnek E, Lansdorp PM. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol 1998; 16: 743– 7. 78. Frenck RW, Jr., Blackburn EH, Shannon KM. The rate of telomere sequence loss in human leukocytes varies with age. Proc Natl Acad Sci U S A 1998; 95: 5607–10. 79. Slagboom PE, Droog S, Boomsma DI. Genetic determination of telomere size in humans: a twin study of three age groups. Am J Hum Genet 1994; 55: 876–82. 80. Baerlocher GM, Rice K, Vulto I, Lansdorp PM. Longitudinal data on telomere length in leukocytes from newborn baboons support a marked drop in stem cell turnover around 1 year of age. Aging Cell 2007; 6: 121–3. 81. Bryder D, Rossi DJ, Weissman IL. Hematopoietic stem cells: the paradigmatic tissue-specific stem cell. Am J Pathol 2006; 169: 338–46. 82. Linton PJ, Dorshkind K. Age-related changes in lymphocyte development and function. Nat Immunol 2004; 5: 133–9. 83. Awaya N, Baerlocher GM, Manley TJ et al. Telomere shortening in hematopoietic stem cell transplantation: a potential mechanism for late graft failure? Biol Blood Marrow Transplant 2002; 8: 597–600.
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7
Catherine M. Flynn & Catherine M. Verfaillie
Cellular Biology of Hematopoiesis
Introduction
Stem cells
Hematopoiesis
Stem cells can be defined based on their obligatory characteristics of (1) self-renewal and (2) differentiation, as well as (3) functional repopulation of a tissue in vivo. Self-renewal is defined as the ability of a cell to divide without maturation and produce progeny identical to the parent cell. This process can be symmetric, wherein a single stem cell gives rise to two daughter stem cells, or asymmetric, generating a single new stem cell and a second daughter cell that has differentiated features. What governs asymmetric versus symmetric cell divisions is not yet known. All stem cells can differentiate, in response to the correct intrinsic and environmental cues, towards progeny of one or more lineages. Depending on the number of cell types that stem cells can generate, they are termed totipotent, pluripotent, and multipotent (Table 7.1). Totipotent stem cells, or the fertilized oocytes, can form a fully functional organism. Pluripotent stem cells differentiate into all cells of the embryo, i.e. the three germ layers – endoderm, ectoderm, and mesoderm – and the germline. Multipotent stem cells can be isolated from various tissues in fetal and adult animals. Such multipotent stem cells, sometimes referred to as adult stem cells or tissue-specific stem cells, differentiate into a limited range of cell types that are specific to the tissue from where the stem cell was obtained [1]. Stem cells are the key ingredient of hematopoietic cell grafts and have been used clinically since 1968 when the first successful BM transplant was performed at the University of Minnesota [2]. Since then, hematopoietic cell transplantation (HCT) has become a well-established treatment modality for malignant and nonmalignant diseases [3]. HSCs, the best-described stem cells, are categorized as multipotent stem cells because they self-renew, differentiate into all the cells of the blood lineage, and reconstitute the hematolymphoid system for the life of an individual.
“Hematopoiesis” is derived from the two words, “(h)aima” for blood and “poiein” meaning to make. Hematopoiesis involves generating an entire hematopoietic and immune system for the life of an individual. The cellular biology of hematopoiesis encompasses three components: the hematopoietic stem cell and its progeny, the environment in which they reside, and the signals and cytokines that regulate their fate. In adults, hematopoiesis occurs in the bone marrow (BM), which supports the lifelong maintenance of stem cells and their regulated trilineage differentiation. The earliest hematopoietic cells of the developing mammalian embryo are detectable in the blood islands in the extraembryonic yolk sac at 2–3 weeks. Subsequently, hematopoietic cells are found within the embryo proper in the gonad–mesonephros region, from where hematopoietic stem cells (HSCs) migrate to the fetal liver and later to the BM. The regulation of HSC fate is complex, and the exact mechanisms that govern HSC self-renewal are not yet fully understood. This process involves factors intrinsic to the HSC and cues from the surrounding microenvironment, which plays a critical role as a supporting structure for HSCs. The BM microenvironment is composed of, aside from HSC progeny, such as macrophages, cells of mesenchymal origin like osteoblasts and adipocytes, and endothelial cells. The mesenchymal lineage cells are purportedly derived from a stem cell different from the HSC, namely the mesenchymal stem cell (MSC), also termed the marrow stromal cell. These three cell types create the HSC niche wherein the HSCs reside, and from which HSCs are released or whereto they home in manipulations aimed at the mobilization and transplantation, respectively, of HSCs. Both HSCs and MSCs continue to be a subject of intense interest and excitement, particularly with the wealth of recent information outlining mechanisms of HSC homing and mobilization, as well as the concept of stem cell plasticity. The focus of this chapter is to discuss HSC biology, HSC regulation, and a critical assessment of the presumed greater potentiality or plasticity of BM stem cells, whether HSCs or MSCs.
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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Stem cell plasticity A number of studies during the last decade have suggested that the therapeutic potential of BM cells may be more extensive than originally thought, because investigators have noted that HSCs and other cells residing in the BM may have differentiation ability beyond their tissuespecific stem cell fate. While conventional dogma dictates that multipotent adult stem cells are irreversibly committed to the organ in which they reside, the cloning of mammals from the nucleus of an adult somatic cell refuted the notion of irreversible commitment, and observations from a number of additional experimental models have challenged this paradigm [4]. Likely more than 1000 reports have suggested that
Cellular Biology of Hematopoiesis
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Table 7.1 Stem cell hierarchy Stem cell
Example
Explanation
Totipotent
Fertilized oocyte
Pluripotent
Embryonic stem cells Embryonic germ cells Inner cell mass of the blastocyst Primordial germ cells of the early embryo Hematopoietic stem cells Skin stem cells Neural stem cells
Cells with the capacity to form mesoderm, endoderm, and ectoderm, including germ cells and extraembryonic tissue Cells with the capacity to form mesoderm, endoderm, ectoderm, and germ cells. Cannot give rise to trophoblast
Multipotent
Cells within various adult organs that have the capacity to self-renew and differentiate into all the organ-specific cell types
stem cells residing in adult tissues can generate, under certain environmental conditions, specialized cell types other than those of the tissue from where the stem cells were obtained, and this even irrespective of the particular germ layer designation (mesoderm, ectoderm, and endoderm). This concept has been termed “stem cell plasticity” and has led to considerable excitement as well as skepticism. If correct, this would defy the developmental biologic principles that lineage-specific stem cells generate only differentiated cells from the tissue of origin, and would signify that adult stem cells may hold much broader therapeutic potential. BM-derived cells have been central to this paradigm shift, as they have been reported to contribute to a number of seemingly unrelated tissues and organs, including the central nervous system [5], skeletal muscle [6], liver [7,8], and cardiac muscle [9], particularly in the context of tissue injury. While evaluating reported plasticity of BM-derived cells, it is important to recognize that fresh adult BM contains multiple stem cell populations that include HSCs, MSCs, and endothelial progenitor cells [10]. In addition, cells with molecular features of hepatic progenitors and other cell types have been described [11]. It is thus possible that somatic stem cells may circulate from their tissue of origin and reside or pass through the BM. Plasticity would then only represent appropriate differentiation of a passenger progenitor/stem cell population. As will be discussed further in the chapter, there is also mounting evidence that, in particular, macrophages derived from HSCs can fuse with a differentiated host cell, creating the false impression that HSCs transdifferentiate in a cell-autonomous fashion to cell lineages other than the hematopoietic lineage [12]. It will be important to further decipher what possible mechanisms underlie BM cell plasticity, whether these be fusion or true transdifferentiation, to determine the clinical implications of the apparent plasticity.
Stem cells in BM HSCs During ontogeny, HSCs can be found in different locations, including the blood islands in the placenta, aorta–gonad–mesonephos region, fetal liver, and BM. In postnatal life, HSCs are found in the BM at a low frequency (1 : 105 cells of mouse BM [13]). At birth, HSCs are also found in the placenta [14,15] and in the peripheral blood of the infant, and hence umbilical cord blood (UCB) [16]. Postnatally, HSCs can also be found at very low frequency in peripheral blood [17], and they circulate at higher frequency following administration of cytokines such as granulocyte colony-stimulating factor [18] or of chemotherapeutic agents like
cyclophosphamide [19]. The observation that HSCs can leave the BM environment and be found in the circulation is of importance when considering possible plasticity of stem cells from tissues other than BM. As HSCs can only be defined based on their functional attributes, i.e. repopulation of the hematopoietic system following transplantation, murine HSCs remain the best characterized at present, chiefly because very accurate functional assays for murine HSCs exist that do not exist for human HSCs. The most accurate enumeration of murine HSCs is done via competitive repopulation assays [20], wherein a population of test cells are co-transplanted with a fixed number of BM cells and repopulation by the test cells is evaluated at 4–6 months following transplantation. To assess the self-renewal ability of the test cells, the BM from the initial recipient is grafted into secondary animals, where again repopulation by the test cells is evaluated. Using such in vivo studies, murine HSCs were found to be highly enriched (1 : 10 cells) within the c-Kit receptor (c-Kit)-positive, T-cell antigen (Thy-1.1)-low, lineage (Lin)-negative, stem cell antigen (Sca-1)-positive (KTLS) fraction of murine BM [21]. Subsequent studies have further enriched murine HSCs by evaluating additional stem cell markers, including CD50 and Flk1 among others [22]. Additional tools to identify murine HSCs include evaluation of the efflux of a number of dyes such as rhodamine (via the multidrug resistance pump) [23] or the DNA-binding dye Hoechst-33342 (via the breast cancer resistance protein) used to define the side population cells, or Sp cells [24]). Isolation of human HSCs has been more challenging owing to the lack of accurate assays such as the reconstitution of lethally irradiated animals. Surrogate in vitro and in vivo assays have been used to screen for biologic activity, including a number of long-term stromal cultures (long-term culture-initiating cells [25], cobblestone area-forming cells [26], myeloid–lymphoid initiating cells [27]) or transplantation into immunodeficient hosts such as the nonobese diabetic severe combined immunodeficiency (NOD-SCID) mouse (SCID-repopulating cell) [28] or the preimmune fetal sheep [29]. However, due to the xenogeneic nature of such transplantation models, accurate assessment of HSC quantity and quality is not possible. As in the mouse, investigators have used fluorescence-activated cell sorter (FACS) separation to enrich for candidate human HSCs. A number of different sorting strategies have allowed enrichment of human HSCs to 1 : 100–500, including selecting for CD34+Thy-1+Lin− [30], CD34+Lin−CD38− [31], CD34+Lin−CD38−ckit+Rho− [32] or CD34+CD38−Lin−Sp [33] cells from BM or UCB [28,34]. Despite the best selection procedures for human HSCs based on cell-surface markers or their ability to extrude dyes, the resultant population remains heterogeneous and contains less than 1% SCID repopulating cells.
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Chapter 7
MSCs The BM stroma plays an important role in sustaining the survival, proliferation, and differentiation of mature blood cells from HSCs. Cells within the BM microenvironment that contribute to this support are, in addition to HSC-derived macrophages, endothelial cells and mesenchymal cells, including osteoblasts and adipocytes. The latter cells are progeny from a stem/progenitor cell, the MSCs. Like HSCs, MSCs are rare [35], comprising 0.01–0.001% of BM cells [36]. MSCs were first described in 1976 [37] as plastic adherent cells from murine BM that, at the clonal level, gave rise to colonies of fibroblast-like cells which could be induced to differentiate to adipocytes, osteoblasts, and chrondrocykes. Owing to their fibroblastic appearance, Friedenstein named these cells “colony-forming fibroblasts” or “CFU-Fs.” Since the initial identification of MSCs in BM, MSCs have also been isolated from a number of other tissues, including UCB, placental tissue, fetal blood and liver, adipose tissue, exfoliated deciduous teeth, amniotic fluid, and adult lung and pancreas [38–43]. MSCs are self-renewing clonal precursors of nonhematopoietic stromal tissues. They are isolated and expanded in culture based on their ability to adhere to plastic in response to factors present in fetal calf serum. However, cultures of MSC are highly heterogeneous with only rare multipotent, uncommitted cells between a sea of more differentiated precursors. MSCs differentiate into cells found in limb bud mesoderm, including osteoblasts, chondroblasts, adipocytes, fibroblasts, smooth muscle-like cells or myofibroblasts, and skeletal muscle cells. However, this differentiation potential is quickly lost from ex vivo expanded MSCs, likely due to the fact that – as is true for HSCs – self-renewing cell divisions of MSCs in vitro occur rarely [44]. Morphologically, cultured MSCs are spindle-like cells with a fibroblast-like appearance. The phenotype of cultured MSCs has been established by a number of investigators as positive for CD44, CD29, CD90, CD105, CD73 but negative for hematopoietic markers CD34, CD45, and CD14, and endothelial markers CD34, CD31, and von Willebrand factor. The phenotype of freshly isolated MSCs is less clear, although enrichment can be obtained by sorting for cells positive for CD105 (SH2) and CD73 (SH3), or for Stro-1brightVCAMbright cells [45–47]. Whether one should even consider MSCs to be “stem cells” depends on how one defines stem cells. If the definition includes the notion of in vivo repopulation of a given tissue, few if any studies have demonstrated that MSCs are true stem cells. What the role is of MSCs within BM is not clear, although they likely are responsible for replacing osteoblasts and adipocytes. MSCs express a wide variety of cell adhesion proteins, interact with endothelial cells, and possess the ability to respond to a wide variety of cytokines and chemokines. Their differentiated progeny secrete a multitude of cytokines and growth factors, which play a role in supporting hematopoiesis. The ability of MSCs to enhance HSC engraftment has been tested and proven in murine models [48,49]. Specifically, co-transplantation of murine MSCs with limiting doses of murine HSCs results in significantly more mice in which hematopoietic reconstitution is seen. Similar results have been observed when human MSCs and HSCs are co-transplanted into immunodeficient mice [50]. This has led to ongoing clinical trials wherein MSCs are co-transplanted with – for instance – haploidentical CD34+ cells or UCB grafts to enhance engraftment [51]. Although MSCs are present in BM harvests, it is unclear whether host mesenchymal cells are replaced by donor-derived cells. Horwitz et al. found that 1–2% of osteoblasts from a patient after HCT were of donor origin [52], and Cilloni et al. [53] showed that 7 of 41 patients who underwent a sex-mismatched leukodepleted transplant displayed donor stromal engraftment. By contrast, a significantly larger number of studies concluded that MSCs, adipocytes, osteoblasts or stromal cells were not
donor derived following HCT. The reason for these discrepancies is not known. It is also not known why MSCs do not engraft. One possible explanation is that, for at least culture-expanded MSCs, over 95% remain within the lung vascular bed and rarely move beyond this capillary bed [54]. As MSCs secrete many cytokines that support hematopoiesis, investigators have examined the possibility that defects in MSCs may contribute to the pathogenesis of hematologic malignancies, including leukemia and myelodysplastic syndrome (MDS). One question is whether the stromal compartment is clonally abnormal. Stromal cultures from patients with MDS, chronic myeloid leukemia (CML), and acute myeloid leukemia have been examined [55–58]. Some have found that stromal cells are devoid of any chromosomal abnormality and are functionally normal, while others have found chromosomal abnormalities in stromal cells. Contaminating HSC-derived macrophages may account for some of the aberrations seen, as, for instance, macrophages (CD14+) in stromal cultures established from CML BM were enriched for Philadelphia chromosome (Ph)-positive cells whereas mesenchymal (CD14−) cells in these same cultures were Ph− [58]. Some reports suggest that endothelial cells in CML patients may be clonally related to the Ph+ HSC clone [59]. However, as has become clear in recent studies, some apparent endothelial progenitors are monocytic in origin [60]. Two recent studies examined the karyotype of hematopoietic cells and stromal cells from patients with MDS [56,57]. In both studies, karyotypic abnormalities were detected in stromal cells and hematopoietic cells, but there was a striking lack of overlap between the karyotypic abnormalities of the hematopoietic and stromal cells. It is not known whether the stromal chromosomal abnormality predates the leukemic process or whether the stromal abnormalities are caused by the same event that caused the initial genetic damage to the HSCs. Whether the stromal cells are initiators, supporters or simply bystanders in the clonal process is not clear. One also needs to be cognizant of the fact that chromosomal alterations may be acquired in culture after several passages, which result in the development of sarcomas when such MSCs are transplanted [61]. The ability of MSCs to secrete cytokines and growth factors may also be exploited for tissue repair. A large number of studies has been published wherein the trophic effects of MSCs on damaged tissues, ranging from radiation-induced damage to the gastrointestinal epithelium, to ischemic renal, cardiac or central nervous system lesions among others, have been described [62–66]. This has led to the contemplation of clinical use of MSCs for therapy of such disorders. A word of caution, however, comes from recent reports that transplantation of MSCs in ischemic tissues may not only have beneficial effects, but also cause aberrant tissue formation. For instance, two recent reports demonstrated that encapsulated structures were found in the infarcted areas of 51% of hearts after injecting MSCs and to a lesser extent unfractionated BM cells [67–69]. An interesting observation is that cultured MSCs can modulate the immune system. Specifically, MSCs are poorly recognized by allogeneic T lymphocytes [70] or by natural killer cells [71], while at the same time inhibiting T-cell [72] and B-cell [73–75] responses. The mechanism underlying this immunomodulatory effect, specifically the inhibition of mixed lymphocyte reactions, is not totally clear, although cell–cell interactions as well as the secretion of soluble T-cell inhibiting factors play a role. This has led to several provoking clinical reports wherein dramatic graft-versus-host disease (GVHD) modulation has been described following MSC administration [51,76,77]. A number of prospective studies are now ongoing testing the efficacy of MSC-based prevention or treatment of GVHD. In addition, the possible usefulness of MSCbased therapies for autoimmune disorders such as inflammatory bowel disease is being evaluated.
Cellular Biology of Hematopoiesis
A number of reports have suggested that MSCs may generate differentiated progeny other than mesenchymal progeny, including endothelial cells, cardiomyocytes, hepatic cells, and neural cells. These studies will be further assessed below.
Plasticity of BM-derived stem cells That BM-derived cells reconstitute the hematopoietic system is well established. Since the late 1990s, numerous reports have appeared concluding that BM-derived cells may also contribute to other tissues, a phenomenon termed plasticity. Some of these surprising results are thought to be due to plasticity of hematopoietic cells, whereas others have been ascribed to plasticity of MSCs. These studies will be discussed in the following paragraphs. While evaluating these studies, we will critically determine to what extent real transdifferentiation has been demonstrated. For that reason, we will here first define criteria that should be fulfilled to demonstrate true plasticity or transdifferentiation. Full proof of plasticity requires the following: 1 A single cell that ordinarily is destined to differentiate towards hematopoietic or mesenchymal progeny can generate the expected progeny as well as cells of another lineage. 2 Differentiated cell types are defined based on morphologic and phenotypic criteria as well as expression of the expected transcripts and proteins, and the acquisition of functional attributes of the unexpected progeny. 3 Finally, the differentiated cell should also integrate into a tissue in vivo and perform the function of the unexpected lineage cell in vivo. When most studies are evaluated based on this set of criteria, relatively few studies have proven that transdifferentiation is occurring. A seminal paper demonstrating the plasticity of BM-derived HSCs was published by Krause et al. in 2001 [78]. In this study, murine, male donor BM cells were HSC enriched by fractionation and lineage depletion, labeled with the membrane intercalating dye PKH26 and injected into irradiated female recipient mice. After 2 days, BM was harvested, bright PKH26 cells were reisolated by FACS, single PKH26 cells were injected into 30 irradiated female hosts, and donor reconstitution was assessed at 5 and 11 months. The HSC-enrichment process, by homing to the BM within 48 hours, identified a population of HSCs capable of durable reconstitution. At 11 months, donor cells contributed 12–86% of the peripheral blood in five mice, and BM from 4 of 5 of these animals could be serially transplanted. Among the five mice who survived to 11 months, HSC-derived progeny could also be found in nonhematopoietic tissues, as proven by presence of the Y chromosome in epithelial cells in the lung, gastrointestinal tract, epidermis, and dermis. Differentiation to the unexpected phenotype was defined based on morphology and expression of cell-type specific antigens, such as cytokeratin 8, 18, and 19. As Y chromosome-positive pulmonary epithelial cells also expressed mRNA for surfactant, it was suggested that these cells had functional properties of alveolar type II pneumocytes. A number of reports subsequently have suggested that the plasticity seen by Krause et al. may not be as extensive. For instance, Wagers et al. [79] evaluated mice grafted with single HSCs (defined as KTLS cells) and found vanishingly rare cells in the brain, liver, and heart that were donor in origin. In this study, donor origin was defined based on the expression of green fluorescent protein (GFP) in the donor HSCs. In addition, they evaluated tissues of parabiotic animals, where wild-type (WT) and enhanced GFP (eGFP) transgenic mice were surgically joined such that they rapidly develop a common, anastomosed circulatory system. As HSCs spontaneously migrate in and out the BM and can be found circulating, one would expect to find unexpected lineage differentiation from HSCs in this model. Even though extensive hematopoietic chimerism was seen, analysis of multiple tissues failed to demonstrate
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significant engraftment of nonhematopoietic tissues by circulating GFPpositive cells. One possible explanation for the discrepancies between the studies was the differing method of identifying donor cells (in situ hybridization of the Y chromosome versus a transgenic marker gene). A subsequent study by Krause et al. reconfirmed the ability of HSCs to transdifferentiate into lung epithelial cells, now using transgenic gene markers [80]. Hence the debate remains whether HSCs in this setting can transdifferentiate into epithelial cells. Since this initial seminal report (and prior to that) a multitude of papers has been published wherein authors describe the possibility that BM-derived stem cells can transdifferentiate. Before the studies are reviewed, we will, however, address possible mechanisms that could explain the perceived and unexpected lineage switch, as well as the many methods that can or have been employed to address plasticity, each with their own caveats.
Possible explanations of plasticity (Fig. 7.1) If plasticity is real, the long-held view that, during development at gastrulation, cells are definitively committed to a single germ layer
(a) Multiple stem cells
=
+
(b) cell fusion
+
=
(c) Trans de-/re-differentiation
(d) Pluripotency
TRENDS in Cell Biology
Fig. 7.1. Possible explanations for the perceived plasticity. (a) Stem cells for a given tissue might exist in an unrelated organ. (b) Perceived plasticity might be caused by the transplanted cells fusing with a host cell of a different lineage, leading to transfer of the genetic information of the transplanted cell to the host-derived cell. (c) Plasticity might occur via de- and redifferentiation, as is seen in cloning or in limb regeneration in amphibians. (d) Cells with pluripotent characteristics might persist even after the initial steps of embryological development. Reproduced with permission from Verfaillie [192]. Copyright (2002), Elsevier.
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(mesoderm, endoderm, ectoderm) would not be totally correct as, in plasticity, tissue-specific stem cells would overcome their definitive commitment to a single germ layer when they were placed in a different and suitable environment. That this might be possible follows from studies as far back as the 1970s in which it was demonstrated that, by transferring the nucleus of Xenopus cells into enucleated, unfertilized Xenopus egg cytoplasm, frogs could be generated with the genetic information of the transferred nucleus. These observations were extended in the 1990s with the cloning of sheep and subsequently other mammals from the nucleus of an adult somatic cell [4]. In addition, dedifferentiation and redifferentiation is a phenomenon known in lower animal species such as newts [81]. How cell fate is altered is not fully understood, although recent studies have shown that overexpression of four transcription factors can reprogram fibroblasts to cells with embryonic stem cell (ESC)-like properties [82,83]. Hence, it is not inconceivable that, in certain circumstances, such reprogramming may occur. What role tissue injury plays in its initiation is controversial. Cells may be summoned to sites of injury and, on arrival in the “damaged” environment, cytokines and external factors may induce changes in gene expression patterns and result in an altered phenotype, and possibly function. Alternative explanations for the perceived plasticity include the fact that tissue-specific stem cells other than HSCs also circulate and home to tissues other than the tissue of origin. In this scenario, neural or liver progenitor/stem cells may be “hiding out’ among the heterogeneous populations of cells existing in the BM. Migration of HSCs from the fetal liver to the BM during development is mediated by a CXCR4– SDF1 gradient, and homing of HSCs to the BM following transplantation is mediated by the same CXCR4–SDF1 gradient [84]. As many stem cells from other tissues also express CXCR4, it is not inconceivable that, during embryogenesis or in postnatal life, stem/progenitor cells other than HSCs may home to the BM [11]. That such cells may act as reserves for tissue regeneration or repair in adult life is tempting to speculate; however, there is no proof for such a hypothesis. A third explanation for plasticity arose in 2002, namely that donor cells can fuse with host cells whether in intact tissue or damaged tissue. Such fusion leads to the generation of 4N cells, which express cell surface and cytoplasmic markers derived from both the donor cell and the cell it fuses with. Terada et al. [85] and Ying et al. [86] found that in co-culture experiments with BM cells or neurospheres and ESCs, tetraploid hybrids could be created that adopted ESC characteristics. Similar findings were subsequently documented in vivo. In an elegant study, Alvarez-Dolado et al. [87] used a Cre–Lox recombination system to detect cell fusion events. Following transplantation of BM cells into lethally irradiated mice, they found that BM-derived cells fused with Purkinje cells, hepatocytes, and cardiomyocytes. Camargo et al. [88] also used a single HSC transplant model to demonstrate that HSCderived hepatocytes occur as a result of cell fusion. Which cell fuses with target cells in vivo has not been addressed in all studies. Nevertheless, two independent groups have demonstrated that HSCs themselves do not fuse with hepatocytes, rather that monocytes generated from HSCs are responsible [12,88]. It was furthermore shown that, even though part of the genetic program of the host cell is preserved, reprogramming by both activating and silencing of genes occurs, so that the hybrid cell resembles the host cell with which the donor cell fused [89]. A final explanation may be that rare, more pluripotent precursors of postnatal stem cells persist. Although pluripotent stem cells are present throughout the body of planaria, such cells were not thought to exist in higher mammalian species. However, as will be discussed below, there is mounting evidence that cells expressing the ESC transcription factors Oct4, Sox2, and Nanog can be isolated from human and murine BM or UCB [90–93]. The frequency of such cells decreases very fast with age.
The natural role of such cells in postnatal tissues is not known. However, the possibility exists that when such cells are placed in a different environment, such as the brain or liver, they have the inherent ability, without need for significant reprogramming, to respond to cues from these new environments and differentiate in a tissue-appropriate manner.
Techniques to assess plasticity One of the most important technical considerations in demonstrating plasticity is to identify without any ambiguity the donor cell in the target organ. A robust, unambiguous molecular marker for the donor cell allows accurate assessment of the clonal relationship of donor cells and facilitates identification of the donor cells following transplantation. A variety of methods has been used to mark the donor cells. For all detection methods, proper fixation, particularly fixative concentration, and staining is essential to ensure maximal signal strength. Donor markers used include the Y chromosome (in sex-mismatched transplantations), membrane or cytoplasmic dyes, labels that incorporate in the DNA, and genetic labels such as β-galactosidase (β-gal) and GFP transgenes. More recently, the advent of conditional genetic marking using the Cre–Lox technology has provided another method of detection. Each of these approaches has advantages and limitations. A recent paper by Brazelton and Blau provides a comprehensive review of stem cell tracking methodology [94]. Equally important when trying to identify a cell whose fate has changed is to definitively prove acquisition of a new cell fate. It is essential to demonstrate that the donor marker and the lineage-specific marker are expressed in the same cell. Cellular morphology can be complex, and it may be difficult to discriminate between two closely associated cells. If tissue sections are examined in two dimensions with standard microscopy, a donor cell may overlay a host cell with the typical host-tissue specific marker, and this may be mistaken for a transdifferentiated donor cell. These events are rare, but the reported frequency of adult stem cells contributing to nonhematopoietic tissues is often equally rare. Methods that allow visualization of thin optical sections and three-dimensional reconstructions of cells in tissue sections are therefore required. The important aspect of the detection of any specific signal in a tissue is the use of appropriate negative controls to evaluate potential confounding factors, including autofluorescence. Careful experimental design and appropriate controls can avoid many of the common pitfalls.
Fluorescent in situ hybridization for the Y chromosome In sex-mismatched transplantation in which male donor cells are transplanted into female recipients, a Y chromosome is present in all intact donor cells. Typically, it is detected using fluorescent in situ hybridization (FISH) with fluorescent probes for specific regions of the Y chromosome. However, the Y chromosome cannot be visualized in all male cells if thin tissue sections that only partially sample the nuclei are assessed, and it is technically difficult to use Y chromosome FISH on thicker sections that contain the full thickness of most cells (greater than 10 μm). Nevertheless, the specificity and sensitivity of Y-chromosome FISH are very high. False-positive results can be caused by cell overlay and from nonspecific binding of the probe. Experience and evaluation of good negative controls in parallel can help to avoid these problems. As discussed earlier, it is critical to demonstrate that the Y chromosome is in the cell of interest, which can be accomplished by threedimensional reconstruction of double-labeled thin sections obtained by confocal microscopy.
Cellular Biology of Hematopoiesis
DNA or cell membrane labels 3
Bromo-deoxyuridine (BrdU) or H-thymidine labels dividing cells by incorporating into DNA during the S phase of cell division and has been extensively used to identify cells transplanted into the brain. However, two recent studies have shown that BrdU is present in the DNA of grafted cells, which can be taken up by actively dividing cells in the injected/injured area if the grafted cells die [95,96]. This then creates the incorrect impression that transplanted cells have adopted a neuronal fate. Membrane dyes such as PKH26 and CM-Dil (3,3′-dioactadecyl-5,5′di(4-sulfophenyl)-oxacarbocyanine, sodium salt, SP-DIOC18) have also been used to mark donor cells [78,97]. Both these dyes are nontoxic fluorescent lipophilic dyes that incorporate into the lipid bilayer of cytoplasmic membranes; hence, the amount of dye per cell diminishes as the cell undergoes cell division, which makes monitoring of proliferating cells difficult. Another pitfall of this marker is that fusion or phagocytosis of a labeled cell with an unlabeled cell results in transfer of the dye to the host cell, and many studies using membrane dyes have not controlled for this possibility [98]. Similarly to other markers, tissue preparation for evaluation is critical as the embedding procedure can influence the detection of fluorescence; for example, PKH26 does not withstand embedding in poly(methyl methacrylate). Another method, used in combination with magnetic resonance imaging, is passive labeling of donor cells with small particles of iron oxide [99]. The label can also be visualized by Prussian blue staining on histologic examination. As with membrane dyes, the label can be diluted so that it becomes no longer visible with cell division, and the possibility exists for false-positive results from microglia that uptake iron particles released from dead cells. Another downside of iron oxide contrast agents is that once cells home to a lesioned area, it becomes difficult to distinguish contrast originating from the stem cells or from bleeding in the damaged area.
Transgenic markers One of the earliest tracking markers was bacterial β-gal. The ROSA26 strain of mice constitutively expresses β-gal in most cells and can be used as a marker in transplantation studies. However, two problems arise with its use: the expression of β-gal as a marker is relatively weak, and it can be difficult to distinguish it from endogenous mammalian β-gal activity [100]. Therefore, when rare events are observed using ROSA cells, it is necessary to exclude unanticipated upregulation of endogenous β-gal in response to the injury itself. Several mammalian cell types, such as Purkinje cells, have substantially higher endogenous β-gal activity than others, and these normal variations can be misleading. Mammalian β-gal can be distinguished by its intracellular location and activity at optimal pH, and techniques on how to optimize β-gal-based tracking in the central nervous system have been recently reviewed [101]. eGFP is a modified protein originating from a naturally occurring fluorophore, and several lines of transgenic mice have been generated that ubiquitously express eGFP [102,103]. However, transgene expression varies among mice, may not be completely ubiquitous, and may weaken with time, age, and tissue differentiation [104]. One can use antibodies to GFP to detect lower levels of GFP expression; however, many anti-GFP antibodies are not totally specific, and several may need to be tested to ensure specificity for the tissue of interest. Other limitations of GFP include its rapid diffusion out of unfixed cells and apparent GFP positivity due to autofluorescence of (injured) tissues. However, careful evaluation of the emission spectrum of GFP compared with autofluorescence may circumvent the latter problem [105]. Some have
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suggested that the presence of GFP in itself may promote differentiation, although abundant evidence suggests that the fluorophore is biologically inert [106]. Others have suggested that decreased GFP expression is a function of cellular differentiation and that expression in “transdifferentiated cells” is decreased, making detection of transdifferentiation difficult [6]. If this were true, it might account for many publications in the literature stating that no donor cells were detected when using GFP as a reporter gene to label BM.
Cre–Lox technology Cre–Lox technology has been a useful addition to the armamentarium to understand cellular plasticity and has been used to address the likelihood of cell fusion as a contributing mechanism. Cre–Lox recombination is a technique used to conditionally turn gene expression on and off in specific cell types and tissues. Cells with Cre recombinase under the control of a ubiquitous or cell-specific promoter are grafted in conditional Cre reporter animals, in which a loxP-flanked stop cassette is located in front of a LacZ or eGFP reporter gene. Only when Cre recombinase excises the floxed stop cassette is the LacZ/eGFP gene expressed. Alvarez-Dolado et al. used this method to show that BM-derived cells could fuse with hepatocytes, cardiomyocytes, and Purkinje cells [87]. Oh et al. showed that intravenously infused progenitor cells from the adult heart could fuse with cardiomyocytes in the setting of myocardial infarction [107], and Reinecke et al. extended this by adding skeletal myoblasts as a clinically relevant cell type that can fuse with cardiac myocytes in vitro and in vivo [108]. This method has failed to detect cell fusion in the pancreas [109]. Keeping these possible explanations and the many methodological pitfalls in mind, the following sections will review papers suggesting differentiation of BM cells into mesodermal, endodermal, and ectodermal cells. We will first discuss studies wherein investigators describe transdifferentiation of HSCs or MSCs into unexpected lineages, and then focus briefly on studies that have suggested that more pluripotent adult stem cells may be responsible for the observed apparent transdifferentiation.
Transdifferentiation of HSCs or MSCs BM-derived cells – mesoderm BM-derived skeletal muscle Already in the late 1990s, some studies suggested that BM-derived cells could contribute to skeletal muscle [110–112]. Ferrari et al. noted that, following muscle damage, more myogenic precursors than expected from resident muscle stem cells, also termed satellite cells, could be found in muscle [110]. To elucidate the source of these additional cells, they chemically injured the tibialis anterior muscle and injected into the limb either whole BM cells or satellite cells containing a LacZ reporter driven by a myogenic promoter. β-gal+ cells were found in the BM cells injected into muscle, even though more βgal+ cells were detected in the satellite cell-injected muscle. The myogenic potential of BM cells appeared to be derived from adherent cells. They also injured hematopoietically reconstituted mice grafted with BM cells from C57/MlacZline (H-2b) mice and found, in 5 of 6 animals, β-gal+ cells in the muscle. Together, these studies suggest that cells present in BM cells can contribute to muscle, and this contribution is enhanced when the muscle is injured. Gussoni et al. [6,113] isolated murine HSCs from WT male mice based on the SP property and transplantated these into female mice with a defect in the dystrophin gene (mdx mice). As expected, hematopoietic reconstitution was seen. In addition, in the recipient muscle,
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Y chromosome-positive cells expressing dystrophin were detected. When Sp cells were isolated from muscle of WT male mice and grafted into mdx mice, radioprotection was seen with a contribution of donor cells to muscle. As studies were not done using single cells, it is not clear whether the same cell that gave rise to hematopoietic reconstitution also contributed to muscle. By contrast, Corbel et al. used single HSC transplantation to address whether HSCs or their progeny contributed to muscle. Single GFP+SPckit+Lin−Sca-1+ (SPKLS) cells from CD45.1 mice were injected into CD45.2 recipients [114]. After 4 weeks, over 30% of blood cells were donor in origin. In these animals, muscle, without and with toxin injury, was evaluated at after 16 weeks for the presence of donor contribution. Donor-derived muscle cells were defined as cells with muscle morphology, encased in laminin displaying the characteristic sarcomeric pattern following antimyosin staining that were GFP+. In the mice reconstituted from a single SPKLS cell, GFP+ cells were detected in the uninjured and injured tibialis anterior muscle of three mice. Damage promoted integration of HSC-derived cells into the regenerating muscle. When BM from the primary transplanted mice was grafted in secondary mice, hematopoietic reconstitution confirmed the self-renewal capability of the original single cell. Moreover, in 1 of the 3 secondary recipients, GFP+ myofibers were also seen, strongly suggesting that the progeny of a single HSC can contribute to the hematopoietic system as well as muscle. The mechanism of contribution, i.e. direct transdifferentiation versus fusion of HSC-derived progeny with myofibers, was not addressed. That human BM contains cells with myogenic potential was shown by identifying rare dystrophin-positive cells in muscle of a 12year-old boy who underwent a BM transplant for immunodeficiency [115]. BM-derived cardiomyocytes Several studies have suggested that cardiomyocytes can be generated from BM, although controversy exists as to the degree and mechanism via which BM cells contribute to improved cardiac function. The cardiomyogenic potential of ESCs from a variety of species including human is well established [116,117]. Whether any other stem cell, except perhaps stem/progenitor cells found in the heart itself [107], can differentiate into bona fide functional cardiomyocytes is still not clear. Fukuda et al. demonstrated that treatment of MSCs with 5-azacytidine gives rise to spontaneously contractile cells with some features consistent with cardiomyocytes [113]. Such MSC-derived cardiomyocyte-like cells formed myotubes with branching fibers and at 4 weeks stained positive for antimyosin, antiactinin and antidesmin. The cells had a cardiomyocyte-like ultrastructure and, by reverse transcription-polymerase chain reaction had cardiomyocyte-specific gene expression. More importantly, electrophysiological studies demonstrated the presence of action potentials consistent with sinus node ventricular action potentials. While the phenotype of these cells derived in vitro at a transcript, protein, and functional level is convincing, no in vivo studies were performed. Toma et al. injected human LacZ-labeled MSCs into the uninjured left ventricle of an adult SCID/beige mouse [118]. At 2 weeks, rare (0.4%) β-gal-, desmin-, cardiac troponin T-, and α-actinin-positive cardiomyocyte-like cells were found. Sarcomeric organization of the contractile proteins in the β-gal+ cells was indistinguishable from that of neighboring recipient cardiomyocytes. Dzau and colleagues performed a number of studies wherein MSCs were grafted into the borderzone surrounding the infarct in mice, and demonstrated functional improvement. The functional improvement was higher when MSCs were genetically modified to express Akt, which was associated with prolonged and more abundant persistence of the grafted cells [119]. However, in subsequent studies, they demonstrated that a
similar effect could be obtained when media conditioned by Akt-transduced MSCs was injected, suggesting strongly that the effect was trophic, i.e. secretion by the MSCs of factor(s), such as secreted frizzled2, chiefly protecting at-risk cardiomyocytes in the ischemic borderzone, and perhaps also inducing recruitment of endogenous progenitors or endothelial cells [120]. Others evaluated whether hematopoietic cells could differentiate into cardiomyocytes. Bittner et al. described that, following male BM cell transplantation into irradiated normal and mdx female mice, not only skeletal, but also cardiac myofibers appeared to be derived from donor BM cells 10 weeks following transplantation [111]. The transdifferentiated phenotype was determined to be skeletal and cardiac muscle based on coexpression of skeletal and cardiac muscle-specific proteins in Y chromosome-positive cells. Jackson et al. transplanted 2000 SP cells from male C57BL/6-Rosa-Ly-5.2 mice into lethally irradiated female C57BL/6-Ly-5.1 mice and after 10 weeks induced a myocardial infarct [121]. Evaluation 2–4 weeks later indicated that 0.02% of the cardiomyocytes were β-gal+α-actinin+CD45− and 3.3% of the endothelial cells were β-gal+Flt-1+CD45− and hence apparently donor derived. Neither study determined the mechanism underlying the apparent transdifferentiation. Orlic and colleagues injected murine GFP+Lin−ckithi BM cells into the borderzone myocardium of female mice several hours after coronary artery ligation [9]. Evaluation 9–14 days afterwards suggested that 68% of the cells in the infarct were GFP+Y chromosome+ cells that coexpressed the early cardiomyocyte transcription factors myocyte enhancer factor-2 and gata-4. In addition, some GFP+Y chromosome+ endothelial cells and smooth muscle cells were also detected. Significant functional improvement was also seen. When Lin−c-kitlo cells were injected, no functional improvement was detected. However, two studies performed subsequently by independent groups demonstrated significantly less contribution of murine BM Lin−ckithi cells to the injured cardiac tissue, and significantly less significant improvement in cardiac function than in the original Orlic et al. report [9]. Murry et al. failed to demonstrate generation of cardiomyocytes from Lin−ckithi cells grafted into the borderzone myocardium, even though some functional improvement was seen [122]. Similarly, following grafting of Lin−ckithi cells from myosin heavy chain (MHC)–LacZ transgenic animals into infarcted hearts of WT mice, no β-gal+ cells could be detected in the heart. Unfractionated BM cells from β-actin– eGFP transgenic mice were also transplanted into lethally irradiated, nontransgenic recipients. Two months after hematopoietic engraftment, a myocardial infarct was induced. Although some eGFP+αMHC+ cells were detected in the borderzone, the apparent transdifferentiated cells were infrequent. Balsam and colleagues transplanted both GFP+Lin− ckithi and GFP+KTLS cells into the infarcted heart of WT mice and could not find cells of donor origin with cardiac characteristics [123]. They also infarcted the heart of WT mice parabiosed with β-actin–EGFP transgenic mice and did not find donor-derived cardiomyocytes in the infarct zone [124]. The latter study did not assess the impact on the cardiac function. Both authors cautioned therefore that the data initially obtained by Orlic et al. [9] might not be sufficient to warrant clinical studies. Nevertheless, a now large number of clinical trials in humans have ensued in which different types of BM cell have been injected following balloon angioplasty into patients who have suffered from a cardiac infarct. Despite initial encouraging results in nonrandomized studies, four studies have been published in 2006 wherein patients were treated in a randomized blind fashion with BM cells or vehicle control, demonstrating little functional benefit for the majority of patients treated with cells [125–128]. Of note, a clinical study in which the hearts of patients were examined at autopsy after sex-mismatched BM transplantation showed that Y
Cellular Biology of Hematopoiesis
chromosome-positive cardiomyocytes contributed 0.23 ± 0.06% of the myocardium, and no Y chromosome-positive cells were seen in gendermatched controls. These data are consistent with the murine studies indicating that very rare human BM cells have the potential to transdifferentiate into cardiomyocytes. Although this has been rarely assessed, the study by Alvarez-Dolado et al. has definitively demonstrated that rare apparent transdifferentiation may be due to cell fusion between donor cells and cardiomyocytes [87]. BM-derived cells – endoderm A number of reports have suggested that BM cells have the potential to generate endodermal tissue. Even though, during embryonic development, the mesoderm and endoderm lie anatomically close to each other and there is considerable crosstalk between these two tissues that enables each to develop, the transdifferentiation between two germ layers would perhaps be even more astounding than that between mesodermal cell types themselves. Petersen et al. were the first to suggest that BM cells contribute to liver regeneration [123]. Hepatic oval cells are regarded as facultative hepatic stem cells that can generate both hepatocytes and cholangiocytes. Their precise origin is not clear, but it is likely that they are derived from canals of Herring or reside as blast-like cells next to the bile ducts. Hepatic oval cells express CD34, Thy-1 and c-kit, markers also present on HSCs. Previous studies had shown that oval cells proliferate when hepatocyte proliferation is suppressed following liver injury. To test whether BM cells could contribute to liver regeneration, female rats were transplanted with BM from syngeneic male rats, and liver injury was induced using a protocol to stimulate oval cell proliferation (2-acetylaminofluorene [2-AAF] and carbon tetrachloride [CCL4]). Thirteen days following transplantation, Y chromosomepositive hepatocytes were detected by in situ hybridization. In a second experiment, irradiated dipeptidyl peptidase-IV (DPPIV)-negative female rats received BM from DPPIV-positive male rats. Liver injury was generated using the 2-AAF–CCL4 protocol. DPPIV expression was observed in oval cells and hepatocytes in the recipient liver, that coexpressed proteins found in mature hepatocytes. Finally, a liver transplant was performed from L21.6 antigen-negative donor rats into allogeneic L21.6-positive rats and hepatic ductular structures containing L21.6+ cells were present in the transplanted liver, suggesting that oval cells were derived from extrahepatic cells. The authors concluded that BM cells can under certain conditions become hepatic oval cells. Similar findings were seen in a murine model [8], and together these results support the concept of a BM or extrahepatic stem cell generating hepatic oval cells. Neither study established the exact phenotype of the “plastic” BM cells or their use in the therapy of liver diseases. Lagasse et al. [7] used an animal model of fatal hereditary tyrosinemia type 1 (fuarylacetoacetate hydrolase [FAH]-deficient mice) in which mutant mice develop progressive liver failure and renal tubular damage unless treated with 2-(2-nitro-4-trifluro-methylebenzyol)-1,3-cyclohexanedione (NTBC). Unfractionated BM or 50 KTLS cells from male FAH–WT β-gal−transgenic mice were injected into lethally irradiated female FAH−/− mice treated with NTBC. Three weeks after transplantation, NTBC was discontinued intermittently to allow selection of the liver repopulating cells. Four out of nine mice grafted with unfractionated BM and 9 of 20 mice grafted with KTLS cells survived and had donor-derived β-gal+FAH+Y chromosome+ cells in the liver with an improvement in liver function. These donor-derived cells coexpressed albumin with β-gal when cultured on a mouse embryonic feeder layer. However, later studies [89,129] demonstrated that the differentiation of HSCs into hepatocytes did not occur in a cell-autonomous manner but following fusion of HSC-derived monocytes with hepatocytes.
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A number of studies have also addressed whether apparent hepatic transdifferentiation occurs in humans. Theise et al. [129] demonstrated that liver tissue examined from male recipients of female livers and female recipients of male BM contained marrow-derived hepatocytes and cholangiocytes using in situ hybridization for the Y chromosome with colabeling with the monoclonal antibody CAM5.2, which identifies hepatocytes and cholangiocytes but not Kuppfer cells, stromal cells or circulating blood cells. The frequency of Y chromosomepositive hepatocytes and cholangiocytes varied but appeared higher in patients with GVHD or liver rejection. Engrafted human cells were most often scattered throughout the parenchyma as isolated cells [130]. Microchimerism for the Y chromosome can occur in the human female liver as a result of transplacental passage of male fetal blood cells during pregnancy; this was excluded as a contributing factor in a similar study by Alison et al. [131]. Both studies conclude that human BM cells can differentiate into mature hepatocytes and cholangiocytes in the absence of severe injury. Korbling et al. published that, following mobilized peripheral blood transplantation, up to 7% of epithelial cells of the liver, gastrointestinal tract, and skin appear donor in origin [132]. The frequency was low, and GVHD did not affect apparent donor cell engraftment in epithelia. Similarly, human hematopoietic progenitor cells transplanted into fetal sheep have been reported to differentiate into hematopoietic cells and hepatocytes [133]. While many studies as outlined have suggested that BM-derived cells may, at low level, contribute to liver, gut or lung tissue, and that this contribution increases in the setting of organ failure, the cell responsible is not known, and whether these phenomena occur outside the setting of cell fusion is not clear. One exception perhaps is the study by Krause et al. [80] demonstrating that contribution to lung epithelium may happen, at least in part, outside the setting of cell fusion [134]. A final tissue in which possible transdifferentiation has been suggested is the pancreas. To address the question of whether a cell within the BM can contribute to pancreatic β cells, Ianus et al. used mice transgenic for the Cre recombinase expression under the mouse Insulin2 promoter crossed into mice ubiquitously expressing the Rosa–Flox–GFP (RosaGFP) transgene (Ins2cre–RosaGFP) [109]. β cells derived from this donor mouse would hence express GFP. Irradiated WT mice were transplanted with Ins2cre–RosaGFP BM and analyzed 4–6 weeks post transplant for pancreatic engraftment. Approximately 1.7–3% of all cells within the pancreas were GFP positive, expressed characteristic β-cell markers (including Hnf3β, Pdx1, Glut2, Insulin1, and Insulin2), and secreted insulin in a glucose-responsive manner at levels similar to that of mature β cells. To exclude fusion as a mechanism, they transplanted Ins2cre BM into irradiated mice with the RosaGFP reporter. As no GFP-expressing β cells were seen, Ianus et al. concluded that the presence of GFP expressing β-cells in the first transplant model was not caused by fusion. In support of these data, Wang et al. reported that neonatal immunocompromised mice transplanted with GFP+ BM cells acquired significant engraftment in pancreatic epithelium (up to 40% with a mean of 4.6%) as well as rare GFP+Ins+ cells (0.03%) [135]. Other studies have questioned these results. Hess et al. demonstrated that BM cells transplanted into a diabetic model could reverse hyperglycemia, but that the mechanism was not direct differentiation to β cells [136]. Within the transplanted BM, the ability to reduce hyperglycemia was accounted for by a c-Kit+ subpopulation [136]. A similar study utilizing the same diabetic model revealed that BM contribution and support during regeneration was likely due to BM-derived endothelial progenitor cells (EPCs) [134]. The donor-derived endothelial cells may have facilitated the proliferation and ultimate recovery of remaining endogenous β cells.
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BM-derived cells – ectoderm Ectoderm is generated from the outer layer of the embryo and produces a surface layer of skin, the epidermis, and the central nervous system. One of the most difficult lineage boundaries to cross is the bridge between mesoderm and ectoderm, yet there are studies suggesting that this too might be possible. Eglitis and Mezey in 1997 were the first to report that BM-derived cells could contribute to, as expected, microglia (F4/80 antigen) but also (unexpected) astroglial (glial fibrillary acidic protein [GFAP]-positive) cells in the adult murine brain [5]. Sublethally irradiated female mice were transplanted with genetically marked male donor cells (enriched for HSCs using 5-flurouracil), the mice were sacrificed at intervals post transplantation, and the brain was examined using a combination of in situ hybridization histochemistry and immunohistochemistry. Doublelabeling analyses revealed that some BM-derived cells acquired glial markers. BM-derived cells in several mice were localized to the subependymal zone, an area known to be neurogenic during development, which would provide cues for glial differentiation. The type of BM cell contributing to astroglial cells was not determined. Mezey et al. transplanted WT BM cells intraperitoneally into PU.1−/− mice to rescue the animal from the typical early postnatal mortality [137]. Brains were examined at 1 and 4 months following transplantation using a combination of in situ hybridization to detect the Y chromosome and immunohistochemistry to visualize the neuronal nuclear marker, NeuN. BM-derived cells were found in the brain of all the animals examined, and the Y chromosome was present in 0.3–2.3% of the NeuN-immunoreactive nuclei. Mezey et al. concluded that BM cells can enter the brain and differentiate into cells that express neuronal markers, supporting the idea that mesodermally derived cells can adopt a neuronal (ectodermal) cell fate. In the same issue of Science, Brazelton et al. documented that when GFP-transgenic BM was grafted in lethally irradiated syngeneic mice, both NF200+ neurons and GFAP+ astrocytes could be found that were GFP+, concluding that cells from BM could generate neuroectodermal cells in vivo [138]. However, as indicated earlier, Wagers et al. were unable to find significant contribution of HSC to cells within the brain, except for rare Purkinje cells [79]. Likewise, Castro et al. could not find the same degree of transdifferentiation from BM to neuronal cells [139]. In a second series of studies, the ability of MSC to differentiate into neurons in vitro or in vivo was evaluated. Agents used to induce neural differentiation in vitro included nonspecific inducers, whereas others used cytokines and growth factors known to induce differentiation of ESCs to neuronal cells or induce differentiation of neural stem cells [140–143]. In most studies, “transdifferentiation” was shown only by the acquisition of neural morphology and the expression of a few neuronor glia-specific proteins or transcripts, which can already be detected at low level in undifferentiated MSCs [144]. A recent study has also suggested that dimethylsulfoxide induces rapid disruption of the actin cytoskeleton, leading to an apparent change in morphology of fibroblasts to cells that appear to extend neurites [145]. A few studies have assessed whether MSC-derived neuron-like cells have the functional attributes of neurons. Kohyama et al. showed that single-cell-derived immortalized BM stromal cells acquired the morphological and surface phenotype of neurons when treated with 5-azacytidine and the BMP2/4 inhibitor Noggin [146]. The resting potential of such cells of −5.0 mV and acquisition of potassium voltage-gated channels suggested the acquisition of some functional features of neurons. Wislet-Gendebien et al. co-cultured MSC-derived nestin-positive cells with cerebellar granule neurons, which led to the expression of astrocyte- and neuron-specific proteins [147]. More importantly, the resting membrane potential increased to −57 mV, at which time the cells also
acquired functional sodium voltage-gated channels, a phenotype similar to that of cells at an intermediate phase of neural development. The fact that MSCs co-cultured with paraformaldehyde-fixed cerebellar granule neurons yielded similar results suggests directed differentiation and not cell fusion. Dezawa et al. expressed the intracellular domain of Notch in MSC [148]. This led to the expression of early neural transcripts, and upon addition of forskolin and basic fibroblast growth factor, more than 95% of MSCs expressed markers for postmitotic neurons, and 50% of the cells acquired outward rectified potassium ion currents, but no voltagegated fast sodium currents. Following exposure to ciliary neurotrophic factor, 40% of the cells expressed genes found in dopaminergic neurons and secreted dopamine following depolarization. When grafted in 6hydroxydopamine treated animals, rotational behavior improved, as is seen with fetal midbrain transplantations. These studies suggest therefore that, under certain conditions, MSCs may acquire not only phenotypic, but also functional characteristics of neuroectodermal cells. Several groups have grafted rodent or human MSCs in vivo in intact or injured brain [149,150]. Although some authors have stated that MSCs transdifferentiate into neuronal cells, these conclusions have in general been based solely on the identification of neuron- (nestin) or glia- (GFAP) specific markers in rare donor cells. Moreover, in many studies, donor origin was determined by evaluation of BrdU or 3Hthymidine incorporated in the donor cells prior to grafting. As discussed earlier, recent studies have shown that such labels, previously thought to be superior over, for instance, membrane dyes, can be transferred from dying grafted cells to endogenously proliferating cells such as astroglia or neural stem/progenitor cells [95]. In addition, no study has excluded conclusively that the presumed neural differentiation is not caused by fusion between MSCs and host brain cells. Finally, no study has demonstrated that presumed MSC-derived neurons are functional. Nevertheless, as is true for grafting of BM cells in the heart, grafting MSCs in the brain may have beneficial effects by secreting trophic factors that enhance vascularization, recruit endogenous progenitors, and/or induce mature neural cell plasticity [151–154]. Endothelial cells in the BM Another cell type present in BM is the endothelial cell. HSCs proliferate and differentiate, and their progeny migrate into the blood circulation lined by endothelium. The endothelium forms the vascular niche within the BM, and at least some HSCs lie in close proximity to it [155]. The vascular niche is thought to promote HSC proliferation and differentiation, whereas the osteoblastic niche may favor quiescence [156]. Explanted endothelial cells can maintain HSCs in culture [157]. Its location at the interface between the BM and the circulation allows the endothelium to regulate transendothelial migration, homing, and mobilization of HSCs [84,158]. It has also been hypothesized that endothelial cells outside the BM medullary cavity may provide a back-up niche when the BM is stressed [159]. The boundaries separating the hematopoietic and vascular developmental programs are somewhat indistinct. HSCs are an accepted entity with a defined hierarchy based on proliferative potential. There remains controversy over the definition of an endothelial progenitor, and a similar organization for endothelial precursors, while postulated, is not proven. There is, at least during development and using in vitro embryonic stem cultures, evidence of a common endothelial and hematopoietic progenitor called a “hemangioblast.” This concept was proposed over 80 years ago when Florence Sabin observed the formation of blood and blood vessels from primitive mesoderm in chick blastoderm [160]. The term “hemangioblast” was coined by P.D.F. Murray in 1932, who noted that cells from the primitive streak of chick embryos formed simultane-
Cellular Biology of Hematopoiesis
ously both endothelial cells and blood islands [161]. Hematopoietic and endothelial cells develop in close proximity within the mammalian yolk sac [162,163], and in the embryo proper, current information suggests the presence of “hemogenic” endothelium in the dorsal aorta as a source of definitive HSCs [164–169]. Investigations in humans have shown similar results [170]. Using murine ESCs, several investigators have isolated hemangioblasts in vitro, which has allowed more extensive evaluation than cells from the embryo proper. ESCs generate embryoid bodies that can be induced to differentiate into hematopoietic lineage [171]. They generate a transient precursor with genes in common with both the hematopoietic and endothelial cell lineages. These transient precursors, referred to as the blast colony-forming cell population, may be an in vitro equivalent of a hemangioblast [168]. Whether circulating endothelial cells originate as outgrowths from vascular endothelium or reside in the circulation or BM is not clear [169]. Whatever the origin (reviewed more extensively in [172]), the endothelium has an intimate relationship with hematopoiesis throughout development. It has been difficult to study this association as EPCs share many phenotypic and functional characteristics with HSCs: they exist in the same fluid microenvironment and respond to the same stimuli. EPCs express KDR (VEGFR1), CD31, and Tie-2 (angiopoietin-1 receptor), ingest acetylated low-density lipoprotein, and bind lectins (a feature of most macrophages). That EPCs are present in BM comes from murine
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and human BM transplantation studies demonstrating that at least some endothelial cells are of donor origin [173]. Whether such endothelial cells could be derived from HSCs rather than EPCs has not been fully addressed. Grant et al. showed that, following transplantation of a single long-term repopulating HSC, perfusable endothelial cells in the retina of a mouse developed following vascular injury [174]. However, recent studies have demonstrated that, aside from EPCs that generate selfrenewing colonies of endothelial cells in vitro, HSC-derived macrophages can also generate cells with features of endothelial cells, such as expression of CD31, von Willebrand factor, and ability to take up acetylated low-density lipoprotein [175]. The difference between the two endothelial cell-like cell progenitors is the time after which they start generating colonies in in vitro cultures, the ability to replate the colonies, and the ability to generate perfused vessels in vivo. The fact that the progeny of HSCs can acquire features of endothelial cells makes evaluation of the possible lineage relationship of HSCs and EPCs in in vivo studies as well as in vitro studies more difficult.
Persistence of more pluripotent adult stem cells? Adult pluripotent stem cells (Table 7.2) There are an increasing number of studies suggesting that cells with greater potency than classical tissue-specific stem cells can be isolated
Table 7.2 Adult pluripotent stem cells isolated from different murine and human sources, with their phenotype and “plasticity” towards alternative dermal lineages Name
Source
Phenotype
Differentiation capacity
MAPC [90]
Murine BM
Endothelium, hepatocytes, neuron-like cells
MIAMI cell [186]
Human BM
hBMSC [97]
Human BM
MASC [93]
Human BM, liver, heart
SSEA-1 cell [91]
Murine BM
VSEL cell [92]
Murine BM
USSCs [187]
Human umbilical cord blood
AFS [188]
Human amniotic fluid
maGSCs [189]
Murine testis
Oct4+, Rex1+ Thy-1.1lo, Sca-1+lo, Flk-1+lo, SSEA-1+, CD13+ CD34−, CD44−, CD45−, CD117−, MHC-I−MHC-II− Oct3a+ Rex-1+ Thy-1+, CD10+, CD29+, CD44+, CD49e+, CD103+ CD34−, CD36−, CD45−, CD54−, CD56−, CD117−, HLA-II− Oct3/4− Thy-1lo, CD105lo, Cd117lo CD29−, CD44−, CD73− Oct4+, Rex1+, Nanog+ Thy-1+, CD13+, CD49b+, CD73+, CD44+, HLA−I+, CD29+, CD105+, Flk-1+, CD49a+ CD14−, CD45−, CD38−, HLA-II−, CD133−, CD117−, CD34− Oct4+, Rex-1+, Nanog+, SSEA-1+ Sca-1+,Thy-1+ CD73+, CD105+, CD44+, CD34−, CD45− Oct4+, Rex1+, Nanog+, SSEA-1+ Sca-1+ CD45− Thy-1+, CD105+,CD13+,CD29+, CD44+, CD49e+ Class-II−, CD34−, CD45−, CD117−, CD33− Oct3/4+,SSEA-4+ Thy-1+, CD44+, CD73+, CD117+, Class-IIlo CD34−, CD45− Oct4+, Rex-1+, Nanog+, SSEA-1+ Thy-1+, Sca-1+lo, CD117+lo, Ter119−, CD34−
Osteoblasts, chondrocytes, adipocytes, pancreatic islets, neural cells
Cardiomyocytes, endothelium, smooth muscle
Osteoblasts, myocytes, endothelium, hepatocytes, neurons, glia
Osteoblasts, chrondrocytes, adipocytes, hematopoietic cells, endothelium, hepatocytes, astrocytes Cardiomyocytes, pancreas, neural cells
Osteoblasts, chrondrocytes, adipocytes, hematopoietic cells, neural cells Osteoblasts, hepatocytes, neural cells
Cardiomyocytes, smooth muscle, endothelium, hepatocyte-like cells, neurons
AFS, amniotic fluid-derived stem cell; BM, bone marrow; hBMSC, human BM-derived multipotent stem cell; maGSC, multipotent adult germline stem cell; MAPC, multipotent adult progenitor cell; MASC, multipotent adult stem cell; MIAMI, marrow-isolated adult multilineage inducible cell; SSEA-1, stage-specific embryonic antigen-1; USSC, unrestricted somatic stem cell; VSEL, very small embryonic-like cell.
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Chapter 7
from BM, UCB, and other tissues. The first report was in 2002, when multipotent adult progenitor cells (MAPCs) were described. MAPCs are isolated from adherent BM cultures under conditions that may not support MSCs, specifically low concentrations of fetal bovine serum (FBS) (2%). Interestingly, isolation of MAPCs from rat and mouse BM requires leukemia inhibitory factor, a cytokine that is also a prerequisite for the isolation of murine ESCs [90]. MAPCs isolated from rodents, swine, and human BM are a clonal population of cells that can be expanded ex vivo without signs of cell senescence. They express the ESC transcription factor Oct4, but not appreciable levels of Nanog or Sox2. By FACS, human MAPCs are characterized as CD13−CD34− CD45−HLA-II−, Thy-1+, and CD45loHLA-Ilo. Under specific in vitro differentiation conditions, they can differentiate into typical mesenchymal cell types as well as endothelium, hepatocyte-like endodermal cells, and neuron and glia-like neuroectodermal cells [90,176–184]. Differentiated cell types including smooth muscle cells, osteoblasts, endothelium (arterial or venous specified endothelium), hepatocyte-like cells, and neuroectodermal-like cells were characterized based on morphology, phenotype, expressed protein, and gene profile, as well as extensive functional characterization. When transplanted in vivo, murine MAPCs can regenerate a complete lymphohematopoietic system [90,185], and some cell lines also contribute to endodermal tissues such as liver, gastrointestinal, and pulmonary endoderm [90]. Undifferentiated or endothelially committed MAPCs also contribute to neovascularization in tumors, or in Matrigel assays [178,183]. Some, but definitely not all, cell lines also contributed in variable degrees to multiple somatic tissues when injected in the blastocyst, although germline transmission has not been observed [90]. In 2004, D’Ippolito et al. described marrow-isolated adult multilineage inducible (MIAMI) cells [186]. These cells are derived from human BM from vertebral bodies by plating on fibronectin at 3% oxygen and 15% FBS for the first 2 weeks; FBS is then decreased to 2%. MIAMI cells, like MAPCs, express Oct3a and Rex1, and can be expanded for 30–50 cell doublings without evidence of senescence or loss of differentiation capacity. By FACS, MIAMI cells are negative for CD34, CD36, CD45, CD117 and HLA-II. In vitro, MIAMI cells can be induced to differentiate to mesodermal cell types as well as cells with phenotypic features of neuroectodermal-like cells and endodermal features (transcripts of pancreatic endoderm), although no functional studies have been done to prove differentiation to ectoderm and endoderm. Yoon et al. purified cells from human BM that could be expanded from single cells for greater than 140 population doublings without senescence and loss of differentiation, named human BM-derived multipotent stem cells (hBMSCs) [97]. By FACS, hBMSCs are negative for CD29, CD44, and CD73, and express low levels of CD105, CD90, and CD117, all surface markers used to characterize mesenchymal cells. In contrast to most cells with apparent greater potency, BMSCs do not express Oct4. They can differentiate into cells with phenotypic features of endothelium, smooth muscle, neural cells, and hepatocytes. However, the investigators did not evaluate the functional properties of the lineage differentiated progeny. When co-cultured with cardiomyocytes in vitro, cells appeared to differentiate into cardiomyocytes, although fusion is, at least in part, responsible for this observation. When hBMSCs labeled with the dye DiL were given as an intracardiac graft in an infarcted heart, multilineage differentiation (endothelium, smooth muscle and cardiac muscle) was seen, which resulted in favorable remodeling and improved cardiac function. Whether the functional improvement was due to apparent transdifferentiation versus trophic factors secreted by BMSCs, as has been seen for hematopoietic cells and MSCs, is not clear. As discussed earlier, the use of DiL for in vivo tracking is not ideal. The authors also did not conclusively demonstrate that the resulting differentiation was
cell autonomous versus fusion between hBMSCs and resident cells in the heart. Multipotent adult stem cells (MASCs) were isolated from adult human BM, liver or heart tissue [93]. These cells were cultured in Mesencult (a medium optimized for human MSC cultures) and subsequently on fibronectin-coated dishes and above stromal cells with 2% FBS. The cell-surface phenotype is that of MSCs, but the cells express Oct4, Rex1, and Nanog transcripts, as well as Oct4 and Nanog protein. Clonally isolated cells differentiate into cells with phenotypic and functional features of osteoblasts, myocytes, endothelial cells, hepatocytes, neurons and glia. Unrestricted somatic stem cells, isolated from human UCBs, are CD45−, HLA class II-negative mononuclear cells with fibroblastic morphology and extended telomere length that can be expanded for 30–50 population doublings [187]. The cells express Oct4 and Rex1 and, in vitro/in vivo, demonstrate differentiation potential to osteoblasts, chondroblasts, adipocytes, and hematopoietic, neural and hepatic cells. As is true for many studies, limited proof exists for acquisition of functional features of the unexpected endodermal and ectodermal cell types. Similarly potent cells have been found in amniotic fluid [188]. In addition, Guan et al. demonstrated that cells with pluripotent features, including teratoma formation and contribution to somatic and germline cells when injected in the blastocyst, could be generated from murine testicular tissue [189]. The latter cells are therefore the only ones that have capabilities similar to those of ESCs. One question that has not been answered is whether the cell populations described above (MAPCs, MIAMI cells, BSSCs, and MASCs) exist in vivo or are created in culture as the result of dedifferentiation. Two recent studies may start to shed light on this. AnjosAfonso and Bonnet have shown that cells expressing stage-specific embryonic antigen-1 (SSEA-1), found on mouse ESCs, can be used to isolate cells from MSC cultures following one passage in vitro [91]. These cells express high levels of Oct4 as well as Nanog and Sox2, and can differentiate following expansion into cells with phenotypic and in vitro functional characteristics of multiple mesodermal cell types, including endothelium, as well as endodermal hepatocyte-like cells and ectodermal lineage cells such as astrocytes. When grafted in the tibia in vivo, pre-MSCs contribute to endothelium, osteoblasts, adipocytes, chrondrocytes, and hematopoietic cells. They further demonstrated that when SSEA-1− cells are isolated that do not express Oct4 and Nanog, these cells cannot be induced to express SSEA-1 or the ESC transcription factors. Kucia et al. recently demonstrated that a homogeneous population of Sca-1+Lin−CD45−CXCR4+ cells can be selected directly from murine BM or human UCBs that express, like the cells identified by AnjosAfonso and like ESCs, SSEA-1, Oct4, Nanog and Rex-1, or SSEA-4 in human UCBs [92,190]. These cells are difficult to culture ex vivo, but by performing co-cultures, phenotypic changes were seen consistent with differentiation to endothelium and possibly pancreatic β cells. No functional confirmation of such differentiation was demonstrated, nor was fusion excluded. The latter two studies suggest that rare cells exist in murine BM with phenotypic features of MAPCs, MIAMI cells, hBMSCs, unrestricted somatic stem cells, and MASCs. It is unknown whether the differentiation ability ascribed to MAPCs, MIAMI cells, hBMSCs, unrestricted somatic stem cells, MASCs, pre-MSCs, multipotent adult germline stem cells or amniotic fluid-derived stem cells is already present in the freshly isolated cells, and hence these cells represent cells with greater potency persisting in vivo into postnatal life, or whether the multilineage differentiation ability is acquired once cells are cultured in vitro, and therefore represent the dedifferentiation of rare Oct4+ cells.
Cellular Biology of Hematopoiesis
Conclusions and future directions This chapter has summarized the significant progress that has been made in defining the properties of stem cells within BM, including HSCs, MSCs, and endothelial progenitor cells. There are continuing advances in understanding the unique biologic properties of these cells and their progeny. This information is leading to increasing numbers of clinical applications for these cells, not only with HSCs, but trials are being initiated with MSCs and EPCs as well. New information highlighting the importance of the BM microenvironment demonstrates that the beneficial effect of these cells extends beyond their direct contribution to the host tissue. The trophic effects of MSCs can be harnessed to increase the potential of existing pharmacologic therapies, especially in ischemic disorders. The immunomodulatory characteristics of MSCs are also being tested in the setting of GVHD, graft rejection, and other immune-mediated disorders. The limitations and potential toxicity of these treatments should, however, be carefully assessed in animal models and clinical trials as translation to humans may reveal unsuspected complications. Many studies have suggested that BM-derived stem cells may differentiate into not only cells of the tissue of origin, but also cells of other
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tissues, a process known as stem cell plasticity. The evidence for transdifferentiation is often confusing, with some studies providing evidence of phenotypic switch and others failing to demonstrate such phenomena. There may be technical reasons contributing to the differences between studies. In addition, the mechanisms underlying the apparent lineage switch are not always clear. Undoubtedly, fusion occurs, especially between cells that are more “fusigenic” by nature. Aside from fusion, there is mounting evidence for rare events of cell intrinsic lineage switch. Studies aimed at understanding the conditions that allow such transdifferentiation may ultimately yield optimization of local signals to induce such fate change. There is also more and more evidence that cells can be cultivated that have (or acquire) greater potency. Further characterization of the potential of these cells to be used for treatment of not only hematopoietic disorders, but also diseases of the central nervous system and liver, among others, may demonstrate that BM cells could be used outside hematologic diseases. Stem cell potential as we know it presently is in its infancy. The huge strides that have been made since the pioneering use of HSCs in human HCT in the 1960s are small steps along the way to fulfilling the possibly much broader, but latent potential of BM-derived cells.
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8
Colleen Delaney & Irwin Bernstein
Expansion of Hematopoietic Stem Cells
Introduction Hematopoietic stem cells (HSCs) are useful in a variety of clinical settings and are routinely available in sufficient numbers from a variety of sources, including bone marrow and mobilized peripheral blood. However, there are instances when the number of available HSCs is insufficient for adequate and/or timely engraftment of the host, in particular when cord blood is used as the source of HSCs for transplantation or in the case of gene therapy, where the frequency of transduced cells is too low. One potential solution to the problem of low stem cell numbers is ex vivo proliferation of the cells prior to transplantation. Extensive research has been done to define the optimal conditions necessary for ex vivo expansion of HSCs, and various expansion techniques have been developed for this purpose [1–7]. However, while there is still a lack of convincing data that pluripotent stem cells increase in number and retain an ability to engraft and sustain multilineage hematopoiesis in a myeloablated recipient following ex vivo expansion, more recent advances in the field, particularly with respect to molecular mechanisms that control HSC amplification, make this goal more realistic. In order to maintain hematopoietic function throughout the life of the organism, pluripotent HSCs are thought to undergo self-renewal, where at least one, possibly identical, progeny remains undifferentiated and capable of long-term, multilineage hematopoietic repopulation, thus preserving the stem cell pool [8]. The differentiated progeny of longterm repopulating cells are thought to include pluripotent, short-term repopulating cells, which in turn generate precursors committed to lymphoid or myeloid differentiation [9,10]. Whereas maintenance of the HSC pool in vivo requires asymmetric division in which only one of the progeny maintains stem cell properties, HSC expansion requires symmetric divisions giving rise to two HSCs (Fig. 8.1). The ability of HSCs to undergo symmetric divisions and expand in numbers has been demonstrated in transplantation studies. However, serial transplant studies have also demonstrated that HSC life span is limited and self-renewal potential diminishes with the age of the animal [11–13]. While several recent studies challenge our concepts of HSC senescence [14–16], the central tenet remains that, in the absence of transforming events, HSCs have finite, though very lengthy, life spans in vivo [17–19]. These “aging” or senescence processes appear to be accelerated in ex vivo
cultures [20]. Nevertheless, studies suggest that HSC progeny formed after several divisions retain the ability to engraft recipients long term, but may actually be somewhat more differentiated with less proliferative capacity that is detectable only under stress conditions. Thus, cells with HSC properties may themselves be heterogeneous, and their self-renewal capacity may depend on the source from which they are derived, with fetal liver HSCs having greater self-renewal capacity than cord blood HSCs, which in turn have greater self-renewal capacity than cells from adult bone marrow [16,21–23]. Substantial effort has focused on the exogenous signals that may be used to favor stem cell self-renewal versus differentiation in order to develop optimal conditions for the ex vivo expansion of stem cells. Most studies have evaluated using combinations of cytokines and/or bone marrow stroma and have met with limited success (reviewed in [3,6,24,25]). The effect of cytokines that support hematopoietic cell survival, proliferation, and differentiation has been extensively studied in vitro, but a significant role for these cytokines in enhancing selfrenewal has not been shown. Consequently, a stochastic model of cellular determination has been suggested in which the fate of hematopoietic precursors is not instructed by soluble cytokines [26,27], but rather by specific interactions between stem and other cells within a particular microenvironment or “stem cell niche.” These interactions are mediated by extrinsic regulators of stem cell fate and are likely to play a key role maintaining numbers of stem cells by regulating their self-renewal and differentiation, now demonstrated by the work of several groups [28–30]. Thus, more recent studies are aimed at identifying intrinsic and extrinsic factors that regulate HSC fate. Clinical trials, which have also mainly evaluated cytokine-driven expansion systems, have not yet provided evidence for stem cell expansion, but have demonstrated the feasibility and safety of ex vivo culturing of stem cells. However, a newer generation of clinical trials, as discussed below, is currently underway evaluating the use of extrinsic regulators of stem cell fate (Notch) and co-culture systems utilizing nonhematopoietic components (mesenchymal stromal cells) of the stem cell niche. Here, we address studies of ex vivo HSC expansion in animals and humans, as well as promising approaches under development.
Preclinical studies: growth factor and stroma-induced expansion Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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In this section, we review work in mice and large-animal models designed to address the ex vivo expansion of HSCs. We will focus on studies that have used in vivo repopulating assays to evaluate stem cell
Expansion of Hematopoietic Stem Cells
HSC
HSC HSC
HSC STR
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Symmetric division
STR Asymmetric division
Fig. 8.1 Maintenance versus expansion of hematopoietic stem cell (HSC) numbers. Symmetric divisions will expand stem cell numbers, whereas asymmetric divisions maintain numbers. The number of HSCs generated may also be affected by cell loss due to apoptosis and, for transplantable stem cells, loss of homing properties. STR, short-term repopulating cell.
expansion as in vitro assays have not always proven informative of HSC function. We then briefly review factors that regulate HSC fate in ex vivo expansion cultures. Murine studies The discovery of transplantable HSCs in long-term cultures of mouse marrow cells (LTMCs) initially described by Dexter [31–33] suggested that HSCs might possibly expand in ex vivo cultures. The HSCs in these LTMCs were dependent on the stromal cells for survival, and their cell cycle status was influenced both positively and negatively by growth factors [34,35], as well as by cellular and noncellular components of the stromal cell layers [36]. However, it was unclear whether HSCs in LTMCs divided or simply remained quiescent. Subsequent studies using gene marking to follow the progeny of individual HSCs demonstrated that HSCs were able to undergo several divisions and still maintain HSC function measured in vivo [37,38]. Evidence for a limited number of in vitro divisions before loss of HSC function also came from studies demonstrating that the progeny of isolated HSCs, formed after one to three in vitro divisions, maintained in vivo repopulating function [39]. However, none of these and other studies [40] demonstrated net gains of HSCs number in these cultures, indicating that divisions were mainly asymmetric. HSC cultured for purposes of ex vivo expansion are altered in obvious as well as subtle ways, including the expression of various cell surface antigens such as receptors that function in cell adhesion and motility. Research is ongoing to better define HSC markers on freshly isolated and subsequently cultured HSCs [41]. Moreover, the impact of stem cell death, loss of homing properties [42,43], and change of cell-cycle status [44–46] that occur during in vitro culture of HSCs and how these influence in vivo repopulating ability have not yet been fully elucidated. For example, both mouse HSCs and human hematopoietic precursors appear to have diminished repopulating function if transplanted during the G1 and S phases of the cell cycle rather than in G0 phase. The combination of cytokines is also critical, with specific hematopoietic growth factors involved in promoting or inhibiting HSC function during ex vivo expansion. Elegant single-cell manipulation studies have demonstrated that interleukin-3 (IL-3) and IL-1 can abrogate the selfrenewal of murine HSCs in vitro [47], in contrast to stem cell factor
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(SCF) and Flt3 ligand (Flt3L), which maintain HSCs [48]. Further, the addition of exogenous thrombopoietin (TPO) to murine LTMCs led to an expansion of marrow repopulating cells [49]. Members of the transforming growth factor (TGF) family and cooperating factors have been demonstrated to inhibit the cell-cycle progression of HSCs in vitro [50–52]. The type of culture medium used also can influence the ex vivo expansion of HSCs. Whereas numerous studies using serum-containing medium have shown maintenance or loss of HSC activity following culture with cytokines, more recent studies using serum-free medium, which allows for a more careful dissection of the interaction of HSCs with defined ligands and avoids the introduction of inhibitory molecules such as TGF-β, have demonstrated enhanced repopulating ability, with some studies documenting up to an eightfold increase of murine marrow HSCs [41].
Large-animal studies To date, human trials of gene therapy and HSC manipulation have not been nearly as successful as experiments in mice might have predicted, and important differences in HSC behavior in vivo in large animals are beginning to be appreciated [53–55]. Thus, studies of stem cell expansion using clinically feasible procedures in larger animals to better mimic human physiology that are now being carried out may provide important insights needed for human clinical application. Thus far, the use of ex vivo cultured cells has mainly led to an enhanced rate of engraftment, but this may have occurred at the expense of long-term repopulating cells. The possibility of enhancing short-term repopulating activity by ex vivo expansion was suggested by two studies of lethally irradiated baboons engrafted with ex vivo, expanded CD34enriched peripheral blood progenitor cells (PBPCs) collected after granulocyte colony-simulating factor (G-CSF) mobilization [56–58]. Significant expansion of precursor cells with in vitro colony-forming activities was documented in both studies, and in one study severe combined immunodeficiency disease (SCID) mouse repopulating cells were also expanded twofold. Animals transplanted with ex vivo expanded cells experienced significantly shorter periods of post-transplant neutropenia, but the recovery of platelet and red cell production was significantly slower, and administration of TPO had no effect on platelet recovery. Although these studies were not designed to assess effects on long-term reconstituting stem cells, the possibility that cytokine-driven expansion may deplete long-term repopulating HSCs in primate models has been suggested by gene-marking studies, in which prolonging the period of in vitro culture worsened rather than improved gene transfer efficiency [59,60]. An increase in the number of precursors at the expense of in vivo marrow reconstituting function has also been observed in cats following ex vivo expansion of marrow cells [61]. In dogs, HSCs and progenitors have been cultured ex vivo for brief periods to promote gene transfer into marrow repopulating cells. The detection of gene-marked progeny in vivo suggests that at least a limited number of cells with HSC function divided in the ex vivo culture [62].
Xenogeneic animal models: transplantation of human cells Immunodeficient mouse xenogeneic transplant models In preclinical studies, xenogeneic transplant models have been developed in which human hematopoietic cells can engraft in immunodeficient mice and then be serially passaged in vivo [21,22,63–69]. These engrafting cells are referred to as SCID repopulating cells (SRCs). At
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present, the relationship of SRCs to HSCs in humans remains to be clarified, but the SRC is generally accepted as the most reliable surrogate marker of an HSC currently available. Nevertheless, human SRC expansion has been assayed in various immune-deficient mouse strains. The necessity for in vivo assessment of ex vivo HSC expansion was emphasized by several studies in which ex vivo cultures of CD34+ cells have resulted in the dramatic increase in numbers of colony-forming cell (CFC) or even long-term culture-initiating cell activity, but with a concomitant decrease in SRCs [70–73]. In contrast, several other studies have suggested a modest increase in engraftment of immunodeficient mice following short-term culture, in which cells were cultured with multiple cytokines or in the presence of stromal cells or stromal cellconditioned medium [6,74–80]. Quantitative studies using limiting dilution SRC assays have confirmed modest two- to fourfold cytokine-induced increases in SRCs. For example, Bhatia et al. and Conneally et al. have cultured CD34+ CD38− cord blood cells in serum-free medium supplemented with SCF, Flt3L, G-CSF, IL-3, and IL-6 and showed expansion of SRCs [65,74]. The study by Bhatia et al., in which cells were cultured for 4 days, found a fourfold increase in CD34+ CD38− cord blood cells, a 10-fold increase in CFCs, and a two- to fourfold increase in SRCs [74] . However, all SRCs were lost after 9 days of culture, despite continued expansion in the total number of cells throughout the culture period. In a similar study, Conneally et al. confirmed that this culture system produced significant increases in CFCs (100-fold) and long-term culture-initiating cells (fourfold), as well as a modest twofold increase in SRCs. Similarly, studies using an IL-6/IL-6-receptor chimera induced significantly higher levels of CD34+CD38− cells and a fourfold increase in SRCs by limiting dilution methods [79,81–84]. Cytokine-induced expansion systems have also utilized several cloned stromal feeder cells and mesenchymal stem cell layers as sources of growth factors, and have been shown to support long-term repopulating cells [85,86]. However, because the cultured cells are in contact with the stromal feeder cells in this type of culture system, it is less advantageous to use this system for clinical application, and noncontact stromalbased culture systems are also being explored as HSC expansion systems [4,87–91]. An example of this type of expansion system was reported by Shih and colleagues [89], in which a murine bone marrow-derived stromal cell line, AC6.21, treated with leukemia inhibitory factor, secreted a factor that resulted in expansion of human HSCs when the HSCs were cultured with the conditioned medium. The expansion occurred in the absence of the stromal cells, and the magnitude of expansion was dependent on the level of activity of the secreted factor. In another example, Lewis et al. cultured umbilical cord blood (UCB) CD34+ with IL-7, SCF, Flt3L, and TPO in a Transwell (where stromal cells are separated from CD34+ cells by a transmembrane) above the murine fetal liver cell stromal feeder line, AFT024, for 7 days and demonstrated the maintenance of long-term repopulating cells in culture using secondary transplants into NOD/SCID mice, as well as secondary and tertiary transplants into fetal sheep [92]. Additionally, the use of conditioned medium from the HS-5 cell line, an immortalized stromal cell line derived from human marrow after transduction with a replication-defective retrovirus containing the human papilloma virus E6/E7 gene, has been shown to support the proliferation of hematopoietic progenitors and maintain CFCs for up to 8 weeks in culture [88,93]. While the above studies documented the expansion of SRCs in shortterm culture conditions only, Piacibello et al. [94] reported maintenance of cord blood CD34+ cell growth for up to 12 weeks in serum-containing medium supplemented with Flt3L, SCF, and IL-6. Additionally, limiting dilution transplants demonstrated a more than 70-fold expansion of SRCs. Although it is not clear whether this approach will be reproducible, Tanavde et al. recently reported the ability to expand and maintain
UCB SRCs for up to 4 weeks [95] using CD34+ from UCB and adult mobilized PBPCs cultured in serum-free medium supplemented with Flt3L, SCF, and TPO with or without IL-6/IL-6-receptor chimera. UCB cells demonstrated extensive in vitro expansion and maintained repopulating ability throughout the 4-week culture period, whereas mobilized PBPCs showed less expansion and no engrafting potential of cultured cells (only fresh cells being engrafted). While the ability to maintain and expand repopulating cells in vitro for extended periods would be beneficial for gene therapy, augmentation of clinical transplantation studies with expanded cells will require shorter expansion times, as time to transplant is critical in the clinical setting. Moreover, there is always the concern that long-term culture of cells may in fact induce transformation of the cultured cells. Fetal sheep xenogeneic transplant model When primitive hematopoietic cells isolated from both cord blood and adult bone marrow are expanded ex vivo and transplanted into fetal sheep, only short-term engraftment of the animals is seen. In their initial study attempting ex vivo expansion of enriched adult human bone marrow cells with in vivo engrafting capabilities, Shimizu and colleagues were able to document the maintenance of cells with engrafting capabilities in fetal sheep for up to 2 months when cultured with KIT ligand, Flt3L, IL-6, and erythropoietin with or without IL-3 [96]. Engraftment was noted in secondary recipients of fresh human cells, but not in recipients of cultured cells, suggesting that the culture conditions used were unable to support or expand long-term engrafting cells. Similar results were demonstrated by McNiece et al. using a two-step culture procedure in which cord blood progenitors cultured in the presence of SCF, megakaryocyte growth and development factor, and GCSF for 7 or 14 days prior to transplant provided no long-term engraftment of fetal sheep [97]. While the expanded cells were capable of early rapid engraftment (threefold over fresh cells), they lacked secondary and tertiary engrafting potential compared with fresh cells, again suggesting that the culture conditions described resulted in the loss of long-term repopulating cells and led to an increase of more mature shortterm repopulating cells. Overall, preclinical studies of human HSC expansion assayed based on repopulating ability in xenogeneic transplant models show promise but must be interpreted with caution, as human NOD/SCID or fetal sheep repopulating ability has not been correlated with non-human primate or human in vivo repopulating ability. In fact, studies that compared repopulation by gene-marked cells from non-human primates in autologous recipients and NOD/SCID mice revealed substantially greater repopulation in mice [98]. Although this finding might reflect detection in mice of a more mature precursor, such as a short-term repopulating cell, other studies have demonstrated serial repopulation by expanded human cells in murine recipients, a function consistent with self-renewing stem cells. Further, while these studies have shown in vitro expansion of SRCs, the results are highly variable, and include numerous reports of loss of stem cell activity after ex vivo culture [73,97,99–101]. Finally, while studies of cytokine-induced expansion suggest the potential for enhancement of short- and long-term repopulating activity, only a few cell divisions at most have been observed. Thus, current efforts are now focused on identifying and exploiting other extrinsic and intrinsic regulators of stem cell fate. Novel approaches for ex vivo HSC expansion using extrinsic and intrinsic regulators of stem cell fate Optimization of cytokine-driven expansion systems has not led to clinically significant HSC expansion, perhaps due to a predominantly permissive, rather than directed, role in determining stem cell fate. The
Expansion of Hematopoietic Stem Cells
search for extrinsic and intrinsic regulators that act directly on human HSCs in regulating cell fate and self-renewal has suggested a role for regulatory molecules active in early development that are important in HSC maintenance and regulation. More recent studies have focused on the regulation of intrinsic signaling pathways by retrovirus-mediated transduction of HSCs, for example expression of homeobox genes. However, culture of cells for clinical application requires the use of extrinsic regulators of cell development including bone morphogenic proteins (BMPs), angiopoietin-like proteins, and Sonic hedgehog (Shh), Wnt, and Notch ligands. Retrovirally mediated overexpression of Hox transcription factors, in particular HoxB4, has led to extensive ex vivo HSC expansion in vitro (3 logs over control cultures) without loss of full in vivo lymphomyeloid repopulating ability [102–105]. Although methods to alter homeobox gene expression in the absence of transducing cells are not available, the self-renewal induced by HoxB4 has suggested the exploration of extrinsic regulators of cell fate involved in embryonic development, such as BMP-4, a member of the TGF-β superfamily, and Shh of the Hedgehog family of proteins, both having been implicated in early hematopoietic development [106,107]. In ex vivo expansion of human cord blood, soluble human BMP-4 has been shown to increase the survival of repopulating blood cells in ex vivo culture [106], while Shh has been shown to induce a few-fold increase in repopulating cells via mechanisms that are dependent on downstream BMP signals [108]. Wnt proteins, which are involved in the growth and differentiation of a variety of primitive tissues, have also been implicated in the regulation of hematopoiesis, possibly exerting their effects through stromal cells [109], and have been shown to stimulate the proliferation of hematopoietic precursor cells [110]. There are also data supporting the presence of an interactive relationship between the various signaling pathways. For example, Notch-1 receptors have been shown to be upregulated in response to Wnt signaling in HSCs [111]. The most extensively studied and successfully utilized extrinsic regulators have been ligands that activate the Notch pathway, with growing data supporting a role of Notch signaling in maintenance and/or self-renewal of HSCs. All four Notch receptors (Notch-1, -2, -3, and -4) identified in vertebrates have been detected in hematopoietic cells [112], and several investigators have reported the expression of Notch-1 and Notch-2 in human CD34+ or CD34+ lineage-negative (Lin−) precursors [112–114], and of the Notch ligands Delta-1 and Jagged-1 in human bone marrow stromal cells and human hematopoietic precursors [115– 118] . Moreover, expression of a constitutively active, truncated form of Notch-1 in murine hematopoietic precursors inhibited differentiation and enhanced self-renewal, leading to the establishment of an immortal cell line that phenotypically resembles primitive hematopoietic precursors [119]. This cell line, depending upon the cytokine context, can differentiate along the lymphoid or myeloid lineage. More recently, expression of constitutively active Notch in murine precursors transplanted in vivo led to an increased stem cell number that was evident in secondary transplantation studies [120]. While these findings point to a role for Notch signaling in the regulation of stem cell self-renewal, expression of activated Notch-1 in human CD34+ cord blood precursors induced only a modest increase in the number of progenitors [121]. In order to affect nontransduced cells, exogenous Notch ligands have been used to induce endogenous Notch signaling in HSCs. Initial studies in mice and humans using soluble or cell-bound Notch ligand revealed limited increases in precursor cell numbers [116,117,121]. However, Varnum-Finney et al. and others [122,123] have demonstrated a requirement for ligand immobilization to induce Notch signaling. Studies performed with hematopoietic precursors cultured in the presence of immobilized Delta-1 demonstrated profound effects on the differentiation of isolated murine marrow precursors, with a multi-log increase in
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the number of stem cell antigen (Sca-1)+ Gr-1− cells with short-term lymphoid and myeloid repopulating ability [124]. Similarly, studies with human cord blood CD34+CD38− precursors cultured in serum-free conditions with immobilized ligand and cytokines resulted in an approximately 100–200-fold increase in the number of CD34+ cells generated compared with control cultures, and included cells capable of repopulating immunodeficient mice [125,126]. Moreover, more recent studies with both murine and human hematopoietic progenitors have demonstrated density-dependent effects of Delta-1 on fate decisions of hematopoietic progenitors, and suggest that optimal ligand densities are required to promote expansion of cells that retain repopulating ability [125,127]. Other investigators have shown similar outcomes. Kertesz et al. demonstrated that culture of murine Lin− hematopoietic progenitor cells with immobilized Jagged-1 resulted in expansion of serially transplantable HSCs, with superior expansion dependent on the combinatorial effect of Notch and cytokine-induced signaling pathways [128]. These data indicate that manipulation of Notch signaling can enhance stem cell self-renewal ex vivo and thereby increase HSC numbers for transplantation. Despite limited evidence of ex vivo HSC expansion with current methodologies, including stroma and stroma-free systems, clinical trials have been conducted with autologous marrow, cytokine-mobilized peripheral blood cells or cord blood cells, and their ability to shorten engraftment periods has been determined. Stroma-free systems provide a clear advantage for clinical use, since they are easier to standardize and maintain for cyclic guanosine monophosphate. However, some studies have used noncontact systems in which stromal cells serve to condition the medium. These trials will be discussed in the next section.
Clinical trials of ex vivo expanded HSCs The first report of a clinical trial using in vitro cultured hematopoietic progenitor cells appeared in 1992. Since then, a number of trials have been carried out with the primary goal of enhancing hematopoietic recovery after high-dose chemotherapy with the infusion of expanded stem cell grafts derived from bone marrow, mobilized peripheral blood or cord blood. Clinical trials using bone marrow and mobilized peripheral blood Table 8.1 summarizes select trials with autologous bone marrow or mobilized PBPCs cultured ex vivo with cytokines and/or stromal elements [129–138]. Studies of expanded mobilized PBPCs were all done in the autologous setting, and included only those patients with sufficient stem cell harvests to allow the use of an aliquot of the apheresis product for expansion. This excluded “poor mobilizers” who clearly stand to benefit from expansion procedures allowing them to undergo treatment with high-dose therapy. However, it is not known whether HSCs derived from such patients are equivalent to those derived from patients with adequate mobilization of HSCs. These trials, primarily in breast cancer patients, assessed enhanced short-term repopulation as a function of decreased time to neutrophil and/or platelet engraftment. With one exception, these studies were unable to address the expansion of longterm repopulating HSCs because patients received nonmyeloablative chemotherapy and/or infusion of noncultured cells. Overall, although the safety and feasibility of stem cell expansion approaches were demonstrated, more rapid engraftment was not observed in patients receiving reduced-intensity preparative regimens. There have, however, been suggestions of ex vivo expansion of short-term repopulating cells in patients who have received myeloablative therapy.
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Table 8.1 Selected bone marrow and mobilized peripheral blood clinical expansion trials
Author/ reference
Conditioning regimen
Infusion expanded cells
Patients
Stem cell source
Cytokines/serum
Notes
Williams et al. (1996) [138]
n=9 Metastatic breast cancer
MPBPC
PIXY321, human serum albumin 1% × 12 days
NM1
Day +1
↑ TNC 26-fold CD15+ post expansion average 29%
Alcorn et al. (1996) [129]
n = 10 Nonmyeloid malignant
MPBPC
SCF, IL-1β, IL-3, IL-6, EPO + autologous plasma × 8 days
M/NM2
Day 0
↑ TNC 21-fold, CFU-GM 139-fold, BFU-E 114-fold (mean) No change in engraftment compared with controls
Bertolini et al. (1997) [131]
n = 10 8 breast cancer, 2 NHL
MPBPC
MGDF, SCF, IL-3, IL-6, IL-11, Flt3L, MIP-1α × 7 days
NM3
Day 0
Study aimed at looking at generating megakaryocytic progenitors
Holyoake et al. (1997) [133]
n=4 2 NHL, 2 MM
MPBPC Expanded cells only
SCF, IL-1β, IL-3, IL-6, EPO + autologous plasma × 8 days
M4
Day 0
No long-term engraftment seen ↑ TNC 8–27-fold, CFU-GM 17–130-fold
Reiffers et al. (1999) [130]
n = 14 MM
MPBPC
SCF, G-CSF, MGDF
M5
Day 0
↑ TNC 34.4-fold, CD34+ cells 2.6-fold, CFU-GM 14.1-fold (median)
McNiece et al. (2000) [134]
2 cohorts 1) n = 10 2) n = 11 Breast CA
MPBPC Cohort 2 – expanded cells only
SCF, G-CSF, MGDF ×10 days
NM6
Day 0
Cohort 1:
Stiff et al. (2000) [137]
n = 19 Breast cancer
BM (~75 mL) – expanded cells only
AastromReplicell Bioreactor Flt3L, EPO, PIXY321, 10% FBS, 10% horse serum × 12 days
NM1
Day 0
TNC 4.8-fold, CFU-GM 4.2-fold, CD34+Lin− cells 0.9-fold, LTC-IC 1.2fold (median) Engraftment times similar to autologous bone marrow transplantation Correlation: cell dose and engraftment
Pecora et al. (2001) [136]
n = 34 Breast cancer
BM (50–100 ml) for expansion + low-dose MPBPC
AastromReplicell Bioreactor Flt3L, EPO, PIXY321 (+TPO in seven cases), 10% FBS, 10% horse serum × 12 days
NM1
Day 0
CD34+Lin− cell number and quantity of stromal progenitors contained in the expanded product correlated with engraftment outcome
Engelhardt et al. (2001) [132]
n = 10 Breast cancer
BM (median 97 mL) – expanded cells only 2 patients: irradiated PBPCs
AastromReplicell Bioreactor Flt3L, EPO, PIXY321, 10% FBS, 10% horse serum × 12 days
NM1
Day 0
↑ TNC 4.5-fold, CFU-GM 18-fold (median) Correlation: CD34+Lin− cells and engraftment
Paquette et al. (2002) [135]
n = 43 Breast cancer
MPBPC
GCSF, SCF, MGDF, 1% human albumin, transferrin × 9–14 days
NM7
Day 0
Varied duration of cultures and starting cell density
↑ TNC 20-fold (median) ↑ CD34+ cells 1.8-fold Cohort 2: ↑ TNC 14-fold (median) ↑ CD34+ cells 2.0-fold TNC/kg post expansion best predictor of time to neutrophil engraftment
BFU-E, burst-forming unit-erythroid; BM, bone marrow; CFU-GM, colony-forming unit-granulocyte–macrophage; EPO, erythropoietin; FBS, fetal bovine syndrome; LTC-IC, longterm culture-initiating cells; M, myeloablative; MGDF, megakaryocyte growth and development factor; MIP, macrophage inhibitory protein; MPBPC, mobilized peripheral blood progenitor cells; MM, multiple myeloma; NHL, non-Hodgkin’s lymphoma; NM, nonmyeloablative; TNC, total nucleated cells. See text for other abbreviations. 1 Cyclophosphamide/carbo/thiotepa (STAMP V). 2 Melphalan/total body irradiation, BEAM, thiotepa/cyclophosphamide, carboplatin/etoposide/melphalan, cyclophosphamide/TBI. 3 Thiotepa/melphalan (breast cancer); mitoxantrone/melphalan (NHL). 4 Cyclophosphamide/total body irradiation (NHL) and busulfan/melphalan (MM). 5 Melphalan ± total body irradiation. 6 Cyclophosphamide/cisplatin/BCNU or taxol/cyclophosphamide/cisplatin or taxotere/melphalan/carboplatin. 7 Cyclophosphamide/carmustine/cisplatin.
Expansion of Hematopoietic Stem Cells
For example, in a study by Holyoake et al. [133], the gain of short-term repopulating cells at the expense of long-term repopulating cells was suggested. In that study, mobilized PBPCs cultured ex vivo with SCF, IL-1β, IL-3, IL-6, erythropoietin, and autologous plasma for 8 days were infused in the absence of noncultured cells into patients that had received myeloablative chemotherapy. None of the four patients showed evidence of long-term hematopoietic recovery, and all required infusions of unmanipulated cryopreserved autologous back-up mobilized PBPCs, after which full hematopoietic recovery was seen. Three patients showed initial neutrophil engraftment that was unsustained, suggesting that short-term repopulating cells may have been generated ex vivo at the expense of long-term repopulating ones, possibly by inducing their differentiation. A shortening of the neutropenic period following high-dose chemotherapy in 21 patients with breast cancer was also shown in a study in which autologous mobilized PBPCs were cultured with SCF, G-CSF, and megakaryocyte growth and development factor for 10 days. The patients in this study were divided into two cohorts: those who received expanded cells only, and those who received both expanded and unmanipulated cells in tandem [134]. Expanded PBPCs resulted in a more rapid neutrophil engraftment (p = 0.02 for cohorts 1 and 2 versus the historical controls), the best predictor of time to neutrophil engraftment was the total number of cells harvested after expansion, and patients receiving more than 4 × 107 cells/kg engrafted by day 8. Reiffers et al. also reported a decreased time to neutrophil engraftment using the same culture conditions to expand autologous mobilized PBPCs for infusion with unmanipulated cells into patients with multiple myeloma after high-dose conditioning. The use of continuous perfusion methods for the expansion of bone marrow has recently been evaluated in three trials utilizing the automated perfusion bioreactor system AastromReplicell [132,136,137]. Notably, these are the only trials that used bone marrow (unselected for CD34+ cells) as the source of stem cells for ex vivo expansion, thereby perhaps including accessory or stromal cells of unclear significance in expansion cultures, and perhaps enhancing the engraftment ability of this expanded cell population. In these studies, breast cancer patients underwent the same conditioning regimen, and cells derived from autologous bone marrow were placed directly into the automated perfusion bioreactor system for expansion with Flt3L, erythropoietin, and PIXY321 cytokines, as well as fetal bovine serum and horse serum. In all trials, cultures were initiated with small amounts of harvested marrow (75– 100 mL). The trials by Stiff et al. and Engelhardt et al. used expanded cells only, while Pecora et al. also infused mobilized PBPCs in low doses. Engraftment was not enhanced but did occur, suggesting the maintenance of HSCs in the bone marrow cultures, and all trials reported a correlation between the time to neutrophil and platelet engraftment with CD34+Lin− cell dose/kg. Stiff et al. reported that only 2 × 105 CD34+ cells/kg of the expanded cells were needed to produce optimal platelet engraftment. This cell dose was lower than the number of cells required for predictable platelet engraftment by day 28 in breast cancer patients undergoing PBPC transplantation with unmanipulated PBPCs, in which the minimal number of CD34+ cells required was reported to be 2–5 × 106/kg [139]. Clinical trials using UCB cells Rationale for use of UCB stem cells for ex vivo expansion With greater than 6000 UCB transplants performed to date since 1988 [140], UCB has emerged as an alternative source of HSC for transplantation. This is especially important for minority patients and patients of mixed ethnicity, for whom UCB is a particularly attractive alternative
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donor stem cell source as it is readily available with no donor attrition and allows reduced stringency in human leukocyte antigen (HLA) matching without an increase in graft-versus-host disease (GVHD). Numerous clinical studies have consistently shown that the total nucleated cell and CD34+ cell doses in cord blood grafts are highly correlated with the rate of neutrophil and platelet engraftment, as well as the incidence of graft failure and early transplant-related complication [141– 149]. Based on these studies, critical cell dose thresholds have been established, and outcomes for patients receiving less than the generally accepted threshold of more than 2.5 × 107 total nucleated cells/kg are significantly inferior in terms of engraftment, transplant-related mortality and overall survival. For patients weighing greater than 35–40 kg, obtaining an adequate cell dose from a single UCB unit may be an impossible hurdle. Delayed engraftment has also been correlated with HLA mismatch at more than two loci, but recent studies have suggested that the impact of HLA disparity on survival can be partially overcome by increasing cell dose [148,150]. Efforts to overcome the obstacle of small cell numbers in UCB grafts by ex vivo cytokine-mediated expansion have not yet demonstrated improved time to engraftment. Infusion of multiple cord blood units as an alternative approach to increase cell numbers is also under investigation. The initial results from this approach are encouraging and have demonstrated the safety of this approach in adults, with improved engraftment and decreased transplant-related mortality, but the significant delay in engraftment has not been abrogated in these patients [151]. Thus, the collective data point to improved outcome following UCB transplantation in patients receiving a higher cell dose, in terms of both CD34+ and total nucleated cell doses, an observation leading to the hypotheses that ex vivo expansion of CD34+ cell numbers or the infusion of more than one unit of cord blood might improve the rate of cord blood engraftment and overall survival. Several lines of evidence suggest that UCB contains a higher frequency of primitive hematopoietic progenitor cells and early committed progenitors than adult bone marrow or peripheral blood [21,68]. There is also increasing evidence that the stem cells isolated from UCB survive longer in culture [95] and may be less mature and have greater proliferative capacity [78,152–154]. Phenotypic analyses of UCB have shown that the more primitive cell population which expresses the CD34 antigen, but not the CD38 antigen, is fourfold more prevalent than in bone marrow or PBPCs, and that this subpopulation has a higher in vitro cloning efficiency than the same population isolated from adult bone marrow [155]. These findings correlate with data from studies using in vitro with bone marrow or PBPCs [156–158]. Second, there are numerous reports of the increased proliferative potential of UCB cells in response to cytokine stimulation. For example, using IL-11, SCF, and G-CSF or granulocyte–macrophage colonystimulating factor, Cairo and colleagues demonstrated an 80-fold increase after a 14-day expansion of UCB versus adult bone marrow [159]. Moreover, compared with adult bone marrow, UCB has been shown to have increased serial in vitro replating efficiency [153] and increased culture life span with increased progenitor cell production [94,156]. Finally, in vivo assays of UCB versus bone marrow have shown that HSCs from UCB, but not adult bone marrow, can engraft NOD/SCID mice without the use of exogenous cytokines [67]. More recently, Rosler et al. showed that expanded cord blood had a competitive repopulating advantage compared with expanded adult bone marrow using an in vivo assay with NOD/SCID mice [22]. A potential contribution to the differences observed between UCB and adult HSCs may arise from the differential response of HSCs from different sources in cytokine-driven expansion systems, leading to variations in cell-cycle status and homing ability of the expanded cells. Other possible sources of stem cells may have even greater proliferative
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potential. For example, murine fetal liver cells have greater proliferation and repopulation potential than HSCs isolated from adult murine bone marrow or peripheral blood [160,161]. Overall, these results suggest that UCB progenitor cells are functionally superior to adult bone marrow, with greater proliferative potential and possibly greater self-renewal capacity. Thus, UCB may represent a more viable target for ex vivo stem cell expansion, a possibility that has led to several clinical studies on the ex vivo expansion of cord blood cells to augment conventional UCB transplantation. Clinical ex vivo expansion trials with UCB UCB expansion trials (summarized in Table 8.2) [162–166] were undertaken to determine whether the delayed engraftment associated with UCB transplantation could be overcome if a portion of the cells from the UCB unit were expanded ex vivo. Like the trials using PBPCs and bone marrow, these initial trials utilized cytokine-based expansion systems as well as newer automated perfusion systems. The choice of exogenous cytokines used for culture has varied, reflecting the still undefined optimal conditions for expansion of the stem/progenitor cell. In all of the initial studies, only a portion of the single cord blood graft was used for expansion, and the expanded cells were infused in addition to unmanipulated cells, as there is a lack of definitive data to suggest that cultured cells retain HSC properties and do not differentiate. There have been no adverse toxicities associated with infusion of the expanded cell product, nor has there been any change in engraftment kinetics. However, only two of the studies have enrolled more than a handful of patients, making it difficult to draw any definitive conclusions. More recently, novel culture systems for the ex vivo expansion of UCB progenitors have begun patient accrual, including a Notch-mediated ex vivo expansion trial at the Fred Hutchinson Cancer Research Center, a trial utilizing a copper-chelating agent and an alternative strategy of coculture of UCB cells with bone marrow mesenchymal stem cells. These will be discussed below. In one of the larger studies to date, Jaroscak et al. reported preliminary data from a phase I trial at Duke University Medical Center undertaken to assess augmentation of UCB transplantation with cells expanded ex vivo in the AastromReplicell Cell Production System [162]. This trial included 28 patients with both malignant and inherited disorders who were conditioned with one of three regimens (Table 8.2), depending on diagnosis. On day 0, a portion of a single unrelated UCB unit was expanded ex vivo in medium supplemented with fetal bovine serum, horse serum, and cytokines (erythropoietin, PIXY321, and Flt3L) for 12 days. The expanded cells were then infused to augment the conventional transplant on day 12 after transplantation. Although expansion of total nucleated cells and CFCs occurred in vitro in all cases, no increase in CD34+Lin− cell number was achieved. In vivo, significant effects on engraftment kinetics were not observed with median time to neutrophil engraftment (absolute neutrophil count [ANC] > 500/μL) of 22 (range 13–40) days. No adverse reactions were observed due to infusion of the cultured cells. This phase I trial was an important contribution to the concept and development of ex vivo expansion of UCB CD34+ cells for use in the clinical setting, since it demonstrates both the safety of reserving an aliquot of an already small cord blood unit for expansion and the feasibility of expansion in a clinical setting. It is also possible that, although the numbers of CD34+Lin- and CD3+ cells were not expanded with this culture system and may have explained the failure to improve engraftment, the delayed infusion (day 10–12 post transplant) of expanded cells may have masked or prevented the benefit of a more rapid engraftment. Newer-generation clinical cord blood expansion trials, discussed below, are currently underway based on a double cord blood model, in which one unit is infused unmanipulated on the day of transplant with a second unit that has been expanded ex vivo.
Shpall and colleagues are currently conducting a number of cord blood expansion trials at the MD Anderson Cancer Center: a cytokinemediated expansion trial and a triethylenephosphoramide (TEPA)-based ex vivo expansion trial. The cytokine-mediated expansion trial is the successor trial to an earlier published trial performed at the University of Colorado [165], with some important changes. In this currently accruing trial, patients are now being randomized to receive either two unmanipulated cord blood units or one unmanipulated unit and one ex vivo expanded unit following high-dose therapy, compared with the previous trial in which only a portion of a single cord blood unit was used for expansion. For those patients randomized to receive expanded cells, the unit identified for ex vivo expansion is thawed, CD133 selected, and placed into a cytokine-mediated expansion system 14 days prior to the day of transplant. Accrual on this trial continues, but analysis of the first cohort of 22 patients enrolled has been performed (Shpall, personal communication). The median time to neutrophil engraftment is 20 and 21 days for patients receiving a conventional unmanipulated double UCB transplant and those receiving an unmanipulated unit and ex vivo expanded unit, respectively. Overall, 30% of patients developed acute grade III–IV GVHD. Based on preclinical data in which Peled et al. demonstrated enhanced expansion of CD34+ UCB progenitors, an additional ex vivo clinical expansion trial is enrolling at the MD Anderson Cancer Center using the copper chelator TEPA (Shpall, personal communication) [167]. Similar to the cytokine-mediated expansion trial, patients with hematologic malignancies are eligible, but all patients in this trial must have identified a single unit of cord blood that has been cryopreserved in two fractions. The smaller of the two UCB fractions is CD133 selected, and expanded ex vivo in the presence of cytokines and TEPA starting 21 days prior to infusion. Ten patients with high-risk malignancies have been treated thus far. The average fold expansion of total nucleated cells placed in culture was 219 (range 2–616), and the median time to engraftment was 28 (range 16–46) days for neutrophils and 48 (range 27–96) days for platelets. Evidence of GVHD during the 180-day study period was found in five of the 10 evaluable patients. Three patients experienced acute GVHD, all grade II, with skin involvement, and two patients developed chronic GVHD, one chronic extensive and one mild limited chronic. One patient with acute GVHD persisted to chronic limited GVHD with skin involvement. All cases were resolved with steroid therapy. There were no incidences of engraftment failure or grades III–IV acute GVHD, and 100-day survival was 90%. Thirty percent of the patients are alive and free of disease at a median 18 months post transplant. At the Fred Hutchinson Cancer Research Center, a phase I trial assessing the potential efficacy of cord blood progenitors cultured in the presence of an engineered form of the Notch ligand Delta-1 in contributing to rapid early engraftment in patients undergoing a high-dose UCB transplant is currently accruing patients. As discussed above, there is accumulating evidence on the role of the Notch signaling pathway in hematopoietic cell fate decisions. This trial has accrued only three patients to date, and while conclusions cannot be made as to the efficacy of this approach, the early results are encouraging (Delaney, unpublished data). Three patients with acute myeloid leukemia have enrolled and received cord blood progenitors that were cultured with Delta-1 following infusion of a second noncultured and unrelated donor cord blood graft. Upon harvest of the cells at day 16 post culture initiation, there was an average fold expansion of CD34+ cells of 116 (±49) and an average fold expansion of total cell numbers of 565 (±179) (Fig. 8.2(a)). The infused CD34 cell dose (defined as the number of CD34+ cells/kg recipient body weight) derived from the ex vivo expanded unit ranged from 1 to 13 million CD34+ cells/kg. In the first two patients, early myeloid recovery was observed, occurring at a time when engraftment due to the infusion of two noncultured
Expansion of Hematopoietic Stem Cells
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Table 8.2 Selected umbilical cord blood clinical expansion trials Author/ reference
Conditioning regimen
Infusion expanded cells
AastromReplicell system Epo, Pixy321, Flt3L × 12 days
CY/TBI or BU/ CY/ATG
Day +12
No significant change in neutrophil engraftment No infusional toxicities 2 deaths prior to engraftment
UCB – sibling 12.5% expanded + 87.5% unmanipulated
G-CSF, TPO, Flt3L, 10% autologous cord blood plasma × 10 days
TBI/thiotepa/CY
Day +1
ANC 310/μL by day +8 Event-free survival now greater than 1 year
n=2 CML (blast crisis and accelerated phase)
UCB (unrelated) 11%, 17% expanded + 89%, 83% unmanipulated cells
AastromReplicell system Epo, Pixy321, Flt3L × 12 days
(1) BU/ATG CY (2) CY/TBI/ATG
Day +12
No infusional toxicities Improved platelet engraftment No change in neutrophil engraftment Lin−, CD34+ cells decreased in one patient, increased 1.7-fold in other patient
Shpall et al. (2002) [165]
n = 37 (25 adult) Stratum A = 25 Stratum B = 12 (split unit) Dx: hematologic malignancy (34), breast cancer (3)
UCB (unrelated) Expanded fraction Cohort A = 40% Cohort B = 60% + unmanipulated cells
G-CSF, SCF, MGDF expanded in Teflon bags × 10 days
Adults: High-dose melphalan/ ATG/TBI Or High-dose melphalan/ ATG/BU Children: CY/TBI/ arabinoside C/ ATG
Stratum (A) Day +10 (B) Day 0
No infusional toxicities No change in engraftment seen No neutrophil engraftment failures
Jaroscak et al. (2003) [162]
n = 28 (26 children) Varied Dx: both malignancies and inherited disorders
UCB (unrelated, one sibling) Expanded + unmanipulated cells
AastromReplicell system PIXY321, Epo, Flt3L, 10% horse serum, 10% FBS × 12 days
Hematologic malignancy TBI/melphalan/ ATG Or BU/melphalan/ ATG Inherited disorders BU/CY/ATG
Day +12
No infusional toxicities No alteration in time to engraftment ↑ TNC 2.4-fold, CFU-GM 82.7-fold, CD34+ 0.5-fold (median)
Shpall et al. (personal communication; see text)
n = 22 (accrual continues)
UCB – double unrelated Randomization: (A) Double unmanipulated versus (B) Expanded + unmanipulated
CD133+ cells G-CSF, SCF, MGDF × 14 days
Myeloid malignancy Flu/BU/ATG Lymphoid malignancy Flu/melphalan/ thiotepa/ATG
Day 0
No infusional toxicities Fold expansion N/A Average time ANC > 500/μL (A) 20 days (B) 21 days
Shpall et al. MDACC (personal communication; see text)
n = 10 (accrual continues)
Single cord blood unit cryopreserved in two fractions: 80%–20%, 60%–40% or 50%– 50% (smaller fraction ex vivo expanded)
CD133+ cells TEPA + Flt3L, TPO, SCF, IL6 × 21 days
Myeloid malignancy Flu/BU/ATG Lymphoid malignancy Flu/melphalan/ thiotepa/ATG
Day +1
No infusional toxicities Fold expansion TNC 219 (mean), range 2–616 Average time to ANC > 500/μL 28 days (16–46 days)
Delaney et al. (personal communication; see text)
n=3 (accrual continues)
UCB – two units Unmanipulated unit Ex vivo expanded unit
CD34+ cells TPO, SCF, Flt3L, IL-3, IL-6 Serum free
CY/TBI/Flu
Day 0
No infusional toxicities Fold expansion TNC 565 (mean), range 210–791 Fold expansion CD34 115 (mean), range 41–210 Average time to ANC > 500/μL 15 days (9–20 days)
Patients
Stem cell source
Cytokines/serum
Stiff et al. (1998) [166]
n=9 Dx: advanced hematologic disorders
UCB (unrelated) Expanded + unmanipulated
Kögler et al. (1999) [163]
n=1 High-risk leukemia
Pecora et al. (2000) [164]
Notes
ATG, antithymocyte globulin; BU, busulfan; CML, chronic myeloid leukemia; CY, cyclophosphamide; Dx, diagnosis; EFS, event-free survival; FBS, fetal bovine serum; TBI, total body irradiation. For other abbreviations, see Table 8.1 and text.
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Patient 1 Patient 2 Patient 3
09
1.0 × 10
08
1.0 × 10
1.0 × 1007 1000000 0
5
10
1.0 × 10 10 1.0 × 10 1.0 × 10
Fold expansion 800
Patient 1 Patient 2 Patient 3
09
700
Fold expansion
Absolute CD34 cell number
1.0 × 1010
Total number of cells
CD34 cell growth
Total cell growth
(a)
08
1.0 × 10 07
600
TNC fold expansion CD34 fold expansion
500 400 300 200 100 0
1000000
15
0
Days in culture
5
10
15
TNC
CD34
Days in culture
Peripheral blood chimerism
(b)
Patient 1
Patient 2
Patient 3
100
100
100
75
75
75
50
50
50
25
25
25
0
0
0 7
14 21 28 42 56 80 100
7
7
14 21 28 42 56 80 100
100
100
100
75
75
75
50
50
50
25
25
25
Not done
0 7
Not done
0
14 21 28 42 56 80 100
CD33
7
14
21
28
CD14
0 7
14 21 28 42 56 80 100
100
100
100
75
75
75
50
50
50
25
25
14
21
28
CD56
25 QNS
7
Percent
0
0
0 14 21 28 42 56 80 100
7
7
14 21 28 42 56 80 100
100
100
100
75
75
75
50
50
50
25
25
25
7
14 21 28 42 56 80 100
21
28
CD3
0
0
0
14
7
14 21 28 42 56 80 100
7
14
21
28
Days post transplant Fig. 8.2 Clinical grade culture of umbilical cord blood (UCB) progenitors with Delta-1ext-IgG for therapeutic application results in significant in vitro expansion of both total nucleated and CD34+ cells. CD34+ cord blood progenitors were selected using the clinical grade Isolex 300i and placed into culture with Delta-1ext-IgG as described. (a) In vitro growth characteristics are presented for three patients enrolled to date, including absolute growth of total nucleated cells (TNC), CD34+ cells, and the resultant fold expansion of both total nucleated and CD34+ cells. Data shown in the bar graph are the mean ± standard error of the mean for the three patients. (b) Peripheral blood was sorted by fluorescence-activated cell sorting into CD3+, CD33+, CD14+, and CD56+ cell fractions to assess for contribution to engraftment from the ex vivo expanded and unmanipulated units (chimerism). Chimerism testing was performed by amplified fragment length polymorphism analysis. The y-axis represents the percent engraftment as contributed by the ex vivo expanded cord blood unit (black), the unmanipulated unit (grey), and the host (white). The x-axis represents time post transplant.
Expansion of Hematopoietic Stem Cells
units would not be expected. Both patients achieved an ANC of 100/μL by day 7 post transplant, a threshold that has been shown to be strongly associated with a survival benefit post allogeneic HCT [168], and an ANC of 500/μL by days 9 and 16 post transplant. A third patient received a more limited CD34 cell dose (1 × 106 cells/kg) due to a less robust in vitro expansion and did not show engraftment before 2 weeks. The CD34 fold expansion (at 41-fold expansion) for this patient was equivalent to the one outlier out of 19 in our preclinical validation runs that resulted in a CD34 fold expansion of less than 50. It is anticipated that a portion of cord blood units will result in less than expected expansion of CD34+ cells based on experience to date. Prediction of units that will expand poorly is not possible at this point prior to ex vivo expansion and suggests a benefit in having pre-expanded units cryopreserved for future use. Nonetheless, the average time to engraftment for all patients (defined as the first of three consecutive days with an ANC > 500/μL) was 15 days, contrasting the expected time to engraftment following a double unmanipulated cord blood transplantation of 23 days, as reported by Barker and colleagues [151]. The kinetics of hematopoietic recovery and the relative contribution of the expanded and unmanipulated cord blood units to engraftment over time were determined by a DNA-based assay for short tandem repeat loci on cells obtained from peripheral blood sorted by fluorescenceactivated cell sorting for CD3+, CD33+, CD56+, CD14+, and CD19+ cell fractions, beginning on day 7 post transplant. These studies revealed that initial myelomonocytic engraftment in these patients was derived from the cultured cells, with subsequent sustained engraftment primarily from the noncultured unit. Analysis of peripheral blood sorted cell fractions revealed loss of contribution to engraftment in all cell populations from the expanded cells by day 21 in two out of three patients transplanted to date; however, there was persistent contribution to engraftment from both the cultured and noncultured cells in the CD14, CD56, and CD19 (data not shown for CD19) sorted cell fractions at day 100 post transplant in one patient (Fig. 8.2(b)). Accrual continues on this study. It is not yet known whether loss of the cultured cord blood unit in marrow and peripheral blood engraftment resulted from loss of stem cell self-renewal capacity due to maturation of the stem cell during culture or yet-to-be-defined immune-mediated mechanisms that allow only cells from a single unit to survive in a double cord blood transplant setting. Our findings are consistent with
97
results seen in most patients undergoing conventional cord blood transplant with two unmanipulated units, where only one unit contributes to sustained donor engraftment, and donor dominance is usually observed early in the majority of patients. However, a clear difference in the expansion setting versus the conventional double cord blood transplant setting is the infusion of two unmanipulated, immunocompetent units versus infusion of an immunocompetent, noncultured unit and a T-celldepleted ex vivo cultured unit, which may allow for rejection of the cultured cells by the noncultured unit.
Conclusion Although optimal conditions for expanding HSC numbers remain undefined, the collective results of studies outlined above suggest that enhanced short-term and long-term repopulating ability may be achievable with cytokine-induced expansion systems. However, these studies further suggest that cytokine-induced effects on HSC self-renewal and expansion are limited, and clinically significant expansion has not been achieved. It is likely that we have not yet identified critical factors and combinations required to induce symmetric HSC self-renewal, and thus current efforts are now focused on identifying and exploiting factors previously shown to regulate stem cell fate in other developing organ systems or during embryogenesis. This approach is beginning to yield promising results, and improved methods for HSC expansion are emerging. Identification of genes and regulatory elements that are responsible for preventing stem cell senescence and driving symmetric divisions of the HSC, and a better understanding of how culturing affects the homing ability of stem cells and of the complex cellular interactions in the bone marrow microenvironment, are all essential to the ultimate success of ex vivo expansion. These areas are discussed in more detail in other chapters. Clinically, ex vivo expansion studies of primitive cells from bone marrow, PBPCs, and UCB have demonstrated the feasibility and safety of ex vivo expansion, and have suggested the enhancement of short-term repopulating cells perhaps at the expense of long-term repopulating cells. Continued development of methods using novel stem cell regulators that are shown to expand HSCs in preclinical studies is necessary, and current trials are underway to assess the safety and efficacy of these approaches, which hold promise for the future of expanding HSC numbers ex vivo.
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67.
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bone marrow: implications for gene therapy. Nat Med 1996; 2: 1329–37. Vormoor J, Lapidot T, Pflumio F et al. Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood 1994; 83: 2489–97. Wang JC, Doedens M, Dick JE. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 1997; 89: 3919–24. Glimm H, Eisterer W, Lee K et al. Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice. J Clin Invest 2001; 107: 199–206. Dorrell C, Gan OI, Pereira DS et al. Expansion of human cord blood CD34(+)CD38(−) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function. Blood 2000; 95: 102–10. Gan OI, Murdoch B, Larochelle A, Dick JE. Differential maintenance of primitive human SCIDrepopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells. Blood 1997; 90: 641–50. Danet GH, Lee HW, Luongo JL et al. Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34(+) cells after ex vivo expansion. Exp Hematol 2001; 29: 1465–73. Mobest D, Goan SR, Junghahn I et al. Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells. Stem Cells 1999; 17: 152–61. Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J Exp Med 1997; 186: 619–24. Gilmore GL, DePasquale DK, Lister J, Shadduck RK. Ex vivo expansion of human umbilical cord blood and peripheral blood CD34(+) hematopoietic stem cells. Exp Hematol 2000; 28: 1297–305. Luens KM, Travis MA, Chen BP et al. Thrombopoietin, kit ligand, and flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34+Thy1+Lin– cells with preserved ability to engraft SCID-hu bone. Blood 1998; 91: 1206–15. Novelli EM, Cheng L, Yang Y et al. Ex vivo culture of cord blood CD34+ cells expands progenitor cell numbers, preserves engraftment capacity in nonobese diabetic/severe combined immunodeficient mice, and enhances retroviral transduction efficiency. Hum Gene Ther 1999; 10: 2927–40. Piacibello W, Sanavio F, Severino A et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999; 93: 3736–49. Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL6, and soluble IL-6 receptor. J Clin Invest 2000; 105: 1013–21.
80. da Silva CL, Goncalves R, Crapnell KB et al. A human stromal-based serum-free culture system supports the ex vivo expansion/maintenance of bone marrow and cord blood hematopoietic stem/ progenitor cells. Exp Hematol 2005; 33: 828– 35. 81. Kimura T, Sakabe H, Tanimukai S et al. Simultaneous activation of signals through gp130, c-kit, and interleukin-3 receptor promotes a trilineage blood cell production in the absence of terminally acting lineage-specific factors. Blood 1997; 90: 4767–78. 82. Kimura T, Wang J, Minamiguchi H et al. Signal through gp130 activated by soluble interleukin (IL)-6 receptor (R) and IL-6 or IL-6R/IL-6 fusion protein enhances ex vivo expansion of human peripheral blood-derived hematopoietic progenitors. Stem Cells 2000; 18: 444–52. 83. Kollet O, Aviram R, Chebath J et al. The soluble interleukin-6 (IL-6) receptor/IL-6 fusion protein enhances in vitro maintenance and proliferation of human CD34(+)CD38(−/low) cells capable of repopulating severe combined immunodeficiency mice. Blood 1999; 94: 923–31. 84. Tajima S, Tsuji K, Ebihara Y et al. Analysis of interleukin 6 receptor and gp130 expressions and proliferative capability of human CD34+ cells. J Exp Med 1996; 184: 1357–64. 85. Emmons RV, Doren S, Zujewski J et al. Retroviral gene transduction of adult peripheral blood or marrow-derived CD34+ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo. Blood 1997; 89: 4040–6. 86. Hanania EG, Giles RE, Kavanagh J et al. Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy. Proc Natl Acad Sci U S A 1996; 93: 15346–51. 87. Bhatia R, McGlave PB, Miller JS et al. A clinically suitable ex vivo expansion culture system for LTC-IC and CFC using stroma-conditioned medium. Exp Hematol 1997; 25: 980–91. 88. Roecklein B, Almaida-Porada G, Torok-Storb B et al. Serial xenogeneic transplantation of ex vivo expanded human CD34+ cells. Blood 1997; 90: 1750a. 89. Shih CC, Hu MC, Hu J et al. A secreted and LIFmediated stromal cell-derived activity that promotes ex vivo expansion of human hematopoietic stem cells. Blood 2000; 95: 1957–66. 90. Verfaillie CM, Catanzarro PM, Li WN. Macrophage inflammatory protein 1 alpha, interleukin 3 and diffusible marrow stromal factors maintain human hematopoietic stem cells for at least eight weeks in vitro. J Exp Med 1994; 179: 643–9. 91. Yildirim S, Boehmler AM, Kanz L, Mohle R. Expansion of cord blood CD34+ hematopoietic progenitor cells in coculture with autologous umbilical vein endothelial cells (HUVEC) is superior to cytokine-supplemented liquid culture. Bone Marrow Transplant 2005; 36: 71–9. 92. Lewis ID, Almeida-Porada G, Du J et al. Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system. Blood 2001; 97: 3441–9. 93. Roecklein BA, Torok-Storb B. Functionally distinct human marrow stromal cell lines immortal-
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of multilineage progenitor cells. Blood 1997; 89: 3624–35. Reya T, Duncan AW, Ailles L et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 2003; 423: 409–14. Kojika S, Griffin JD. Notch receptors and hematopoiesis. Exp Hematol 2001; 29: 1041–52. Milner LA, Kopan R, Martin DI, Bernstein ID. A human homologue of the Drosophila developmental gene, Notch, is expressed in CD34+ hematopoietic precursors. Blood 1994; 83: 2057–62. Ohishi K, Varnum-Finney B, Flowers D et al. Monocytes express high amounts of Notch and undergo cytokine specific apoptosis following interaction with the Notch ligand, Delta-1. Blood 2000; 95: 2847–54. Jones P, May G, Healy L et al. Stromal expression of Jagged 1 promotes colony formation by fetal hematopoietic progenitor cells. Blood 1998; 92: 1505–11. Karanu FN, Murdoch B, Gallacher L et al. The notch ligand jagged-1 represents a novel growth factor of human hematopoietic stem cells. J Exp Med 2000; 192: 1365–72. Karanu FN, Murdoch B, Miyabayashi T et al. Human homologues of Delta-1 and Delta-4 function as mitogenic regulators of primitive human hematopoietic cells. Blood 2001; 97: 1960–7. Li L, Milner LA, Deng Y et al. The human homolog of rat Jagged1 expressed by marrow stroma inhibits differentiation of 32D cells through interaction with Notch1. Immunity 1998; 8: 43–55. Varnum-Finney B, Xu L, Brashem-Stein C et al. Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med 2000; 6: 1278–81. Stier S, Cheng T, Dombkowski D et al. Notch1 activation increases hematopoietic stem cell selfrenewal in vivo and favors lymphoid over myeloid lineage outcome. Blood 2002; 99: 2369–78. Carlesso N, Aster JC, Sklar J, Scadden DT. Notch1-induced delay of human hematopoietic progenitor cell differentiation is associated with altered cell cycle kinetics. Blood 1999; 93: 838– 48. Varnum-Finney B, Wu L, Yu M et al. Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. J Cell Sci 2000; 113(Pt 23): 4313–18. Vas V, Szilagyi L, Paloczi K, Uher F. Soluble Jagged-1 is able to inhibit the function of its multivalent form to induce hematopoietic stem cell self-renewal in a surrogate in vitro assay. J Leukoc Biol 2004; 75: 714–20. Varnum-Finney B, Brashem-Stein C, Bernstein ID. Combined effects of Notch signaling and cytokines induce a multiple log increase in precursors with lymphoid and myeloid reconstituting ability. Blood 2003; 101: 1784–9. Delaney C, Varnum-Finney B, Aoyama K et al. Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood 2005; 106: 2693–9. Ohishi K, Varnum-Finney B, Bernstein ID. Delta1 enhances marrow and thymus repopulating ability of human CD34(+)CD38(−) cord blood cells. J Clin Invest 2002; 110: 1165–74. Dallas MH, Varnum-Finney B, Delaney C et al. Density of the Notch ligand Delta1 determines
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143. Gluckman E. Current status of umbilical cord blood hematopoietic stem cell transplantation. Exp Hematol 2000; 28: 1197–205. 144. Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. N Engl J Med 1997; 337: 373–81. 145. Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996; 335: 157–66. 146. Laughlin MJ, Barker J, Bambach B et al. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001; 344: 1815–22. 147. Rocha V, Gluckman E. Clinical use of umbilical cord blood hematopoietic stem cells. Biol Blood Marrow Transplant 2006; 12(1 Suppl 1): 34– 41. 148. Rubinstein P, Carrier C, Scaradavou A et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998; 339: 1565–77. 149. Wagner JE, Rosenthal J, Sweetman R et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease. Blood 1996; 88: 795–802. 150. Wagner JE, Barker JN, DeFor TE et al. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood 2002; 100: 1611–18. 151. Barker JN, Weisdorf DJ, DeFor TE et al. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 2005; 105: 1343–7. 152. Broxmeyer HE, Hangoc G, Cooper S et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci U S A 1992; 89: 4109–13. 153. Lu L, Xiao M, Shen RN et al. Enrichment, characterization, and responsiveness of single primitive CD34 human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential. Blood 1993; 81: 41– 8. 154. Mayani H, Dragowska W, Lansdorp PM. Characterization of functionally distinct subpopulations of CD34+ cord blood cells in serum-free longterm cultures supplemented with hematopoietic cytokines. Blood 1993; 82: 2664–72. 155. Hao QL, Thiemann FT, Petersen D et al. Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population. Blood 1996; 88: 3306–13. 156. Hows JM, Bradley BA, Marsh JC et al. Growth of human umbilical-cord blood in longterm haemopoietic cultures. Lancet 1992; 340: 73–6. 157. Mayani H, Lansdorp PM. Thy-1 expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood. Blood 1994; 83: 2410–17. 158. Steen R, Tjonnfjord GE, Egeland T. Comparison of the phenotype and clonogenicity of normal
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9
Edwin M. Horwitz
Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation
Introduction Hematopoietic cell transplantation (HCT) is well established as therapy for hematologic malignancies as well as many nonmalignant disorders. Over the last several years, the spectrum of diseases that may be treated with HCT has dramatically expanded, increasing the importance of this therapeutic modality and extending HCT beyond the traditional bounds of hematology and oncology. HCT is founded on the principle that hematopoietic stem cells (HSCs) can be intravenously infused, will home to and engraft in the stem cell niche within the bone marrow microenvironment, and will then proliferate and differentiate to repopulate all lineages of the blood. The cells that comprise the microenvironment are thought to support, and possibly regulate, hematopoiesis. However, one cellular constituent of the microenvironment, the marrow mesenchymal stromal cells (MSCs), may have quite complex biologic functions that can be exploited in the development of primary or adjunct cell therapy with HCT or in regenerative medicine. MSCs are spindle-shaped, plastic-adherent cells isolated from bone marrow as well as other tissue sources [1]. First recognized nearly 40 years ago [2], MSCs have emerged as one of the most rapidly evolving areas of research in HCT. The trilineage differentiation of MSCs, capable of giving rise to osteoblasts, chondrocytes, and adipocytes in vitro, led investigators to hypothesize a vast differentiation potential and focus research on the regenerative capacity of MSCs in an effort to repair diseased or damaged tissue [3]. More recently, MSCs have been recognized to possess striking immunomodulatory properties, and it is this characteristic of MSCs that has generated the greatest interest among physicians and investigators in the field of HCT [4,5]. Indeed, MSCs produce an extensive array of immunomodulatory cytokines as well as a variety of other, known and likely undiscovered, biochemical mediators. The secretion of these biologic response modifiers may well prove to be the most biologically and clinically important property of MSCs. While MSC research continues along many avenues, this chapter will focus on the biology and early clinical applications of MSCs in close relation to HCT.
“MSC” [2]. He showed that these cells were precursors for osteoblasts [2] and comprised the hematopoietic microenvironment [6], and that a fraction of the population, cells he termed “CFU-F”, possessed a high proliferative potential demonstrated by forming clonal colonies when plated at low density [7]. Friedenstein went on to demonstrate the extensive expansion potential, showing that single cells could regenerate an entire population of cells, suggesting that individual cells were precursors for fibroblasts in vivo [8,9]. However, Friedenstein did not suggest that the marrow stromal fibroblasts that he identified were stem cells. The first formal presentation of the concept that a nonhematopoietic stem cell might reside within the bone marrow microenvironment was by Maureen Owen [10]. She proposed that the marrow stroma consisted of a lineage of cells analogous to the hematopoietic system. She then expanded this theory and proposed a stromal stem cell, with fibroblastic, osteogenic, and adipocytic cells being the terminally differentiated phenotypes [11,12]. The term “mesenchymal stem cell” was proposed by Arnold Caplan, who hypothesized that this cell could differentiate to a wide variety of mesenchymal tissues and proposed a broad lineage “tree” suggesting that the mesenchymal stem cell could give rise to bone, cartilage, muscle, hematopoietic-supportive stroma, tendon, ligament, adipose, and other connective tissues [13]. Data demonstrating the identification of MSCs by reactivity with the monoclonal antibodies SH2 and SH3, the considerable self-renewal capacity, and the multilineage differentiation potential in vitro supported the notion that these cells were a homogeneous population of stem cells [14]. However, more recent data demonstrated that the adherent cells isolated from marrow are a heterogeneous population of cells [15,16], although a nonhematopoietic stem cell may exist within this cell population [17]. Many other investigators, too numerous to recount here, have made important contributions to the scientific development of MSCs. At present, MSCs are generally accepted to be a heterogeneous population of cells with diverse biologic properties suggesting a remarkable potential as cell therapy. While the expansion and differentiation potential of MSCs has yet to make a substantial impact in regenerative medicine, the cytokine-generating and immunomodulatory properties promise to contribute to HCT.
Brief history of MSCs The notion that a mesenchymal progenitor cell with osteogenic differentiation potential resided within bone marrow was considered in the latter 19th century. However, it was the pioneering work of Alexander Friedenstein that first identified the cells that now bear the designation Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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Nomenclature The nomenclature of these cells merits clarification. MSCs have been designed by a wide variety of terms including “marrow stromal cells,” “marrow stromal fibroblasts,” and most often “mesenchymal stem cells,” which was the basis for the abbreviation MSC. While the specific properties of MSCs studied in the laboratory may vary depending on the methods of isolation and culture expansion, the assortment of terms, in general, refer to the same heterogeneous population of cells.
Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation
The term “stem cell,” however, implies specific biologic properties. Our general concept of a stem cell evolved from our understanding of the HSC. Till and McCulloch suggested that the stem cell could be defined as a cell with extensive self-renewal capacity and the potential to terminally differentiate into two or more lineages [18]. While MSCs can, indeed, undergo extensive self-renewal and trilineage differentiation to osteoblasts, chondrocytes, and adipocytes in vitro, true “stemness” is certainly much more complex and may be best defined operationally. As this idea has become increasingly recognized, many investigators have suggested that convincing data are lacking to support the notion that the unfractionated population of plastic-adherent cells from bone marrow designated as MSCs are properly considered as stem cells [19]. These thoughts are not intended to dispel the notion of a mesenchymal stem cell. Such a stem cell most assuredly exists and may, indeed, reside within the adherent population of cells that we designate MSCs or some other cell population. Notably, the antibody STRO-1, which identifies CFU-Fs from freshly harvested human bone marrow, was generated from CD34+ nonadherent cells [20]. Recently, Anjos-Afonso et al. showed that a subset of murine MSCs that expressed stage-specific embryonic antigen-1 (SSEA-1) was capable of single-cell expansion followed by multilineage differentiation in vivo, suggesting that SSEA1+ MSCs may be primitive stem/progenitor cells [17]. These data notwithstanding, the entire unfractionated population of MSCs isolated by plastic adherence does not seem to meet the criteria of a homogeneous population of stem cells. Therefore, in 2005, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy recommended that the term “mesenchymal stromal cell” was a more appropriate designation for this heterogeneous population of cells, maintaining the abbreviation MSC while reserving the term “mesenchymal stem cell” for a subset of these (or other) cells that demonstrate stem cell activity in vivo by clearly stated criteria [1].
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Table 9.1 Summary of criteria to identify mesenchymal stromal cells 1. Adherence to plastic in standard culture conditions 2. Phenotype Positive (≥95% +) CD105 CD73 CD90
3. Differentiation: (by staining of in vitro cell culture)
Negative (80) 85 (45) 78 (24)
77 (45) 73 (53) 96 (39) 88 (35) 42 (58) 75 (39)
0 (>80) 8 (>80) 8 (>80) 0 (>80) 7 (>80) 0 (>80)
Data were pooled from more than 20 experiments with totals of 15–39 mice for each strain combination. Donor marrow cells (4 × 106) were T-cell depleted and injected intravenously along with unseparated (whole) T cells or T-cell subsets (1 × 106 cells) into appropriate lethally irradiated (8–10 Gy) recipient mice. (Adapted from [56].)
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intensity of GVHD [91], nor does injecting the host mice with anti-CD4 antibody [97]. These findings imply that the capacity of CD8+ cells to mediate GVHD to mHAs does not necessarily require help from CD4+ cells. Yet, in some strain combinations, like B6 → BALB.B, the CD8+ cells will only mediate GVHD in the presence of CD4+ cells [98]. The criteria which dictate CD8+ cell dependency on exogenous help appear to involve the cytokines available to APCs and their ability to express the 4-1BB ligand molecule, which can bind to its counterpart (4-1BB; CD137) on CD8+ T cells and serve as a co-stimulator for activation and survival [99]. In an effort to investigate the heterogeneity of the T-cell response across a donor–recipient pair disparate at multiple mHA loci, the repertoire of responding CD8+ T cells in the B6 → BALB.B (8.25Gy) GVHD model was analyzed by TCR Vβ CDR3-size polymerase chain reaction spectratyping. This response was found to be oligoclonal in nature, involving T cells from seven Vβ families [79]. With the exception of a single Vβ family, these responses were also found after transplantation of B6 CD8+ T cells into irradiated CXBE mice, a recombinant inbred strain that is derived from a cross between B6 and BALB/c mice and therefore expresses a subset of the mHA found in the BALB.B strain. This overlap in the CD8+ T-cell response repertoire suggested that the phenomenon of competitive immunodominance was not a factor in the recognition of mHAs involved in the development of GVHD. In addition, it was demonstrated that lethal GVHD could be induced by the transplantation of T cells from a single purified B6 CD8+ Vβ family into either irradiated BALB.B or CXBE mice, supporting their involvement in disease pathogenesis and suggesting a possible hierarchy of GVHDmediating specificities within the responding T-cell population [79].
Role of CD4+ cells In the case of the CBA → B10.BR combination and certain other combinations, CD4+ cells play no obvious role in GVHD. The helper function of these cells is not required, and transferring even high doses of purified CD4+ cells generally fails to cause GVHD. Nevertheless, in two of six combinations tested from this panel, namely B10.D2 → BALB/c and B10.D2 → DBA/2, purified CD4+ cells do cause a high incidence of lethal GVHD [90,100] (Table 14.1). This is also true in the B6 → BALB.B combination, in which CD4+ T cells are required for CD8mediated GVHD and can cause GVHD on their own account [98]. Why CD4+ cells mediate GVHD in only a limited number of mHA-different strain combinations is obscure. Insight into this problem has been generated from studying the B6 CD4+-mediated GVHD response in BALB.B versus CXBE recipients, where BALB.B, but not CXBE, mice succumb to lethal GVHD despite sharing multiple mHAs. A comparison of the responding CD4+ TCR Vβ repertoires from the thoracic duct lymph of irradiated BALB.B and CXBE recipients was made at the early stages of GVHD development (5–6 days) [101]. The spectratype analysis revealed overlapping utilization of nine Vβ families. In addition, the unique skewing of two Vβ families in the B6 → BALB.B response suggested the recognition of BALB.B mHAs not expressed in the CXBE recipients. Interestingly, the B6 → CXBE strain combination also exhibited unique skewing of two other Vβ families, which may hint at “immunodominance effects,” since one would have expected them to also be present in the B6 → BALB.B response. Direct evidence that Vβ families found skewed by spectratype analysis were involved in the pathogenesis of GVHD came from immunohistochemical staining of lingual epithelial tissue from BALB.B recipients, which revealed a high correlation between several of the skewed Vβ families and those with increased representation in the epithelial tissue infiltrates [101]. Further proof that the skewed Vβ families were indeed involved with GVHD was provided upon transplantation of those CD4+
Vβ families into irradiated BALB.B recipients, which resulted in a significant level of lethal GVHD. In contrast, mice that received transplants of the unskewed Vβ families survived with minimal symptoms of disease [101]. Applying the above observations concerning differences in responding Vβ repertoires, the capacity of B6 CD4+ T cells to induce lethal GVHD in BALB.B but not CXBE recipients was further examined. Transplantation of highly enriched populations of the two B6 CD4+ Vβ families, that were uniquely skewed in the B6 → BALB.B response, i.e. Vβ2 and Vβ11, were indeed able to induce lethal GVHD in BALB.B recipients. These data suggested that the BALB.B unique responses contribute in a way that either qualitatively or quantitatively enhances the severity of GVHD. Subsequent data suggested that these cells might preferentially infiltrate the small bowel of BALB.B recipients, thus increasing their importance to the systemic severity of GVHD (Fig. 14.3) [102]. Further studies revealed that the B6 Vβ11 CD4+ T cells, alone, could mediate severe GVHD, and immunohistochemical staining of host lingual and intestinal epithelial tissues supported their capacity to infiltrate typical GVHD-associated target areas [80]. To further characterize the specific CD4+ Vβ11+ T cells involved in this anti-mHA response, TCR Vβ spectratype analysis was performed, and indicated that six Vβ chains were used by this reactive population [80]. Probing deeper into this limited Vβ response, using a nonpalindromic adaptor polymerase chain reaction method, it was found that there was dominant use of the TRAV13–TRAJ16 transcript combination [103]. Then, using laser capture microdissection, the identical TRAV– TRAJ nucleotide sequence was found in areas dominated by the infiltrating Vβ11(+) CD4(+) T cells during the development of GVHD, both in the rete-like prominences of the dorsal lingual epithelium and the ileal crypts of the small intestine [103]. These combined results provided evidence that a restricted repertoire of T-cell specificities, presumably recognizing a correspondingly low number of mHAs, was sufficient for the induction of severe GVHD. In order to investigate whether the scope of the mHA-driven alloresponse changes during the development of GVHD, the responding B6 T-cell repertoire was examined between days 7 and 40 post HCT in the B6 → BALB.B strain combination. The results indicated that for the CD8 response, eight Vβ families were consistently skewed throughout the period of observation. It is interesting that skewing in four of those Vβ families had not been detected earlier in the thoracic duct population when examining the single day 5 time point [79]. In addition, skewing of three other Vβ families appeared to develop at later stages post transplant, which could indicate weaker anti-mHA responses, responses to antigens revealed via tissue destruction, or responses to nonalloantigen, such as to infectious agents. Similar findings were obtained for the CD4 analysis. Eight Vβ families were skewed throughout the course of GVHD development, all of which had been detected earlier in the thoracic duct lymph at day 6 post transplant [101]. Four additional Vβ families were transiently skewed at later time points. Furthermore, elimination of the eight consistently skewed Vβ families from the CD4+ T-cell donor inoculum delayed the onset in the appearance of most of the remaining four responsive Vβ families. The reason for this delayed appearance is unclear but would be consistent with them resulting from tissue damage, which would likely be slowed in the absence of dominant GVHD effector cells. These complex relationships in the development of anti-mHA GVHD are in obvious need of further investigation. One further aspect of the Vβ repertoire studies that remains unclear is the redundant capacity of a single mHA to drive the response of multiple Vβ families via cross-reactive TCR-binding sites. Insight gained into this issue will provide clues as to the minimum number of mHAs required to elicit GVHD in an MHC-matched combination and
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Fig. 14.3 GVHD potential of B6 anti-BALB.B Vβ2+ and 11+ CD4+ T cell responses. BALB.B and CXB-2 mice were lethally irradiated and injected with 2 × 106 B6 anti-thy1-depleted bone marrow (ATBM) cells alone, or in combination with either 2 × 107 unseparated B6 CD4+ T cells, an equal number of Vβ2- and 11-depleted (Vβ2/11−) CD4+ T cells, or 1.1 × 106 Vβ2and 11-enriched (Vβ2/11+) CD4+ T cells (mean purity 91%). (a) Survival of transplanted recipients. (BALB.B CD4 versus BALB.B Vβ2/11+; p ≥ 0.14). (b) Body weights for each group normalized as the mean ± standard error of the mean % initial body weight during sequential 1-week periods. HCT, hematopoietic cell transplantation. (Adapted from [84], with permission.)
why some mHA dominate the responses. Overall, the study of Vβ representation in a heterogeneously responding T-cell population in this acute GVHD model has begun to provide valuable insight into the scope of the allogeneic anti-mHA T-cell response.
Approaches for the separation of GVHD and graft-versus-leukemia responses The particular goal of any effort in this area is to provide a mechanism for reducing GVHD potential without sacrificing GVL or the more generalized graft-versus-tumor capability of donor T cells. Various
Fig. 14.4 Vβ7-enriched CD4+ T cells mediate a graft-versus-leukemia response with minimal induction of graft-versus-host disease in an allogeneic bone marrow transplantation model. Lethally irradiated (B6 × DBA/2)F1 mice were injected with 2 × 106 B6 anti-thy1-depleted bone marrow (ATBM) cells and 2.5 × 105 MMB3.19-primed Vβ7-enriched (82%) B6 CD4+ T cells or an equal number of unfractionated CD4+ T cells. The mice received MMB3.19 challenge (2 × 104; intraperitoneally) 1 day post bone marrow transplantation. Spectratype analysis had previously indicated that B6 Vβ7+ T cells were reactive to the MMB3.19 tumor cells, but not to stimulation by host (B6 × DBA/2)F1 alloantigen. (Adapted from [86], with permission).
strategies have been utilized to minimize GVHD in murine models, such as focusing on providing only CD8+ [104–107] or CD4+ [108, 90] T-cell subsets, with or without retention of GVL activity. The subset approach for separation of the two processes has also been studied for donor lymphocyte infusion models [109]. Restricting donor T cells to effector/memory subpopulations, either CD62L− or CD44hi, has also proved capable of preventing GVHD and often without impairment of GVL responses [110–113]. Likewise, and possibly related to the induction of memory cells, allogeneic sensitization of donor T cells to either leukemia cells or host cells can amplify GVL responses with reduced GVHD [114]. Results for all of these investigations may vary, depending upon the histoincompatibility involved and the specific tumor type used. Other approaches involve ex vivo tolerization of alloreactive T cells [115,116], separation of donor T cells based on functional phenotypes [117,118], and cytolytic effector mechanisms [119,120]. Cytokines, their promoters or their inhibitors have also been administered to tip the balance of donor T-cell responses towards GVL [121–127]. Establishing mixed chimerism with nonmyeloablative conditioning regimens has also been used as a means of reducing GVHD effects and allowing GVL responses with either donor-derived [128] or recipient-derived [129] delayed donor lymphocyte infusion. The alloreactive specificity of the donor T cells, as well as tissuespecific expression of host mHA or tumor-specific antigens, can also lead to a more targeted immunotherapeutic approach for minimizing GVHD development [130–132]. Vβ spectratype analysis has also been used to identify antihost alloreactive and antileukemia-specific responses and guide selected immunotherapy for the latter with GVHD minimization (Fig. 14.4) [133]. The high inverse correlation between the clinical incidence of GVHD and the rate of leukemic relapse has long suggested that antihost alloreactivity may provide the dominant GVL effect [134]. Therefore, strategies that would involve permitting the initiation of GVHD, presumably with an associated concurrent GVL response that could then be brought under control to avoid full pathologic development of disease, would be highly desirable. As mentioned earlier, several studies using CD4+CD25+ Treg or other suppressor type cells have shown some promise in accomplishing this goal [44,46–48].
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Immunopathology As for anti-H-2 GVHD, the effector mechanisms involved in GVHD to mHAs are still not fully understood. The histopathology of GVHD mediated by CD4+ cells and CD8+ cells is quite similar in the major target tissues of the skin, liver, and intestinal tract, although gut pathology is more prominent in recipients of CD4+ cells [96]. In the skin, epidermal cytotoxicity is induced by both subsets, albeit by different pathways, but dermal fibrosis seems to be a unique aspect of CD8-mediated GVHD. Mast cell degranulation occurs in the skin early after transplantation of either subset, even before lymphocyte infiltration is detected [135]. In addition, TNF-α (from either mast cells or T cells) does not appear to play a role in initial keratinocyte damage associated with early GVHD stages, but is involved in later CD4-mediated pathology, once the cells have infiltrated into the epidermal layers. In the case of CD8+ cells, it would seem that tissue damage, at least in the skin, reflects primarily direct CTL activity, although cytokines other than TNF-α may contribute to pathology. It has also been established that apoptosis has a central role in epithelial injury in acute GVHD [136], and that the primary targets of attack are phenotypically and antigenically distinctive epithelial cells within the basal layer of the skin and squamous mucosa [137,138], potentially corresponding to cytokeratin-15-expressing follicular stem cells (Plate 14.1) [139]. These cells are confined to rete ridges in the skin and
rete-like prominences in the dorsal tongue, and their discovery raises questions about their central role in driving the GVHD response forward. In all, the course and kinetics of pathogenesis in GVHD to mHAs may depend on several variables, including the strength of the individual CD4+ and CD8+ cell responses, the levels of expression of antigens in target tissue and the fluctuations of cytokines, both locally and systemically.
Summary Studies in murine models have provided many important insights into the pathophysiology of GVHD directed across MHC and/or mHA disparities. We now have a workable understanding of the T-cell subsets that can be involved in any given situation of GVHD and clues as to their mechanisms of action. These and future studies in the mouse can help guide us in the development of novel approaches to selectively reduce the risk of GVHD while allowing for the generation of GVL responses that can counteract the relapse of leukemia.
Acknowledgment The writing of this chapter was supported by grants HL55593 and HL75622 from the United States Public Health Service.
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73. Goulmy E. Minor histocompatibility antigens: from T cell recognition to peptide identification. Hum Immunol 1997; 54: 8–14. 74. Korngold R, Sprent J. Lethal graft-versus-host disease after bone-marrow transplantation across minor histocompatibility barriers in mice. Prevention by removing mature T cells from marrow. J Exp Med 1978; 148: 1687–98. 75. Hamilton BL, Bevan MJ, Parkman R. Antirecipient cytotoxic T lymphocyte precursors are present in the spleens of mice with acute graftversus-host disease due to minor histocompatibility antigens. J Immunol 1981; 126: 621–5. 76. Korngold R, Leighton C, Mobraaten LE, Berger MA. Inter-strain graft-versus-host disease T cell responses to immunodominant minor histocompatibility antigens. Biol Blood Marrow Transplant 1997; 3: 57–64. 77. Blazar BR, Roopenian DC, Taylor PA, Christianson GJ, Panoskaltsis-Mortari A, Vallera DA. Lack of GVHD across classical, single minor histocompatibility (miH) locus barriers in mice. Transplantation 1996; 61: 619–24. 78. Perreault C, Jutras J, Roy DC, Filep JG, Brochu S. Identification of an immunodominant mouse minor histocompatibility antigen (MiHA). T cell response to a single dominant MiHA causes graft-versushost disease. J Clin Invest 1996; 98: 622–8. 79. Friedman TM, Gilbert M, Briggs C, Korngold R. Repertoire analysis of CD8+ T cell responses to minor histocompatibility antigens involved in graft-versus-host disease. J Immunol 1998; 161: 41–8. 80. DiRienzo CG, Murphy GF, Jones SC, Korngold R, Friedman TM. T-cell receptor Valpha spectratype analysis of a CD4-mediated T-cell response against minor histocompatibility antigens involved in severe graft-versus-host disease. Biol Blood Marrow Transplant 2006; 12: 818–27. 81. Berger MA, Korngold R. Immunodominant CD4+ T cell receptor Vb repertoire involved in graftversus-host disease responses to minor histocompatibility antigens. J Immunol 1997; 159: 77–85. 82. Howell CD, Li J, Roper E, Kotzin BL. Biased liver T cell receptor Vb repertoire in a murine graft-versus-host disease model. J Immunol 1995; 155: 2350–8. 83. Brochu S, Baron C, Hetu F, Roy DC, Perreault C. Oligoclonal expansion of CTLs directed against a restricted number of dominant minor histocompatibility antigens in hemopoietic chimeras. J Immunol 1995; 155: 5104–14. 84. Perreault C, Roy DC, Fortin C. Immunodominant minor histocompatibility antigens: the major ones. Immunol Today 1998; 19: 69–74. 85. Korngold R, Wettstein PJ. Immunodominance in the graft-vs-host disease T-cell response to minor histocompatibility antigen. J Immunol 1990; 145: 4079–88. 86. Nevala WK, Wettstein PJ. H4 and CTT-2 minor histocompatibility antigens: concordant genetic linkage and migration in two-dimensional peptide separation. Immunogenetics 1996; 44: 400–4. 87. Malarkannan S, Shih PP, Eden PA et al. The molecular and functional characterization of a dominant minor H antigen, H60. J Immunol 1998; 161: 3501–9. 88. Malarkannan S, Horng T, Eden PA et al. Differences that matter: major cytotoxic T cellstimulating minor histocompatibility antigens. Immunity 2000; 3: 333–44.
89. Wettstein PJ. Immunodominance in the T-cell response to multiple non-H2 histocompatibility antigens. II. Observation of a hierarchy among dominant antigens. Immunogenetics 1986; 24: 24–31. 90. Korngold R, Sprent J. Variable capacity of L3T4+ T cells to cause lethal graft-versus-host disease across minor histocompatibility barriers in mice. J Exp Med 1987; 165: 1522–64. 91. Korngold R, Sprent J. Lethal GVHD across minor histocompatibility barriers: nature of the effector cells and role of the H2 complex. Immunol Rev 1983; 71: 5–29. 92. Jaffee BD, Claman HN. Chronic graft-versus-host disease (GVHD) as a model for scleroderma. I. Description of model systems. Cell Immunol 1983; 77: 1–12. 93. Ferrara J, Guillen FJ, Sleckman B, Burakoff SJ, Murphy GF. Cutaneous acute graft-versus-host disease to minor histocompatibility antigens in a murine model. Histologic analysis and correlation to clinical disease. J Invest Dermatol 1986; 123: 401–6. 94. Rappaport H, Khalil A, Halle-Pannenko O, Pritchard L, Dantcher D, Mathe G. Histopathologic sequence of events in adult mice undergoing lethal graft-vs-host reaction developed across H2 and/or non H2 histocompatibility barriers. Am J Pathol 1979; 96: 121–43. 95. Charley MR, Bangert JL, Hamilton BL, Gilliam JN, Sontheimer RD. Murine graft-versus-host skin disease. A chronologic and quantitative analysis of two histologic patterns. J Invest Dermatol 1983; 81: 412–17. 96. Murphy GF, Whitaker D, Sprent J, Korngold R. Characterization of target injury of murine acute graft-versus-host disease directed to multiple minor histocompatibility antigens elicited by either CD4+ or CD8+ effector cells. Am J Path 1991; 138: 983–90. 97. Korngold R. Lethal graft-versus-host disease in mice directed to multiple minor histocompatibility antigens. Features of CD8+ and CD4+ T-cell responses. Bone Marrow Transplant 1992; 9: 355–64. 98. Berger M, Wettstein PJ, Korngold R. T cell subsets involved in lethal graft-versus-host disease directed to immunodominant minor histocompatibility antigens. Transplantation 1994; 57: 1095–102. 99. Kwon B, Moon CH, Kang S, Seo SK, Kwon BS. 4-1BB: still in the midst of darkness. Mol Cells 2000; 10: 119–26. 100. Hamilton BL. L3T4-positive T cells participate in the induction of graft-vs-host disease in response to minor histocompatibility antigens. J Immunol 1987; 139: 2511–15. 101. Friedman TM, Statton D, Jones SC, Berger MA, Murphy GF, Korngold R. Vb spectratype analysis reveals heterogeneity of CD4+ T-cell responses to minor histocompatibility antigens involved in graft-versus-host disease: correlations with epithelial tissue infiltrate. Biol Blood Marrow Transplant 2001; 7: 2–13. 102. Jones SC, Friedman TM, Murphy GF, Korngold R. Specific donor Vbeta-associated CD4 T-cell responses correlate with severe acute graft-versushost disease directed to multiple minor histocompatibility antigens. Biol Blood Marrow Transplant 2004; 10: 91–105. 103. DiRienzo CG, Murphy GF, Friedman TM, Korngold R. T-cell receptor V(alpha) usage by
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Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect 118. Fowler DH, Breglio J, Nagel G, Eckhaus MA, Gress RE. Allospecific CD8+ Tc1 and Tc2 populations in graft-versus-leukemia effect and graftversus-host disease. J Immunol 1996; 157: 4811–21. 119. Hsieh MH, Varadi G, Flomenberg N, Korngold R. Leucyl-leucine methyl ester-treated haploidentical donor lymphocyte infusions can mediate graft-versus-leukemia activity with minimal graftversus-host disease risk. Biol Blood Marrow Transplant 2002; 8: 303–15. 120. Schmaltz C, Alpdogan O, Horndasch KJ et al. Differential use of Fas ligand and perforin cytotoxic pathways by donor T cells in graft-versushost disease and graft-versus-leukemia effect. Blood 2001; 97: 2886–95. 121. Sykes M, Harty MW, Szot GL, Pearson DA. Interleukin-2 inhibits graft-versus-host diseasepromoting activity of CD4+ cells while preserving CD4- and CD8-mediated graft-versus-leukemia effects. Blood 1994; 83: 2560–9. 122. Krijanovski OI, Hill GR, Cooke KR et al. Keratinocyte growth factor separates graft-versusleukemia effects from graft-versus-host disease. Blood 1999; 94: 825–31. 123. Hill GR, Teshima T, Gerbitz A et al. Differential roles of IL-1 and TNF-alpha on graft-versus-host disease and graft versus leukemia. J Clin Invest 1999; 104: 459–67. 124. Teshima T, Hill GR, Pan L et al. IL-11 separates graft-versus-leukemia effects from graft-versushost disease after bone marrow transplantation. J Clin Invest 1999; 104: 317–25. 125. Reddy P, Teshima T, Hildebrandt G et al. Pretreatment of donors with interleukin-18 attenuates acute graft-versus-host disease via STAT6 and preserves graft-versus-leukemia effects. Blood 2003; 101: 2877–85.
126. Korngold R, Marini JC, de Baca ME, Murphy GF, Giles-Komar J. Role of tumor necrosis factoralpha in graft-versus-host disease and graftversus-leukemia responses. Biol Blood Marrow Transplant 2003; 9: 292–303. 127. Durakovic N, Radojcic V, Powell J, Luznik L. Rapamycin promotes emergence of IL-10-secreting donor lymphocyte infusion-derived T cells without compromising their graft-versusleukemia reactivity. Transplantation 2007; 83: 631–40. 128. Pelot MR, Pearson DA, Swenson K et al. Lymphohematopoietic graft-vs.-host reactions can be induced without graft-vs.-host disease in murine mixed chimeras established with a cyclophosphamide-based nonmyeloablative conditioning regimen. Biol Blood Marrow Transplant 1999; 5: 133–43. 129. Rubio MT, Kim YM, Sachs T, Mapara M, Zhao G, Sykes M. Antitumor effect of donor marrow graft rejection induced by recipient leukocyte infusions in mixed chimeras prepared with nonmyeloablative conditioning: critical role for recipient-derived IFN-gamma. Blood 2003; 102: 2300–7. 130. Anderson LD Jr, Savary CA, Mullen CA. Immunization of allogeneic bone marrow transplant recipients with tumor cell vaccines enhances graft-versus-tumor activity without exacerbating graft-versus-host disease. Blood 2000; 95: 2426– 33. 131. Fontaine P, Roy-Proulx G, Knafo L, Baron C, Roy DC, Perreault C. Adoptive transfer of minor histocompatibility antigen-specific T lymphocytes eradicates leukemia cells without causing graftversus-host disease. Nat Med 2001; 7: 789–94. 132. Meunier MC, Delisle JS, Bergeron J, Rineau V, Baron C, Perreault C. T cells targeted against a
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15
Megan Sykes
Mechanisms of Tolerance
Introduction Immune tolerance to a set of antigens can be defined as a state in which the immune system does not mount a destructive response to organs or tissues expressing those antigens but is capable of responding normally to foreign antigens. Tolerance may occur in several different immunologic states. Tolerance can be “operational,” in which, for example, a graft is accepted without immunosuppressive therapy but a second graft from the same donor is rejected. In a slightly more robust form of tolerance, a second graft of the same type from the same donor would be accepted, but a more immunogenic graft from that donor would be rejected. In an even more robust type of tolerance, any organ bearing the same set of alloantigens as the original graft would be accepted. Finally, tolerance may be systemic, meaning that the animal’s entire immune system is tolerant to that set of antigens, so that a response cannot be induced to them either in vivo or in vitro. In allogeneic hematopoietic cell transplantation (HCT), persistent graft survival and the absence of graft-versus-host disease (GVHD) depend on a state of mutual tolerance of the donor to the host (graftversus-host, or GVH, tolerance), and of the host to the donor (hostversus-graft, or HVG, tolerance). Unfortunately, this state is not always achieved, and GVHD or failure of engraftment, respectively, ensues. Development of tolerance in the HVG direction requires host conditioning that either eliminates mature host immune cells, creating an immunologic “clean slate,” or permits pre-existing T cells to be rendered tolerant by the donor hematopoietic cells. Since newly developing T and B lymphocytes have a unique capacity to be rendered tolerant by antigens they encounter during their maturation, especially on hematopoietic cells, these lymphocytes can be educated to recognize an engrafted donor and host antigens as “self,” so that a state of donor- and hostspecific tolerance results. The ability of hematopoietic stem cells (HSCs) to induce a state of donor-specific tolerance suggests an additional potential application for HCT, namely the induction of organ allograft acceptance without the need for chronic immunosuppressive therapy. Such a state of immune tolerance would circumvent several major limitations to the practice of organ and tissue transplantation. In particular, improvements in immunosuppressive therapy have greatly augmented early graft survival, but these have had little impact on late graft loss, which is due in large part
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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to chronic rejection. Moreover, a high incidence of malignancies and opportunistic infections as well as drug-specific metabolic and other toxicities severely limit the tolerability of long-term chronic immunosuppressive therapy. The induction of donor-specific immune tolerance would avoid these complications while also preventing chronic rejection. Reliable, nontoxic methods of achieving allogeneic HSC engraftment across major histocompatibility complex (MHC) barriers will be required before HCT can be routinely used for the induction of organ or tissue allograft tolerance. However, recent progress in experimental systems has brought this approach into pilot clinical trials. A shortage of allogeneic donors has become another major limitation to solid organ transplantation, resulting in considerable interest in xenotransplantation (i.e. transplantation of organs from other species) to augment organ and tissue availability. However, it has become increasingly clear that the immunologic barriers to xenografts are even stronger than those resisting allografts, making it unlikely that an acceptable amount of nonspecific immunosuppressive therapy could be used to prevent xenograft rejection. Thus, it will be especially critical to develop methods of inducing donor-specific tolerance across xenogeneic barriers in order to make xenotransplantation efficacious. Because of its demonstrated efficacy in experimental models, and because of recent developments in our understanding of the mechanisms by which HCT can induce allo- and xenotolerance, this approach may have the greatest potential to make tolerance induction a routine part of clinical organ transplantation. This chapter will first provide a general discussion of mechanisms of T-cell tolerance, and then discuss these mechanisms in the context of specific models associated with induction of HVG tolerance in the absence or presence of HCT. The mechanisms by which both mature T cells pre-existing in allogeneic HCT inocula as well as T cells developing de novo from donor hematopoietic cells can be rendered tolerant of host antigens will also be reviewed.
Mechanisms of T-cell tolerance Three major mechanisms have been proposed to explain the induction and/or maintenance of T-cell tolerance to self or alloantigens: clonal deletion, clonal anergy, and active suppression. For developing T cells, these processes take place in the thymus, which is the central organ for T-cell development. Hence, induction of tolerance among developing thymocytes is referred to as “central,” as distinguished from the “peripheral” tolerance that may develop among already mature T cells when they encounter antigen in the peripheral tissues. It will become apparent to the reader from the discussion that follows that overlapping
Mechanisms of Tolerance
mechanisms have been implicated in models involving both central and peripheral tolerance induction. Clonal deletion T-cell receptors (TCR), consisting of uniquely rearranged α and β chains, known as αβTCR, are the cell surface heterodimers that recognize complexes of MHC antigens plus peptide. The thymus plays a critical role in the development of tolerance by a clonal deletion mechanism. Deletion occurs during T-cell maturation when the avidity of an interaction between an immature thymocyte and an antigen-presenting cell (APC) in the thymus is sufficiently high to induce apoptotic cell death [1]. The concept of avidity includes variations in the intensity of T-cell signaling resulting from variations in TCR affinity and density and ligand density, and from interactions of accessory molecules with their ligands on APCs. High avidity of the overall T cell–APC interaction is due, at least in part, to a relatively high affinity interaction between a rearranged αβTCR and a self-peptide–MHC complex presented by an APC in the thymus. Several marrow-derived cell types, including dendritic cells (DCs), B cells, and thymocytes, as well as nonhematopoietic cells of the thymic stroma, have the capacity to induce intrathymic tolerance by both deletional and nondeletional mechanisms, but DCs may be the most potent deletional APCs in the thymus (reviewed in [2]). Evidence for clonal deletion as a mechanism of tolerance was originally obtained by using monoclonal antibodies (mAbs) that are specific for certain V-gene products contributing to the β chain of the αβTCR [3]. Certain Vβ gene products selectively recognize “superantigens” encoded by endogenous retroviruses in the genome of mammalian species. Superantigens bind to a unique site on the class II MHC molecule. If a particular Vβ is capable of recognizing a superantigen, developing thymocytes with TCR using these Vβ undergo deletion [3]. These observations allowed the original demonstration that self-tolerance occurs largely through an intrathymic deletional mechanism, and that tolerance to marrow donors also occurs through a similar mechanism when allogeneic bone marrow transplantation (BMT) is performed in mice [3]. Later data supporting clonal deletion as a major mechanism for induction of self-tolerance utilized TCR transgenic mice expressing TCRs specific for antigens expressed naturally in the animal or by bone marrow grafts. Deletion is not unique to immature stages of T-cell development. Peripheral deletion has been described for mature T cells upon exposure to antigen in vivo [4]. Activation-induced cell death, which leads to partial deletion of expanded T-cell clones, is a normal physiologic consequence of high levels of T-cell activation. Deletion is also the natural consequence of cross-presentation by lymph node DCs of self-antigen expressed in parenchymal tissues to CD8+ cytotoxic lymphocytes (CTLs), under noninflammatory conditions [5]. Peripheral CD8 cells may undergo clonal deletion through “exhaustion” in the presence of a large, persistent antigen load, which may explain the anergy and deletion observed for host-reactive CTLs in the context of GVHD [6]. As discussed below, exposure to alloantigens in the presence of co-stimulatory blockade can lead to peripheral deletion of alloreactive T cells by mechanisms that are incompletely understood. In addition, veto cells and “double-negative” T cells (see below) can delete alloreactive CTL precursors in the peripheral tissues. T-cell anergy Studies on mechanisms of tolerance often include functional data, such as limiting dilution analyses to quantify CTLs, helper T cells or T cells proliferating in response to antigen. However, the failure to detect T
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cells with a particular allospecificity using this approach does not distinguish between clonal deletion and functional inactivation, or so-called “anergy.” Anergy may result when T cells encounter antigens without receiving adequate accessory or co-stimulatory signals. Anergic lymphocytes cannot be fully activated by encounter with the antigen that its clonally distributed surface receptor (surface immunoglobulin [Ig] in the case of the B-cell receptor, and the TCR in the case of the T cell) specifically recognizes. In the case of T cells, anergy is associated with a lack of proliferation and interleukin-2 (IL-2) production, and can usually, but not always, be overcome by providing exogenous IL-2. T-cell anergy is associated with altered signaling and tyrosine phosphorylation patterns [7]. Numerous methods of inducing T-cell anergy in vitro have been described, many of which involve blocking of molecular interactions between T-cell surface molecules and their ligands on APCs. Some of these interactions are termed “co-stimulatory signals” because they provide signals to the responding T cell, other than those transmitted through the TCR–CD3 complex, that are essential to obtaining a full state of activation [7,8]. These molecules, including CD28, inducible co-stimulator, OX40, 4-1BB, and several additional co-stimulatory pathways, have been targets of attempts to induce tolerance by presenting allo- or xenoantigens without adequate co-stimulation (see below). T-cell anergy has, in some instances, been associated with TCR downregulation [4,9], and a similar phenomenon has been associated with B-cell anergy [10]. In addition to the encounter of antigen without co-stimulation, T cells may also be rendered anergic by an encounter with peptide ligands for which they have low affinity [1]. An overall low avidity interaction between a T cell and an APC may also result in anergy [11]. It appears that, in addition to mature peripheral T cells, thymocytes are also susceptible to anergy induction, and this can be induced by antigens presented on both hematopoietic and nonhematopoietic stromal [12] cells. Anergy can be reversed in vivo and can be overcome by infection or by removal of antigen. Anergy can be induced in vivo by nonhematopoietic cells. In hematopoietic chimeras prepared with recipient myeloablation followed by allogeneic BMT, all marrow-derived cells are of donor origin, so that clonal deletion of thymocytes recognizing host-specific antigens is incomplete [12]. T cells bearing these host-reactive TCRs can be anergized by thymic epithelial cells [13] or by antigens encountered extrathymically [14–16]. T-cell clones with specificity for particular alloantigens can exist without causing destruction of tissue bearing those antigens [11,13,14,16]. In some instances, T cells with receptors specific for the in vivo tolerated antigen can be stimulated to respond to the antigen in vitro [13,14,17] or may even induce a state of inflammation that does not lead to organ destruction in the absence of additional stimuli [11]. In other systems, the T cells are fully or partially refractory to stimulation through their TCRs in vitro [11,14]. However, anergy is not the only mechanism that can account for the failure of T cells with self-reactive or alloreactive TCRs to cause autoimmunity or graft rejection, respectively. In the situation where T cells respond to antigens in vitro but not in vivo, they seem to be able simply to ignore antigens presented in the periphery [9]. This may be due to the presentation of these antigens by “nonprofessional APCs” that are unable to activate T cells, or it may reflect a failure of recipient T cells to migrate to the antigen-bearing tissue [18]. The level of peripheral antigen expression, the recency of T-cell emergence from the thymus [9], and the presence or absence of proinflammatory cytokines and costimulatory molecules within peripheral tissues may all influence the decision of a T cell to ignore or respond to peripheral antigens. T-cell activation may even occur within a tissue without initiating the whole cascade of organ damage [11]. However, proinflammatory stimuli, as
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may be present due to infection, may overcome such precarious states [9]. Some experimental models in which transplantation tolerance has been induced include T-cell anergy in association with a persistent suppressive T-cell population [19]. However, in most of these models, including those involving co-stimulatory blockade and other approaches designed to render APCs non-costimulatory, anergy has not been shown clearly to be the sole mechanism maintaining tolerance in vivo. Suppression A third mechanism, active suppression, has also been implicated in the induction and maintenance of self- and allotolerance. Suppressive activity has been attributed to both T cells and non-T cells, and may be antigen-specific or nonspecific. In general, the models in which suppressor T cells have been implicated involve exposure to antigen in the presence of a pre-existing immune response to the antigen. When, on the other hand, all mature lymphocytes are eliminated prior to administration of the antigen, tolerance may be due mainly to clonal deletion or anergy mechanisms and not to active suppression [20]. In the ensuing discussion, we categorize mechanisms of suppression in terms of the degree of specificity of their ability to suppress immune responses. Specific suppression While functional evidence for the existence of specific suppressor cells had been obtained in several transplantation models [21,22], until the last decade there had been little success in identifying or cloning suppressive cells, or in understanding the mechanisms by which they suppressed. In early studies, anti-idiotypic recognition, which is the recognition of determinants specifically expressed by a rearranged T cell or Ig receptor, was implicated [21,22]. However, apparent idiotypic specificity can be difficult to distinguish from suppression by T cells with the same specificity as the T cell being suppressed. The importance of regulatory phenomena in self-tolerance and regulation of autoimmunity, and in certain transplantation models, has recently become very clear. Some of these suppressive cell types are briefly described in the ensuing paragraphs. T cells comprise several major subsets of regulatory cells. Regulatory T cells (Tregs) can be classified as “naturally arising” and “antigen driven” [23,24]. Naturally arising Tregs are generated in the postnatal thymus and constitutively express CD25 (IL-2 receptor α chain) as well as CTLA4 [25]. A deficiency in “Treg” numbers in neonatally thymectomized mice permits the development of multiorgan autoimmune disease in susceptible strains [25]. Tregs require nonhematopoietic thymic stromal cells for their selection, and expression of the forkhead winged helix transcription factor 3 (FoxP3) is essential for full differentiation and maintenance of their regulatory cell features, including failure to produce IL-2 and dependence on paracrine IL-2 [26]. While it has been suggested that Tregs are positively selected on cortical epithelial cells of the thymus [27], recent studies suggest that the thymic medulla may be the major site of their positive selection [28]. Once in the periphery, these Tregs can be expanded in the presence of the positively selecting peptide antigen [29]. While some studies have suggested that the TCR repertoire of Tregs leaving the thymus is skewed toward greater affinity for self-antigens than that of conventional T cells, recent studies challenge this conclusion and suggest that the repertoire and specificities of both subsets may be quite similar [30]. Tregs play an important role in preventing autoimmune disease throughout adult life, as illustrated by the development of severe autoimmunity when FoxP3+ cells are selectively depleted in vivo. Their importance in preventing autoimmunity in humans is illustrated by congenital FoxP3 deficiency, which results in immune dysregulation, polyendocri-
nopathy, enteropathy, X-linked (IPEX) syndrome [31]. The regulatory cells are themselves hyporesponsive to TCR-mediated stimulation alone, and require IL-2 for their development and expansion [32]. IL-15 can also promote their expansion [33]. Transforming growth factor-β (TGFβ) also plays an important role in maintenance of their function via maintenance of FoxP3 expression [26]. Suppression seems to require cell-to-cell contact [25]. CD25+CD4+ Tregs express the memory T cellassociated CD45 isoform (CD45RC+ in the rat, CD45RBlow in the mouse). Although CD4 cells have been the best-studied targets of regulation by these cells, they can also suppress CD8 T-cell reactivity. While the Tregs show specificity for the antigen to which they are inducing suppression, the effector mechanism of suppression is non-antigen specific [25]. The immunosuppressive drug rapamycin appears to allow Treg survival and expansion while suppressing effector T-cell responses [34]. Antigen-driven Tregs include murine and human CD4 cells stimulated through their TCRs in the presence of IL-10. These “Tr1” cells produce high levels of IL-10 and TGF-β without making IL-4, distinguishing them from T helper cells type 2 (Th2) [35]. These cells, which do not constitutively express FoxP3, suppress antigen-specific responses in vitro and can suppress inflammatory bowel disease syndrome induced by naïve CD4 cells in mice. Immature or specialized IL-10-secreting DCs promote Tr1 differentiation [35]. In addition to Tr1 cells, antigen exposure can also result in the differentiation of CD25−CD4 cells to FoxP3+ Tregs [36–38]. This differentiation appears to be critically dependent on TGF-β and is inhibited by the presence of IL-6, which, in combination with TGF-β, promotes the differentiation of proinflammatory IL-17-producing Th17 effector cells. Recent studies showing that the transcription factor nuclear factor of activated T cells (NFAT), which has long been known to interact with the activating transcription complex activator protein-1 upon TCR stimulation, can alternatively interact with FoxP3 to turn on the “Treg” program that includes CD25 and CTLA4 upregulation, suppression of IL-2 production and suppressive function [39]. Thus, the choice of transcription partner by NFAT may determine the regulatory versus effector differentiation pathway of a T cell. It has previously been suggested that T-cell polarization to the Th2 subset that secretes IL-4 and IL-10 could promote allograft acceptance [40]. For practical purposes, an immune response that is itself nondestructive and inhibits the development of destructive Th1 responses could provide a powerful means of ensuring graft acceptance. However, only limited data exist to implicate an active role for Th2 cells in tolerance induction [41,42]. It is clear that Th2 responses are not always benign, as Th2 cells and Th2-associated cytokines can mediate or contribute to allograft rejection [43,44]. The original T cells associated with a suppressive network were CD8+. Recently, several groups have reported that FoxP3+ CD8 cells, which may also express CD25, can mediate suppressive activities [37]. A subset of suppressive human CD8+CD28− T cells that upregulate the inhibitory ligands immuoglobulin-like transcript-3 and -4 expressed on APCs, rendering them tolerogenic, has also been described [45]. NKT cells (T cells that express natural killer [NK] cell-associated markers and may utilize an invariant TCR-α chain) are another subset of T cells with regulatory activity, which may be mediated in part by Th2-type cytokines. These cells have been reported to suppress autoimmune diseases, apparently by suppressing interferon-γ (IFN-γ) production via an IL-4-dependent mechanism. The role of these cells in various transplantation models is discussed below. A double-negative, NK1.1− T-cell population that suppresses skin graft rejection by CD8 T cells with the same TCR has been described in a TCR transgenic mouse model [46], but the importance of this cell population in the setting of polyclonal T-cell responses remains to be determined.
Mechanisms of Tolerance
Veto cells Alloresponses can be downregulated by “veto” activity, which may be mediated by several different T-cell types. “Veto” cells inactivate CTLs recognizing antigens expressed on the veto cell surface [47]. Several types of cell have been reported to have veto activity, including CTLs [47] and various bone marrow subpopulations, including CD34+ cells and their progeny [48]. CD34 cells mediate this activity through a tumor necrosis factor-α (TNF-α)-dependent mechanism, whereas CD8 T cells may act through a Fas–Fas ligand-dependent pathway [49]. Veto activity has also been ascribed to marrow cells with characteristics of NK or lymphokine-activated killer cells, which can be used to promote GVH tolerance and facilitate allogeneic marrow engraftment in murine BMT models [50]. Murine studies also suggest that veto cells might contribute to the donor-specific transfusion (DST) effect [51], wherein prior administration of donor blood induces subsequent hyporesponsiveness to that donor. One suggested mechanism for the veto effect involves triggering of a CTL through a surface class I MHC molecule while it is also activated through its TCR, resulting in apoptosis of the responding CTL [52]. This class I-mediated signal could result from binding of a T-cell’s class I molecules to CD8 molecules on the veto cell. However, not all veto cells express CD8, so this mechanism cannot explain all veto phenomena. Veto activity has been also suggested to involve the immunosuppressive cytokine TGF-β [53]. Other suppressive cell populations Non-antigen-specific suppressive activity has been previously attributed to “natural suppressor” cells, which were originally described as suppressive non-T, non-B cells that were detected in sites of hematopoiesis after BMT, in lymphoid tissues in the presence of GVHD, and in normal bone marrow [54]. Such suppressive populations proved to be heterogeneous, including NKT cells [55,56] and hematopoietic progenitors [57]. Overall, the abundance of suppressive populations results in a strong inhibitory effect of bone marrow on immune responses in vitro [54,58]. Suppressive populations found in lymphoid tissues in association with GVH responses include a myeloid progenitor cell population whose activity depends on IFN-γ and nitric oxide [59]. Suppressive myeloid progenitor cells have also been detected in the setting of sepsis and traumatic stress, and immature myeloid cells have been associated with cancer-induced immunosuppression in humans and mice. Other cell types with the capacity to downregulate T cell responses, such as mesenchymal stem cells and specially manipulated DCs, are discussed in more detail elsewhere in this textbook. Thus, while a plethora of phenotypes and activities for regulatory cells has been described in recent years, much remains to be learned about the relative importance of each of these and the circumstances in which they can be optimally generated and utilized to improve transplant outcomes.
Mechanisms of B-cell tolerance The above discussion has focused mainly on the induction of T-cell tolerance. If T-cell tolerance is achieved, T-cell-dependent antibody responses do not occur. In this setting, tolerance of mature B cells may occur via a deletional process associated with lack of T-cell help [60]. However, T-cell-independent B-cell responses are also important in settings where natural antibodies (NABs) are present (i.e. antibodies that are present in the absence of known antigen exposure), such as in ABO blood group-mismatched transplantation and xenotransplantation. Several mechanisms of B-cell tolerance have been described. Autoreactive developing B cells undergo developmental arrest, and recombination activating gene-dependent light-chain receptor editing then occurs. Formation of a non-autoreactive Ig receptor allows the B cell to survive;
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if such a rearrangement does not occur, the B cell undergoes apoptosis [61]. Encounter of immature B cells with soluble antigen expressed at relatively low levels leads to B-cell anergy [10]. The induction of B-cell anergy versus activation may be dependent on antigen concentration [10]. Antigen expressed only on cell membranes in the periphery has, in some instances, been associated with a deletion of B cells [10,62]. Anergy is a major mechanism of self-tolerance of newly formed B cells [63]. However, interaction with membrane-bound self-antigen can activate anergic B cells, breaking their tolerance [64]. Cells of the B-1 subset, which produce NABs responsible for xenograft hyperacute rejection [65], may undergo apoptosis when their surface Ig is crosslinked by cell-bound antigen [66].
Approaches to inducing HVG transplantation tolerance The potential benefits of inducing HVG tolerance in the setting of organ transplantation were described in the introductory section. To recapitulate briefly, these benefits include the avoidance of chronic immunosuppressive therapy, the prevention of chronic rejection, and facilitation of the ability to use xenogeneic (i.e. from other species) organ and tissue sources, such as the pig. Thus, induction of HVG tolerance has become a major goal of research in transplantation. In this section, we will discuss some experimental approaches to inducing tolerance and will divide the discussion into efforts to induce peripheral tolerance versus those aimed at achieving HSC engraftment and central T-cell tolerance. However, it should become apparent by the end of the discussion that tolerance cannot always be readily characterized as peripheral versus central, and that a combination of central and peripheral tolerance mechanisms may ultimately prove to be most practical and effective for the induction of HVG tolerance. Approaches to peripheral tolerance induction Tolerance of pre-existing, peripheral T cells has been induced in numerous animal models, often by mechanisms that are not fully understood. Evidence for anergy has been obtained in a few models [67], and recently, a role for regulatory cells has been found in many (see below). Peripheral deletion of donor-reactive T cells has been implicated [68] and directly demonstrated [69–72] in some models. It is important to note that many of the strategies for inducing tolerance to primarily vascularized rodent allografts such as hearts and kidneys rely heavily on the capacity of the organ graft itself to control the immune response, and that the tolerance thereby achieved is not systemic. Most of these tolerance protocols are not effective when tested with less “tolerogenic” grafts, such as primary skin allografts across full MHC barriers. Strategies that are effective for primarily vascularized allografts in rodents are often ineffective in large animals and man. Thus, a thorough understanding of the mechanisms involved in tolerance induction and successful extension to large-animal models are important criteria to fulfill before these approaches can be attempted in humans as a substitute for chronic immunosuppressive therapy. Some experimental approaches to tolerance induction are summarized below. DST, modified APCs or administration of autologous cells transduced with allogeneic MHC genes In rodents, infusion of donor leukocytes around the time of transplant can lead to graft prolongation and, in some cases, operational tolerance [73]. In other species, DST has been less effective as the only graftprolonging therapy, but it has shown beneficial effects, even in humans [74]. However, infusion of donor cells is also associated with the risk of sensitizing the recipient to donor alloantigens. Moreover, improve-
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ments in graft outcomes with modern immunosuppression, combined with the ability to treat uremia-associated anemia with erythropoietin instead of transfusion, have ended the practice of transfusion prior to transplantation. B cells and T cells in DST, both of which can present antigen in a non-costimulatory manner, may contribute to their immunomodulatory effect [75]. Administration of autologous bone marrow transduced with a single donor MHC antigen promotes tolerance to other antigens expressed by the cardiac or renal allograft donor in mice [76] and miniature swine models, respectively [77]. Several mechanisms of tolerance have been implicated in murine DST recipients, including veto activity leading to deletion of donor-reactive T cells [51], regulatory CD4 cells [78], and a role for the Th2 cytokines IL-4 [79] and IL-10 [78]. The extension of tolerance in these models from the antigens transduced into autologous cells to additional antigens expressed on the donor organ is a form of “linked suppression” (see below). The introduction of alloantigen on tolerogenic APCs can contribute to the acceptance of allografts [80]. Stimulation with immature DCs, or with DCs modified in certain ways, for example with cytokines [81], with transduced genes that promote T-cell death or tolerance [82] or with rapamycin [83], can favor the activation of Tregs over effector T cells. Attempts to intentionally immunize with “tolerogenic” DCs have been associated with prolonged heart, islet, and kidney allograft survival in rodent models, usually without permanent survival [84]. The combination of rapamycin-treated DCs and a short course of posttransplant rapamycin has recently permitted long-term survival of vascularized cardiac allografts in mice [83]. Thus, both mature and immature DCs, under various conditions, can favor graft acceptance. The mechanisms by which DCs promote unresponsiveness after differentiation under various conditions vary. Indoleamine 2,3 dioxygenease is an enzyme that catalyses tryptophan catabolism. Its production is induced by a signal transducer and activator of transcription-1 (STAT1)-dependent signal mediated by IFN-γ, which may, paradoxically, be produced by Tregs [85], which thereby promote DC-induced tryptophan depletion [86]. Plasmacytoid (type I IFN-producing) DCs have been implicated in the generation of IL-10-producing Tregs [87] via a mechanism associated with plasmacytoid DC expression of the costimulatory molecule inducible costimulator [88]. Costimulatory blockade, with or without infusion of donor lymphocytes Additional signals besides those resulting from TCR stimulation are required for T-cell activation. This costimulatory signal, or “signal 2,” must be provided to prevent the development of T-cell anergy [7]. While the B7 (CD80, CD86)–CD28 costimulatory interaction was the first to be defined, it is now known that multiple molecular pathways may costimulate T cells, many through Ig and TNF receptor family members [89]. Efforts to induce tolerance by blocking the B7–CD28 co-stimulatory pathway have enjoyed success in rodents receiving vascularized allografts under cover of CTLA4Ig [90], a fusion protein containing the B7-binding portion of CTL antigen-4 (CTLA4), a high-affinity T-cell ligand for B7. CTLA4Ig blockade of CD28 binding to both CD80 and CD86, combined with the tolerogenic capacity of primarily vascularized organ allografts in rodents, can lead to long-term graft acceptance. Signaling through the interaction of CD40 on APCs and B cells with its T-cell ligand (CD40L, CD154, which is expressed transiently on activated T cells) plays an important role in activating APCs so that they can optimally co-stimulate T cells. Signaling through CD40 leads to upregulation of B7 expression, as well as other costimulatory molecules, class II MHCs, and cytokines such as IL-12, and induces Ig class switching by B cells. This APC activation via CD40 is a major facet of CD4 T cell-mediated “help” for CTL generation [91]. Administration of
DST under cover of anti-CD154 mAb can prevent islet allograft rejection [92]. In thymectomized mice, the combination of DST and antiCD154 permits long-term skin graft survival in fully MHC-mismatched recipients, but this approach is less successful in euthymic mice [93]. DST in combination with anti-CD154 permits deletion of peripheral donor-reactive CD8 T cells [94]. Although CD4+ regulatory cells promote CD8 tolerance in this model [95], they are of insufficient potency or duration to prevent rejection by donor-reactive new thymic emigrants [94]. Xenogeneic skin graft prolongation is also observed with DST and anti-CD40L [96]. Cardiac allograft survival is markedly prolonged in the presence of anti-CD40L [97], but chronic graft arteriosclerosis still occurs [98]. Graft prolongation with anti-CD40L or CD28 blockade has been associated with a Th2-dominant immune response [99], which may promote graft arteriosclerosis [98]. The combination of CTLA4Ig and anti-CD40L antibody can markedly prolong the survival of primary skin allografts [100], renal allografts in large animals [101], and xenografts [102], but without inducing tolerance. Functional tolerance has been achieved with this combination only in rodent models of cardiac or islet allografts [103] or islet xenografts [104], but not in the more stringent models of primary skin grafting or large-animal vascularized organ or islet allografts [105–107]. Linked suppression and “infectious tolerance,” in which tolerant CD4+ T cells can render naïve T cells unresponsive in secondary recipients, have been demonstrated in several models involving co-stimulatory blockade. These phenomena are largely attributable to Tregs [108]. Costimulatory blockade more effectively suppresses CD4-mediated than CD8 cell-mediated alloreactivity [105,109], although the extent of CD8 cell-mediated resistance is genetically determined. A short course of rapamycin with anti-CD40L and CTLA4Ig, has, quite remarkably, permitted long-term acceptance of primary skin allografts across full MHC barriers in mice in certain strain combinations [110]. The combination of rapamycin, anti-CD154, and DST has also led to markedly prolonged islet allograft survival in non-human primates [111]. The beneficial effect of rapamycin may relate to its ability to allow the selective expansion of Tregs [34,112]. A recent protocol involving the combination of rapamycin, cytolytic IL-15–Fc fusion protein, and agonist IL-2 protein has been reported to lead to graft survival in stringent models by causing the death of activated effector T cells while sparing Tregs [113]. The mechanisms of hyporesponsiveness induced by organ transplantation with costimulatory blockade are only partly understood. The greater success in achieving acceptance of primarily vascularized allografts in rodents using a short course of costimulatory blockade or any of a number of different immunosuppressive agents suggests that these types of graft are highly tolerogenic. As noted above, comparable results usually have not been achieved in large-animal models of organ grafting, limiting the clinical applicability of these approaches. This disparity between results in large and small animals may reflect, in part, the inability of CD8 T cells to reject vascularized allografts in rodents without CD4 T-cell help, combined with the tendency of such grafts to activate Tregs, which then predominate over the suppressed effector T-cell response. Thus, many different forms of relatively mild and shortterm immunosuppression have successfully allowed the long-term survival of such grafts in rodents but not in large animals or man. Calcineurin inhibitors such as cyclosporine and FK506 can block graft prolongation induced by costimulatory blockade [100], possibly by preventing IL-2-induced priming for activation-induced cell death [110] and by preventing de novo T cell conversion to Tregs [112]. Consistently, IL-2 has been shown to play an essential role in the induction of tolerance with CTLA4Ig and cardiac allografts [114]. However, Fas and TNF-α receptors do not appear to be required for tolerance induction with this method.
Mechanisms of Tolerance
Despite the observation that Th1-associated cytokines are associated with graft rejection, IFN-γ plays a critical role in graft prolongation induced with combined CD40/CD28 co-stimulatory blockade [114], DST with CD28 blockade [114], and DST with CD40 blockade [95]. IFN-γ has several known mechanisms for downmodulating T-cell responses, including the promotion of activation-induced cell death, the blockade of T-cell differentiation to a proinflammatory Th17 phenotype, and the induction of indoleamine 2,3 dioxygenease production by DCs (see above). Antibodies to T cells and adhesion molecules Nondepleting anti-T cell mAbs, with or without T-cell-depleting mAbs, have been used to induce skin graft tolerance across minor or MHC barriers, and this has led to infectious tolerance mediated by Tregs [108]. Combination of such mAbs with DST can lead to permanent cardiac allograft survival in mice. Similar results have been achieved by blockade of 1eukocyte function-associated antigen-1 and intercellular adhesion molecule-1 (ICAM-1) interactions, which, in addition to promoting cell–cell adhesion, transmit costimulatory signals to T cells. LFA-1/ ICAM-1 blockade has also been combined with anti-CD154 or blockade of very late antigen-4 and vascular cell adhesion molecule (VCAM) interactions to achieve cardiac allograft prolongation. Other mAbs that have been used in rodent heart graft models include combinations of anti-CD2 and anti-CD3 mAbs [115], anti-CD2 alone [116], and antiCD2 plus anti-CD28 [117]. Various mechanisms, including Th2 deviation, have been implicated. Anti-CD45RB mAb, especially in combination with anti-CD154 or rapamycin, can promote allograft survival through a mechanism that is dependent on CTLA4 upregulation by T cells and Tregs [118]. Total lymphoid irradiation Total lymphoid irradiation (TLI) has also been evaluated as immunosuppression for organ transplantation and has been successful in about one third of baboons studied. In clinical transplantation, TLI has been reported to be beneficial, although it is cumbersome and toxic, especially in the case of cadaver-donor transplantation. Donor-specific tolerance has been demonstrated in a small number of patients in whom immunosuppressive therapy was terminated following kidney transplantation under cover of TLI. One of these patients was studied 12 years after immunosuppression withdrawal, and shown to have active anti-donor mixed lymphocyte reaction and no evidence for microchimerism [119]. TLI used in conjunction with antithymocyte globulin has been shown to induce tolerance to organs in several large-animal models [120]. The relationship between peripheral T-cell tolerance and central tolerance In addition to inducing central tolerance, the thymus can promote tolerance of T cells that are already in the periphery at the time of organ grafting. In a pig model in which class I MHC-mismatched kidneys are accepted under cover of a short course of cyclosporine, the thymus appears to play a role in the induction of tolerance among pre-existing peripheral T cells [121]. The thymus also promotes peripheral tolerance when soluble donor MHC antigens are injected intrathymically or intravenously in a rat model, as removal of the thymus before or within the first few days of allografting results in rejection of the allograft [122]. Migration of DCs from the periphery to the thymus can lead to deletion of thymocytes recognizing antigens these DCs present [123]. While this mechanism would explain the tolerization of newly developing T cells, it does not explain the role of the thymus in tolerizing pre-existing peripheral T cells. There are several possible explanations for the latter phenomenon:
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1 T cells that are activated in the periphery by the organ allograft may recirculate to the thymus, as has been described [124], and encounter donor antigen there, perhaps on graft-derived DCs, which may then inactivate the T cells. This mechanism has been implicated in the rat soluble antigen injection model described above [125]. 2 The migration of donor antigen to the thymus may result in the development of Tregs that specifically recognize the donor antigen, migrate to the periphery and downregulate the activity of destructive alloreactive T cells. The role of the thymus in the development of Tregs has already been discussed. However, recent evidence suggests that only antigens produced by thymic epithelial cells, and not those produced by hematopoietic cells, promote the production of Tregs recognizing them [28]. Nevertheless, active regulatory cell populations have been implicated in both the rat and pig models discussed above. 3 Recent thymic emigrants that encounter antigen in the periphery may differentiate into suppressive rather than rejecting T cells. The role of the allograft itself in inducing tolerance in the above models in which a pre-existing peripheral T-cell repertoire must be rendered tolerant should again be emphasized. Transferable tolerance is not induced by intrathymic marrow injection alone without an organ allograft in rats [126]. In contrast to models involving intrathymic injections or short-term immunosuppressive treatments and immediate solid organ transplantation, pure intrathymic deletional tolerance induced by mixed allogeneic chimerism (discussed below) is not dependent on the continued presence of an organ allograft [127]. In fact, this tolerance does not depend on the continual presence of any donor antigen in the periphery, as long as there is a continuous supply of donor antigen to the thymus [128].
Achieving HVG tolerance with HCT Resistance to engraftment of HSCs Two major factors influence the engraftment of HSCs. The first is sometimes referred to as “space” in the hematopoietic system. The mechanism by which myelosuppressive host treatment promotes marrow engraftment is not fully understood, and could include both the creation of physical niches and the upregulation of cytokines and other molecules that transmit signals to promote hematopoiesis. It is probably the species specificity of some of these interactions that accounts for the competitive advantage enjoyed by recipient marrow over xenogeneic marrow. An understanding of these physiologic barriers is likely to be critical to the success of the approach of using xenogeneic HCT to induce xenotolerance. Once these are understood, genetically modified donors of other species, such as pigs, could be engineered such that their HSCs would be better able to compete for hematopoietic function in a human marrow microenvironment. In syngeneic BMT recipients, a mild myelosuppressive treatment such as a low dose of total body irradiation (TBI) is required to make physiologic “space” for the engraftment of marrow cells given in numbers similar to those which could be obtained from marrow of living human allogeneic marrow donors [129]. However, this requirement can be overcome by the administration of very high doses of marrow [130,131]. While HSCs injected intravenously readily home to marrow niches [132], the efficiency of this process may be enhanced by direct injection into the bone marrow cavity [133]. The second factor limiting alloengraftment is the host immune system. Because of this immune resistance, allogeneic HCT can be performed successfully only in immunosuppressed recipients. Not surprisingly, T cells of the CD4 and CD8 subsets resist engraftment of class II- and class I-mismatched marrow grafts, respectively. It is also noteworthy that CD4 cells weakly resist engraftment of class I-disparate marrow and
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that CD8 cells quite markedly resist class II-disparate marrow grafts in mice [134,135]. NK cells, which may kill target cells due to the absence of self-MHC molecules on allogeneic marrow cells [136], resist allogeneic marrow engraftment in mice, and are responsible for the ability of A × B F1 recipients to resist engraftment of AA or BB parental marrow. NK cells of both humans and mice express clonally distributed surface receptors that recognize specific class I molecules. This recognition transmits an inhibitory signal to the NK cell, thereby preventing the NK cell from killing “self” class I-bearing targets [136]. While the ability of NK cells to resist the engraftment of pluripotent HSCs in mice is rather limited [137,138], NK cells might pose a more significant barrier to human leukocyte antigen (HLA)-mismatched human marrow engraftment, in which stem cell and progenitor cell numbers in the donor inoculum may be more limited. Such a role might become apparent in the future as more reduced-intensity conditioning regimens are used in the setting of HLA-mismatched transplantation. However, a clear role for NK cells in resisting alloengraftment has not yet been established for large animals or man. GVH-reactive NK cells have been reported to promote donor engraftment in mice and to mediate graft-versus-leukemia (GVL) effects against myeloid leukemias in man [139], and the presence or absence of NK cells in patients with severe combined immunodeficiency may determine whether or not cytotoxic host conditioning is needed in order to achieve engraftment of haploidentical HCT [140]. NK cells are tolerized by the induction of mixed chimerism [141], consistent with the observed long-term stability of the chimerism. Such tolerance requires that each functional NK cell receives adequate inhibitory signals from two completely disparate sets of MHC molecules on donor and host cells. This “tolerance” cannot be explained by changes in the expression of inhibitory or activating MHC-recognizing (Ly49) receptors [142]. Several observations suggest that anergy of NK cells chronically encountering cells lacking inhibitory ligands is responsible for their tolerance in mixed chimeras. Tolerance is broken when donor and host NK cells are separated in vitro [143]. Moreover, tolerance is specific for the donor and recipient in the allogeneic situation [142], but is associated with global unresponsiveness in the presence of mixed xenogeneic chimerism [144]. Inhibitory Ly49 receptors are quite broad in their class I allorecognition, and recognition of even fully allogeneic class I molecules can confer some protection from NK-mediated marrow destruction compared with that observed for cells lacking class I MHC. Because of the increased disparity of xenogeneic compared with allogeneic MHC molecules, NK cells likely receive less inhibitory signal from xenogeneic than allogeneic cells. Thus, “tolerance” that reflects anergy induction by repeated encounter with cells lacking inhibitory ligand would be associated in this situation with a global functional NK defect, as has been observed. Consistently, this global lack of NK function is similar to that observed in the presence of chimerism from class Ideficient marrow [145]. Humoral mechanisms can also mediate resistance to marrow engraftment. Antibodies that inhibit engraftment can exist without prior immunization (so-called “natural” antibodies, or NABs). For example, NABs that recognize ABO blood group determinants or xenoantigens, and that exist in normal sera, can resist the engraftment of hematopoietic cells that express the determinants recognized by the NAB. Antibodies against donor antigens can also be present due to presensitization, usually from prior blood product infusions in HCT recipients. Animal studies suggest that the effect of NABs on engraftment can be overcome by the administration of sufficiently high marrow doses [146], and in man, deleterious consequences of ABO-incompatible BMT can usually be avoided with adequate red blood cell depletion from the marrow product. Occasionally, plasmapheresis may be needed to remove such antibodies if they are present at particularly high titer [147]. The presence of antibodies
resulting from presensitization to donor alloantigens, on the other hand, is associated with a high incidence of marrow graft failure in humans and mice. A permissive environment for engraftment of allogeneic marrow can exist under several physiologic and artificially induced conditions, as outlined below. The mechanisms by which HCT induces tolerance in each of these models is discussed. HCT in developmentally immunodeficient recipients Hematopoietic engraftment has been achieved without conditioning in developmentally immunoincompetent recipients. This was first observed in Freemartin cattle (fraternal twins sharing a placental circulation) [148]. Since the prenatal diagnosis of a number of congenital diseases has become possible, the injection of HSCs to preimmune fetuses is an attractive approach to inducing tolerance for postnatal organ transplantation from the same allogeneic or xenogeneic donor. While renal allograft tolerance has recently been achieved with this approach in a largeanimal model [149], the timing and purity of HSC injection are critical in balancing the tendencies for rejection and GVHD, and chimerism and tolerance have been variable in other animal models [150]. Animals can also be rendered tolerant by injection of alloantigens shortly after birth. Several mechanisms have been invoked to explain the observed HVG tolerance (reviewed in [151]). Lasting microchimerism has been detected in some studies and may be essential for the maintenance of tolerance. Evidence to support an intrathymic deletional mechanism has been obtained in some, but not other, strain combinations. However, the presence of microchimerism does not always predict skin graft tolerance in recipients of allogeneic lymphocytes perinatally, and nontolerant animals can still maintain microchimerism following rejection of donor skin grafts. Thus, it is not surprising that several additional mechanisms have been implicated in rodents in which neonatal tolerance has been induced. These include the following: 1 The action of specific suppressor T cells [151]. Tolerance cannot be easily broken by the infusion of nontolerant host-type lymphocytes in animals rendered tolerant at birth, and this has been attributed to the presence of suppressive T-cell populations. Specific suppressor T cells have been detected, and Tregs have been implicated [152]. 2 Neonatal mice have a tendency to produce Th2 responses, which have been implicated in donor-specific skin graft acceptance [41,153]. 3 The ability of allogeneic spleen cell infusions to induce tolerance may reflect the high ratio of non-costimulatory APCs (resting T and B cells) in donor inocula to recipient T cells in the neonate, rather than any particular susceptibility to tolerance induction at this time of life [154]. HCT in immunocompetent recipients following high-dose conditioning Marrow chimerism and tolerance can be achieved in otherwise normal adult animals if immunodeficiency and “space” are artificially created by lethal TBI. While the toxicity associated with this approach makes it unusable for the purpose of organ allograft tolerance induction in humans, studies in animals have provided important information on the immunobiology of HCT and tolerance induction. In murine recipients of T-cell-depleted (TCD) allogeneic BMT, lymphohematopoietic repopulation is largely (95–99%) allogeneic; a small population of host cells (mainly T cells) survives permanently in such animals. If TCD allogeneic and TCD syngeneic marrow are co-administered, mixed allogeneic chimerism ensues, i.e. lymphohematopoietic cells of both donor and recipient type coexist permanently in the survivors [155]. These mixed chimeras show no evidence of GVHD and show specific tolerance, both
Mechanisms of Tolerance
in vivo and in vitro, to donor antigens [155], i.e. “systemic” tolerance is achieved. Animals reconstituted with fully MHC-mismatched TCD allogeneic marrow alone demonstrate a similar pattern of specific unresponsiveness but tend to show poorer immunocompetence both in vivo and in vitro, possibly due to a mismatch between the MHC restriction imposed by the host thymus and the MHCs of donor-type APCs in full chimeras across complete MHC barriers. Another potential advantage of mixed chimerism over full allogeneic chimerism is that hematopoietic cells are most efficient at inducing clonal deletion of T cells in the thymus [3]; thus, a continuous source of host-type hematopoietic cells assures that host-reactive T-cell clones will not emerge from the thymus [127], whereas such cells can emerge in animals reconstituted with TCD allogeneic marrow alone [12]. The importance of antigen on hematopoietic cells for the induction of tolerance among newly developing T cells in humans is illustrated by studies in patients with severe combined immunodeficiency receiving HLAmismatched BMT and in whom donor T-cell, but not B-cell or myeloid, engraftment develops initially. Newly developing donor T cells from these patients exhibit reactivity toward donor class II MHC antigens. This reactivity disappears if the engraftment of donor class II-bearing cells later occurs [156]. Clonal deletion is the predominant mechanism of HVG tolerance in lethal TBI-treated recipients of TCD allogeneic BMT [3,12]. In vivo evidence suggests that suppressor cells are not involved in the maintenance of tolerance to donor antigens, regardless of whether TCD allogeneic marrow cells are given alone or with TCD syngeneic cells. Tolerance can be readily broken in such chimeras by administering a relatively small number of nontolerant recipient-type spleen cells [157], indicating the absence of any potent suppressive activity. Results of in vitro co-culture studies supported this conclusion. As discussed earlier, one important difference between TBI/BMT recipients and neonatally tolerized animals is that TBI leads to the elimination of most pre-existing T-cells. Thus, the number of remaining alloreactive T cells may be too small to provide an adequate stimulus for the activation and expansion of regulatory T-cell populations. In other words, activation of Tregs may require the presence of alloreactive effector cells, which is not the case if there is complete T-cell ablation followed by the intrathymic deletion of donor-reactive cells. In peripheral tolerance models, in contrast, mature donor-reactive T cells already exist in the periphery, and the observed difficulty in breaking tolerance by the infusion of nontolerant host-type cells is consistent with the presence of potent suppressive mechanisms. When, on the other hand, “pure” deletional tolerance is induced in animals in which the pre-existing peripheral T-cell response has been fully ablated, the absence of suppressive cell populations makes it easy to abolish tolerance by the infusion of nontolerant host-type lymphocytes [128,157,158]. In contrast to the above chimeras prepared with lethal TBI followed by reconstitution with TCD allogeneic marrow, animals receiving nonTCD allogeneic cells are highly resistant to the breaking of tolerance by the administration of nontolerant host-type lymphocytes [157]. This resistance is dependent upon the GVH reactivity of those T cells [157]. Rather than implying the existence of regulatory mechanisms, this finding suggests that GVH-reactive T cells persist long-term in these chimeras and eliminate administered nontolerant host-type lymphocytes before they can “take” and eliminate donor cells. Studies in animal models have addressed the possibility that, following lethal irradiation and reconstitution with exhaustively TCD autologous marrow, tolerance might be induced by expression of antigen on nonhematopoietic organs grafted simultaneously with the TCD autologous marrow. This approach was, however, insufficient to induce tolerance to MHC mismatched skin allografts in rodents or to MHC-mismatched vascularized grafts in large-animal studies. Similarly, in vivo treatment
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with depleting anti-T-cell mAbs alone does not permit induction of skin allograft tolerance across MHC barriers in mice. Thus, alloantigen seems to be required within the lymphohematopoietic system for the most complete tolerization of newly developing T cells, probably reflecting the unique ability of alloantigen expressed on marrow-derived cells to induce clonal deletion in the thymus. An alternative to the induction of mixed chimerism in lethally irradiated recipients has been to reinfuse autologous marrow that is genetically modified to express an allogeneic MHC gene. This approach has led to donor-specific hyporesponsiveness in a class I-mismatched mouse model and with class II gene transduction in a pig model [77]. While it would be technically cumbersome to modify each individual transplant recipient’s marrow with all of the MHC genes of any given cadaveric organ donor, the tolerance-inducing capacity of the organ graft itself can induce spreading to other graft antigens of the tolerance that is induced by a single donor MHC gene introduced into the recipient marrow [77]. The discussion so far would suggest that induction of mixed chimerism may be the most straightforward way of ensuring donor- and hostspecific tolerance across MHC barriers. However, the original methods for producing mixed allogeneic chimeras in mice were not directly applicable to man, because of the toxicity of lethal TBI and the high risk of GVHD and graft failure encountered when HCT is attempted across MHC barriers in man. HCT following lethal TBI would not be justifiable as a means of inducing tolerance for the purpose of organ transplantation, for which less toxic chronic immunosuppressive therapy is available. If HCT, therefore, is to be used as a means of inducing donorspecific organ allograft or xenograft tolerance, it will be essential to develop less toxic and more effective methods of rendering recipients permissive for engraftment than those which are currently available. Conditioning regimens are needed which specifically eliminate the host elements that resist alloengraftment without producing generalized toxic effects. Some approaches to achieving this goal are discussed below. HCT in immunocompetent recipients following conditioning with immunosuppressive but nonmyeloablative protocols Recently, several HCT protocols have been developed that involve myelotoxic and/or immunosuppressive, but not myeloablative, conditioning regimens. The term “nonmyeloablative” is used in this chapter to describe regimens that are known to leave sufficient host hematopoiesis intact to avoid significant cytopenia without a bone marrow graft or if the donor graft fails. These criteria have been demonstrated for certain regimens used in animals and patients, but not for all reduced-intensity regimens. Since host HSCs survive after nonmyeloablative conditioning, mixed chimerism develops when allogeneic marrow is administered. Such regimens include TLI [159], sublethal TBI [160], administration of cyclophosphamide following sensitization with allogeneic donor antigens [161], and the use of mAbs against host T cells in combination with other modalities [162]. HCT has also been performed in unconditioned patients receiving organ transplantation under conventional immunosuppressive drug therapy. Low levels of chimerism have been achieved, but withdrawal of immunosuppressive therapy has not been made possible with this approach [163,164]. However, the incidence of chronic rejection may be decreased in this setting [164]. A brief description of some nonmyeloablative or reduced-intensity conditioning approaches follows. Sublethal TBI Induction of mixed chimerism using fractionated sublethal TBI and allogeneic BMT can lead to donor-specific tolerance across complete
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MHC barriers in mice [160]. Donor T cells that need not have alloreactivity against the recipient promote alloengraftment in this model, and donor T cells maintain tolerance [160]. In dogs, a low dose of TBI, in combination with post-transplant cyclosporine and mycophenolate mofetil, has been associated with mixed chimerism and tolerance across minor, but not major, histocompatibility barriers [165].
Recently, combinations of chemotherapeutic agents have been used as reduced-intensity conditioning in patients with hematologic malignancies who were considered to be at high risk from complications of conventional high-dose conditioning. Thus far, these approaches have not been combined with organ transplantation in man. Anti-T-cell antibodies
TLI Another early approach to achieving mixed hematopoietic chimerism and donor-specific tolerance involved the use of TLI plus BMT. The long bones are shielded during the radiation preparative regimen, so BMT results in mixed chimerism rather than full donor reconstitution, and recipient T cells are not fully ablated. Mice treated in this way have been shown to be both resistant to GVHD and tolerant to donor skin grafts (reviewed in [159]). NKT cells producing Th2-type responses predominate following TLI, and this Th1 to Th2 shift may play a role in permitting graft acceptance and preventing GVHD. TLI as the sole conditioning modality for BMT has only variably permitted the induction of mixed chimerism and tolerance in large animals, and this has often been associated with considerable toxicity. A trial of donor BMT with fractionated TLI in high-risk patients receiving renal allografts was not associated with chimerism, and significant toxicity was also observed [166]. A recent trial of HLA-mismatched allogeneic HCT in patients conditioned with TLI and antithymocyte globulin has demonstrated transient induction of chimerism, but withdrawal of immunosuppressive therapy was associated with rejection of donor kidneys grafted at the same time (reviewed in [120]). Nevertheless, remarkably low rates of acute GVHD have been observed in patients receiving HLA-identical HCT for treatment of hematologic malignancies using TLI plus antithymocyte globulin-based conditioning [167]. Pharmacologic agents Cyclophosphamide has long been a staple of high-dose conditioning regimens and of less-intense conditioning regimens for HCT to treat aplastic anemia. Pre-HCT cyclophosphamide has more recently been used in combination with other agents in reduced-intensity conditioning regimens for HCT to treat hematologic malignancies. As is discussed below, a nonmyeloablative HCT/kidney allograft regimen involving cyclophosphamide conditioning has been the first successful attempt at the intentional induction of organ allograft tolerance in humans [168]. In rodents, high doses of cyclophosphamide administered several days following inoculation with large numbers of allogeneic marrow and/or spleen cells permit microchimerism and donor-specific tolerance across multiple minor histoincompatibilities and selected MHC disparities. Additional host treatment with a monoclonal anti-T-cell antibody [162], with sublethal TBI or with fludarabine and low-dose TBI [169], permits the induction of tolerance across complete MHC barriers. Sensitization followed by administration of cyclophosphamide causes selective destruction of host T cells driven into cell cycle in response to alloantigen, so that pre-existing donor-reactive T-cell clones are eliminated [162], and newly developing T cells can mature in the presence of donor marrow-derived antigen in the thymus. While thymic and peripheral chimerism can be demonstrated in animals receiving the regimen involving cyclophosphamide alone, the levels of such chimerism are extremely low, reflecting the fact that cyclophosphamide is relatively ineffective at producing marrow “space.” Evidence has been obtained for intrathymic clonal deletion as a mechanism of tolerance in this model. However, chimerism was shown to disappear with time, and anergy was reported as a mechanism of longer-term tolerance. Post-transplant cyclophosphamide has recently been applied clinically in a reduced-intensity regimen for haploidentical HCT [170].
Early experimental efforts to combine BMT with T-cell depletion to eliminate host cells that mediate alloresistance involved polyclonal sera (antilymphocyte serum). A fraction of mice receiving antilymphocyte serum and skin allografts several days prior to administration of marrow from the same allogeneic donor could be rendered specifically tolerant of donor antigens in the presence of certain MHC disparities [171]. The addition of rapamycin to the regimen significantly increases the incidence of tolerance [172]. Complex mechanisms, including suppressor cells of donor origin, were implicated in this model, and more recent studies have demonstrated that T-cell depletion by antilymphocyte serum is associated with sparing of Tregs [172,173]. A similar approach has been reported to prolong renal allograft survival in large animals, with the best results being achieved when the donor and recipient share a DR class II MHC allele [174]. In the primate studies, a veto cell population has been implicated. Conventional marrow doses are administered in this model without host myelosuppression, so that high levels of donor HSC engraftment are not achieved. Moreover, T-cell depletion is incomplete, so the involvement of regulatory cells in tolerance is therefore not surprising. In vivo depletion of host CD4+ and CD8+ T cells along with TBI (at least 6 Gy) permitted engraftment of allogeneic marrow and induction of skin graft tolerance across complete MHC barriers [17]. Adding a high dose of thymic irradiation (7 Gy) to the regimen permitted engraftment of fully MHC-mismatched allogeneic marrow in animals receiving only 3 Gy TBI. Thymic irradiation is needed because thymocytes become coated with mAbs but, unlike T cells in the peripheral lymphoid compartment, are not eliminated. The low dose (3 Gy) of TBI is necessary for the creation of marrow “space.” Permanent mixed chimerism and donor- and host-specific tolerance are reliably induced across complete MHC barriers using this regimen (Fig. 15.1) (reviewed in [20]). This approach has been extended to a xenogeneic (rat→mouse) combination by adding anti-NK1.1 and anti-Thy1.2 mAbs to the conditioning regimen [175]. These mAbs are needed to deplete recipient NK cells and γδ T cells [176], both of which pose a much more significant barrier to engraftment of xenogeneic than allogeneic HCT. Allogeneic tolerance has been achieved in a non-human primate model using this nonmyeloablative approach to inducing mixed chimerism [177]. In both the allogeneic [128](reviewed in [20]) and the xenogeneic (reviewed in [179]) rodent systems, intrathymic clonal deletion, rather than peripheral suppression or anergy, is the major mechanism inducing and maintaining long-term donor-specific tolerance. Accordingly, donor class IIhigh cells are present in the thymuses of these animals at all times, including 10 days post-BMT, when the first wave of thymopoiesis is underway. Furthermore, tolerance can be broken by depleting donor cells with mAbs after stable chimerism has been established, and this loss of tolerance correlates with the de novo appearance in the periphery of T cells bearing donor-reactive TCRs [128]. However, if the host thymus is removed before depletion of donor hematopoietic cells, or if donor-depleted spleen cells from chimeras are transferred to syngeneic athymic mice, donor-specific tolerance is maintained, and cells with donor-reactive TCR do not appear in the periphery [128]. These results demonstrate that intrathymic chimerism is essential and sufficient to maintain ongoing deletional tolerance in long-term mixed allogeneic chimeras, whereas peripheral chimerism is not required for the maintenance of tolerance. Since persistent antigen is required to maintain
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Fig. 15.1 Nonmyeloablative conditioning regimen allowing the induction of mixed allogeneic chimerism and specific transplantation tolerance across complete major histocompatability barriers in mice. BMT, bone marrow transplantation; CML, chronic myeloid leukemia; mAb, monoclonal antibody; MLR, mixed lymphocyte reaction; TBI, total body irradiation.
anergy, tolerance cannot be explained by a peripheral anergy mechanism. Moreover, the ease with which new thymic emigrants break tolerance after donor cell depletion by mAbs or with infusion of nontolerant recipient lymphocytes [128] is strong evidence that suppression does not play a significant role in maintaining long-term tolerance. Thus, this is a relatively pure model of ablation of pre-existing peripheral and intrathymic mature T cells followed by lifelong central, deletional tolerance. The potential toxicity of the regimen shown in Fig. 15.1 can be further minimized by replacing thymic irradiation with a second injection of anti-donor mAbs on day –1, and deletional tolerance is again attained. These thymus-directed host treatments are needed to eliminate pre-existing donor-alloreactive thymocytes and also to create “thymic space.” Thymic engraftment is regulated independently of marrow engraftment, and specific measures are required to permit donor progenitors to repopulate the recipient thymus at high levels, even in syngeneic mice (reviewed in [180]). In this allogeneic BMT model, TBI can be eliminated from host conditioning by giving a very high dose of donor marrow cells, still achieving durable multilineage mixed chimerism and systemic donorspecific tolerance, all in association with the lasting presence of donor APCs in the thymus and intrathymic deletion of donor-reactive host thymocytes. Thus, high levels of allogeneic hematopoietic repopulation and central deletional tolerance may be achieved with a conditioning regimen that excludes myelosuppressive treatment. Hematopoietic cell engraftment and donor-specific tolerance have been achieved across a full haplotype barrier using a similar nonmyelosuppressive regimen plus high stem cell doses in a large animal porcine model [180]. Based on the mouse model in Fig. 15.1, a primate model for tolerance induction has recently been developed. Since effective T-cell-depleting mAbs are not available for use in primates, polyclonal antithymocyte globulin and a short (28-day) course of cyclosporine are used in their place. A high percentage of cynomolgus monkeys receiving class I- and II-mismatched marrow with this protocol develop transient mixed chimerism and donor kidney allograft acceptance [177]. Several other regimens involving various combinations of anti-T-cell antibodies, irradiation, and immunosuppressive drugs have also permitted the achievement of mixed chimerism in mice (reviewed in [181]). Depleting and nondepleting anti-T-cell mAbs can also be used to induce tolerance in association with conventional-dose BMT without irradiation, although tolerance is then only reliably achieved across minor
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Days Fig. 15.2 Renal function (measured by serum creatinine [creat]) and cyclosporine levels (measured by a monoclonal antibody assay) in two patients (Pt 1, Pt 2) with multiple myeloma who received combined kidney and bone marrow transplantation from human leukocyte antigen-identical sibling donors following nonmyeloablative conditioning with cyclophosphamide, pre- and post-transplant antithymocyte globulin, and thymic irradiation. Cyclosporine (CyA) was the only pharmacologic immunosuppressive agent given post-transplant, and it was discontinued in the third month post-transplant. Both patients have sustained normal creatinine levels (now at 7 years and 9 years) following discontinuation of cyclosporine, indicating the presence of tolerance to their kidney allografts. Partial and complete remissions of the multiple myeloma were achieved in the two patients, and no GVHD has occurred. (Reproduced from Buhler LH, Spitzer TR, Sykes M et al. Induction of kidney allograft tolerance after transient lymphohematopoietic chimerism in patients with multiple myeloma and end-stage renal disease Transplantation 2002; 74: 1405–9, with permission.)
histocompatibility barriers, and lasting high levels of chimerism are not achieved [17]. In this setting, clonal anergy rather than deletion may be the major mechanism of tolerance [17]. The successful induction of tolerance in a primate model using nonmyeloablative conditioning to induce mixed chimerism [177], combined with murine studies demonstrating the utility of mixed chimerism followed by delayed donor leukocyte infusions (DLIs) as an approach to achieving GVL effects without GVHD [182–184], has been used as the basis for a clinical trial of mixed chimerism induction followed by DLI for the treatment of patients with hematologic malignancies [185,186]. The experience with this relatively nontoxic protocol provided an opportunity to evaluate the potential of this approach to induce transplantation tolerance in patients with a hematologic malignancy, multiple myeloma, and consequent renal failure. Six patients received a simultaneous nonmyeloablative bone marrow transplant and renal allograft from an HLA-identical sibling, and have accepted their kidney graft without any immunosuppression for follow-ups as long as 9 years. This study describes the first intentional achievement of organ allograft tolerance in humans. Three of the six patients have had prolonged complete remissions of their myelomas [168] (Fig. 15.2). This is especially surprising since chimerism in four of these six patients (including two with prolonged complete remissions of their myelomas) was only transient [168]. These data raise the possibility that transient chimerism followed by marrow rejection, which was evidenced by sensitized anti-donor T-cell responses in some of these patients [168], could lead to antitumor responses, and this hypothesis has been supported by data in a mouse model [187]. The renal allograft tolerance achieved in patients who lost chimerism suggests that the kidney graft itself may participate in tolerance induc-
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tion and/or maintenance after chimerism has played its initial role. The detection of sensitized T-cell responses to donor minor histocompatibility antigens expressed on hematopoietic cells with unresponsiveness to donor renal tubular epithelial cells in these patients [168] raises the possibility that tolerance may be specific for minor antigens expressed on the kidney graft itself. In the primate model described above, chimerism is also transient, but both BMT and early renal transplantation have been shown to be essential for tolerance induction [177]. Because T-cell depletion is only partial in these models, it is clear that the pure central, deletional tolerance described above in murine models has not yet been achieved with nonmyeloablative conditioning in large animals or humans. Nevertheless, the promising results obtained in these patients have provided an important proof of principle. Recently, this approach has been extended to a pilot study in patients without malignant disease with renal failure from other causes, who receive HLA-mismatched combined kidney and BMT. A systemic state of donor-specific tolerance was achieved in 4 out of 5 patients. These four patients have been off all immunosuppression for periods of more than 1 year to almost 5 years, providing the first successful intentional achievement of organ allograft tolerance across HLA barriers (178). The mechanisms of T-cell elimination using antibody treatments are not entirely clear. Animals treated with T-cell-depleting mAbs after thymectomy show a partial but significant recovery of CD4+ and CD8+ T cells over a period of several weeks, suggesting that some T cells escape depletion and expand due to lymphopenia and/or antigenic stimuli. Many studies have shown that the initially recovering T cells in large animals and humans predominantly express the “memory” phenotype, consistent with lymphopenia-driven expansion, but also consistent with selective resistance of memory cells to antibody-mediated depletion. T-cell depletion with at least some mAbs is independent of complement, but this pathway seems to be involved in T- and B-cell depletion with other mAbs. Co-stimulatory blockade with HCT Both thymic irradiation and host T-cell depleting mAbs in the conditioning regimen shown in Fig. 15.1 can be replaced by co-stimulatory blockade [69]. All preconditioning can be eliminated by giving a high dose of fully MHC-mismatched donor marrow followed by a single injection of each of two costimulatory blockers [188] or repeated injections of anti-CD40 ligand [189]. This ability to replace recipient Tcell depletion with costimulatory blockade to allow bone marrow engraftment is encouraging for several reasons. First, it has been dif-ficult to achieve T-cell depletion with antibodies in large animals and humans that is as exhaustive as that achieved in the above rodent models, perhaps due to the use of inadequate doses or suboptimal reagents. Second, if sufficiently exhaustive T-cell depletion could be achieved in humans, T-cell recovery from the thymus might be dangerously slow, especially in older individuals, since thymic function diminishes with age (reviewed in [190]). The ability to minimize the degree and duration of T-cell depletion by replacing some [109] or all [69,188,189] of the T-cell depleting antibodies with co-stimulatory blockers is therefore encouraging. As in other protocols achieving sustained mixed chimerism, long-term tolerance is maintained by intrathymic deletion in mixed chimeras prepared with costimulatory blockade [69,109,188]. However, since alloreactive T cells are abundant in the peripheral repertoire at the time of BMT with these regimens, these peripheral T cells must be rendered tolerant by BMT under cover of costimulatory blockade. Initial tolerance of peripheral T cells involves specific deletion of donor-reactive CD4 [71] and CD8 [72] T cells. This specific peripheral deletion of CD4 cells only requires failure of the CD154–CD40 interaction and does not require antibody-mediated clearance of CD154+ cells [191]. In mice receiving BMT with anti-CD154 and a low dose of TBI, CD8 deletion
occurs within 1–2 weeks and requires CD4 cells that do not have characteristics of “natural” Tregs [72]. Unlike models involving “adaptive” Tregs, CD4 cells are not required for maintenance of tolerance after this initial 2-week period [72]. Thus, while CD4 cells are clearly required for CD8 tolerance in this model, there is currently no evidence that this regulatory function of CD4 cells involves a specific subset of cells that is differentiated to mediate suppression. Deletion of donor-reactive CD4 cells occurs more slowly, over 4–5 weeks, and is preceded by a state of anergy [71,191]. Regulatory cells do not appear to play a major role in maintaining the long-term tolerance induced by anti-CD154 with BMT, since tolerance and chimerism are obliterated by the infusion of relatively small numbers of nontolerant recipient-type spleen cells in this model and linked suppression is not observed [71]. Since HSC engraftment ensures complete central deletional tolerance in these long-term chimeras [69,70,109,188], and specific peripheral deletion is quite complete, there may be insufficient donor-reactive T cells present to induce the expansion and maintenance of specific regulatory cells. However, some models using CTLA4Ig and anti-CD154 in combination with allogeneic BMT may be associated with less-complete deletion of donor-reactive T cells and involve regulatory mechanisms [192,193]. GVHD does not occur in the rodent models discussed above, despite the use of unmodified donor bone marrow cells. This is most readily explained by the continued presence of the T-cell-depleting or costimulatory blocking antibodies in the serum of the hosts at the time of BMT. These levels are sufficient to prevent alloreactivity by the relatively small number of mature T cells in the donor marrow. There are similarities and differences in the mechanisms of tolerance induced by anti-CD154 in combination with BMT versus DST. Only the BMT model allows engraftment of HSCs and intrathymic deletion as a mechanism of long-term tolerance [109,188]. Thus, in contrast to BMT, DST and anti-CD40L allows long-term skin graft survival only in thymectomized mice [95]. The inability to maintain tolerance in the presence of a thymus indicates the failure to establish central tolerance in the absence of substantial chimerism. In addition, the inability to resist breaking of tolerance by new thymic emigrants in this model argues that powerful peripheral regulatory mechanisms are not operative in these animals, even though regulatory mechanisms may play a role in the initial suppression of CD4-mediated alloresponses [95]. Concurrent viral infections have a deleterious effect on the induction of mixed chimerism under cover of co-stimulatory blockade [194]. Another concern is that successful induction of allogeneic tolerance in the presence of viral infections may also lead to tolerance to the virus and failure to clear it. These results suggest that de novo systemic viral infections should be avoided during the period of BMT and tolerance induction, and have important implications for the practice of BMT with this approach. Application of mixed chimerism for the induction of xenogeneic tolerance Considerable difficulties have been encountered in achieving high levels of engraftment and hematopoietic function from highly disparate species. Xenogeneic hematopoiesis can be enhanced by the administration of donor-specific cytokines, and spontaneous seeding of the thymus with tolerance-inducing cells from the donor has been detected in pig cytokine-transgenic mice receiving porcine HCT, resulting in the achievement of donor-specific tolerance [195]. An alternative approach involves the removal of the recipient thymus and its replacement with a xenogeneic thymus. Donor-specific skin graft tolerance across the discordant pig to mouse species barrier has been achieved with this approach [196]. Normal, immunocompetent mice that are thymectomized and treated with T-cell-depleting mAbs before porcine thymus grafting recover T cells in the xenogeneic thymic grafts. These cells repopulate the periphery and are competent to resist infection [197]. Tolerance to both donor
Mechanisms of Tolerance
and host by intrathymic deletional mechanisms is observed, and this deletion reflects the presence of class IIhigh cells from both species within the graft [196,198]. Since MHC restriction is determined by the MHC of the thymus, and porcine MHC is entirely responsible for positive selection of murine T cells in this model [199], it was surprising that T cells that differentiated in xenogeneic thymus grafts were able to respond to peptide antigens presented by host MHC [197]. However, the excellent immune function achieved in humans receiving HLA-mismatched allogeneic thymic transplantation for the treatment of congenital thymic aplasia (DiGeorge syndrome) suggests that this “restriction incompatibility” may not be a major obstacle to the achievement of adequate immune function [200]. Perhaps this high level of crossreactivity, even between species, reflects the fact that MHC reactivity is inherent in unselected TCR sequences [201]. Importantly, it has been demonstrated that human T cells can also develop in porcine thymic grafts (in immunodeficient mice), and that these T cells show specific tolerance toward the MHC of the xenogeneic thymus donor [202]. Induction of B-cell tolerance using HCT As mentioned above, mixed chimerism can also induce tolerance across xenograft barriers. Pigs are widely believed to be the most suitable xenogeneic donor species for transplantation to humans, but transplantation from this species is impeded by the presence in human sera of NABs that cause hyperacute rejection of porcine vascularized xenografts. In humans, the major specificity recognized by NABs on porcine tissues is a ubiquitous carbohydrate epitope, Galα1-3Galβ1-4GlcNAc-R(αGal). Humans lack a functional α1-3Gal transferase (galactosyltransferase; GalT) enzyme, as do GalT knockout mice, which also make anti-αGal NABs. Both pre-existing and newly developing B cells producing anti-αGal antibodies are tolerized by the induction of mixed chimerism in GalT knockout mice receiving αGal-expressing allogeneic or xenogeneic marrow. The induction of mixed xenogeneic chimerism prevents hyperacute rejection, acute vascular rejection, and cell-mediated rejection of primarily vascularized cardiac xenografts. Long-term chimeras produced in GalT knockout mice lack anti-Gal surface Ig-bearing cells in the spleen, and show tolerance in enzyme-linked immunosorbent spot assays, suggesting that clonal deletion and/or receptor editing tolerizes B cells developing after BMT. However, tolerance develops by 2 weeks post-BMT, even in mice that are presensitized to Gal antigens, despite the fact that the conditioning regimen does not deplete antibodyproducing cells. In GalT−/− mice rendered tolerant by the induction of mixed chimerism 2 weeks earlier, cells with anti-Gal receptors are present in the spleen but do not produce anti-Gal (reviewed in [203]). This rapid tolerance of NAB-producing cells in mice rendered mixed chimeric with BMT is due to an anergy mechanism [204]. The cells producing anti-Gal and most IgM NABs in mice belong to the B-1b subset [65], and their tolerization requires the expression of complement receptors on recipient nonhematopoietic cells [205] by mechanisms that are incompletely understood. An alternative approach for tolerizing B cells toward the Gal epitope involves retroviral transduction of the GalT gene into autologous HSC. Administration of marrow transduced in this manner to lethally irradiated GalT knockout mice led to tolerance to the Gal epitope [206]. Establishing molecular chimerism involves modification of autologous BM stem cells, avoiding the difficulties in achieving engraftment of pig HSCs in primates. However, this approach has only been evaluated in lethally irradiated mice, and it is not yet known whether or not a transduced, transplanted BM population could compete sufficiently with host hematopoietic cells to give significant long-term Gal expression in recipients of a more clinically relevant, nonmyeloablative conditioning regimen. Even if this approach were to be successfully applied, non-
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Gal-reactive xenoreactive NABs and cell-mediated rejection would remain to be overcome. The significance of these non-Gal antibodies has become apparent since the recent development of αGal knockout pigs using nuclear transfer [207–209]. In contrast, all types of rejection can be avoided with a single treatment by induction of mixed xenogeneic hematopoietic chimerism.
Preventing GVHD by depleting or tolerizing mature donor T cells in the HCT inoculum: is GVH tolerance a desirable goal? In order to avoid GVHD, the de novo development of host-reactive T cells from progenitors in the marrow must be prevented, and the GVH reactivity of pre-existing mature T cells in the marrow inoculum must be inhibited. While avoidance of GVH alloreactivity is not always desirable (see below), many strategies for preventing GVHD induced by mature T cells in the HCT inoculum involve depletion or tolerization of pre-existing GVH-reactive T cells. Tolerization of mature T cells in donor marrow could have both beneficial and harmful effects. The beneficial effects of GVH alloreactivity include GVL/graft-versus-lymphoma effects and promotion of alloengraftment. However, T cells that lack GVH reactivity also have the potential, if given in sufficient numbers, to promote alloengraftment (see Chapter 11), possibly by the veto mechanism described above [210]. Additionally, non-GVH-reactive T-cell populations can confer immunity to pathogens, thereby avoiding the high risk of opportunistic infection associated with global T-cell depletion. Induction of global GVH tolerance is highly desirable in the use of HCT for the treatment of nonmalignant diseases, such as the induction of transplantation tolerance (discussed above), and the treatment of inborn hematologic, immunologic, and metabolic disorders. However, when hematologic malignancy is present, GVH tolerance can be associated with reduced GVL effects, since GVH alloresponses are potent and largely responsible for the GVL response. One way to circumvent this deleterious aspect of GVH tolerance would be to preserve tumor antigenspecific responses while responses to normal host antigens were inhibited. Several candidate tumor-specific antigens have recently been identified (see Chapter 18), and CTL activity can be generated against them, generating interest in the idea of tolerizing GVH-reactive T cells while maintaining tumor-specific responses. However, the frequency of tumor antigen-specific T cells pre-existing in a given T-cell repertoire is even lower than that against multiple minor histocompatibility antigens, and the generation of meaningful tumor-specific responses is likely to necessitate donor presensitization along with in vitro expansion of tumor-specific effector cells, which might limit the homing capacity of injected cells. Such prolonged cultures are impractical for routine use in the setting of HCT, in which leukemia-reactive cells must eliminate exponentially expanding leukemic cells. In theory, the less risky strategy of generating tumor-specific responses from autologous T cells could achieve similar outcomes. However, T-cell immunity may be markedly impaired in the tumor-bearing host, and the use of immunologically unimpaired allogeneic donors is therefore attractive. Minor histocompatibility alloantigens expressed by lymphohematopoietic cells (including leukemias and lymphomas) but not by the epithelial GVHD target tissues may also be targeted using in vitro expanded CTLs in order to achieve GVL without GVHD. Several human minor histocompatibility antigens have demonstrated this pattern of expression (see Chapters 12 and 18]. However, GVH disparities for some of these same minor antigens (e.g. HA-1) have been associated with an increased incidence of GVHD. Recently, it was shown that administration of primed T cells specific for a single immunodominant class I-restricted minor histocompatibility antigen could mediate GVL without GVHD.
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Avoidance of GVHD was dependent on the absence of GVH-reactive T cells with additional specificities in the donor inoculum [211]. If such T cells are present, the response to immunodominant minor histocompatability antigens may induce APC activation and cytokine production, which activates T-cell responses to additional minor histoincompatibilities that are shared by GVHD target organs. However, the potency of antitumor effects in HCT is greatest in the setting of MHC disparity [212–215], in which GVH alloresponses are most potent. The potent ability of the anti-MHC GVH alloresponse to mediate GVL effects reflects the extraordinarily high frequency of T cells recognizing MHC alloantigens that is present in the unprimed Tcell repertoire. T cells reactive with an allogeneic MHC determinant may represent as many as 2% or more of the total T-cell population, whereas T cells reactive with a peptide–self-MHC complex are several orders of magnitude less frequent. Several reasons for this high frequency of allogeneic MHC-reactive T cells have been identified. One is the large number of different “nonself” peptide–MHC complexes presented by an allogeneic MHC molecule (i.e. high number of different determinants), resulting in recognition by a large number of different T-cell clones. The second reason is the large number of MHC molecules on an allogeneic cell. This may lead to recognition by large numbers of TCRs that are sufficiently independent of the peptide component for their recognition of the MHC– peptide complex that they bind with relatively low affinity to the MHC molecules regardless of the peptides to which they are complexed. In these instances, the high density of MHC determinants compensates for the low affinity of the TCRs recognizing them. In contrast, since minor histocompatibility antigens are peptides, the number of minor antigen– MHC complexes on a given APC will be more limited, perhaps resulting in recognition only by TCRs with relatively high affinity. As is discussed later in this section, a number of experimental strategies have been developed that permit exploitation of the GVH alloresponse for GVL effects while avoiding GVHD. Specific depletion or tolerization of mature GVH-reactive T-cells present in the donor inoculum at the time of HCT Several approaches have been developed in efforts to tolerize GVHreactive mature T cells within hematopoietic cell grafts, including blocking of adhesive and co-stimulatory interactions of T cells with recipient APCs. One of these approaches was evaluated in a pilot clinical trial with some success at reducing acute GVHD [216], but insufficient numbers of patients have been transplanted with this approach to allow an assessment of protective immunity from infection or tumor relapse. Additional approaches involve the selective elimination, rather than tolerization, of GVH-reactive T cells. These approaches preserve T-cell populations that may react to tumor-specific antigens and pathogens and perhaps promote engraftment. This goal can be achieved by stimulating donor T cells with host antigens, then depleting the activated cells using antibodies or immunotoxins directed against cell surface markers associated with recent activation, such as CD25 and CD69. Early clinical results with this approach suggest that GVHD is reduced, though not eliminated, and that such inocula may confer some anti-infectious immunity to the recipients [217]. Use of suppressive T cell populations to induce GVH tolerance CD25+CD4+ T cells generated by in vitro culture with alloantigen and anti-CD154 have been shown to suppress GVHD and to tolerize naïve and presensitized T cells via a regulatory cell population [218]. Additionally, donor Tregs can inhibit GVHD while preserving some GVL activity [219], although it is unclear whether or not the Tregs reduce the magnitude of GVL effects. While several groups are now exploring the
possibility of infusing fresh or expanded Tregs clinically, expanding the cells ex vivo has proved to be difficult. One important benefit of Treg infusion in an experimental model is protection of the recipient lymphoid tissues from GVH-associated damage, resulting in improved antiinfectious immunity [220]. In mice, NKT cells in the marrow or in the lymphoid tissues after TLI treatment also have the ability to suppress GVHD, apparently via an IL-4-dependent mechanism [221], while GVL activity is preserved. As is discussed above, this approach has recently been applied in patients receiving HLA-identical HCT with strikingly low rates of acute GVHD [167]. However, it remains to be determined to what extent GVL effects are preserved with this approach. In addition to the NKT cells, T cells lacking NK-cell markers that are resident in murine bone marrow have a reduced capacity to produce GVHD, with preserved ability to mediate GVL [222]. Approaches to preventing GVHD that do not involve induction of tolerance Cytokine manipulation Considerable interest was generated in the 1990s by the notion that GVHD-inducing T cells had a Th1 (IFN-γ- and IL-2-producing) phenotype and that Th2 (IL-4-, IL-5-, and IL-13-producing cells) cells could suppress GVHD, and some evidence supported this possibility. Similarly, CD8 cells producing type 1 cytokines mediated severe GVHD in MHC class I-mismatched or parent into F1 hybrid combinations, whereas type 2 cytokines did not [223]. Meanwhile, Th2 cytokines mediated GVL and promoted donor marrow engraftment [223]. However, infusion of ex vivo polarized donor Th2 cells did not produce a measurable reduction in GVHD in a clinical trial [224]. In fact, the role of Th1- and Th2-associated cytokines in HCT and transplantation in general is rather complex. Systemic administration of Th1-type cytokines such as IL-2, IL-12 or IFN-γ shortly after BMT in mice can actually reduce the severity of GVHD [225–227] while preserving the GVL effect [228,229]. Furthermore, T cells from IFN-γ-deficient donors have an increased capacity to cause acute GVHD in lethally irradiated recipients (reviewed in [230]). While the mechanism by which IFN-γ inhibits graft rejection and GVHD is not fully understood, possible mechanisms have already been discussed above. Further studies have shown that Th2 and Th2-associated cytokines are also capable of contributing significantly to GVHD pathology of particular tissues. Other approaches A number of additional approaches to ameliorating GVHD do not involve initial tolerance induction of donor T cells in the HCT graft. One such approach is to transduce donor T cells with suicide genes, such as herpes simplex virus thymidine kinase, so that the alloresponse can be turned off at will, hopefully after tumor has been eradicated [231]. Initial clinical success with this approach has been somewhat limited, in part due to the fact that ganciclovir, the drug used to kill thymidine kinase-transduced T cells, is also needed clinically to treat cytomegalovirus reactivation, which is a common and serious complication of HCT. Furthermore, the transduction of GVH-reactive T cells may be incomplete, resulting in escape of some clones from suicide after treatment has been initiated. Finally, the manipulations involved in the transduction process itself might render the donor T cells less effective at eliminating leukemias when transferred back to the patient. The development of new vectors and the use of genes other than thymidine kinase may lead to further improvements in the utility of this approach. Another approach is to attempt to block the early inflammatory mediators of GVHD, such as IL-1 or TNF-α. While some of these approaches have been evaluated in pilot clinical trials, none has yet been shown to have
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a durable effect on GVHD. Recently, keratinocyte growth factor has been shown to ameliorate GVHD without blocking GVL, perhaps by reducing the disruption of the intestinal barrier associated with myeloablative conditioning and by preventing some of the thymic injury and consequent immune damage associated with GVHD (see Chapter 16). Delayed donor T-cell administration DLIs can mediate powerful GVL effects in patients with chronic myeloid leukemia, lymphomas, and multiple myeloma who have relapsed following allogeneic HCT (see Chapter 72). In established mixed allogeneic chimeras, which are immunologically tolerant of their original marrow donor’s antigens, a GVH reaction takes place after administration of DLI, resulting in conversion of the state of mixed hematopoietic chimerism to a state of full donor chimerism. This conversion to full chimerism is due to a GVH reaction directed primarily against recipient MHC alloantigens. Remarkably, this powerful GVH alloreaction is not associated with any clinically significant GVHD, even though recipient alloantigens are expressed on many cell types and donor T cells are given in numbers that would cause rapidly lethal GVHD in freshly irradiated recipients [182,183,215,232]. This demonstration that GVH reactions could be confined to the lymphohematopoietic system suggested an approach to separating GVHD from GVL effects. Since hematologic malignancies reside largely in the lymphohematopoietic system, the result suggested that GVH reactions might be confined to this system and eliminate tumor cells without entering the epithelial GVHD target tissues. Indeed, GVL is achieved without GVHD when delayed DLIs are given to established murine mixed allogeneic chimeras [184,215]. Recent studies have revealed mechanisms by which delayed DLI may mediate GVL without GVHD. Antihost MHC alloreactivity clearly mediates the most potent GVL effects [184,215]. Moreover, GVHreactive donor T cells become activated and proliferate markedly in mixed chimeric recipients of DLI [215,232]. However, these cells do not migrate to GVHD target tissues (epithelial tissues such as skin, intestines, and liver) unless there are inflammatory signals in those tissues [232]. The recipient’s recovery from conditioning-induced tissue injury markedly reduces the tendency of GVH-reactive donor T cells that are activated by host alloantigens in the lymphoid tissues to leave the lymphohematopoietic system and cause GVHD [232]. Provision of a local toll-like receptor stimulus promotes the entry of such cells into the skin and induces localized GVHD [232], indicating that tissue inflammation provides a critical checkpoint for T-cell recruitment to GVHD target tissues. Such inflammatory signals can be provided by the tissue injury induced by conditioning therapy and by toll-like receptor activation [232], which may be induced by infection or by conditioning itself. Both high-dose and reduced-intensity conditioning are associated with very early production of chemokines in the GVHD target tissues, which is followed by immigration of T cells that then elicit a further cascade of chemokines that amplifies the response [233]. Adhesion molecules that promote leukocyte infiltration through the microvasculature of these tissues are also likely to be upregulated by conditioning. Lethal TBI and cyclophosphamide are known to upregulate the proinflammatory cytokines IL-1, IL-6, and TNF-α [234–236], which can upregulate endothelial cell E-selectin, P-selectin, ICAM-1, and VCAM1 [237,238]. Proof of principle has been obtained that delayed DLI can mediate GVH responses confined to the lymphohematopoietic system in patients receiving nonmyeloablative HCT with in vivo or ex vivo T-cell depletion of the initial donor inoculum, who achieve mixed chimerism without GVHD [186,239]. However, some patients achieving initial mixed chimerism from a TCD donor inoculum nevertheless develop GVHD following administration of delayed DLI. In general, delayed DLI is somewhat variably associated with GVHD in patients, but to a lesser
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degree than would be expected in freshly conditioned recipients of similar cell numbers [240,241]. The greater ease in avoiding GVHD in mice compared with humans receiving delayed DLI after TCD HCT may reflect the more robust thymic T-cell recovery that occurs in mice than in heavily pretreated human adults receiving TCD HCT. This thymic recovery confers protection from infection, avoiding toll-like receptor stimuli, and also allows the recovery of regulatory T-cell populations that downregulate GVH reactions. The ability of Treg to inhibit DLI-induced GVHD has been demonstrated in mouse models [242– 244]. The potent antitumor responses seen in patients with advanced lymphoid malignancies who received the transplant approach described above (mixed chimerism induction using TCD HCT and nonmyeloablative conditioning, followed by delayed DLI [245]) led to the hypothesis that an initial state of mixed chimerism might permit more powerful graft-versus-tumor effects to be achieved from the GVH alloresponse than is possible in full allogeneic chimeras. To test this hypothesis, the GVL effects of DLI were compared in mixed versus full allogeneic chimeras inoculated with a host-type T-cell lymphoma in a murine model. Remarkably, DLI led to only a slight prolongation of survival in leukemic full allogeneic chimeras, whereas they led to almost complete protection, with 80–100% survival, in mixed chimeras receiving the same tumor inoculum (Fig. 15.3). This markedly more potent GVT effect of DLI in mixed compared with full chimeras was shown to be dependent on the expression of host class I and class II MHC molecules on recipient-type hematopoietic cells [184,215]. GVHD was not observed in the mixed chimeras with these potent GVL effects from DLI [184]. These data are not inconsistent with an earlier report showing a critical role for recipient hematopoietic APCs in initiating GVHD [246]; in that report, the donor T cells were given to freshly lethally irradiated mice, whereas the donor T cells were given as delayed DLI in the studies comparing mixed with full chimeras [184]. The complete absence of GVHD in mixed chimeras enjoying potent GVL effects of DLI dramatically illustrates the power to separate GVHD and GVT effects by confining the anti-MHC alloresponse to the lymphohematopoietic system. GVH tolerance of T-cells developing from progenitors in the marrow As has been discussed above, the mechanism of GVH tolerance in lethally irradiated recipients of TCD allogeneic marrow alone (i.e. “full” chimeras) appears mainly to involve T-cell anergy rather than clonal deletion [12]. In contrast, mixed chimeras show excellent clonal deletion of T cells reacting against host antigens [127,247], undoubtedly due to the intrathymic presence of host-type marrow-derived cells in mixed chimeras [127]. However, partial clonal deletion of host-reactive T cells has been described in lethally irradiated mice receiving allogeneic BMT alone [248,249], which may reflect the weaker ability of thymic epithelial cells (compared with hematopoietic cells) to induce intrathymic deletion [250–252]. Perturbations in the thymic environment, such as the destruction of thymic epithelial elements observed in GVHD [253,254], or thymic injury induced by treatment with cyclosporine [255,256], can impair processes leading to tolerance [257,258]. Defective production of Tregs may contribute to autoimmunity in this setting.
Summary and future directions It is clear that HCT has not yet met its full potential to provide a cure for lymphohematopoietic malignancies or to treat nonmalignant diseases. There is a need for improved methods of exploiting the immuno-
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Fig. 15.3 Superior donor leukocyte infusion (DLI)-mediated graft-versus-leukemia effects in mixed compared with full allogeneic chimeras. Mixed and full chimeras were prepared by lethally irradiating B6 mice and reconstituting them with either T-cell-depleted allogeneic (B10.A) bone marrow cells alone (to produce full chimeras), or a mixture of T-cell-depleted B6 plus B10.A marrow (to produce mixed [Mix] chimeras). DLIs were administered on day 56 postbone marrow transplantation, and this step was followed by intravenous injection of 500 EL4 (a B6 T-cell lymphoma) cells, on day 63 (↓). (a) Full chimeras receiving DLI and EL4 cells (䊏; n = 20) showed a slight but statistically significant prolongation of survival compared with full chimeras receiving tumor cells only (䊐; n = 12; p < 0.001). Full chimeras receiving (䉲; n = 14) or not receiving DLI (䉮; n = 7) showed no treatment-related mortality. (b) Mixed chimeras receiving (◊; n = 11) or not receiving (䉫; n = 8) DLI showed no treatment-related mortality. Mixed chimeras receiving DLI and EL4 cells (䊉; n = 25) had a significantly improved survival compared with mixed chimeras receiving EL4 alone (䊊; n = 14; p < 0.0001). (c) Mixed chimeras receiving DLI and EL4 cells (䊉; n = 25) had a significantly improved survival compared with full chimeras receiving EL4 +DLI (䊏; n = 20; p < 0.0001). (Reproduced from Mapara MY, Kim YM, Wang SP, Bronson R, Sachs DH, Sykes M. Donor lymphocyte infusions mediate superior graft-versus-leukemia effects in mixed compared to fully allogeneic chimeras: a critical role for host antigen-presenting cells. Blood 2002; 100: 1903–9, with permission.)
therapeutic aspects of HCT without causing GVHD. In view of the new strategies and improved understanding of these phenomena that are developing, it seems likely that this challenge can be met and that HCT will ultimately be made available to all who could benefit from it, including those lacking an HLA-matched or closely matched donor. In addition, advances in the ability to achieve engraftment of hematopoietic and thymic tissue without ablative treatment of the host, and the demonstration of efficacy in primate models, have brought these approaches to initial clinical application for the induction of solid organ graft tolerance. Improvements in the ability to overcome HVG resistance to HLAmismatched HSC engraftment without increasing host toxicity should
further broaden the applicability of this approach. It will also be essential to extend this ability to xenogeneic marrow and organ transplantation, which presents additional immunologic and physiologic hurdles, to overcome the critical organ shortage that currently limits the number of life-saving organ transplants that can be performed. A major goal of transplant immunologists therefore now will be to extend new laboratory findings, first to additional preclinical models in large animals, and then to clinical protocols. In addition, it will be important to continue basic research into the mechanisms by which tolerance is induced and maintained in rodent model systems, so that these mechanisms can be further exploited.
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200. Markert ML, Kostyu DD, Ward FE et al. Successful formation of a chimeric human thymus allograft following transplantation of cultured postnatal human thymus. J Immunol 1997; 158: 998–1005. 201. Zerrahn J, Held W, Raulet DH. The MHC reactivity of the T cell repertoire prior to positive and negative selection. Cell 1997; 88: 627–36. 202. Nikolic B, Gardner JP, Scadden DT, Arn JS, Sachs DH, Sykes M. Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC. J Immunol 1999; 162: 3402–7. 203. Ohdan H, Sykes M. B cell tolerance to xenoantigens. Xenotransplantation 2003; 10: 98–106. 204. Kawahara T, Shimizu I, Ohdan H, Zhao G, Sykes M. Differing mechanisms of early and late B cell tolerance induced by mixed chimerism. Am J Transplant 2005; 5: 2821–9. 205. Shimizu I, Kawahara T, Haspot F, Bardwell PD, Carroll MC, Sykes M. B-cell extrinsic CR1/CR2 promotes natural antibody production and tolerance induction of anti-alpha-Gal-producing B-1 cells. Blood 2006; 109: 1773–81. 206. Bracy JL, Iacomini J. Induction of B-cell tolerance by retroviral gene therapy. Blood 2000; 96: 3008–15. 207. Phelps CJ, Koike C, Vaught TD et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science 2003; 299: 411–14. 208. Kolber-Simonds D, Lai L, Watt SR et al. Production of {alpha}-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proc Natl Acad Sci U S A 2004; 101: 7335–40. 209. Chen G, Qian H, Starzl T et al. Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys. Nat Med 2005; 11: 1295–8. 210. Reich-Zeliger S, Zhao Y, Krauthgamer R, BacharLustig E, Reisner Y. Anti-third party CD8+ CTLs as potent veto cells: coexpression of CD8 and FasL is a prerequisite. Immunity 2000; 13: 507– 15. 211. Fontaine P, Roy-Proulx G, Knafo L, Baron C, Roy DC, Perreault C. Adoptive transfer of minor histocompatibility antigen-specific T lymphocytes eradicates leukemia cells without causing graft-versus-host disease. Nat Med 2001; 7: 789–94. 212. Sykes M, Sachs DH. Genetic analysis of the antileukemic effect of mixed allogeneic bone marrow transplantation. Transplant Proc 1989; 21: 302224. 213. Aizawa S, Sado T. Graft-versus-leukemia effect in MHC-compatible and -incompatible allogeneic bone marrow transplantation of radiation-induced, leukemia-bearing mice. Transplantation 1991; 52: 885–9. 214. Beatty PG, Anasetti C, Hansen JA et al. Marrow transplantation from unrelated donors for treatment of hematologic malignancies: effect of mismatching for one HLA locus. Blood 1993; 81: 249–53. 215. Chakraverty R, Eom HS, Sachs J et al. Host MHC Class II+ antigen-presenting cells and CD4 cells are required for CD8-mediated graft-versusleukemia responses following delayed donor leukocyte infusions. Blood 2006; 108: 2106–13. 216. Guinan EC, Boussiotis VA, Neuberg D et al. Transplantation of anergic histoincompatible
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James L.M. Ferrara & Joseph H. Antin
The Pathophysiology of Graft-Versus-Host Disease
Acute GVHD pathophysiology: a three-step model Acute graft-versus-host disease (GVHD) results from complex interactions between donor T cells and host tissues in an inflammatory milieu. The pathophysiology of acute GVHD can be considered as a three-step process involving both the innate and adaptive immune systems (Fig. 16.1). The three steps are: (1) tissue damage to the recipient by the radiation/chemotherapy pretransplant conditioning regimen; (2) donor T-cell activation and clonal expansion; and (3) cellular and inflammatory factors. This schema underscores the importance of mononuclear phagocytes and other accessory cells to the development of GVHD after complex interactions with cytokines secreted by activated donor T cells. In step 1, the conditioning regimen (irradiation and/or chemotherapy) leads to damage and activation of host tissues throughout the body and the secretion of the inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1). These cytokines enhance donor T-cell recognition of host alloantigens by increasing the expression of major histocompatibility complex (MHC) antigens and other molecules on host antigen-presenting cells (APCs). An important aspect of the mucosal injury in the gastrointestinal tract is the resultant leakage of endotoxins into the systemic circulation. In step 2, host APCs present alloantigen in the form of a peptide– human leukocyte antigen (HLA) complex to the resting T cells. Costimulatory signals are required for T-cell activation, and these signals further activate APCs and further enhance T-cell stimulation. Donor T-cell activation is characterized by cellular proliferation and the secretion of cytokines, including IL-2 and interferon-gamma (IFN-γ). IL-2 expands the T-cell clones and induces cytotoxic T lymphocyte (CTL) responses, whereas IFN-γ primes mononuclear phagocytes to produce TNF-α and IL-1. Regulatory T cells (Tregs) that can act as a powerful brake on the GVH reaction also expand at this time. In step 3, effector functions of mononuclear phagocytes and neutrophils are triggered through a secondary signal provided by mediators, such as lipopolysaccharide (LPS), that leak through the intestinal mucosa damaged during step 1. Release of inflammatory cytokines stimulates host tissues to produce inflammatory chemokines that direct activated T cells and other effector cells such as mononuclear cells and granulocytes into target organs where they adhere due to increased adhesion molecule expression. This mechanism results in the amplification of local tissue injury, other cellular effectors and further promotion of a
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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proinflammatory response which, together with CTLs, leads to target tissue destruction. It should be noted from the outset that all these steps do not carry equal weight in the pathogenesis of acute GVHD. The pivotal interaction occurs in step 2, where host APCs activate allogeneic donor T cells. The subsequent cytokine cascade is clearly important, but blockade of individual cytokines may not reverse established GVHD when other cellular effectors such as CTLs are present. GVHD can also occur when no conditioning of the host has occurred (e.g. transfusion-associated GVHD).
Step 1: Effects of conditioning in hematopoietic cell transplantation The first step of acute GVHD starts before donor cells are infused. Prior to hematopoietic cell transplantation (HCT), a patient’s tissues have been damaged, sometimes profoundly, by underlying disease and its treatment, infection, and transplant conditioning. High-intensity chemoradiotherapy characteristic of HCT conditioning regimens activates host APCs that are critical to the stimulation of donor T cells infused in the stem cell inoculum. These important effects help explain a number of unique and seemingly unrelated aspects of GVHD. For example, a number of clinical reports have noted increased risks of GVHD associated with advanced stage leukemia, certain intensive conditioning regimens, and histories of viral infections [1]. Total body irradiation is particularly important in this process because it activates host tissues to secrete inflammatory cytokines, such as TNF-α and IL-1 [2], and it induces endothelial apoptosis that leads to epithelial cell damage in the gastrointestinal tract [3]. Injury to the gut is transient and self-limited after autologous HCT. However, after allogeneic HCT, further damage by GVHD effectors amplifies gastrointestinal tract and systemic GVHD by allowing the translocation of microbial products such as LPS into the systemic circulation. This scenario helps to explain the increased risk of GVHD associated with intensive conditioning regimens. The relationship between conditioning intensity, inflammatory cytokines, and GVHD severity has been confirmed by animal models [4] and clinical observations [1].
Step 2: Donor T-cell activation and cytokine secretion Donor T-cell activation Donor T-cell activation occurs during the second step of acute GVHD. Murine studies have demonstrated that host APCs alone are both
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Conditioning: Tissue Damage
(I) Host APC activation
Host tissues
Small intestine
TNF-a IL-I LPS
LPS
Mf Host APC
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TNF-g Donor T-cell
(II) Donor T-cell activation
Target cell apoptosis Thr
CD4 CTL
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TNF-a IL-I CD8 CTL
CD8 CTL
(III) Cellular and inflammatory effectors
Fig. 16.1 The pathophysiology of acute graft-versus-host disease (GVHD). GVHD pathophysiology can be summarized in a three-step process. In step 1, the conditioning regimen (irradiation, chemotherapy or both) leads to the damage and activation of host tissues, especially the intestinal mucosa. This allows the translocation of lipopolysaccharide (LPS) and other inflammatory stimuli from the intestinal lumen to the circulation, stimulating the secretion of the inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) from host tissues. These mediators increase the expression of major histocompatibility complex (MHC) antigens and adhesion molecules on host antigen presenting cells (APCs), enhancing the recognition of MHC and minor histocompatibility antigens by mature donor T cells. In step 2, donor T-cell activation is characterized by a predominance of T helper type 1 (Th1) cells and the secretion of interferon-gamma (IFN-γ), which activates mononuclear phagocytes. Regulatory T cells (Tregs) limit the proliferation and clonal expansion of activated donor T cells. In step 3, effector functions of activated mononuclear phagocytes are triggered by the secondary signal provided by LPS and other stimulatory molecules that leak through the intestinal mucosa damaged during steps 1 and 2. Activated macrophages, along with cytotoxic T lymphocytes (CTL), secrete inflammatory cytokines that cause target cell apoptosis. CD8+ CTLs also lyse target cells directly. Damage to the gastrointestinal tract in this phase, principally by inflammatory cytokines, amplifies LPS release and leads to the “cytokine storm” characteristic of severe acute GVHD. This damage results in the amplification of local tissue injury and further promotes an inflammatory response.
necessary and sufficient to stimulate donor T cells [5,6]. Donor T cells migrate to secondary lymphoid organs, such as Peyer’s patches, lymph nodes, and the spleen, where they first encounter host APCs [7]. In murine models of GVHD to MHC differences between donor and host, robust donor T-cell proliferation is observed in the spleen as early as day 3 after HCT, preceding the engraftment of donor stem cells [5,7]. After allogeneic HCT, both host- and donor-derived APCs are present in secondary lymphoid organs. T-cell receptors (TCRs) of donor T cells can recognize alloantigens on either host APCs (direct presentation) or donor APCs (indirect presentation). During direct presentation, donor T cells recognize either the peptide bound to allogeneic MHC molecules or the foreign MHC molecules themselves [8]. During indirect presentation, T cells respond to the peptides generated by degradation of the allogeneic MHC molecules that are presented on self-MHCs [9]. In GVHD to minor histocompatibility antigens (mHAs), direct presentation is dominant because APCs derived from the host rather than from the donor are critical [6]. Several laboratories have identified that naïve (CD62L+) T cells cause experimental GVHD whereas memory (CD62L−) T cells do not, although memory stem cells may be involved [10,11]. In the majority of HLAidentical HCT, GVHD is induced by mHAs, which are peptides derived from polymorphic cellular proteins that are presented on the cell surface
by MHC molecules [12]. Because the genes for these proteins are located outside the MHC, two siblings often have many different peptides in the MHC groove. In this case, different peptides presented by the same MHCs are recognized by donor T cells and lead to GVHD. In mice, the actual number of so-called “major minor antigens” that can potentially induce GVHD is likely to be limited. In humans, of five previously characterized mHAs (HA-1, -2, -3, -4, and -5) recognized by T cells in association with HLA-A1 and -A2, mismatching of HA-1 alone was significantly correlated with acute grade II–IV GVHD. Theoretical models also predict substantial benefits if multiple minor loci could be typed [13]. Several mHAs are encoded on the male-specific Y chromosome and are associated with an increased risk of GVHD when male recipient cells are transplanted from female donors [14]. mHAs with tissue expression limited to the hematopoietic system are potential target antigens of graft-versus-leukemia (GVL) reactivity [15], and separation of GVHD and GVL by using CTLs specific for such antigens is an area of intense research [16]. Adhesion molecules mediate the initial binding of T cells to APCs. TCR signaling after antigen recognition induces a conformational change in adhesion molecules, resulting in higher-affinity binding to the APCs [17]. Full T-cell activation also requires co-stimulatory signals provided by APCs in addition to TCR signals. Two primary co-
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stimulatory pathways signal through either CD28 or TNF receptors. Currently, there are four known CD28 superfamily members expressed on T cells: CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), inducible co-stimulator, and programmed death-1. In addition, there are four TNF receptor family members: CD40 ligand (CD154), 4–1BB (CD137), OX40, and herpes simplex virus glycoprotein D or herpesvirus entry mediator. The best-characterized co-stimulatory molecules, CD80 and CD86, deliver positive signals through CD28 that lower the threshold for T-cell activation and promote T-cell differentiation and survival, while signaling through CTLA-4 is inhibitory [18]. The most potent APCs are dendritic cells (DCs); however, the relative contribution of DCs and other semiprofessional APCs, such as monocytes/macrophages and B cells, to the development of GVHD remains to be elucidated. Donor APCs may also amplify GVHD, but in the skin, Langerhans cells of host origin appear to be essential for the activation of donor T cells [18,19]. Signaling through Toll-like receptors (TLRs) and through other innate immune receptors such as NOD may act as “danger signals” and activate host APCs, amplifying the donor T-cell response [20,22]. DCs can be matured and activated during HCT by: (1) inflammatory cytokines; (2) microbial products such as LPS and the dinucleotide CpG entering the systemic circulation from intestinal mucosa damaged by conditioning; and (3) necrotic cells that are damaged by recipient conditioning. All of these stimuli may be considered “danger signals” [20] and may make the difference between an immune response and tolerance [23]. When T cells are exposed to antigens in the presence of adjuvant such as LPS, the migration and survival of T cells are dramatically enhanced in vivo [24]. The effect of age in enhancing the allostimulatory activity of host APCs may also help explain the increased incidence of acute GVHD in older recipients [25]. The elimination of host APCs by activated natural killer (NK) cells can prevent GVHD [26]. This suppressive effect of NK cells on GVHD may have relevance in humans: HLA class I differences driving donor NK-mediated alloreactions in the GVH direction mediate potent GVL effects and produce higher engraftment rates without causing severe acute GVHD [26,27].
Cytokine secretion by donor T cells T-cell activation involves multiple, rapidly occurring intracellular biochemical changes, including the rise of cytoplasmic free calcium and activation of protein kinase C and tyrosine kinases [28]. These pathways in turn activate the transcription of genes for cytokines such as IL-2, IFN-γ, and their receptors. Cytokines secreted by activated T cells are often classified as T-helper type 1 subset (Th1) (secreting IL-2 and IFNγ) or T-helper type 2 subset (Th2) (secreting IL-4, IL-5, IL-10, and IL-13) [29]. The role of Th17 cells, a recently described functional Tcell subset, has not yet been clarified in GVHD [30]. Several factors influence the ability of DCs to instruct naïve CD4+ T cells to secrete Th1 or Th2 cytokines. These factors include the type and duration of DC activation along with the DC : T-cell ratio and the proportions of DC subsets present during T-cell interactions [31]. Differential activation of Th1 or Th2 cells has been evoked in the immunopathogenesis of GVHD and the development of infectious and autoimmune diseases. Although this dichotomy has many exceptions, in both settings activated Th1 cells: 1 amplify T-cell proliferation by secreting IL-2; 2 lyse target cells by Fas–Fas ligand (FasL) interactions; 3 induce macrophage differentiation in the bone marrow by secreting IL-3 and granulocyte macrophage colony-stimulating factor; 4 activate macrophages by secreting IFN-γ and by their CD40–CD40 ligand interactions; 5 activate endothelium to induce macrophage binding and extravasation;
6 recruit macrophages by secreting monocyte chemoattractant protein-1 [32]. During step 2 of acute GVHD pathophysiology, IL-2 has a pivotal role in both controlling and amplifying the immune response against alloantigens. IL-2 induces the expression of its own receptor (autocrine effect) and stimulates proliferation of other cells expressing the receptor (paracrine effect). IL-2 is secreted by donor CD4+ T cells in the first several days after GVHD induction [33]. In some studies, the addition of low doses of IL-2 during the first week after allogeneic bone marrow transplantation enhanced the severity and mortality of GVHD [34]. The precursor frequency of host-specific IL-2 producing cells predicts the occurrence of clinical acute GVHD [35,36]. Monoclonal antibodies (mAbs) against IL-2 or its receptor can prevent GVHD when administered shortly after the infusion of T cells [37,38], but this strategy was only moderately successful in reducing the incidence of severe GVHD [39]. Cyclosporine (CSP) and tacrolimus dramatically reduce IL-2 production and effectively prevent GVHD. IL-15 is another critical cytokine in initiating allogeneic T-cell division in vivo [40], and elevated serum levels of IL-15 are associated with acute GVHD in humans [41]. IL-15 may, therefore, also be important in the clonal expansion of donor T cells in step 2. IFN-γ is another crucial cytokine that can be implicated in the second step of the pathophysiology of acute GVHD. Increased levels of IFN-γ are associated with acute GVHD [42,43], and a large proportion of Tcell clones isolated from GVHD patients produce IFN-γ [44]. In animals with GVHD, IFN-γ levels peak between days 4 and 7 after transplantation before clinical manifestations are apparent. CTLs that are specific for mHAs and produce IFN-γ also correlate with the severity of GVH reaction in a skin-explant assay of human disease [45]. Experimental data demonstrate that IFN-γ modulates several aspects of the pathophysiology of acute GVHD. First, IFN-γ increases the expression of numerous molecules involved in GVHD, including adhesion molecules, chemokines, MHC antigens, and Fas, resulting in enhanced antigen presentation and the recruitment of effector cells into target organs [46]. Second, IFN-γ alters target cells in the gastrointestinal tract and skin so that they are more vulnerable to damage during GVHD; the administration of anti-IFN-γ mAbs prevents gastrointestinal tract GVHD [47], and high levels of both IFN-γ and TNF-α correlate with the most intense cellular damage in the skin [45]. Third, IFN-γ mediates GVHD-associated immunosuppression seen in several experimental HCT systems in part by the induction of nitric oxide (NO) [48–50]. Fourth, IFN-γ primes macrophages to produce proinflammatory cytokines and NO in response to LPS [51]. At early time points after HCT, IFN-γ may paradoxically reduce GVHD by enhancing Fas-mediated apoptosis of activated donor T cells [52,53]. Both cell-mediated and inflammatory cytokine GVHD effector mechanisms can sometimes be inhibited if donor T cells produce fewer Th1 cytokines [54]. Furthermore, cell mixtures of Th2 donor cells with an otherwise lethal inoculum of allogeneic bone marrow and T cells also protect recipient mice from LPS-induced lethality, demonstrating the ability of Th2 cells to modulate Th1 responses after allogeneic transplantation [55]. Polarization of donor T cells toward a Th2 phenotype by pretreating HCT donors with granulocyte colony-stimulating factor also results in less severe GVHD [56]. It should be noted, however, that systemic administration of Th2 cytokines IL-4 or IL-10 as experimental prophylaxis of GVHD was either ineffective or toxic [57,58]. On the other hand, administration of Th1 cytokines can also reduce GVHD. High doses of exogenous IL-2 early after bone marrow transplantation protect animals from GVHD mortality [59]. It has been suggested that IL-2 mediates its protective effect via inhibition of IFNγ [42]. However, injection of IFN-γ itself can prevent experimental GVHD [60], and neutralization of IFN-γ results in accelerated GVHD
The Pathophysiology of Graft-Versus-Host Disease
in lethally irradiated recipients [61]. These paradoxes may be explained by the complex dynamics of donor T-cell activation, expansion, and contraction. Activation-induced cell death is a chief mechanism of clonal deletion and is largely responsible for the rapid contraction of activated T cells following an initial massive expansion [62]. Thus, the complete absence of IFN-γ may result in an unrestrained expansion of activated donor T cells, leading to accelerated GVHD, which may be of particular importance in recipients of intensified conditioning [4]. Similarly, administration of IFN-γ-inducing cytokines, such as IL-12 or IL-18, protects mice from GVHD in a Fas-dependent fashion [53]. Thus, moderate amounts of Th1 cytokines production after donor T-cell expansion may amplify GVHD; extremes in production (either low or high), particularly during T-cell expansion, may hasten the death of activated donor T cells, aborting T-cell expansion and reducing GVHD. Regulatory T cells In both humans and mice, Treg deficiency results in immune dysregulation and autoimmunity, as characterized by the human IPEX syndrome resulting from loss of function mutations of FOXP3 [63]. A similar condition occurs in scurfy mice, a strain that lacks the transcription factor FOXP3 [64]. FOXP3 appears to function as a master control gene for the development and function of natural Tregs, which normally constitute approximately 5–10% of the circulating CD4+ T cell population. Tregs constitutively express markers associated with activated/ memory T cells: CD25 (IL-2 receptor), glucocorticoid-induced TNF-α receptor, CD152 (CLTA-4), CD103, CD62L, and neuropilin-1; and downregulate the expression of CD45RB and CD127 [65,66]. Tregs suppress both innate and the adaptive immune functions [67–69] by producing inhibitory cytokines (IL-10 and transforming growth factorbeta) as well as by cell contact-dependent inhibition of APC function and direct cytotoxicity against antigen-presenting B cells [68,70]. Additional purified CD4+CD25+ Treg populations can suppress the proliferation of conventional T cells and prevent GVHD [71,72]. Tregs do not themselves induce GVHD, and the small numbers of Treg cells present in a graft appear to be overwhelmed by the large number of conventional donor T cells present. The use of calcineurin inhibitors and thymic injury from GVHD may also interfere with the development of adequate numbers of Treg cells that can control GVHD [71,73–75]. NK1.1+ T cells may also possess regulatory function, and both peripheral blood and marrow NK1.1+ T cells can prevent GVHD by their IL-4 secretion [76].
Step 3: Cellular and inflammatory effectors The pathophysiology of acute GVHD culminates in the generation of multiple cytotoxic effectors that contribute to target tissue injury. At a minimum, step 3 includes: (1) several inflammatory cytokines; (2) specific antihost CTL activity using Fas and perforin pathways; (3) large granular lymphocytes or NK cells; and (4) NO. Significant experimental and clinical data suggest that soluble inflammatory mediators act in conjunction with direct cell-mediated cytolysis by CTL and NK cells to cause the full spectrum of deleterious effects seen during acute GVHD. As such, the effector phase of GVHD involves aspects of both the innate and adaptive immune response, and the synergistic interactions of components generated during steps 1 and 2.
Cellular effectors The Fas/FasL and perforin/granzyme (or granule exocytosis) pathways are the principal effector mechanisms used by CTLs and NK cells to lyse their target cells [77,78]. Following recognition of a target cell through TCR–MHC interaction, CTLs secrete perforin and insert it into
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the target cell membrane, forming “perforin pores” that allow granzymes to enter the cell and induce apoptosis through various downstream effector pathways [79]. Ligation of Fas results in the formation of the deathinducing signaling complex and the subsequent activation of caspases [80]. A number of ligands on T cells also possess the capability to trimerize TNF-α receptor (TNFR)-like death receptors (DR) on their targets, such as TNF-related apoptosis-inducing ligand (TRAIL : DR4,5 ligand) and TNF-like weak inducer of apoptosis (TWEAK : DR3 ligand) [81,82]. The involvement of these pathways in GVHD has been tested by utilizing donor cells that are genetically deficient in each molecule. Lethal GVHD occurs even in the absence of perforin-dependent killing, demonstrating that the perforin/granzyme pathway plays a significant, but not exclusive, role in target organ damage. CD4+ CTLs preferentially use the Fas/FasL pathway during acute GVHD, while CD8+ CTLs primarily use the perforin/granzyme pathway, consistent with other conditions involving cell-mediated cytolysis [18]. Fas is a TNF-receptor family member that is expressed by many tissues, including GVHD target organs. Inflammatory cytokines such as IFN-γ and TNF-α can increase the expression of Fas during GVHD [83]. FasL expression on donor T cells is also increased during GVHD [84,85]. Elevated serum levels of soluble FasL and Fas have also been observed in some patients with acute GVHD [86,87]. FasLdefective donor T cells cause markedly reduced experimental GVHD in liver, skin, and lymphoid organs [88,89]. The Fas/FasL pathway is particularly important in hepatic GVHD, consistent with the marked sensitivity of hepatocytes to Fas-mediated cytotoxicity in models of murine hepatitis [90]. Fas-deficient recipients are protected from hepatic GVHD, but not from GVHD in other target organs [91]. Administration of anti-FasL (but not anti-TNF) mAbs significantly blocked hepatic GVHD damage occurring in one model [92], whereas the use of FasL-deficient donor T cells or the administration of neutralizing FasL mAbs had no effect on the development of intestinal GVHD in several studies [93,94].
Inflammatory effectors In the effector phase of acute GVHD, inflammatory cytokines synergize with CTLs, resulting in the amplification of local tissue injury and the development of target organ dysfunction in the transplant recipient. The cytokines TNF-α and IL-1 are produced by an abundance of cell types involved in innate and adoptive immune responses, and they have synergistic and redundant activities during several phases of acute GVHD. A central role for inflammatory cytokines in acute GVHD was confirmed by a recent murine study using bone marrow chimeras in which either MHC class I or MHC class II alloantigens were not expressed on target epithelium but on APCs alone [5]. GVHD target organ injury was induced in these chimeras even in the absence of epithelial alloantigens, and mortality and target organ injury were prevented by the neutralization of TNF-α and IL-1. These observations were particularly true for CD4-mediated acute GVHD but also applied, at least in part, for CD8mediated disease. A critical role for TNF-α in the pathophysiology of acute GVHD was first suggested almost 15 years ago because mice transplanted with mixtures of allogeneic bone marrow and T cells developed severe skin, gut, and lung lesions that were associated with high levels of TNF-α messenger RNA in these tissues [95]. Target organ damage could be inhibited by infusion of anti-TNF-α mAbs, and mortality could be reduced from 100% to 50% by the administration of the soluble form of the TNF-α receptor [2]. Extensive experimental data further suggest that TNF-α is involved in the multistep process of GVHD pathophysiology. TNF-α can:
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1 cause cachexia, a characteristic feature of GVHD; 2 induce activation of DCs, thus enhancing alloantigen presentation; 3 recruit effector T cells, neutrophils, and monocytes into target organs through the induction of inflammatory chemokines; 4 cause direct tissue damage by inducing apoptosis and necrosis [96]. TNF-α is also involved in donor T-cell activation directly through its signaling via TNFR1 and TNFR2 on T cells; TNF–TNFR1 interactions promote alloreactive T-cell responses [97], whereas TNF–TNFR2 interactions are critical for intestinal GVHD [98]. TNF-α plays a central role in intestinal GVHD in murine and human studies [92,95]. TNF-α also seems to be an important effector molecule in GVHD in skin and lymphoid tissue [94,95,99] and can contribute to hepatic GVHD, probably by enhancing effector cell migration to the liver via the induction of inflammatory chemokines. Studies in animals have demonstrated that neutralization of TNF-α alone or in combination with IL-1 resulted in a significant reduction of hepatic GVHD [5,100]. An important role for TNF-α in clinical acute GVHD has also been suggested by multiple studies. Elevations of TNF-α protein (serum) and messenger RNA (peripheral blood mononuclear cells) have been measured in patients with acute GVHD and other endothelial complications, such as hepatic veno-occlusive disease, now termed sinusoidal obstructive syndrome [101–103]. IL-1 is another proinflammatory cytokine that contributes to acute GVHD toxicity. Secretion of IL-1 appears to occur predominantly during the effector phase of GVHD in the spleen and skin, two major GVHD target organs [104]. Experimental models implicated IL-2 in GVHD pathophysiology, and direct inhibition of IL-1 activity with IL-1 receptor antagonist looked promising in mice and in phase II clinical trials. However, an attempt to use IL-1 receptor antagonist to prevent acute GVHD in a randomized trial was not successful [105]. It is likely that redundancy of inflammatory pathways bypasses the direct inhibition of IL-1, reducing the clinical benefit of this approach. Finally, macrophages can also produce significant amounts of NO as a result of activation during GVHD. Several studies have shown that NO contributes to the deleterious effects of GVHD on target tissues and specifically to GVHD-induced immunosuppression [50,106]. NO also inhibits repair mechanisms in target tissue by inhibiting the proliferation of epithelial stem cells in the gut and skin [107]. In humans and rats, development of GVHD is preceded by an increase of NO oxidation products in the serum [108].
Toll-like receptors and innate immunity As alluded to above, components of the HCT conditioning regimen (in particular total body irradiation) and the secretion of type 1 cytokines (specifically IFN-γ) prime mononuclear phagocytes to produce inflammatory cytokines. However, the actual secretion of soluble cytokines occurs after primed macrophages are triggered or stimulated by a second signal. This stimulus may be provided through TLRs by LPS and other microbial products that have leaked through an intestinal mucosa damaged initially by HCT conditioning regimens during step 1. Since the gastrointestinal tract is known to be particularly sensitive to the injurious effects of cytokines [95,109], damage to the gastrointestinal tract during the effector phase can lead to a positive feedback loop wherein increased translocation of LPS results in further cytokine production and progressive intestinal injury. Thus, the gastrointestinal tract may be critical to propagating the “cytokine storm” characteristic of acute GVHD; increasing experimental and clinical evidence suggests that damage to the gastrointestinal tract during acute GVHD plays a major role in the amplification of systemic disease [97]. This conceptual framework underscores the role of LPS in the development of acute GVHD as suggested by several groups [51,109,110].
LPS is a major structural component of Gram-negative bacteria and is a potent stimulator of cellular activation and cytokine release [111]. LPS shed from bacteria that comprise normal bowel flora can elicit a broad range of inflammatory responses from macrophages, monocytes, and neutrophils. In particular, LPS may stimulate gut-associated lymphocytes and macrophages [51]. Following allogeneic HCT, LPS accumulates in both the liver and spleen of animals with GVHD prior to its appearance in the systemic circulation [110]. Elevated serum levels of LPS have been shown to correlate directly with the degree of intestinal histopathology occurring after allogeneic HCT [109,112]. A genetic mutation in the TLR-4 (Tlr4) gene makes mice resistant to LPS, and transplantation with LPS-resistant donor cells results in a significant reduction of TNF-α levels, gastrointestinal tract histopathology, and systemic GVHD compared with animals receiving LPSsensitive HCT [109]. In addition, transplantation from deficient (CD14−/−) donors that are insensitive to LPS stimulation in vitro resulted in a major reduction in gastrointestinal tract GVHD and improved longterm survival compared with normal controls [113]. Early animal studies showed that death from GVHD could be prevented if transplanted mice were given antibiotics to decontaminate the gut [114]. After clinical HCT, endotoxemia is associated with intestinal injury, hepatic toxicity, acute GVHD, and fever in the absence of bacteremia [112,115]. Accordingly, Gram-negative gut decontamination during HCT has also been shown to reduce GVHD [116], and the intensity of this decontamination can predict severity of GVHD [117–119]. Naturally occurring antibody titres to a rough-mutant strain of Escherichia coli J5, which protect humans and animals from septic shock, are also associated with a decreased incidence of acute GVHD after allogeneic SCT [120]. Molecules that act as competitive inhibitors to LPS at the cell surface and block nuclear factor-kappa B activation and nuclear translocation block the biologic response to LPS and result in decreased inflammatory cytokine release and reduced GVHD severity while preserving potent GVL effects and improved leukemia-free survival [113]. Traffic of cellular effectors Regulation of effector cell migration into target tissues during the development of GVHD occurs in a complex milieu of chemotactic signals where several chemokine receptors may be triggered simultaneously or successively. Inflammatory chemokines expressed in inflamed tissues are specialized to recruit effector cells, such as T cells, neutrophils, and monocytes [121]. Chemokine receptors are differentially expressed on subsets of activated/effector T cells. Upon stimulation, T cells can rapidly switch chemokine receptor expression, acquiring new migratory capacity [24,122]. The involvement of inflammatory chemokines and their receptors in GVHD has been recently investigated in mouse models of GVHD. Macrophage inhibitory protein-1 alpha recruits CCR5+CD8+ T cells into the liver, lung, and spleen during GVHD [123,124], and levels of several chemokines are elevated in GVHD-associated lung injury [125]. DCs in the lymph node activate lymphocytes and induce a profile of adhesion molecules and chemokine receptors that direct the activated cells to migrate back to the organ of DC origin. For example, activated T cells that leave a lymph node in the skin express cutaneous lymphocyte antigen [126]. T cells expressing cutaneous lymphocyte antigen only home back to the skin where they may mediate cutaneous GVHD, but they do not traffic to other organs. Similarly, T cells exiting mesenteric lymph nodes express α4β7 (LPAM-1), which is necessary for homing to gastrointestinal tract and gut-associated lymphoid tissues [127], but they do not express cutaneous lymphocyte antigen. Lymphocytes homing to the small intestine bear chemokine receptors for the chemokine thymus-expressed chemokine (CCL25) that is produced in the small intestine, but not in the skin. Thus, cells bearing the appropriate cell
The Pathophysiology of Graft-Versus-Host Disease
surface adhesion receptors for the gastrointestinal tract (e.g. LPAM-1) and chemokine receptors (e.g. CCR25) undergo firm adhesion followed by transmigration from the vessel into the appropriate organ.
GVHD prevention: from animal models to clinical practice The development of strategies for GVHD prophylaxis and therapy has suffered from a limited understanding of the underlying pathophysiology of GVHD. This conceptual limitation led to approaches that were based on an “oncologic” model of GVHD, i.e. the notion that since GVHD is dangerous and life-threatening, management requires the utilization of high-dose immunosuppressant regimens. High doses of multiple immunosuppressive agents have been used much like we use combination chemotherapy to treat malignancies. While there is often reasonable control of the clinical manifestations of GVHD, this “shotgun” approach lacks elegance and is usually associated with opportunistic infections, lymphoproliferative disorders or relapse of the underlying malignancy. Indeed, it is often infection while GVHD is in remission that results in mortality. The objective should be to reduce the nonspecific tissue damage associated with GVHD, while allowing effective immunologic recovery. The three-phase model described above can be simply summarized. GVHD is an exaggerated and dysregulated response of a normal immune system (that of the donor) to tissue damage that is intrinsic to transplantation. The donor’s immune system reacts as if there is a massive and uncontrolled infection, and its efforts to deal with this injury result in the clinical manifestations of GVHD. It is quite possible that the tissue injury intrinsic to the administration of high-dose chemoradiotherapy initiates the breakdown of mucosal barriers, allowing endotoxin into the tissues. TLRs on DCs bind to endotoxin and activate signal transduction pathways that lead to DC maturation and induce inflammation [128]. The upregulation of co-stimulatory molecules, MHC molecules, adhesion molecules, cytokines, chemokines, prostanoids, and other inflammatory mediators prime and trigger the attack on target tissues, fueling the fire. Thus, it is likely that components of cytokine storm [129], the danger hypothesis [20], and more traditional notions of adaptive and innate immunity [130] all apply. Moreover, with our improved understanding of inhibitory pathways that regulate and suppress inflammation, it may be possible to enhance regulatory mechanisms rather than attempt to engender immunologic paralysis.
Step 1: Reduced-intensity conditioning regimens The three cardinal organs affected by GVHD are the skin, intestinal tract, and liver. The common thread would seem to be exposure to the environment. Skin and gut have very obvious barrier functions and a well-developed reticuloendothelial system. DCs resident in these organs present antigens to naïve T cells, which then have alterations in cell surface characteristics that cause them to return to the organ that originated the specific DC. Similarly, the liver is the first line of defense downstream of the gut. One would expect the lung to be a similar target; however, its less intense exposure to organisms, particularly Gramnegative rods, probably reduces its primacy as a target organ. Nevertheless, recent studies clearly demonstrate pulmonary injury that is likely to reflect GVHD. All of these organs are rich in DCs, a probable prerequisite for generating the injurious cytokines as well as directing the attack of the adaptive immune system. They are all subject to injury from conditioning, and breaches of the barrier will allow organisms or endotoxin into the circulation. One prediction of this model is that less-intense conditioning regimens would be associated with less GVHD. This effect was initially
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observed after donor lymphocyte infusions. The T cells were administered without pharmacologic GVHD prophylaxis, and the resultant GVHD was less frequent and severe than would be expected in the absence of immunosuppressive drugs. It could be inferred that reducedintensity conditioning regimens might similarly be associated with less severe GVHD, both through less cellular injury per se and through a reduction in endotoxin exposure by virtue of less mucosal injury. Available data suggest that the rate of GVHD after reduced-intensity conditioning regimens is similar to the rate seen after conventional dose conditioning [131,132]. Interestingly, the onset of GVHD is delayed and an overlap syndrome of acute and chronic GVHD is more prevalent. This observation suggests that the initial tissue injury of high-dose regimens speeds the development of GVHD and reduced-intensity procedures seem to have little effect on chronic GVHD. Thus, while data on both reducing colonization with bacteria and reducing tissue injury with less-intense regimens are suggestive and support the basic concepts described above, the effect is insufficient to control GVHD adequately in human transplantation.
Step 2: Modulation of donor T cells Reduced T-cell numbers The advent of monoclonal technology in the 1980s led to the logical conclusion that if a pharmacologic reduction in T-cell numbers could partially control GVHD, albeit at the risk of drug-induced toxicity, then eliminating the cells with mAbs, immunotoxins, lectins, CD34 columns or physical techniques, might be more effective (reviewed by Ho and Soiffer in [133]). Furthermore, the ability to identify and target specific T-cell subsets opened the possibility that the graft could be engineered to control GVHD while allowing immunologic recovery as well as GVL. A typical unmanipulated marrow transplant entails the infusion of about 107 T cells/kg of recipient weight. Establishment of a clear dose–response relationship is difficult since the risk of acute GVHD depends on factors beyond simple dosing, for example minor histocompatibility disparities, virus exposure, disease and stage, donor age, recipient age, etc. Studies of counterflow elutriation showed that 106 T cells/kg plus CSP resulted in a GVHD rate similar to that of CSP alone, but 5 × 105 T cells/kg plus CSP resulted in an overall rate of 22%, and only limited skin involvement was observed [134]. After lectin depletion of T cells in the absence of supplemental immunosuppression, a T-cell dose of around 105/kg was associated with complete control of GVHD [135]. More recently, the combination of very high stem cell numbers and CD3 T-cell numbers of less than 3 × 104/kg allowed haploidentical transplantation without GVHD [136]. Global T-cell depletion (TCD) using a broad panel of mAbs has been largely abandoned because of the risk of graft failure [137]. However, it is clear that even depletion of T-cell subsets must be undertaken with caution. Depletion of CD4+ T cells or CD8+ T cells alone can be associated with a high graft failure rate [138]. However, depletion of CD5+ or CD6+ T cells seems to be associated with a low graft failure rate [139,140]. The presumption is that radioresistant T cells that survive the initial conditioning are responsible for graft rejection. When the stem cell source is rich in T cells, the GVH reaction will further reduce the residual population capable of alloreactivity, thus decreasing graft rejection. To some degree, the higher graft failure rates may be controlled by increasing the intensity of the conditioning regimen [141], adding back T cells [134], or with additional immunosuppressants [142]. Additional problems associated with T-cell depletion (TCD) include a higher incidence of Epstein–Barr virus-induced lymphoproliferative disorders, loss of the GVL effect with a consequent increase in relapses and slower immunologic recovery due to reduced passive transfer of
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immunocompetent donor T cells. These concerns also have been approached by increasing the conditioning intensity, delayed T-cell add-back [143], rituximab [144] or the infusion of specific T-cell lines [145]. However, in no case has there been an improvement in survival that can be definitively attributed to TCD. A large multicenter study of unrelated donor HCT similarly showed no improvement in outcomes after TCD [146]. Another interesting and novel approach to restore T-cell function without GVHD is to start by removing T cells from the initial graft using a technique such as CD34 selection. This step is followed by reacting donor and recipient lymphocytes in a mixed lymphocyte reaction in the presence of an anti-IL-2 receptor immunotoxin. This agent removes only activated T cells since it binds to CD25. The remaining resting cells are then infused to enhance immunologic recovery [147]. Alternatively, studies in mice have accomplished a similar effect, depleting donor cells activated in a mixed lymphocyte culture with antibodies to the activation antigen CD69 and immunomagnetic bead sorting [148]. Finally, both antithymocyte globulin and anti-CD52 therapy (alemtuzumab) deplete T cells in vivo. Antithymocyte globulin appears in some studies to reduce both acute and chronic GVHD [149]. Alemtuzumab also reduces acute and chronic GVHD but at the expense of high relapse rates and graft failure [150].
Reduced T-cell activation The introduction of CSP in the late 1970s was a significant advance in GVHD prevention. More recently a similar agent, tacrolimus, has been shown to provide similar control of GVHD. Both drugs inhibit T-lymphocyte activation, although the exact mechanism of action is not known. Both drugs bind to immunophilins: CSP binds cyclophilin, and tacrolimus binds FKBP12. The complex of drug and binding protein, calcium, calmodulin, and calcineurin is then formed, and the phosphatase activity of calcineurin is inhibited. This effect may prevent the dephosphorylation and translocation of nuclear factor of activated T cells, a nuclear component thought to initiate gene transcription for the formation of lymphokines (such as IL-2 and IFN-γ). The net result is the inhibition of T-lymphocyte activation. As a single agent, CSP was about as effective as methotrexate (MTX) [151]. However, in combination with MTX, there was a significant reduction in the incidence of GVHD and an improvement in survival [152]. Subsequent trials of tacrolimus and MTX compared with CSP and MTX showed no advantage for either combination [153,154]. The addition of prednisone to the conventional two-drug regimen resulted in similar rates of GVHD and no improvement in survival [155]. DCs appear to be critical to the development of acute GVHD [6]; therefore, interference with DC function would probably prevent GVHD. The goal would be to inhibit function selectively or temporarily until transplanted DC precursors had engrafted and become functional. Campath is an anti-CD52 mAb that has been extensively used for the prevention of GVHD, primarily in Europe. It is a promiscuous antibody, targeting B cells, T cells, monocytes, and other cells. Interestingly, it also appears to target DCs, and when the drug was administered prior to transplantation, host DCs were selectively depleted and the donor DCs repopulated the recipient [156], although the sensitivity of Langerhans cells to depletion is controversial [157]. These observations are difficult to separate from concomitant TCD, but they may be consistent with the theme that facilitation of DC turnover may ameliorate GVHD.
Reduced T-cell proliferation The first generally prescribed GVHD preventive regimen was the administration of intermittent low-dose MTX as developed in a dog
model by Thomas and colleagues [158]. Another regimen developed by Santos and based on cyclophosphamide [159] was never widely adopted. The principle of this approach is to administer a cell-cycle specific chemotherapeutic agent intermittently immediately after the transplant, when the T cells will have started to divide after exposure to allogeneic antigens. This was an effective approach compared with no prophylaxis [160]; however, GVHD control was incomplete. Subsequently, the addition of antithymocyte globulin or prednisone or both resulted in incremental improvement in the GVHD rate but no improvement in survival [161]. Long courses of MTX proved to be associated with a higher risk of interstitial pneumonitis and mucositis, and were abandoned with the advent of CSP. One concern regarding the use of MTX is that the enhanced mucositis might be associated with increased transfer of endotoxin across the mucosa. Ultimately, the course of MTX was abbreviated and combined with a T-cell activation inhibitor, such as CSP or tacrolimus. The use of cyclophosphamide immediately after HCT may also prevent both acute and chronic GVHD [162]. Mycophenolate mofetil (MMF) is another useful agent that may prevent GVHD with less mucositis and myelotoxicity than MTX. It is the prodrug of mycophenolic acid, a selective inhibitor of inosine monophosphate dehydrogenase, and inhibits the de novo pathway of guanosine nucleotide synthesis. Since T lymphocytes are dependent on the de novo synthesis of purines, mycophenolic acid inhibits the proliferative responses of T cells to both mitogenic and allogeneic stimulation. Myeloid and mucosal cells can utilize salvage pathways, so the drug is less toxic to mucosa and myeloid recovery. Mycophenolic acid also prevents the glycosylation of glycoproteins that are involved in adhesion to endothelium and may reduce recruitment of leukocytes into inflammatory sites. MMF does not inhibit the activation of T cells as such, but blocks the coupling of activation to DNA synthesis and proliferation [163]. Many trials of the combination of MMF with CSP or tacrolimus have shown reliable but incomplete control of GVHD when used in conjunction with a calcineurin inhibitor. Sirolimus is a macrocyclic lactone immunosuppressant that is similar in structure to tacrolimus and CSP. All three drugs bind to immunophilins; however, sirolimus complexed with FKBP12 inhibits T-cell proliferation by interfering with signal transduction and cell-cycle progression. Sirolimus (rapamycin) affects lymphocyte activation at a later stage than either CSP or tacrolimus, and activation stimuli that resist inhibition to the latter agents have been shown to be sensitive to sirolimus. Sirolimus has been shown to inhibit antibody-dependent cellular cytotoxicity and the cytolytic effects of NK cells and IL-2 lymphokine-activated killer cells. The sirolimus–FKBP12 complex binds to mammalian target of rapamycin (mTOR), which blocks IL-2-mediated signal transduction pathways that prevent G1/S phase transition. This effect is mediated through a complex pathway involving the inhibition of ribosomal protein synthesis at several levels, as well as effects on transcription and translation [164]. The drug has similar effects on proliferation of T cells induced by IL-2, IL-4, IL-7, IL-12, and IL-15. Interestingly, it may also interfere with co-stimulation [165] by inhibiting CD28-mediated blocking of inhibitor of nuclear factor kappa B and translocation of c-Rel to the nucleus [166]. Sirolimus contributes to massive T-cell apoptosis if signals 1 and 2 of T-cell activation are blocked, an effect that is not seen with CSP [167]. This effect may be related to its ability to inhibit the expression of bcl-2 and BAG-1 [168,169]. Sirolimus completely inhibits the kinase activity of the cdk4–cyclin B and cdk2–cyclin E complexes despite normal expression of these proteins and, as a consequence, blocks the hyperphosphorylation of the retinoblastoma gene product. Interestingly, sirolimus has inhibitory effects on integrin-mediated signal transduction [170], and dermal microvascular injury in a Hu-SCID transplantation model [171].
The Pathophysiology of Graft-Versus-Host Disease
Sirolimus has excellent antirejection activity in organ transplantation [170], and combination therapy with sirolimus and tacrolimus appears to be extremely effective in human organ allografting and murine models of marrow grafting. Since it acts through a separate mechanism from the tacrolimus–FKBP complex (and CSP–cyclophilin complex), which inhibits calcineurin, and thus T-cell activation, sirolimus is synergistic with both tacrolimus and CSP. While both tacrolimus and sirolimus bind to FKBP12, there appear to be adequate binding sites for both molecules. Therefore, in contrast to expectations, the drugs are not competitive, and in fact they appear to be synergistic. Since the sirolimus–mTOR complex does not bind to calcineurin, sirolimus also is free of nephrotoxicity and neurotoxicity, making combination therapy appealing. Clinical trials indicate that sirolimus is a promising addition to the GVHD armamentarium in both related and unrelated donor HCT [172].
Step 3: Blockade of inflammatory stimulation and effectors Reduced exposure to organisms Another hypothesis that flows from this model is that reduction of intestinal colonization with bacteria could prevent GVHD. The first indication that elimination of exposure to microorganisms could prevent GVHD was in germ-free experiments in mice, where GVHD was not observed until the mice were colonized with Gram-negative organisms [114]. Later, gut decontamination and use of a laminar airflow environment was associated with less GVHD and better survival in patients with severe aplastic anemia [116]. Similarly, studies of intestinal decontamination in patients with malignancies have shown less GVHD in some [118] but not all [173] studies. However, despite the power of a large number of patients in registry analyses [173], it is possible that the heterogeneity of care in the large number of participating centers obscured an important effect. Viral infections, particularly herpesviruses, may also be associated with GVHD [174]. Blockade of inflammatory effectors As alluded to above, an important role for TNF-α in clinical acute GVHD was suggested by studies demonstrating elevated levels of TNFα in the serum of patients with acute GVHD and other endothelial complications such as sinusoidal obstructive syndrome [101,102,175]. Importantly, the appearance of the increased levels was predictive of the severity of complications and overall survival. Patients with higher serum TNF-α levels during the conditioning regimen (pre-HCT) had a 90% incidence of grade II or greater acute GVHD and an overall mortality of 70%, compared with a mortality of 20% in patients without TNFα elevations [102]. Murine mAbs or F(ab)′2 fragments directed at TNF-α produced partial responses either as therapy for steroid-resistant GVHD [176] or as prophylaxis [177]. A phase II trial of etanercept, a chimeric fusion protein consisting of the extracellular ligand-binding portion of the human TNFR linked to the Fc portion of human immunoglobulin G1, has recently shown significant efficacy as primary treatment for acute GVHD when added to systemic steroids (Fig. 16.2). Almost 70% of patients had complete resolution of their GVHD within 1 month, compared with 33% of patients treated with steroids alone [178]. Blockade of chemokines may prevent the recruitment, localization, and activation of lymphocytes during GVHD. Prevention of lymphocyte entry into secondary lymphoid organs, acquisition of appropriate homing and adhesion molecules, or modification of their entry into target tissues could all reduce GVHD [179–181]. Such molecules as FTY720 [182,183] and alefacept [184–186] as well as new chemokine inhibitors may allow
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selective targeting of organ-specific lymphocytes, although as yet little clinical experience has accumulated.
Chronic GVHD It is important to recognize that chronic GVHD was originally defined temporally rather than clinically or pathophysiologically. The initial clinical reports of chronic GVHD used descriptions of clinical problems occurring at least 150 days after HCT [187]. By convention, many transplant clinicians and scientists use day 100 after stem cell infusion as a convenient divider between acute and chronic GVHD. However, it should be recognized that acute manifestations of GVHD may occur after day 100, and problems typically associated with chronic GVHD may occur before day 100. Thus, it is preferable to consider the symptom and signs per se rather than the timing of the onset of clinical findings. The National Institutes of Health consensus conference on chronic GVHD recognizes four categories of GVHD [188] (see Chapter 87). The lack of animal models that reproduce the critical aspects of chronic GVHD has limited our ability to address the pathophysiology of this complication. Chronic GVHD is often considered an autoimmune disease because of distinctive similarities to various autoimmune disorders, especially collagen vascular disease. The relationship is difficult to prove clinically, but experimental studies have demonstrated an autoimmune aspect of chronic GVHD pathophysiology [189]. Indeed, autoantibody production is commonly observed after transplantation, and some of the clinical manifestations of chronic GVHD are similar to those of scleroderma, lichen planus, and other autoimmune diseases. T cells from animals with chronic GVHD produce unusual patterns of cytokines, such as IL-4, or IFN-γ in the absence of IL-2 [190–192]. These cytokines can stimulate collagen production by fibroblasts, which can be further amplified by IL-4. Interestingly, there is often an eosinophilia as an early manifestation of chronic GVHD, further supporting the notion that cells with a Th2 phenotype are important mediators of this problem [193]. One mechanism to induce autoimmune attack might be failure of normal regulatory mechanisms in the setting of thymic damage or immunosuppressants or both. Failure of Tregs results in autoimmune phenomena increasingly implicated in both acute and chronic GVHD [191,192,194–197]. Moreover, Tregs are implicated in preventing autoantibody production [198]. The notion that autoimmune attack can be induced by calcineurin inhibitors after autologous or syngeneic HCT has been observed in animals by preventing the development of Tregs [199]. In clinical trials, the manipulation of CSP to engender autoimmunity produced limited GVHD and limited GVL responses [200,201]. However, thymic injury and GVHD in the context of allogeneic HCT is more ominous, and one randomized trial suggested that calcineurin inhibitors might increase the risk of chronic GVHD [152]. Another explanation of autoimmunity may be that the massive apoptosis of T cells early in acute GVHD overwhelms the macrophage/DC system, resulting in autoantigen presentation [202]. The autoreactive cells of chronic GVHD are associated with a damaged thymus, which may be injured by acute GVHD, by the conditioning regimen, or subject to age-related involution and atrophy. Thus, the normal ability of the thymus to delete autoreactive T cells and to induce tolerance may be impaired. In addition, data are accumulating to support the concept that B cells may contribute to chronic GVHD. The anti-CD20 mAb rituximab has been used clinically to treat severe autoimmune disorders including autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura, and now several series indicate that anti-B-cell therapy is an interesting approach to treating chronic GVHD [203–205]. Alloantibodies can facilitate the cross-presentation of antigens to effector T cells, thereby amplifying T-cell responses to mHAs [206,207]. Donor B
Chapter 16
B. Related donor recipients
C. Unrelated donor recipients
100 90 80 70 60 50 40 30 20 10 0
100 90 80 70 60 50 40 30 20 10 0
p1,500 3.6 ×
109 107 106 104 104 108
Individual human genome SNP frequency Estimated number of CNVs per individual genome
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1/300 base pairs >100
CNV, copy number variant; SNP, single nucleotide polymorphism. Adapted with permission from Mullaly and Ritz [194].
sequences that, in the setting of MHC-matched allogeneic HCT, could potentially stimulate and serve as targets for donor T- and B-cell responses that could mediate GVT activity. Dissection of GVT responses at the cellular and molecular level is, indeed, beginning to show that GVT in any one HCT recipient is likely to be mediated by a broadly focused immune response directed at both polymorphic alloantigens and nonpolymorphic, over- or aberrantly expressed self-antigens on recipient tumor cells. The diversity of this response is likely to be one of the keys to the remarkable potency of the GVT effect. Nonetheless, posttransplant persistence or recurrence of disease remains a major cause of treatment failure after allogeneic HCT, and developing a more comprehensive understanding of the GVT response is an urgent research priority. Recognition of the enormous diversity in the genome demonstrates that there is abundant polymorphism to be exploited, but also suggests that whole-genome approaches to understanding GVT will likely provide the key to its successful exploitation.
Strategies for exploiting the GVT response Early attempts to manipulate the GVT effect and decrease the relapse rate after allogeneic HCT were motivated by the recognition that GVT is mediated by cells contained in or derived from the hematopoietic cell graft, and that exogenous immune suppression, although it suppresses GVHD, also suppresses GVT. Complete omission of GVHD prophylaxis in the MHC-matched sibling BMT setting led to severe and hyperacute GVHD, and the increase in GVHD-related mortality precluded the identification of any decrease in the relapse rate [139]. The prophylactic administration of donor buffy-coat cells after MHC-identical sibling BMT, with a standard course of methotrexate alone as GVHD prophylaxis, was also associated with an increased rate of severe, and often fatal, acute GVHD, and also prevented the detection of any effect on the relapse rate [140]. Characterization of T- and B-cell immune responses associated with GVT activity after HCT or DLI, and identification of their antigenic targets, has demonstrated that the targets of GVT and GVHD responses are not completely overlapping. Many current efforts to manipulate GVT are consequently focused on the development of therapeutic strategies for enhancing donor immune responses against defined antigens that are selectively or exclusively expressed on recipient tumor cells, and not widely expressed on tissues that are targets for GVHD. In addition, characterization of the extensive sequence and
Identifying genomic loci associated with GVT activity The identification of extensive sequence and structural variation in the human genome suggests exciting possibilities for exploiting genomic diversity to enhance GVT, but also poses several challenging new questions. What fraction of the polymorphic loci in the genome can elicit T- or B-cell responses that can contribute to GVT? Are there specific autosomal loci at which donor–recipient disparity makes a measurable contribution to GVT activity? Are there genetic variants in the donor or the recipient that influence GVT activity in a manner that is independent of donor–recipient matching or mismatching? The advent of microarraybased genotyping technologies that permit simultaneous determination of SNP genotype and of copy number at hundreds of thousands of unique sites distributed throughout the genome has raised the prospect of using whole-genome association analysis to address these questions in a comprehensive fashion. A genome-wide, case-control approach to the identification of loci associated with GVT could potentially facilitate the identification of polymorphic loci at which donor–recipient mismatching is significantly associated with decreased risk for relapse, and could identify genetic variants in the donor or in the recipient that, independent of donor–recipient matching or mismatching, are also associated with decreased relapse risk. MHC genotypically identical sibling pairs represent a transplant population that is uniquely suited to the application of whole-genome association analysis for the identification of loci associated with GVT activity. Full siblings share 50% of their genomes and, on average, are expected to be genetically identical for both alleles at 25% of their genomes. If risk of relapse is in fact measurably influenced by donor– recipient disparity at specific autosomal loci, it is reasonable to hypothesize that HCT recipients who relapse post transplant will, on average, show more genetic identity with their donors for both chromosomes at such putative “GVT loci” than recipients who do not relapse. Thus, in the case of MHC-identical siblings, where donors and recipients are selected for genetic identity for both alleles within the MHC, this hypothesis predicts that HCT recipients who relapse post transplant will show more genetic identity for both alleles with their donors than the expected 25% at GVT loci, and patients who do not relapse post transplant will show less genetic identity for both alleles with their donors than the expected 25% (Fig. 18.5). This hypothesis is currently being tested in a population of 1000 MHC-identical sibling donor–recipient pairs – 500 “cases” in which the recipient relapsed, and 500 “controls” in which no relapse occurred – who underwent T-replete allogeneic HCT for the treatment of a myeloid malignancy at a single transplant center. Donor selection For patients for whom several potential hematopoietic cell donors can be identified, deliberate selection of a donor who is mismatched with the recipient at one or more “GVT loci” represents one potential strategy for enhancing GVT. In the T-cell-depleted haploidentical transplant setting, for example, where alloreactive donor NK cells are primary mediators of GVT responses, selection of a donor with a specific KIR genotype and appropriate MHC class I mismatch with the recipient can take advantage of predicted donor KIR–recipient MHC incompatibilities [141]. Since most HCT recipients will have several potential haploidentical donor candidates from which to choose – including parents, siblings, and children – donor selection based on donor–recipient KIR–MHC
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Relapse (cases) No relapse (controls)
Fraction identical at both alleles
1.00
GVT locus Δ/2 0.25 Δ/2
6p
Genome position
Fig. 18.5 Identification of a putative “graft-versus-tumor (GVT) locus” through assessment of donor–recipient genetic identity for both alleles at a large number of polymorphic loci distributed across the entire genome. Major histocompatibility complex (MHC)-identical sibling donor–recipient pairs are genotyped at hundreds of thousands of polymorphic loci distributed across the genome using high-density oligonucleotide arrays. Although the average fraction of donor–recipient pairs who have identical genotypes at any given polymorphic locus outside the MHC is expected to be 0.25, the observed fraction will fluctuate around this mean value. At a hypothetical “GVT locus,” the observed fraction of donor–recipient pairs with identical genotypes in a population in which the recipient relapsed (“cases”) is expected to be significantly higher than the observed fraction of pairs with identical genotypes in a population of pairs in which the recipient did not relapse (“cases”), with an effect size of Δ.
MHC-matched sibling donors of patients with multiple myeloma have been safely vaccinated with purified idiotype protein harvested from their respective siblings, and cellular and humoral idiotype-specific immune responses were subsequently detected in the donors as well as in the sibling recipients to whom they donated bone marrow [145–147]. CML patients with post-transplant relapse have been safely vaccinated with BCR–ABL fusion peptides, and CD4+ BCR–ABL fusion peptidespecific T-cell responses were subsequently detected in several patients [148]. A vaccine containing the nonameric PR1 peptide derived from the proteinase-3 and elastase proteins in incomplete Freund’s adjuvant has been administered to patients with AML, CML, and myelodysplastic syndrome in the allogeneic HCT as well as nontransplant settings, and has been associated with durable complete remissions in several patients [149], and WT1-derived peptide vaccines administered to patients with AML in the nontransplant setting have elicited clinical responses [150,151]. Leukemic cells from CML [152] and AML [153– 155] patients acquire characteristics of dendritic cells and stimulate CTL responses when cultured in vitro with selected cytokines, suggesting that such cells could be used for a cellular leukemia vaccine. A cellular vaccine consisting of autologous leukemic blasts admixed with skin fibroblasts transduced with adenoviral vectors encoding human IL-2 and CD40L was administered to patients in remission after HCT, and stimulated donor CD8+ and CD4+ T cell responses to the recipient leukemic blasts [156]. Preliminary results of other vaccination trials with wholecell vaccines, including a trial in AML patients undergoing reducedintensity HCT of vaccination with autologous, irradiated AML cells transduced with the granulocyte–macrophage colony-stimulating factor (GM-CSF) gene, have appeared in abstract form, with final results expected in the near future. Adoptive cellular therapy – donor lymphocytes
genotypes to optimize GVT activity will likely become an increasingly important consideration in haploidentical, T-cell-depleted HCT. Donor selection based on donor–recipient KIR–MHC genotypes could also conceivably prove to be useful in mismatched unrelated donor HCT, but retrospective studies have not consistently identified an association between donor–recipient KIR ligand mismatching and relapse rate in this setting [51,142–144]. For the minority of patients for whom multiple related or unrelated MHC-matched donor candidates are identified, donor selection based on donor–recipient disparity at non-MHC loci awaits the unambiguous identification of loci at which mismatching has a statistically significant effect on post-transplant relapse. The subject of donor selection is more extensively reviewed in Chapter 48. Donor or recipient vaccination Active immunotherapeutic approaches to enhancing GVT based on pretransplant vaccination of HCT donors or post-transplant vaccination of recipients are currently being investigated at many transplant centers. Vaccination strategies can be employed to augment donor T-cell responses to specific recipient mHAs, or alternatively to tumor-specific or tumor-associated antigens expressed on recipient tumor cells. The immunogens currently being evaluated in clinical trials include whole proteins, specific peptides or peptide mixtures, DNA, and whole cells, including tumor cells and dendritic cells, some of which are genetically or biochemically modified to enhance their immunogenicity. A variety of adjuvants are also being administered with the immunogens. The results of published trials demonstrate that vaccination is in general safe and can elicit frequent humoral or cellular vaccine-specific immune responses, and occasional clinical responses.
The remarkable potency of DLI for the treatment of recurrent malignancy after HCT, and its frequent serious toxicities – most notably GVHD and myelosuppression [14,16,17] – have generated intense interest in the development of more selective adoptive cellular therapies that have greater antitumor efficacy with less toxicity to normal, nonmalignant tissues. Strategies for exploiting GVT that are based on the adoptive transfer of donor-derived cells permit ex vivo selection and manipulation of the cellular product, and potentially of the donor from whom it is derived, prior to infusion, as well as independent manipulation of the recipient in order to increase the safety and the efficacy of adoptive therapy. Adoptive transfer approaches also allow the genetic characterization or marking of cells prior to infusion to facilitate precise monitoring of the in vivo persistence and trafficking of infused cells. Administration of DLI in escalating doses and selective depletion of CD8+ cells from donor lymphocyte products prior to infusion have been performed in an attempt to harness the antileukemic effect of DLI while minimizing the toxicity attributable to GVHD. Sequential administration of escalating doses of CD3+ donor lymphocytes has proven to be an effective approach to the treatment of recurrent CML post transplant [157,158]. Doses in the range of 1–2 × 107 CD3+ cells/kg recipient weight have been associated with high response rates and low rates of clinically significant GVHD. Patients who do not respond to an initial dose at this level often respond to subsequent higher doses, at which the risk of GVHD is greater. Based on the hypothesis that the therapeutic efficacy of DLI for CML is attributable to the activity of CD4+ T cells, and that the GVHD associated with DLI is attributable to the activity of infused CD8+ T cells, donor lymphocyte products selectively depleted of CD8+ T cells have been used to treat recurrent CML and multiple myeloma post transplant [56,57]. Complete responses were observed in a majority of the patients with CML and many myeloma patients, with
The Human Graft-versus-Tumor Response – and How to Exploit It
rates of GVHD that were lower than would be expected based on historical controls. Whether the lower rates of GVHD were attributable to the depletion of GVHD-causing CD8+ T cells from the DLI products administered in these studies, or simply due to a lower mean dose of cells administered, cannot be determined from the available data. Concomitant administration of cytokines with DLI and ex vivo activation of DLI products prior to infusion have been explored as means to enhance the antitumor efficacy of adoptive therapy with donor lymphocytes. IL-2 administration during or after DLI has dose-dependent effects that range from prolonging the in vivo survival and persistence of adoptively transferred cells at low doses to nonspecific activation of both CD3+ T cells and NK cells at higher doses [15]. A reproducibly significant enhancement of the antitumor efficacy of DLI by administration of IL-2 has yet to be demonstrated. A retrospective analysis suggested that administration of IFN-α did not significantly improve the efficacy of DLI for treatment of CML recurrence [14], but DLI and IFN-α combined with GM-CSF has produced clinical responses in patients who were refractory to DLI or to DLI and IFN-α [152]. IFN-α can, however, elicit durable tumor regression in patients with persistence of metastatic renal cell carcinoma after reduced-intensity allogeneic HCT and DLI [23,105]. Whether the effect of IFN-α in this setting is attributable to a primary effect on activation or expansion of donorderived effector cells in the host, or alternatively to a direct antitumor effect on recipient tumor cells, is unknown. Infusions of donor lymphocytes activated and expanded ex vivo via CD3–CD28 co-stimulation has been used in combination with chemotherapy and conventional DLI to treat patients with a variety of aggressive malignancies whose tumors recurred or persisted post transplant. Complete remissions were seen in eight of 18 patients treated in one phase I trial [159], suggesting that further studies of ex vivo activated DLIs are warranted. Adoptive cell therapy with antigen-specific effector cells The low frequency of T cells in unselected donor lymphocyte products that are reactive with recipient tumor cells, combined with the presence in such products of alloreactive cells that contribute to GVHD, has stimulated interest in adoptive therapy with products manipulated ex vivo to selectively expand the subsets of effector cells reactive with recipient tumor cells. Infusion of donor-derived CTL lines generated by repetitive in vitro stimulation of donor peripheral blood mononuclear cells with recipient CML cells was followed by a complete remission in a CML patient with a post-transplant recurrence of accelerated-phase disease who had not responded to unselected DLI [160]. Although the contribution of the ex vivo manipulated cells to elimination of residual leukemia in this case could not be unambiguously established, the results demonstrate that adoptive therapy with products that are enriched for tumor-reactive cells will likely be more effective than infusion of unselected donor lymphocytes. Techniques for large-scale ex vivo expansion of antigen-specific T cell clones [161] are now being used to support clinical trials evaluating the safety and efficacy of adoptive cell therapy with homogeneous populations of cloned CD8+ and CD4+ T cells specific for antigens selectively or exclusively expressed on recipient tumor cells. Three broad categories of target antigens expressed on tumor cells are being investigated: polymorphic mHAs encoded by genes that are expressed on normal and malignant hematopoietic cells but not widely expressed in nonhematopoietic tissues, tumor-specific antigens, and nonpolymorphic tumorassociated antigens that are encoded by normal cellular genes and over- or aberrantly expressed in tumor cells. There are advantages and limitations associated with adoptive cell therapy directed at each of these three categories of target antigen, and thus they will be discussed separately.
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Defined mHAs that are expressed selectively in normal and malignant hematopoietic cells include those encoded by the HA-1 (KIAA0223) [89], MYO1G [73,104], HB-1 [79], UTY [162,163], BCL2A1 [82], CENPM [164], ECGF1 [165], P2RX5 [166], and HMSD [167] genes. It is likely that many more such mHAs with expression limited to hematopoietic cells remain to be identified [77]. The applicability of adoptive T-cell therapy with CD8+ T cells specific for MHC class I-restricted mHAs, however, is restricted to HCT recipients who (1) express the MHC class I allele that presents the mHA(s) to CD8+ T cells, and (2) are appropriately discordant with their donors for expression of the mHA (donor negative, recipient positive). Therefore, for mHAs that are presented by uncommon MHC class I alleles or are created by SNPs for which the minor allele frequency is low, these restrictions imply that eligibility for adoptive T-cell therapy targeting such antigens will be limited to a very small minority of HCT recipients. For any candidate hematopoietic-specific mHA, the proportion of HCT recipients who would be eligible for T-cell therapy targeting that antigen can be readily estimated from Internet resources which provide the allele frequencies in the four HapMap populations of most common (minor allele frequency ≥0.01) human SNPs (http://www.hapmap.org), as well as the frequency of various MHC alleles in several ethnic and racial groups (http://www.ashi-hla.org/publicationfiles/archives/prepr/motomi.htm). Tumor-specific antigens that could potentially be targeted with adoptive T-cell therapy to enhance GVT activity include fusion peptides such as BCR–ABL [168] and PML–RARα [169] that are created by tumorspecific chromosomal translocations, the Ig idiotype of B-lymphoid tumors [170], and novel peptide sequences created by the FLT3-internal tandem duplication mutation that is common in AML [171,172]. Many other potential tumor-specific antigens that could be targeted with adoptive cell therapy are likely to exist. Comprehensive genetic sequence analysis of breast and colorectal tumor cells, for example, has demonstrated that such tumors carry, on average, 90 mutations that generate amino acid substitutions and could therefore generate tumor-specific antigens [173]. The clinical utility of adoptive therapy targeting antigens created by tumor-specific mutations, however, will be highly dependent on the frequency with which those mutations recur in tumor cells from different patients. A recent comprehensive genetic analysis of pediatric ALL has shown that mutations causing nonsynonymous changes in the coding sequence of the PAX5 gene are common in tumors from different patients [174], suggesting that “shared” tumor-specific antigens that could be targeted in multiple different patients will, indeed, be identified. Antigens derived from nonpolymorphic proteins that are over- or aberrantly expressed in tumor cells and not widely expressed in nonhematopoietic tissues represent a third class of potential target for adoptive T-cell therapy to enhance GVT. The most extensively characterized antigens in this class include the PR1 peptide encoded by the proteinase3 and elastase genes and presented by HLA-A*0201 [120,121] and several different epitopes encoded by the WT1 gene [175,176]. Genes such as HOXA9 and BMI1 that are selectively expressed in normal hematopoietic and leukemic stem cells and whose products perform critical nonredundant functions in leukemic stem cells [177,178] also represent attractive candidates for T-cell therapy. A peptide encoded by BMI1 and recognized by CD8+ T cells has been identified [179], and is undergoing further evaluation as a potential target for GVT therapy. Adoptive cell therapy directed at nonpolymorphic antigens encoded by normal, unmutated genes that are overexpressed in tumor cells has potential limitations imposed by the expression of these genes, at some level, in normal tissues. Suppression of nonmalignant hematopoiesis might occur, for example, if normal hematopoietic progenitor cells also present the antigen to T cells, as has been suggested for an epitope encoded by WT1 [151]. Alternatively, expression of the antigen in
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normal tissues might lead to a deletion or functional inactivation of T cells with high avidity for the antigen, leaving only low-avidity cells with limited reactivity against tumor cells. Adoptive therapy with regulatory T cells Several subsets of human T cell with the capacity to suppress the in vitro activity or function of effector CD8+ or CD4+ T cells or of NK cells have been described, and there is evidence that these cells can regulate immune responses in vivo (reviewed in [180]) (see Chapter 15). The role of such cells in GVHD and GVT after allogeneic HCT in humans is poorly defined. Studies in murine systems, however, have suggested that adoptive transfer of CD4+CD25+ regulatory T cells can selectively inhibit GVHD but preserve GVT activity after MHC-mismatched HCT [181]. These studies have suggested that analogous strategies based on adoptive transfer of T cells with regulatory function to preserve GVT while inhibiting GVHD in human HCT could soon be developed. Adoptive therapy with genetically modified cells Genetic modification of donor lymphocytes to increase the safety and the efficacy of adoptive cell therapy is an active area of investigation in which significant progress has already been made. Donor lymphocytes transduced with multicomponent suicide genes combining a selectable marker with the Herpes simplex virus thymidine kinase gene, to permit in vivo ablation of adoptively transferred cells with acyclovir or ganciclovir, have been administered to HCT recipients in both the T-celldepleted, haploidentical [182,183] as well as T-replete, MHC-identical [184] transplant settings to treat post-transplant relapse or Epstein–Barr virus-related lymphoproliferative disease. The rationale for adoptive therapy with suicide gene-transduced cells is to permit administration of effector cells that are likely to have a potent antitumor effect, albeit with a risk for GVHD, and that can subsequently be ablated if clinically significant GVHD does, in fact, develop. The antitumor efficacy of suicide gene-transduced donor lymphocytes has been significantly greater in T-cell-depleted compared with T-replete settings due to the immunogenicity of the transgenes, which elicit CD8+ transgene-specific T-cell responses that dramatically limit the in vivo persistence of adoptively transferred, transgene-bearing cells in Treplete HCT recipients [182,184–186]. Although T-cell responses to transgene products have also been detected after transfer of transduced cells to recipients of T-cell-depleted HCT, GVT activity attributable to adoptively transferred cells has been observed in the majority of recipients [183,186]. Tumor regression was temporally correlated with the development of GVHD in most responding patients, and GVHD was largely controlled by the subsequent administration of ganciclovir. Novel suicide genes encoding human FAS or caspase death domains fused to a modified FK506-binding protein domain, which can be activated by a drug that dimerizes the FK506-binding protein domain, have been developed [187,188], and are currently being evaluated in clinical trials. Genetic modification of T cells is also being explored as a means to create GVT effector cells expressing high-affinity receptors for defined tumor-specific or tumor-associated antigens. Genes encoding chimeric antigen receptors comprising the antigen-binding domains of Igs fused to cytoplasmic signaling domains of the T-cell receptor (TCR)–CD3 complex, for example, have been constructed and introduced into polyclonal T cells from peripheral blood via retroviral transduction [189]. Transduced cells expressing the chimeric immunoreceptor demonstrate potent MHC-unrestricted cytotoxicity against cells expressing the target antigen, and can be expanded ex vivo with retention of chimeric receptor expression and antigenic specificity, for use in adoptive therapy.
Clinical trials of adoptive therapy with T cells transduced with a chimeric receptor specific for CD19 for the treatment of CD19-expressing B-lineage malignancies [190] are currently in progress. Effector T cells can also be rendered specific for tumor-specific or tumor-associated antigens through transfer of genes encoding the α- and β-chains of TCRs with defined specificity and affinity. T cells expressing transgenic TCRs retain the MHC restriction of the T cell from which the receptor was derived. The therapeutic potential of this approach was demonstrated in a recent clinical trial in patients with metastatic melanoma [191]. T cells expressing a transgenic αβ TCR specific for the melanoma-associated antigen encoded by the glycoprotein 100/PMEL17 (SILV) gene were adoptively transferred into patients with melanoma after the administration of lymphodepleting chemotherapy, followed by an infusion of IL-2. Durable engraftment of genetically engineered T cells at levels exceeding 10% of peripheral blood lymphocytes was seen in a majority of the patients for at least 2 months after T-cell transfer, and in two patients persistence of transduced cells was seen for 1 year after transfer, accompanied by objective regression of metastatic disease. TCR genes encoding a receptor specific for an HLA-B52-restricted H-Y antigen encoded by UTY have been transferred into peripheral blood T cells, and transduced cells exhibit HLA-B52-restricted UTY peptidespecific cytotoxicity [163]. Transduced cells can also be expanded ex vivo with retention of their antigenic specificity, and can potentially be used in adoptive therapy of male patients with leukemia or other tumors expressing UTY. In vivo persistence of adoptively transferred GVT effector cells will likely be required for optimal antitumor efficacy, and several different strategies for improving persistence and establishing durable antitumor immunity are currently being explored [192]. Genetic modification of human CD8+ T-cell clones to express chimeric cytokine receptors that deliver an IL-2 signal upon ligation of GM-CSF can endow them with GM-CSF-dependent growth, such that antigen recognition and consequent GM-CSF secretion will drive expansion of the T cells in an autocrine fashion [193]. Transfer into GVT effector cells of genes that enable resistance to immune suppressive agents commonly used in the allogeneic HCT setting, such as corticosteroids and mycophenolate mofetil, is also an active area of investigation, and such studies, if successful, will facilitate the use of adoptive cell therapy in patients suffering from or at risk for GVHD. Further development of T-cell-depleted transplant platforms in which alloreactive donor T cells are selectively removed from the hematopoietic cell graft prior to infusion will likewise facilitate the development of adoptive therapy in transplant settings where there is a high prevalence of GVHD with unmodified, T-replete grafts. It is anticipated that the development of T-cell-depletion strategies for selectively removing alloreactive donor cells that might potentially contribute to GVHD will alleviate, if not completely eliminate, the problem of poor immune reconstitution observed in recipients of nonselectively T-depleted grafts. Further characterization of the properties and function of the different subsets of human memory T cells, particularly the effector and central memory subsets, will likely provide valuable insight into the requirements for establishing sustained GVT activity via adoptive transfer.
Conclusion The GVT effect associated with allogeneic HCT is the clearest example of effective immunotherapy in humans. Genetic disparity between donor and recipient is the key to the therapeutic efficacy of HCT but is also the root of GVHD, its primary limitation. Cellular and molecular dissection of GVT responses has demonstrated that GVT after MHC-matched HCT is initiated by donor CD8+ and CD4+ T cells that recognize recipient mHAs encoded by polymorphic genes, and that this
The Human Graft-versus-Tumor Response – and How to Exploit It
alloresponse likely recruits additional effector cells recognizing tumorspecific or tumor-associated antigens encoded by nonpolymorphic genes that are over- or aberrantly expressed in recipient tumor cells. The GVT response in most HCT recipients is therefore likely to comprise a broadly focused immune response directed at a large number of polymorphic and nonpolymorphic target antigens. Selective expression of some GVT target antigens in recipient hematopoietic cells and recipient tumor cells,
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with limited expression in nonhematopoietic tissues, may allow the development of therapeutic strategies for enhancing GVT activity without inducing or aggravating GVHD. Characterization of the extensive sequence and structural variation in the human genome suggests that there are likely to be a large number of polymorphic protein and peptide sequences at which immunotherapy to augment GVT could potentially be directed.
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19
Miriam Merad, Matthew P. Collin & Edgar G. Engleman
Dendritic Cells in Hematopoietic Cell Transplantation
Introduction: the dendritic cell network Dendritic cells (DCs) are hematopoietic cells that belong to the antigenpresenting cell (APC) family, which also includes B cells and macrophages. The two main DC subsets are conventional DCs (DCs) and plasmacytoid DCs (PDCs). Although Langerhans cells (LCs) in the skin were described in 1868, their role as APCs was not appreciated until 1973 when Steinman and Cohn identified DCs in mouse spleen as potent stimulators of the primary immune response (reviewed in [1]). Shortly thereafter, several groups reported the presence of DCs in nonlymphoid tissues of rodents and humans, and demonstrated early evidence that these cells contribute to heart and kidney transplant rejection (reviewed in [2]). However, the low number of DCs in vivo, the paucity of markers that distinguish them from monocytes/macrophages, and the problems involved in purifying these cells made for slow progress in the understanding of DC biology. In the 1990s, the development of methods to isolate DCs from blood [3,4] and to generate DC-like cells from bone marrow (BM) or monocytes in vitro [5] led to an explosive growth of the DC field. DC heterogeneity DCs are characterized as cells that express the hematopoietic marker CD45 but lack most markers of T-cell, B-cell, monocyte, and natural killer (NK) cell lineages, and constitutively express major histocompatibility complex (MHC) class II molecules [6]. In mice, expression of the integrin and complement receptor CD11c/C3b is often used in association with the markers cited above to distinguish DCs. Several subsets of DC have been described in vivo based on their surface markers, their location in tissues or their progenitors [7]. Classification of DCs based solely on their surface phenotype is problematic as DC surface markers are modulated during their maturation, as discussed later. Instead, distinguishing DCs based on their anatomic location is likely to be more relevant as it reflects the nature of the antigenic milieu in which DCs develop. Tissue DCs can be divided into two major subsets: those present in lymphoid tissues including the spleen, lymph nodes (LNs), and thymus, and those present in peripheral nonlymphoid tissues. Among DCs in nonlymphoid tissues, those located at the inter-
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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face with the external environment, such as the skin and the mucosa, can be distinguished from those located in internal organs such as the liver. Also, in vitro-derived DCs will be described as they have been widely used to study DC functions. It is important to note that the markers on human and mouse DCs overlap but are not identical (Table 19.1). DCs that occupy skin and mucosal tissues display distinct phenotypes and functions that are well adapted to the microenvironment in which they reside. Cutaneous DCs (Fig. 19.1) (reviewed in [8]) The skin represents the largest interface between the external environment and the internal tissues, and one of its major roles is to provide immune function at this critical site. The most superficial layer of the skin, the epidermis, provides the first immune barrier against foreign invasion. The dermis, separated from the epidermis by the basement membrane, supports the vascular network that supplies the avascular epidermis with nutrients. DCs are present on both sides of the basement membrane and play a critical role in the defense against pathogens. These include epidermal DCs, also called LCs, named after the Austrian medical student Paul Langerhans who first identified the cells in the human epidermis. The other population of DCs in the skin, located in the dermis, is called dermal or interstitial DCs. LCs account for 3–5% of total cells in mouse and human epidermis, with approximately 700 LCs/mm2. In the mouse epidermis, two populations of hematopoietic cells coexist, including LCs and a population of T cells that express the γδ T-cell receptor (γδ T cells). γδ T cells are absent from human epidermis, where LCs represent by far the largest population of epidermal hematopoietic cells. In both species, LCs form a tight network of cells constituting the first barrier to the external environment. Human and mouse LCs express the hematopoietic marker CD45, MHC class II molecules, the C-type lectin receptor langerin/CD207, and its associated pathognomonic Birbeck granules. LCs also express the adhesion molecule, E-cadherin, that is also expressed on keratinocytes and participates in homotypic adhesion. Similar to LCs, dermal DCs express the hematopoietic marker CD45 as well as MHC class II molecules, while they are negative for langerin and E-cadherin. Human, but not murine, dermal DCs also express the c-type lectin DC-SIGN and the coagulation factor XIIIa, although both markers can also be expressed on macrophages. LCs also differ from dermal DC in the factors necessary for their development, as LCs are the only DC population absent from mice deficient in transforming growth factor-β [8]. In contrast to all other DC populations, LCs are maintained by local radioresistant
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Dendritic Cells in Hematopoietic Cell Transplantation Table 19.1 Phenotype of dendritic cells in mice and human CD45
Lineage markers
CD11c
MHC class II
CD11b
CD8α
Langerin
Siglec-H
BDCA2
BDCA4
Mouse DC
+
−
++
+
CD8− subset: +
CD8+ subset: +
−
−
−
Human DC Mouse PDC Human PDC
+ + +
− B220low Ly6Clow CD4
+ +/− −
+ + +
+/− − −
− Activated PDC −
Epidermal LC: ++ CD8+ subset + Epidermal LC: ++ − −
+ −
+
+
DC, dendritic cell; LC, Langerhans cell; MHC, major histocompatability complex; PDC, plasmacytoid dendritic cell.
(a)
(b)
(c)
Murine skin cross-section
Human epidermal sheet
Epidermis LC
Migrating LC
Dermal DC MHC II Langerin
(d)
Dermis
CD11c/Langerin/DAPI
Human LCs
CD1a/DAPI
Human dermal DCs
Keratinocyte
T cell
Fig. 19.1 Cutaneous dendritic cells (DCs) in mice and man. (a) Epidermal sheet isolated from a C57BL/6 mice and stained for major histocompatibility complex (MHC) class II molecules and langerin. Langerhans cells (LCs) coexpress MHC class II and langerin, and form a very tight network of cells that contrast with dermal DCs, which are more sparsely distributed. (b) Cross-section of murine skin stained with anti-CD11c, antilangerin and DAPI (which stains nuclei) to reveal LCs and dermal DCs. LCs co-express CD11c and langerin, while dermal DCs express CD11c but not langerin. LCs are present exclusively in the epidermis, but migrating LCs on their way to the LNs can also be found in the dermis. (c) Human epidermal sheet stained with anti-CD1a antibody. Human LCs express CD1a; they represent the major hematopoietic cell population in the epidermis, where they form a tight network of cells constituting the first immunologic barrier with the environment. (d) Epidermal and dermal sheets were isolated from human skin maintained in culture for 2 days to allow the LCs and dermal DCs to migrate into the medium. Migrating cells are stained with Giemsa. Arrows show a contaminating keratinocyte in the epidermis and a contaminating T cell in the dermis. Figure includes material from M. Bogunovic and M. Merad (unpublished data) and Collin (unpublished data).
proliferative precursors that have taken up residence in the skin prior to birth [9], and we will discuss later in this chapter how these attributes may affect transplant immunity (Fig. 19.1). Mucosal DCs (Fig. 19.2) (reviewed in [10]) Type II mucosal surfaces are covered by stratified squamous epithelia, which share many common features with the skin and include those surfaces that cover the cornea, the oroesophageal cavity and the vaginal cavity. Mucosal DCs present in stratified epithelia other than skin are also called LCs. Corneal LCs express CD11c but little or no MHC class
II in the steady state, while oroesophageal LCs express MHC class II and langerin. Vaginal LCs express MHC class II, CD11c, F4/80, DEC205, and langerin but no Birbeck granules, whereas submucosal DCs, the counterpart to dermal DCs in the skin, reside beneath the basement membrane of type II mucosal epithelia and express CD11c and CD11b but no langerin. Type I mucosal surfaces are covered by simple epithelia including those that cover the small and large intestines, the upper female reproductive tract, and the pseudostratified epithelia of the respiratory tract. Type I mucosal surfaces contain mucus secreting cells called goblet
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(a)
Chapter 19
Murine gut cross-section
MHC class II-DAPI-CX3CR1
(b)
Murine gut serosa
MHC class II
cells, and mucosa-associated lymphoid tissue (MALT) that includes Peyer’s patches in the small intestine and isolated lymphoid follicles in the colon. Intestinal DCs, the most widely studied mucosal DCs, are enriched in the lamina propria (the tissue beneath the epithelium), in the MALT and in mesenteric LNs. DCs present in MALT and mesenteric draining LNs are very similar to those present in other lymphoid tissues. Lamina propria DCs include CD11b+F480+CD8α− and CD11b−F480− CD8α+ DCs and PDCs. CD8α+ DCs are usually absent from nonlymphoid tissues other than intestine, and some groups have suggested that the CD8α+ DCs present in the intestine reflect a contamination from lymphoid tissues as it is difficult to purify lamina propria DCs free of contaminating lymphoid tissue-resident DCs. In addition to lamina propria DCs, a large population of DCs is also present in the gut muscularis layer and the serosa at the interface with the peritoneum, and the exact role of these DCs in gut and peritoneal immunity, as well as their migration properties, remains to be examined [11] (Fig. 19.2). Lymphoid tissue DCs (Fig. 19.3) Lymphoid tissue-resident DCs are the most studied DC population in mice and include thymic, spleen, and LN DCs. Lymphoid tissue-resident DCs express MHC class II and CD11c, and can be further subdivided into two major subsets that include CD11b+CD8− DCs and CD11b−CD8+ DCs [7]. CD8+ DCs express low levels of langerin and are the only DC population able to cross-present cell-associated antigens to CD8+ T cells in mice, a process discussed later in this chapter. However, the clinical relevance of the CD8 marker on DCs in mice remains unclear, as it is absent on human DCs. Very similar types of DC exist in MALT and in the mucosa-draining LNs [10]. Although some investigators have tended to group these populations, different lymphoid organs are likely to contain DCs of different origin. In mice, the majority of thymic DCs express CD8, and initial studies suggested that thymic DCs are generated in situ from thymic lymphoid precursors [7]. Myeloid precursors can also give rise to CD8+ thymic DCs [12]. However, studies of DC ontogeny are based mainly on results obtained from the adoptive transfer of purified precursors into irradiated recipient mice, and it remains to be determined whether these results also apply in the steady state. Thymic DCs play a critical role in the induction of negative selection, while they seem dispensable for the induction of positive selection [13]. In the setting of allogeneic hematopoietic cell transplantation (HCT), it is likely that the host or donor nature of DCs that populate the thymus plays a critical
Fig. 19.2 Gut dendritic cells (DCs). (a) Crosssection of the colon isolated from transgenic CX3CR1–GFP mice. CX3CR1 is a marker of DCs in the gut. The section is stained with anti-major histocompatibility complex (MHC) class II antibody and DAPI. Gut DCs are MHC class II+ CX3CR1/GFP+. (b) Gut serosa whole mount stained with anti-MHC class II antibody. Serosal DCs form a network of cells that resembles the Langerhans network in the epidermis. Figure courtesy of M. Bogunovic and M. Merad (unpublished data).
role in the post-transplant immune response. Although DCs charged with peripheral antigens traditionally have been thought to end their life in draining LNs after only hours, a recent study showed that in vitroderived, systemically administered DCs distributed to the thymus, spleen, and LNs, where they survived for more than 40 days [14]. The spleen is populated mostly by blood DCs. Blood DCs transport blood-borne antigens such as plasma components, proteins expressed by circulating cells, and foreign antigens that breach the blood circulation. LN DCs are more heterogeneous in origin as they include blood DCs that enter the LNs through high endothelial venules as well as tissue DCs that enter the draining LNs through the lymphatics [1]. DCs present in the LNs should reflect the tissue environment from which DCs originate. For example, LCs and dermal DCs are present in skindraining LNs but are excluded from mesenteric LNs. Although careful studies are still needed to distinguish blood DCs from tissue-derived DCs in LNs, several groups use CD8 as a marker of blood-derived DCs based on the argument that CD8 is not expressed on peripheral nonlymphoid DCs [15]. Despite its absence from human DCs, the CD8 marker currently occupies a central stage in DC research as CD8+ DCs are very well equipped to cross-present antigens [15]. Based on these data, some groups have hypothesized that tissue DCs play mainly a ferrying role dedicated to the transport of tissue antigens to the draining LNs, while blood DCs are responsible for cross-presentation of cell-associated antigens to CD8+ T cells [15]. However, it can be misleading to use phenotypic expression as an indication of DC ontogeny as cell surface antigens are strongly modulated during the DC lifecycle and it remains possible that CD8 is induced during DC migration to lymphoid organs, which would explain why it is expressed only by lymphoid organ DCs. Another possibility is that tissue DCs and lymphoid CD8+ DCs derive from separate precursors, and that tissue DCs are dedicated to acquiring antigens, migrating to the draining LNs, and directly presenting processed antigens to T cells, while lymphoid DCs alone have the specialized machinery required to cross-present cell-associated antigens to T cells. PDCs (extensively reviewed in [16]) Similar to DCs, PDCs express MHC class II molecules constitutively and lack CD3, CD19, CD14, and CD56 lineage markers. Although human PDCs express very low-to-undetectable levels of CD11c, they express CD4 and CD45RA antigens, the c-type lectin receptor BDCA2, and BDCA4, a neuronal receptor often used to isolate PDCs in vivo.
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Dendritic Cells in Hematopoietic Cell Transplantation
BONE MARROW
BLOOD
SPLEEN CD8+ DC
CLP Flk2+ pDC CMP Flk2+
CD8– DC
DCp
?
CDP Flk2+CD115+ Monocyle
Inflammatory CD8– DC DCp CD11c– MHCII+ Inflammation
DRAINING LYMPH NODES CD8+ DC
Blood monocyte During embryogenesis
CD8– DC
DCp
Embryonic precursor
Interstitial DC
UV
Interstitial DC
Langerhans cells
PPERIPHERAL TISSUES (GUT, LUNG, DERMIS?)
Via lymphatics
EPIDERMIS Radioresistant precursor
Langerhans cells
Fig. 19.3 Origin of dendritic cells (DCs) in mice. This cartoon illustrates a hypothetical view of DC differentiation pathways in mice. Embryonic hematopoietic precursors give rise to Langerhans cells (LCs) in the skin. LCs are maintained by radioresistant hematopoietic progenitors that have taken residence in the skin during steady-state conditions. After severe skin injury, local LC progenitors are eliminated and LCs are repopulated by circulating monocytes. Common myeloid precursor (CMP) and common lymphoid precursor (CLP) give rise to DCs, but the DC differentiation potential is restricted to the Flk2 (fms-like thyrosine kinase 2, the receptor for Flt3L)-positive populations in mice and humans. A common DC/plasmacytoid DC precursor (CDP) was recently identified in the bone marrow as an Flk2+CD115+MHC II+ cell. This precursor likely derives from Flk2+ CMP and Flk2+ CLP, although this has still not been confirmed. CDPs give rise to plasmacytoid DCs (pDC) and to DC precursors (DCp) in the blood (MHC II+CD11c−) that can further differentiate in lymphoid organs into CD8+ and CD8− DCs. Lymphoid organ DCp divide in situ for three to four cycles. In contrast to lymphoid organ DCs, it is still unclear if CDPs participate in DC homeostasis in nonlymphoid tissues. On the other hand, monocytes have been shown to give rise to DCs in nonlymphoid tissue including the gut and the lung, and potentially the dermis, while they do not give rise to splenic DCs in the steady state. However, in inflamed conditions, monocytes can be induced to differentiate into splenic DCs and into epidermal LCs. Lymph nodes (LNs) and mucosa-associated lymphoid tissue contain DCs that originate from the blood (i.e. are CDP progeny) in addition to DCs migrating from peripheral nonlymphoid tissue. For example, skin-draining LNs but not the mesenteric LNs contain LC-derived DCs, while both LNs contain CD8+ DCs. MHC, major histocompatibility complex; UV, ultraviolet.
Human PDCs also express high levels of the interleukin-3 (IL-3) receptor (CD123). Murine PDCs express low levels of the integrin CD11c, express low levels of the lineage markers CD45RA/B220+ and ly6C/GR1+ molecules, lack CD11b, and express plasmacytoid DC antigen-1 (PDCA1) and siglec-H, a member of the sialic acid binding immunoglobulin-like lectin (Siglec) family, recently identified as a specific surface marker of PDCs in mice. In the steady state, PDCs are generated in BM, circulate in the blood, enter LNs through high endothelial venules, and accumulate in the paracortical T-cell-rich areas of LNs. Thus, the migration pattern of PDCs closely resembles that of lymphocytes but is clearly distinct from
that of DCs. Migration of PDCs into LNs is greatly enhanced when the LNs drain a site of inflammation. PDCs are absent from nonlymphoid peripheral tissue such as the skin in the steady state, but they can be found in these tissues when they are inflamed. In vitro-derived DCs and PDCs In humans, CD34+ progenitor hematopoietic cells are commonly used to derive LC- and dermal DC-like cells [6]. Monocytes are also a common source of DC-like cells, referred to as monocyte-derived DCs. Granulocyte–macrophage colony stimulating factor is the most common cytokine used to obtain DCs in vitro, although it is dispensable for DC
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development in mice in the steady state [7]. Transforming growth factorβ is required to obtain LC-like cells in vitro in humans and epidermal LCs in vivo in mice [8]. Murine BM cells can also be used to obtain DC-like cells in the presence of granulocyte–macrophage colony stimulating factor either alone or in combination with tumor necrosis factor [6]. PDCs can be generated from BM cells in cultures enriched with the cytokine fms-like thyrosine kinase ligand-3 [7]. Although in vitroderived DCs differ from in vivo-generated DCs, these culture systems have been extremely valuable for analysing the molecular basis of DC function. DC functions In contrast to most cell populations, DCs are defined by a set of properties that include functional as well as phenotypic properties. The ability to take up, process, and present antigens, to migrate selectively through tissues, and to prime naïve T-cell responses are defining DC properties. The superior ability of DCs, particularly myeloid DCs, to stimulate T cells became clear in the late 1970s from studies that compared the ability of DCs, B cells, and macrophages to stimulate a primary mixed lymphocyte reaction, which could be used as a model for graft rejection if T cells were isolated from the donor graft and DCs from the recipient. Normally, mixed lymphocyte reactions are carried out with equal number of stimulator (APCs) and responder (T cells), but only one DC is necessary to activate 100–3000 T cells, consistent with the concept that DCs are specialized to initiate immunity [1]. Elegant studies have subsequently shown that mice deficient in DCs are defective in initiating adaptive T-cell responses [17], while mice that genetically lack macrophages (Csf-1op/op) are not [18]. Now it is clear that DCs form a specialized system to prime T cells not only to mismatched MHC proteins, but also to minor histocompatibility antigens (mHAs) and foreign proteins. More recently, several studies have demonstrated that, in addition to inducing immunity, DCs can play a critical role in the induction of central and peripheral tolerance, a function that might be interesting to utilize in the setting of allogeneic HCT. Although it seems counterintuitive that potent APCs induce tolerance, it is now clear that induction of tolerance as well as immunity requires two critical functions that are performed uniquely by DCs [1]. First, the antigen sampling and migratory capacities of DCs effectively allow naïve T cells to come into contact with peripheral antigens that they would otherwise not encounter. Indeed, cell-derived antigens located in diverse anatomic sites in the body must be found and recognized by T cells that circulate in the blood stream. This task is made more difficult by the low density of specific antigen–MHC complexes on the surface of most cells (3 log reduction after consolidation therapy) and poor molecular responders, who had a higher MRD level at both time points [45]. The probability of 2-year survival was 48% for the good molecular responders compared with 0% for the poor molecular responders. Krampera et al. demonstrated that frequent immunophenotypic MRD measurement in the first year of treatment is a useful outcome predictor for adult T-cell-ALL patients [46]. MRD-positive patients prior to consolidation therapy had a probability of relapse at 2 years of 82%, compared with 39% in MRD-negative patients. In a larger study of 102 adolescent and adult ALL patients, MRD at day 35 was the most powerful independent prognostic parameter by multivariate analysis [47]. Patients in morphologic complete remission (CR) and a low MRD level (10 years after CR) have the same clonal IGH V-D-J gene rearrangement as at diagnosis [160]. How can such examples of dormancy be explained? Perhaps prolonged immune surveillance, most relevant after HCT, can control the expansion of a leukemia clone. Or, as in the example of t(8;21) noted above, the MRD assay may detect signal in a cell lineage not involved in the leukemia. Lastly, a residual leukemia cell may be “preleukemia,” not having all the mutations necessary for malignancy, or “postleukemia,” whereby additional mutations have rendered the clone relatively inert. Regardless, it is likely that the clinical definition of “cure” does not mean the elimination of all cells involved in the leukemia
The Detection and Significance of Minimal Residual Disease
process. The further study of dormancy may clarify what it takes to “cure” leukemia. Lack of standardization of the MRD assays, nomenclature, and clinical reporting The need for uniform or standardized MRD testing is critical to facilitate precise comparisons between new treatment strategies. This is particularly true in PCR-based assays. The problematic areas of gene nomenclature and clinical molecular reporting are being actively addressed by the pertinent international scientific professional societies in collaboration with the health-care professionals who utilize molecular testing that impact patient management. We need to be proactive and join in with this concerted effort to develop a uniform approach for quantifying and reporting MRD. To avoid future nomenclature issues, especially in reference to genes, their transcripts, and the resultant proteins, scientists are requested to follow the gene names and symbols provided the Human Genome Organization (HUGO) gene nomenclature (reader is referred to http://www.gene.ucl. ac.uk/nomenclature). Guidances for clinical molecular laboratory reporting, including FISH, PCR, mutations, deletions, and other genetic variations testing, have been proposed to assess the clinical relevance of MRD testing and facilitate precise comparisons among clinical trials.
Conclusion Today’s technologic advances in laboratory science are providing the tools to design therapeutic strategies according to the molecularly
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defined features of hematopoietic malignancies and the presence or absence of MRD. But much more work remains to be done in this area. The detection of MRD is strongly associated with relapse. MRD monitoring is being melded into the design of clinical trials, and is likely to improve the speed and efficacy of these trials. Many research avenues bear further investigation. The treatment of MRD in almost all settings is still under investigation, and cries out for large-scale studies. The recognition of “dormancy” may allow us to adopt therapies to achieve that state, rather than persist in high-dose therapy designed to eliminate leukemia, which may be impossible. In many leukemia subtypes, there are few good MRD markers. Further research (e.g. gene expression and proteomics studies) is expected to identify new markers for MRD assays, and provide insights into the cell signaling pathways that can be exploited as therapeutic targets. Recent genomic characterization studies of the hematopoietic disorders by highdensity microarrays (containing ~350,000 genetic loci) are unscrambling the recurrent theme between cancer and cellular differentiation. These assays have led to the discovery of deletions and loss-of-function mutations in genes known to be involved in regulating the differentiation of blood cells. It will be through these high-density molecular approaches applied to large patient cohorts that recurrent molecular aberrant patterns will lead to systematic strategies to tackle the underlying genetic causes of these disorders with the eradication of MRD. Without question, MRD detection is key in patient management. It is our responsibility to adopt standardized MRD techniques within clinical trials to clarify its role and greatly improve the pace at which therapeutic strategies are developed and tested.
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70% of acute myeloid leukaemia patients – results from a single-centre study. Br J Haematol 2004; 125: 590–600. Trka J, Kalinova M, Hrusak O et al. Real-time quantitative PCR detection of WT1 gene expression in children with AML: prognostic significance, correlation with disease status and residual disease detection by flow cytometry. Leukemia 2002; 16: 1381–9. Weisser M, Kern W, Rauhut S et al. Prognostic impact of RT-PCR-based quantification of WT1 gene expression during MRD monitoring of acute myeloid leukemia. Leukemia 2005; 19: 1416–23. Scholl S, Loncarevic IF, Krause C, Kunert C, Clement JH, Hoffken K. Minimal residual disease based on patient specific Flt3-ITD and -ITT mutations in acute myeloid leukemia. Leuk Res 2005; 29: 849–53. Beretta C, Gaipa G, Rossi V et al. Development of a quantitative-PCR method for specific FLT3/ ITD monitoring in acute myeloid leukemia. Leukemia 2004; 18: 1441–4. Stirewalt DL, Willman CL, Radich JP. Quantitative, real-time polymerase chain reactions for FLT3 internal tandem duplications are highly sensitive and specific. Leuk Res 2001; 25: 1085–8. Kottaridis PD, Gale RE, Langabeer SE, Frew ME, Bowen DT, Linch DC. Studies of FLT3 mutations in paired presentation and relapse samples from patients with acute myeloid leukemia: implications for the role of FLT3 mutations in leukemogenesis, minimal residual disease detection, and possible therapy with FLT3 inhibitors. Blood 2002; 100: 2393–8. Shih LY, Huang CF, Wu JH et al. Internal tandem duplication of FLT3 in relapsed acute myeloid leukemia: a comparative analysis of bone marrow samples from 108 adult patients at diagnosis and relapse. Blood 2002; 100: 2387–92. Gorello P, Cazzaniga G, Alberti F et al. Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations. Leukemia 2006; 20: 1103–8. Perego RA, Marenco P, Bianchi C et al. PML/ RAR alpha transcripts monitored by polymerase chain reaction in acute promyelocytic leukemia during complete remission, relapse and after bone marrow transplantation. Leukemia 1996; 10: 207– 12. Roman J, Martin C, Torres A et al. Absence of detectable PML-RAR alpha fusion transcripts in long-term remission patients after BMT for acute promyelocytic leukemia. Bone Marrow Transplant 1997; 19: 679–83. Elmaagacli AH, Beelen DW, Kroll M, Trzensky S, Stein C, Schaefer UW. Detection of CBFbeta/ MYH11 fusion transcripts in patients with inv(16) acute myeloid leukemia after allogeneic bone marrow or peripheral blood progenitor cell transplantation. Bone Marrow Transplant 1998; 21: 159–66. Elmaagacli AH, Beelen DW, Trenschel R, Schaefer UW. The detection of wt-1 transcripts is not associated with an increased leukemic relapse rate in patients with acute leukemia after allogeneic bone marrow or peripheral blood stem cell transplantation. Bone Marrow Transplant 2000; 25: 91–6. Gaiger A, Schmid D, Heinze G et al. Detection of the WT1 transcript by RT-PCR in complete
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Chapter 26
remission has no prognostic relevance in de novo acute myeloid leukemia. Leukemia 1998; 12: 1886–94. Osborne D, Frost L, Tobal K, Liu Yin JA. Elevated levels of WT1 transcripts in bone marrow harvests are associated with a high relapse risk in patients autografted for acute myeloid leukaemia. Bone Marrow Transplant 2005; 36: 67–70. Ogawa H, Tamaki H, Ikegame K et al. The usefulness of monitoring WT1 gene transcripts for the prediction and management of relapse following allogeneic stem cell transplantation in acute type leukemia. Blood 2003; 101: 1698–704. Feller N, Jansen-van der Weide MC, van der Pol MA, Westra GA, Ossenkoppele GJ, Schuurhuis GJ. Purging of peripheral blood stem cell transplants in AML: a predictive model based on minimal residual disease burden. Exp Hematol 2005; 33: 120–30. Venditti A, Maurillo L, Buccisano F et al. Pretransplant minimal residual disease level predicts clinical outcome in patients with acute myeloid leukemia receiving high-dose chemotherapy and autologous stem cell transplantation. Leukemia 2003; 17: 2178–82. O’Brien SG, Deininger MW. Imatinib in patients with newly diagnosed chronic-phase chronic myeloid leukemia. Semin Hematol 2003; 40(2 Suppl 2): 26–30. Kantarjian H, Talpaz M, Cortes J et al. Quantitative polymerase chain reaction monitoring of BCR-ABL during therapy with imatinib mesylate (STI517; Gleevec) in chronic-phase chronic myelogenous leukemia. Clin Cancer Res 2003; 9: 160–6. Hughes TP, Kaeda J, Branford S et al. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003; 349: 1423–32. Hughes T, Deininger M, Hochhaus A et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 2006; 108: 28–37. Branford S, Rudzki Z, Walsh S et al. Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis. Blood 2003; 102: 276–83. Muller MC, Gatterman N, Lahaye T et al. Dynamics of BCR-ABL mRNA expression in first-line therapy of chronic myelogenous leukemia patients with imatinib or interferon alpha/ara-C. Leukemia 2003; 17: 2392–400. Cortes J, Talpaz M, O’Brien S et al. Molecular responses in patients with chronic myelogenous leukemia in chronic phase treated with imatinib mesylate. Clin Cancer Res 2005; 11: 3425–32. Marin D, Kaeda J, Szydlo R et al. Monitoring patients in complete cytogenetic remission after treatment of CML in chronic phase with imatinib: patterns of residual leukaemia and prognostic factors for cytogenetic relapse. Leukemia 2005; 19: 507–12. Branford S, Rudzki Z, Harper A et al. Imatinib produces significantly superior molecular
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responses compared to interferon alfa plus cytarabine in patients with newly diagnosed chronic myeloid leukemia in chronic phase. Leukemia 2003; 17: 2401–9. Merx K, Muller MC, Kreil S et al. Early reduction of BCR-ABL mRNA transcript levels predicts cytogenetic response in chronic phase CML patients treated with imatinib after failure of interferon alpha. Leukemia 2002; 16: 1579–83. Wang L, Pearson K, Ferguson JE, Clark RE. The early molecular response to imatinib predicts cytogenetic and clinical outcome in chronic myeloid leukaemia. Br J Haematol 2003; 120: 990–9. Hughes TP, Morgan GJ, Martiat P, Goldman JM. Detection of residual leukemia after bone marrow transplant for chronic myeloid leukemia: role of polymerase chain reaction in predicting relapse. Blood 1991; 77: 874–8. Lion T, Henn T, Gaiger A, Kalhs P, Gadner H. Early detection of relapse after bone marrow transplantation in patients with chronic myelogenous leukaemia. Lancet 1993; 341: 275–6. Miyamura K, Tahara T, Tanimoto M et al. Long persistent bcr-abl positive transcript detected by polymerase chain reaction after marrow transplant for chronic myelogenous leukemia without clinical relapse: a study of 64 patients. Blood 1993; 81: 1089–93. Pichert G, Roy DC, Gonin R et al. Distinct patterns of minimal residual disease associated with graft-versus-host disease after allogeneic bone marrow transplantation for chronic myelogenous leukemia. J Clin Oncol 1995; 13: 1704–13. Roth MS, Antin JH, Ash R et al. Prognostic significance of Philadelphia chromosome-positive cells detected by the polymerase chain reaction after allogeneic bone marrow transplant for chronic myelogenous leukemia. Blood 1992; 79: 276–82. Sawyers CL, Timson L, Kawasaki ES, Clark SS, Witte ON, Champlin R. Molecular relapse in chronic myelogenous leukemia patients after bone marrow transplantation detected by polymerase chain reaction. Proc Natl Acad Sci U S A 1990; 87: 563–7. Radich JP, Gehly G, Gooley T et al. Polymerase chain reaction detection of the BCR-ABL fusion transcript after allogeneic marrow transplantation for chronic myeloid leukemia: results and implications in 346 patients. Blood 1995; 85: 2632–8. Costello RT, Kirk J, Gabert J. Value of PCR analysis for long term survivors after allogeneic bone marrow transplant for chronic myelogenous leukemia: a comparative study. Leuk Lymphoma 1996; 20(3–4): 239–43. Radich JP, Gooley T, Bryant E et al. The significance of bcr-abl molecular detection in chronic myeloid leukemia patients “late,” 18 months or more after transplantation. Blood 2001; 98: 1701– 7. van Rhee F, Lin F, Cross NC et al. Detection of residual leukaemia more than 10 years after allogeneic bone marrow transplantation for chronic myelogenous leukaemia. Bone Marrow Transplant 1994; 14: 609–12. Mackinnon S, Barnett L, Heller G. Polymerase chain reaction is highly predictive of relapse in patients following T cell-depleted allogeneic bone marrow transplantation for chronic myeloid leukemia. Bone Marrow Transplant 1996; 17: 643–7.
129. Branford S, Hughes TP, Rudzki Z. Monitoring chronic myeloid leukaemia therapy by real-time quantitative PCR in blood is a reliable alternative to bone marrow cytogenetics. Br J Haematol 1999; 107: 587–99. 130. Lin YT, Lin DT, Jou ST, Lin KS, Lin KH. Allogeneic bone marrow transplantation for Philadelphia chromosome-positive chronic myelogenous leukemia in childhood. J Formos Med Assoc 1997; 96: 320–4. 131. Mensink E, van de Locht A, Schattenberg A et al. Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real-time quantitative RT-PCR. Br J Haematol 1998; 102: 768–74. 132. Mughal TI, Yong A, Szydlo RM et al. Molecular studies in patients with chronic myeloid leukaemia in remission 5 years after allogeneic stem cell transplant define the risk of subsequent relapse. Br J Haematol 2001; 115: 569–74. 133. Olavarria E, Kanfer E, Szydlo R et al. Early detection of BCR-ABL transcripts by quantitative reverse transcriptase-polymerase chain reaction predicts outcome after allogeneic stem cell transplantation for chronic myeloid leukemia. Blood 2001; 97: 1560–5. 134. Preudhomme C, Chams-Eddine L, Roumier C et al. Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using an in situ RT-PCR assay. Leukemia 1999; 13: 818–23. 135. Lin F, van Rhee F, Goldman JM, Cross NC. Kinetics of increasing BCR-ABL transcript numbers in chronic myeloid leukemia patients who relapse after bone marrow transplantation. Blood 1996; 87: 4473–8. 136. Caballero D, Garcia-Marco JA, Martino R et al. Allogeneic transplant with reduced intensity conditioning regimens may overcome the poor prognosis of B-cell chronic lymphocytic leukemia with unmutated immunoglobulin variable heavychain gene and chromosomal abnormalities (11q– and 17p–). Clin Cancer Res 2005; 11: 7757–63. 137. Gribben JG. Salvage therapy for CLL and the role of stem cell transplantation. Hematology Am Soc Hematol Educ Program 2005: 292–8. 138. Ritgen M, Stilgenbauer S, von Neuhoff N et al. Graft-versus-leukemia activity may overcome therapeutic resistance of chronic lymphocytic leukemia with unmutated immunoglobulin variable heavy-chain gene status: implications of minimal residual disease measurement with quantitative PCR. Blood 2004; 104: 2600–2. 139. Sorror ML, Maris MB, Sandmaier BM et al. Hematopoietic cell transplantation after nonmyeloablative conditioning for advanced chronic lymphocytic leukemia. J Clin Oncol 2005; 23: 3819–29. 140. Rawstron AC, Kennedy B, Evans PA et al. Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy. Blood 2001; 98: 29–35. 141. Esteve J, Villamor N, Colomer D, Montserrat E. Different clinical value of minimal residual disease after autologous and allogeneic stem cell transplantation for chronic lymphocytic leukemia. Blood 2002; 99: 1873–4. 142. McSweeney PA, Niederwieser D, Shizuru JA et al. Hematopoietic cell transplantation in older patients with hematologic malignancies: replac-
The Detection and Significance of Minimal Residual Disease
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not associated with adverse prognosis. Leukemia 2007; 21: 143–50. Jagannath S, Richardson PG, Sonneveld P et al. Bortezomib appears to overcome the poor prognosis conferred by chromosome 13 deletion in phase 2 and 3 trials. Leukemia 2007; 21: 151–7. Sagaster V, Ludwig H, Kaufmann H et al. Bortezomib in relapsed multiple myeloma: response rates and duration of response are independent of a chromosome 13q-deletion. Leukemia 2007; 21: 164–8. Stewart AK, Bergsagel PL, Greipp PR et al. A practical guide to defining high-risk myeloma for clinical trials, patient counseling and choice of therapy. Leukemia 2007; 21: 529–34. Bensinger WI, Buckner CD, Anasetti C et al. Allogeneic marrow transplantation for multiple myeloma: an analysis of risk factors on outcome. Blood 1996; 88: 2787–93. Martinelli G, Terragna C, Zamagni E et al. Molecular remission after allogeneic or autologous transplantation of hematopoietic stem cells for multiple myeloma. J Clin Oncol 2000; 18: 2273–81. Barlogie B, Kyle RA, Anderson KC et al. Standard chemotherapy compared with high-dose chemoradiotherapy for multiple myeloma: final results of phase III US Intergroup Trial S9321. J Clin Oncol 2006; 24: 929–36. Cavo M, Bandini G, Benni M et al. High-dose busulfan and cyclophosphamide are an effective
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27
Howard M. Shulman, Robert C. Hackman & George E. Sale
Pathology of Hematopoietic Cell Transplantation
Introduction The hematopoietic cell transplantation (HCT) setting presents pathologists with four major types of complication: toxicities resulting from pretransplant cytoreductive conditioning regimens, immunologic problems (rejection or graft-versus-host disease [GVHD]), infections and malignancy. In the last few years, substantial changes have taken place in the practice of HCT, including the use of reduced-intensity conditioning regimens and the inclusion of older allogeneic recipients and those with autoimmune disorders or with substantial pretransplant comorbidities. There is also increased use of mismatched and/or unrelated HCT donors, different immunosuppression regimens, resulting in a change in the presentation of GVHD, and recognition of additional lateterm complications.
Pretransplant evaluation Lymphoreticular system Before a patient is accepted for HCT, the referral evaluation is used to confirm the diagnosis, review available protocols, and identify potential complications that may influence transplant decisions. The pathologist reviewing material from the referring institution will need relevant flow cytometric, immunohistologic, molecular, and cytogenetic data as well as details of recent therapy. Prior to undergoing HCT for hematologic malignancies, the marrow is evaluated to determine remission/relapse status. Of the various modalities utilized, multiparameter flow cytometry is among the most important because it samples thousands of cells and has the ability to detect as few as 0.01% malignant cells, even without having a prior immunophenotype signature for comparison [1–3]. In patients with lymphoma, review of the diagnostic material is especially important if the initial diagnosis was based on small needle biopsies. Following transplantation, the availability of slides showing the lymphoma immunohistology is very helpful when evaluating small lymphoid aggregates in the marrow or possible extramedullary relapse in surgical specimens. The pretransplant subclassification of myelodysplastic syndromes by World Health Organization criteria into aggressive compared with milder forms (e.g. refractory anemia with excess blasts versus refractive
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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anemia) relies predominantly on the marrow blast count. Post-transplant survival for both de novo and secondary myelodysplastic syndrome/ transformed acute myeloid leukemia is related to the disease stage (blast count), cytogenetics, conditioning regimen, and donor relationship [4]. Determining the pretransplant immunophenotypic signature of the malignant population is extremely valuable in separating some chronic myeloproliferative disorders from myelodysplastic disorders, monitoring minimal residual disease, and detecting early post-transplant relapse. Identification of the JAK2V617F and other novel mutations aids in the proper classification of myeloproliferative disorders [5]. Aplastic anemia requires precise evaluation of marrow cellularity and individual lineages to verify eligibility for HCT, and also to rule out myelodysplasia or paroxysmal nocturnal hemoglobinuria insidiously simulating primary aplasia. Lymphomas require review of previous diagnostic biopsies in order to evaluate possible marrow involvement. Detailed evaluation of myelofibrosis may be needed to distinguish chronic myeloproliferative disorders from acute panmyelosis, myelodysplastic syndromes, and metastatic disease. Studies may involve multiple marrow biopsies stained for reticulin and trichrome, as well as molecular testing of BCR–ABL1 fusion transcripts, JAK2V617F, and other novel mutations. Severe myelofibrosis portends slower engraftment but is not a contraindication to HCT [6]. Magnetic resonance imaging of the pelvis and femur helps assess the extent and distribution of myelofibrosis and other disease [7]. Several new agents have affected pretransplant evaluation. Most patients with chronic myeloid leukemia receive imatinib mesylate (Gleevec) or other more recent tyrosine kinase inhibitors which are effective in producing clinical or cytogenetic remissions. Their pretransplant marrows may be morphologically normal or even hypoplastic and display a normal myeloid-to-erythroid ratio with a low blast count. Granulocyte–macrophage colony-stimulating factor and other cytokines may cause confusing left shifts in hematopoietic cells. Bisphosphonates given for multiple myeloma may thicken trabecular bone in pretransplant marrow biopsies. Hepatic disease Pretransplant hepatic disease has a substantial influence on the choice of conditioning regimen and donor [8]. Patients at increased risk of developing sinusoidal obstructive syndrome (SOS), formerly called hepatic veno-occlusive disease, include those with inflammatory liver diseases demonstrating sinusoidal fibrosis, significant necroinflammatory activity or bridging fibrosis. Specific diseases include steatohepatitis, sinusoidal fibrosis associated with extramedullary hematopoiesis,
Pathology of Hematopoietic Cell Transplantation
prior toxicity from gemtuzumab ozogamicin (Mylotarg) (Plate 27.1) and rarely Gleevec [9], active chronic viral hepatitis, and amyloidosis. Poorly compensated cirrhosis is a contraindication for HCT because of the prohibitive risk of developing SOS after most high-dose regimens. Even compensated cirrhosis has a high likelihood of hepatic decompensation after reduced-intensity regimens [10].
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fibrosis), yet the associated clinicopathologic entity may be highly characteristic of toxicity. Other changes due to toxicity, particularly those in the mucous membranes [13], skin, gastrointestinal tract, and lung, cause diagnostic difficulties because of their clinical or histologic overlap with GVHD and infection (Plates 27.5–27.8) Studies of these organs with serial biopsies demonstrate the diffuse distribution of conditioning injury and marked lessening of these changes with time (Plate 27.9).
Post-transplant hematopoietic evaluations High-dose conditioning produces massive destruction of the host marrow, resulting in acute serous myelitis without necrosis of the bony trabeculae (Plate 27.2). The myelitis includes edema, hemorrhage, loss of most marrow elements except plasma and mast cells, and relative predominance of damaged and frequently necrotic fat associated with iron-rich macrophages. This damage gradually resolves over the first 4–6 weeks as colonies of donor myeloid, megakaryocytic, and erythroid precursors develop. Lymphoid cells are loosely scattered and sparse. Rarely, marrow necrosis occurs after intense cytoreduction or at the onset of leukemic relapse. Assessment of aspirated marrow cellularity should include particle sections because overall cellularity cannot be accurately judged from aspirate smears. Biopsies may be small or artifactually disrupted. Engraftment is usually simultaneous in the three major cell lines, although there can be considerable lag in one or more lineages. Failure or loss of donor engraftment may be manifest by the decline or absence of peripheral counts and marrow hypocellularity. In rare patients with GVHD and absence of marrow hematopoiesis, there is apparently only lymphoid engraftment. Graft rejection has no specific histologic features but may be defined as transient engraftment with proliferation of at least one cell line followed by its loss. Rejection following trilineage engraftment occasionally involves loss of only a single line, analogous to pure red cell aplasia. Although rejection usually results in hypocellularity, this may not be true in patients being treated for some nonmalignant conditions such as thalassemia or immunodeficiency. Proof of mixed chimerism or rejection may require confirmation by flow cytometric sorting of CD33 and CD3 cells followed by fluorescence in situ hybridization studies for X and Y chromosomes (if there is a host–donor gender disparity) or by amplified fragment-length polymorphisms. Despite numerous advances in the application of HCT, relapse remains the major cause of mortality. With the use of hematopoietic growth factors, it is not unusual to encounter rare circulating benign myeloblasts. Multiparameter flow cytometry can usually distinguish benign from malignant blasts and also identify rare aberrant blasts long before morphologic relapse is detectable [2,3]. Post-transplant lymphoproliferative disorder (PTLD) has a spectrum of characteristic plasmacytoid to frankly immunoblastic features associated with positive immunohistochemistry (IHC) stains for Epstein–Barr virus (EBV). PTLD lymphoma should be suspected in the setting of adenopathy or worsening of gastrointestinal symptoms despite treatment for acute GVHD (Plates 27.3 and 27.4) [11]. Serial monitoring of plasma EBV DNA by polymerase chain reaction (PCR) is used to detect increasing viral copy numbers, thus allowing early intervention [12].
Toxicity from pretransplant cytoreductive therapy The cytoreductive therapy given for treatment of underlying malignancy, marrow ablation, and immune suppression may cause widespread multiorgan dysfunction and atypical changes. The frequency, the organ systems involved, and the time of onset are related to both the type and the overall intensity of the conditioning protocol. The histopathology within some affected organs, such as the heart, may be disease “nonspecific” (edema, hemorrhage, necrosis of myocytes, and interstitial
Hepatic SOS (veno-occlusive disease) The most frequent life-threatening regimen-related toxicity involves a clinical syndrome of jaundice, weight gain, ascites, and painful hepatomegaly that typically develops in the first few weeks after HCT. In earlier editions, this syndrome was called hepatic veno-occlusive disease, referring specifically to the obliterative lesions within the lumina of small hepatic venules (Plate 27.10) [8,14]. However, based on more recent experimental and molecular studies, the authors have proposed renaming the syndrome “sinusoidal obstructive syndrome” because the primary site of injury occurs in the sinusoids before clinical signs appear. The term SOS reflects the proximate cause, toxic injury to the zone 3 sinusoidal and venular endothelium related to specific drug metabolites, and to intracellular depletion of glutathione stores. Secondary events are: a decrease in nitric oxide level associated with vasoconstriction; deposition of coagulants evinced by factor VIII staining within the widened venular subendothelial zone and the perivenular zone corresponding to the pores that drain the sinusoids into the small venules (Plate 27.11); and subsequently the deposition of extracellular matrix and collagens by activated and proliferated hepatic stellate cells (Plate 27.12). Liver biopsy and autopsy specimens reveal that the histology of injury to the sinusoids, hepatocytes, and venules may be rapidly progressive. The trichrome stain, which illuminates both parenchymal and connective tissue liver components, is critical for recognizing the diagnostic sinusoidal and venular injuries, while other connective tissue stains (e.g. Verhoeff-van Gieson, reticulin, and immunohistologic stains) are complementary. Examination of serial sections may be needed to identify characteristic histologic changes that may not be present in all levels. The sequence of damage derived from experimental and clinical studies consists of death and detachment of sinusoidal endothelial cells followed by their downstream embolization, leading to sinusoidal obstruction along with deposition of fibrin and procoagulants in the pores which drain into the venules, and hemorrhage in zone 3 and the space of Disse (Plates 27.10, 27.11, and 27.13). Immunostaining with CD31 demonstrates a loss of endothelium in the perivenular sinusoids and the central venule (Plate 27.14). Frank necrosis of perivenular hepatocytes, demonstrated by their absence of cytoplasmic cytokeratin immunostaining, is often more widespread and severe than the extent of venular injury. A consequence of the sinusoidal obstruction, elevated sinusoidal pressure, ischemia, and fragmentation of hepatocyte cords is dislodgement of clusters of liver cells, including hepatocytes, which may flow in a retrograde fashion into portal veins or embolize through disrupted pores into the damaged central venules (Plate 27.15). Within 3 weeks of high-dose conditioning, the deposition of sinusoidal reticulin is accompanied by a marked linear sinusoidal pattern of α smooth muscle actin staining for hepatic stellate cells and increases in monocytes/Kupffer cells at the interface of ischemic hepatocyte necrosis (Plate 27.16) [8,14]. Later changes include collagenous obliteration of the venules, thickening of the outer wall of the hepatic venules (phlebosclerosis) (Plate 27.17), extinction of zone 3 hepatocytes, and fibrotic replacement of the sinusoids adjacent to the terminal hepatic venules (zone 3) of the liver acinus (Plate 27.12), In prolonged and fatal SOS, the liver may take on a pattern of reversed cirrhosis with fibrotic linkage between destroyed perivenular zones.
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Correlation of histologic findings with clinical signs Several additional pericentral (zone 3) lesions that follow cytoreductive therapy are associated with SOS. Two studies have demonstrated that zone 3 sinusoidal fibrosis and phlebosclerosis, a nonocclusive perivenular fibrosis, are also associated with the same clinical features as SOS (Plate 27.17) [8]. An example of the histologic spectrum is a rapidly developing syndrome of SOS that has developed among some HCT patients treated with gemtuzumab ozogamicin (Mylotarg), a humanized anti-CD33 monoclonal antibody conjugated to calicheamicin. The associated histology of gemtuzumab ozogamicin-associated SOS differs slightly from SOS occurring after high-dose cytoreductive conditioning in displaying more sinusoidal fibrosis than venular occlusion (Plate 27.1). Late-developing nodular regenerative alterations are not associated with symptoms of early liver toxicity. They most likely are secondary to disturbances in hemodynamics and serve as an indicator of compensatory repair and regeneration of hepatocytes in different zones of the liver acinus [15]. Several other conditions that may result in an SOS-like picture from diminished sinusoidal blood flow with increased sinusoidal resistance are viral hepatitis with bridging fibrosis, occult cirrhosis, and steatohepatitis, both alcohol and nonalcohol-related.
cells of a lesion tends to favor GVHD, although it is by no means specific. A combined histologic, ultrastructural, IHC study of human upper endoscopic mucosal biopsies found both TNF-α and Fas expressed in the infiltrate and a correlation of the number of apoptotic cells with the day 90 transplant-related mortality [20]. Mucosal FOXP3 regulatory T cells are numerically reduced in gastrointestinal GVHD [21]. TUNEL and XY fluorescence in situ hybridization staining of skin biopsies from sex-mismatched HCT recipients has demonstrated both keratinocytes and dermal endothelial cells of donor origin, most numerous in the areas of severe GVHD damage, and only in the patients with GVHD. However, the implications for any subsequent repair of GVHD-damaged tissues are unclear since the switch from recipient to donor phenotype was present only in biopsies taken early and not later in the course of GVHD [22]. The use of IHC for routine diagnosis of GVHD is limited by the sparseness of the lymphoid infiltrates, additional delay and expense, lack of control data for other inflammatory conditions in the transplant milieu, and variations in the GVHD effector cell immunophenotype. There are limited data comparing the apoptosis marker caspase-3 to hematoxylin and eosin (H&E) stains on both GVHD and relevant non-GVHD inflammatory conditions [23]. The use of IHC with lymphoid, myeloid or EBV markers is indicated when deciding whether a post-transplant infiltrate is a recurrent hematologic malignancy or an EBV PTLD.
Graft-versus-host disease Pathogenetic studies
Surgical pathology of acute GVHD
A discussion of the pathogenesis and pathology of GVHD is detailed in several chapters of this edition and illustrated in several books devoted to this topic [16,17]. Pathologic studies have contributed to this body of knowledge by elucidating the selective epithelial targets in the afferent stage and cellular response in the efferent stages. In the afferent stage, cytokines released by tissue damage from conditioning upregulate the expression of minor or major histoincompatibilities on targeted cells which are presented to donor T cells. Preferential target cells reside in subregions of epithelium where cytokeratin 15 (K15) epithelial stem cells or their early progeny are located (e.g. the parafollicular bulge of the hair follicle and rete ridges of skin, and the laterobasal and neck regions in the crypt cells of gut) (Plates 27.18–27.20). In an experimental system using murine lingual epithelium, which resembles human cutaneous rete ridges, it has been shown that the genes that regulate apoptotic vulnerability are differentially expressed in a basal layer subpopulation distinguishable by K15 [18]. The classical concept for the efferent stage in GVHD is that donor cytotoxic lymphocytes attack host cells, including some epithelial cells of skin, intrahepatic bile ducts, and gut. Electron microscopic and immunohistologic data demonstrate lymphocyte epithelial attachments and activation of infiltrating cytotoxic lymphocytes in human biopsy material. Ultrastructural studies of human rectal biopsies of GVHD demonstrate cell-mediated cytotoxicity with close contact to targeted enterocytes. Additional nonlymphoid cells of the innate immune system also play a role in the effector stage of GVHD, as two French studies have demonstrated that activated eosinophils [19] and apoptotic granulocytes [20] (along with tumor necrosis factor-alpha [TNF-α] and Fas) are found in the lamina propria in gastrointestinal GVHD.
Tissue biopsies are used as a primary biomarker for establishing the diagnosis of GVHD, for assessment of activity to monitor therapy, and as a tool for prognosis. Preferably, the pathologists should be familiar with the changing histologic spectrum of GVHD [24,25], and have an awareness of relevant clinical data and questions surrounding a biopsy. Protocols for guiding histologic evaluation, and clinical information relevant for interpretation from skin, liver, gut, and mucosal biopsy sites, are available online at the American Society of Blood and Marrow Transplantation (http://www.asbmt.org/cGvHD_Guidelines.htm). The classification and criteria for the clinical and histologic diagnosis and staging of acute and chronic GVHD have undergone a number of modifications based upon the consensus of expert panels supplemented by evaluation of the available peer reviewed literature that was codified at the National Institutes of Health (NIH) consensus conference in 2005 [25,26]. This classification and the time of onset of acute GVHD have a bearing on the choice and duration of treatment for GVHD and the outcome [27,28]. In the current milieu, the artificial separation of acute from chronic GVHD based on the time of onset before or after day 100 is no longer tenable. Recipients of reduced-intensity conditioning regimens with a late taper of immunosuppression, and recipients of donor lymphocyte infusions at various intervals post HCT, may present with a clinical picture of acute GVHD several months after transplantation. Some patients may present with chronic features within 50–60 days post HCT. Lastly, there can be an overlap syndrome with acute and chronic features [24,26]. In the liver and gut (excluding cicatricial webs in the upper esophagus), there is no clear histologic demarcation between acute and chronic GVHD. The NIH pathology consensus committee’s minimal histologic criteria for GVHD by organ system are reproduced in Table 27.1 [25]. These guidelines may vary slightly within different institutions and could change with prospective clinicopathologic studies. While the basic pathology of GVHD is apoptosis and eventually destruction of selective targeted epithelia with or without fibrosis, there are a number of factors that can influence or cause difficulty in interpretation. These include residual toxicity from conditioning, immunosuppressive treatment that blunts inflammation, a key indicator of activity, co-existent infections, and drug reactions that can mimic GVHD. The timing of the biopsies,
Immunohistology In general, T-cell infiltrates, predominately CD8, have been found in IHC studies of skin biopsies. Those few studies that have studied blood and tissue ratios of T-cell subsets simultaneously have found parallel CD4:CD8 ratios. Later studies of GVHD in the skin and lip have shown that the infiltrates are cytotoxic T cells as defined by CD8, the cytotoxic marker TIA-1, and perforin. Similarly, DR-expression in the epithelial
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Table 27.1 Histologic criteria for GVHD by organ system Organ or System
Minimal Criteria for Active GVHD*
Specific Criteria for Chronic GVHD†
Skin, any stage
Apoptoses in epidermal basal layer or lower malphigian layer or outer root sheath of hair follicle or acrosyringium ± lichenoid inflammation ± vacuolar change ± lymphocytic satellitosis
Skin lichen planus-like
Skin sclerotic Skin morpheic
Skin fasciitis Liver Gastrointestinal
Oral mucosa and conjunctiva Minor salivary or lacrimal gland
Global assessment of dysmorphic or destroyed small bile ducts ± cholestasis, lobular and/or portal inflammation Variable apoptotic criteria (≥ l/piece) in crypts
Combination of epidermal orthorkeratosis, hypergranulosis, and acanthosis with lichenoid changes ± syringitis of eccrine units ± panniculitis Collagenous deposition with thickening throughout the papillary dermis, or pan-dermal collagenosis ± panniculitis Clinically focal or localized lesion predominated by sclerosis in the lower reticular dermis or along the dermal-hypodermal border ± epidermal and appendigeal involvement Fibrous thickening of fascial septa with adjacent inflammation ± panniculitis Ductopenia, portal fibrosis, and chronic cholestasis reflect chronicity but are not specific for chronic GVHD Destruction of glands, ulceration, or submucosal fibrosis reflect long-standing disease but are not specific for chronic GVHD
Lymphocytic infiltration of mucosa with variable apoptosis‡
Lung
Infiltration and damaged intralobular ducts, fibroplasia in periductal stroma, and inflammation with destruction of acinar tissue§ Obliterative bronchiolitis: dense eosinophilic scarring beneath the respiratory epithelium, resulting in complete fibrous obliteration or some degree of luminal narrowing¶
* Conditions that result in lesser degrees of change include immunosuppressive treatment, biopsy very soon after the onset of signs, suboptimal or small tissue sample, insufficient scrial sectioning, confounding infection, drug reaction, or inflammatory conditions. † Once the diagnosis of chronic GVHD has been established or after immunosuppressive treatment, the histologic manifestations of active disease may meet only minimal diagnostic criteria for activity. ‡ Inflammation of the oral mucosa and within the minor salivary glands may persist from prior chemoirradiation or prior inflammation. The distinction between acute and chronic GVHD requires the addition of distinctive oral manifestations [9]. § The distinction of past acinar destruction and fibrosis from ongoing chronic GVHD activity can be difficult and relies on assessing lobules that are not completely fibrotic. Fibroplasia, with acinar and periductal inflammation and features of damage to ducts, such as vacuolar change, lymphocytic exocytosis, nuclear dropout, dyspolarity, or apoptosis, indicates chronic GVHD activity. ¶ Obliterative bronchiolitis [48] should be distinguished from bronchiolitis obliterans–organizing pneumonia [49], which is also associated with GVHD but has a different clinicopathologic presentation and a more favorable outcome. Reproduced from [25], with permission.
sampling error (especially with gut biopsies), and technical factors can cause a false-negative histologic assessment. Further, the diagnostic standards for minimal criteria for GVHD are inexact regarding the minimum required number of apoptotic epithelial cells, whether their presence is necessary in mucosal biopsies with inflammation, the number of serial sections needed to exclude a false-negative diagnosis, and the extent of dysmorphic change for a positive diagnosis in salivary and bile ducts. As a result, histologic examination may not always be the gold standard for the diagnosis. In reality, the histologic changes, especially in patients undergoing immunosuppressive treatment for GVHD, are often not pronounced; a biopsy can be interpreted as GVHD even though there may be confounding conditions and even though florid histologic alterations are absent. The pathology report can convey these issues by integrating the histologic findings with the relevant clinical details in a comment and by inserting qualifying phrases, whose implication is understood within that institution, into the final diagnosis (e.g. “possible,” “consistent with,” “favor,” etc.). In the final analysis, the decision
to treat for GVHD is made by the clinician based on a number of factors including the stage of a malignancy, desirability of a graft-versus-tumor effect, clinical stage of the target organ abnormality, rapidity of progression, toxicity of treatment, and histologic findings [28]. Grading of GVHD The initial histologic grading system by Lerner et al. was devised in 1974, with conditions far different from those of today. The scheme had four histologic grades for acute GVHD in the skin, liver, and gut, which attempted to use the chronologic progression of injury as a scale of severity based on the amount of inflammation, apoptosis, and degree of tissue damage [29]. In fact, GVHD histology is a snapshot in time such that mild (grade I) or severe (grades III–IV) changes may primarily be a reflection of timing and duration of activity (stage) and degree of immunosuppressive treatment. The Lerner scheme and other subsequent histologic grading schemata for GVHD were created without first being verified against useful clinical endpoints. Nonetheless, a histologic grade with a high level
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of inflammatory and/or apoptotic activity is highly relevant information that will impact the current treatment decisions [25]. Evidence-based observations indicate that time to recovery is proportionate to the severity (stage) of the destruction of the gut mucosa and/or bile ducts [8,25]. Whereas skin histologic grading schemata have limited independent prognostic value in the current milieu, the stage of cutaneous GVHD, acute or chronic, carries important therapeutic implications. Skin The cutaneous histologic grading system of Lerner et al. has provided a useful framework for discussing the minimal diagnostic criteria and the specificity of the histology for GVHD. Grade I is superficial perivascular dermatitis with epidermal basal cell vacuolization; grade II is an interface dermatitis with scattered apoptotic or dyskeratotic keratinocytes in the basilar or lower spinosum layers sometimes in close contact with the infiltrating lymphoid cells (lymphocytic satellitosis) (Fig. 27.1, Plate 27.19); grade III has extensive apoptosis with reticular degeneration, destruction of the basal layer with nuclear dyspolarity and atypia, and suprabasilar bulla formation (Plates 27.18 and 27.21); and grade IV is represented by ulceration of the epidermis (Plate 27.22) [29]. Distinction of GVHD from a variety of conditions is problematic and not always
Fig. 27.1 Skin, acute graft-versus-host disease, day 30. Marked lichenoid reaction involving the epidermis and acrosyringium with intraepidermal lymphocytes, apoptosis, and destruction of rete ridges. (Hematoxylin and eosin.)
resolved by using grade II change for the minimal diagnostic criteria [24,30–32]. The use and timing of the skin biopsy for diagnosing GVHD has wide institutional variation. At one extreme, some centers avoid using skin biopsies based on studies [30,31] that concluded that acute GVHD is histologically indistinguishable from other causes of rashes, viral infections, drug reactions, engraftment syndrome associated with lymphocyte recovery [33,34], and toxicity from the pretransplant cytoreductive conditioning [30–32]. The conclusion of Kohler et al. [30] that the skin biopsy has limited diagnostic utility cannot be easily corroborated because of differences in institutional practices, the basis for selection of patients for skin biopsy, and the uncertainty of clinically defining GVHD based on jaundice, diarrhea, and the extent of the rash. A second general criticism of these negative studies pertains to the relative prevalence of the putative non-GVHD causes and whether they can be distinguished from GVHD. The syndrome of painful cutaneous acral erythema involving the palms and soles is a distinct clinical entity caused by cytoreductive conditioning (Plate 27.23) [35]. Histologically, this toxic injury to the epithelium cannot be clearly distinguished from GVHD (Plates 27.5 and 27.18). Both may display varying degrees of epidermal keratinocyte atypia, vacuolar damage, dyskeratosis, apoptosis, and interface inflammation [31,32]. Depending on the particular high-dose conditioning regimen used, these authors [31,32] have advocated waiting 20–30 days before using the skin biopsy. The maculopapular rash attributed to engraftment with early lymphocyte recovery consists of scant superficial perivascular lymphocytic inflammation with little or no keratinocyte apoptosis that spontaneously improves without treatment or following withdrawal of current medications [33,34]. Although various viral etiologies, including HHV-6, have been suggested for some of these early rashes, there is little or no clinical or immunohistologic evidence to support this claim. Drug hypersensitivity, especially a rash with an atypical pattern or distribution for GVHD, should be considered a possibility. Tissue eosinophils in the infiltrate are not an indication of a drug eruption as they are often found in GVHD as well [36]. Drug eruption seems an unlikely explanation for most early rashes given the usual patient’s exposure to a multitude of drugs and the resolution of the rash without their discontinuation. There is no simple answer to this conundrum of when to use skin biopsies to diagnose early GVHD. The management of the early skin rashes is influenced by a set of variables including the sex and degree of donor match, whether conditioning is high dose, desirability of graftversus-tumor effect, extent of rash, rapidity of progression, and protocol requirements. In a recent informal survey of several tertiary centers, the experienced clinicians estimated that up to 60% of all patients undergoing myeloablative conditioning for an allogeneic HCT develop an early skin rash, of which they estimate 60% are from GVHD. In a study with a large cohort of 809 consecutive patients, 265 (33%) developed clinical grades II–IV GVHD. Twenty-seven percent of these had an early-onset acute GVHD manifested by day 14, referred to as hyperacute GVHD. Eighty-eight percent of the patients with hyperacute GVHD had severe skin involvement with a higher nonrelapse mortality, especially in recipients of mismatched unrelated or matched unrelated donors [28]. In previous editions, we advocated obtaining serial skin biopsies to aid in the interpretation of early skin rashes: GVHD was considered likely if the changes progressed without treatment or remained grade II or higher. Firoz et al. addressed this issue in a study using a decision analysis model to estimate the value of skin biopsy in the evaluation of a hypothetical case of a morbilliform rash covering 60% of the body surface post allogeneic stem cell transplant [37]. Ten experts provided their estimates of the prevalence of GVHD, the sensitivity and specificity of the skin biopsy, and the potential outcomes from treatment or observation. Only 25% of the experts chose an intervention consistent
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with their estimates of the prevalence, test characteristics (histologic results), and outcome evaluations. In this decision analysis modeling, the best patient outcomes occurred if biopsy was done when the prevalence of GVHD was less than 30% whereas treatment for GVHD was given without biopsy if the prevalence of GVHD was over 30%. When skin biopsies are obtained to monitor GVHD treatment, the cellular infiltrate may be minimal and apoptosis infrequent (Plates 27.21, 27.22, 27.24, and 27.25). It is important to review serial sections looking for apoptosis along the outer root sheath of hair follicles, the basilar layer of the epidermis, and at the tips of rete ridges. An eczematous pattern of spongiosis with abundant lymphocytes and little or no apoptosis suggests a drug reaction rather than GVHD. On histologic grounds alone, a severe drug eruption producing a clinical picture of Stevens– Johnson syndrome or toxic epidermal necrolysis with suprabasilar bullous formation and denudation can be difficult to distinguish from severe GVHD.
Gastrointestinal The initial sites of injury in the gut are focused on the regenerative or stem cell areas (i.e. the neck region of the gastric glands, the basal layer of the esophagus, and the lower lateral basilar portion of the crypts in the small and large bowel) (Plate 27.20). In humans, gut involvement is often diffuse, evinced by the gross and radiographic studies that demonstrate the simultaneous diffuse changes of mucosal edema with effacement of the normal mucosal folds involving the stomach and entire small and large bowel (Plates 27.26–27.28). In several studies of upper endoscopic biopsies, epithelial necrosis and crypt abscesses from the small intestines were always associated with diarrhea and usually with similar histologic changes in the rectal biopsy. On the other hand, some patients with positive gastric biopsies may present with only nausea and vomiting. Endoscopic biopsies are obtained to diagnose GVHD, monitor the response to immunosuppressive therapy, and to identify infections. The location, timing, and frequency of endoscopic biopsies are highly dependent on institutional bias and past experience with complications. In one study, the gastric biopsy provided the highest diagnostic yield even when the predominant symptom was diarrhea [38]. Based solely on histology, a recent study found that the combination of rectal biopsy and gastric biopsy provided the highest diagnostic yield for GVHD [39]. A recent pediatric study advocated first using the less invasive rectal biopsy alone to avoid the procedural risks of upper endoscopy [40]. In general, upper endoscopic biopsies are more difficult to interpret than rectal biopsies because of the disagreement on the minimal diagnostic threshold criteria for apoptosis and the overlap with reactive and inflammatory conditions. There are also differing opinions on whether a biopsy from the duodenum [19], gastric fundus [41] or antrum [38] provides the greatest sensitivity. Of greater importance is that the negative predictive value of an endoscopic biopsy is potentially affected by the endoscopist’s sampling and the number of serial sections examined. In acute GVHD, edema of the intestinal wall is readily apparent to the radiologist who performs imaging tests of the intestine and to the endoscopist who can also see mucosal erythema, friability, and microerosions. Edema and erythema are not often appreciated by the pathologist whose specimens are limited to tissue above the muscularis mucosal (Plates 27.26 and 27.27). Likewise, the diagnosis of GVHD by histology is based on findings that the endoscopist cannot see, and the diagnosis of GVHD by direct inspection of mucosa is based on findings that the pathologist cannot appreciate (Plate 27.28) [42]. Thus the gold standard for gastrointestinal GVHD may not be histology alone [25,38], but a composite of clinical signs and symptoms, endoscopic appearance, histologic findings, and absence of infection by culture and histology. That is, the
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inability of histology to demonstrate typical findings of GVHD does not mean that GVHD is not present in the gastrointestinal tract. Spectrum of gastrointestinal GVHD The sequence of damage in gut GVHD has been conceptualized into a four-tier grading system that is a modification of the original Lerner histologic grading schema [29]. 1 Grade I. Individual cell necrosis in basal and lateral crypts, with sparse lymphocytic infiltrate (Fig. 27.2, Plates 27.27–27.29). The minimal criterion is a single or rare apoptotic crypt cell, referred to as an exploding crypt cell. These alterations may involve only a few scattered crypts and can be missed unless the specimen contains properly oriented biopsies with multiple serial sections. In the grade I lesion, the exploding crypt cell may not have an accompanying lymphocytic infiltrate. Histologic grade I does not mean that the entire gut has the same grade; it is not uncommon for gastric and/or rectal biopsy histology to underestimate the severity of GVHD in the midgut, particularly the ileum. 2 Grade II. Apoptotic crypt abscess, in which the dilated walls of the apoptotically damaged crypt are lined by thin epithelium devoid of mucin, are infiltrated by lymphocytes. The crypt abscesses contain apoptotic debris. A scattering of lymphocytes, neutrophils, and eosinophils may be present in the interstitium, crypt walls, and crypt abscesses [13,17]. They may be present in considerable numbers, especially after withdrawal of immunosuppression in a fully engrafted recipient (Fig. 27.3, Plates 27.20 and 27.29). 3 Grade III. Crypt loss resulting in stretches of mucosa devoid of crypts with some focal ulceration (Plate 27.30) 4 Grade IV. There is mucosal denudation, and widespread ulceration with loss of the surface epithelium and crypts (Fig. 27.4, Plates 27.31
Fig. 27.2 Colonic graft-versus-host disease, grade I. A single crypt displays individual epithelial cell apoptosis (“exploding crypt cell”).
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Fig. 27.3 Colonic graft-versus-host disease, grade III. Destruction of crypts with formation of crypt abscesses containing apoptotic debris.
damage ranging from rare individual exploding crypt cells to widespread apoptosis, both single and confluent within a single crypt (Plate 27.29). In practice, identifying the minimal diagnostic threshold for grade I can require analyzing many serial sections. Focally enhanced gastritis, a focal mixed lymphohistiocytic infiltrate with or without neutrophils surrounding a small group of foveolae or glands without apoptosis, is associated with GVHD but, by itself, is not diagnostic [43]. The cumulative damage in grades II and III GVHD is typically widespread, whereas in grade IV there may be no crypts left to demonstrate the apoptotic destruction of the earlier grades (Fig. 27.4). All four grades may occur in different concurrent samples. In contrast to the colon and small bowel, the stomach rarely develops diffuse grade IV changes. Grade IV gut GVHD is slow to heal and often refractory, even with secondary immunosuppressive treatment or aggressive surgical management of obstruction (Plate 27.31). The inability to re-epithelialize, even with prolonged secondary treatment for gut GVHD, may result from a loss of the mucosal stem cells (Plate 27.32). Serial colonoscopic biopsies, obtained to assess response to treatment for severe GVHD of the lower gut, may show regenerating crypts without apoptosis lying in close proximity to grade II or III changes. A complete or partial resolution may take months. Rarely, refractory gut GVHD may develop collagenous deposits within the lamina propria, submucosa, and serosa, or a segmental intestinal stenosis with ulceration and fibrosis confined to the mucosa and submucosa (Plates 27.31 and 27.33). Differential diagnosis
Fig. 27.4 Grade IV graft-versus-host disease (GVHD) in a day 48 duodenal biopsy. Mucosal ulceration with loss of villous epithelium and subtotal destruction of crypts. The features that indicate the cause as GVHD are the two small remaining degenerative crypts showing apoptosis and infiltration by lymphocytes and eosinophils. Concurrent biopsies from the stomach and colon had less severe damage: focal grade III and grade II changes, respectively.
and 27.32). This results in an empty lamina propria devoid of surface epithelium and crypts. There may be granulation tissue and scattered areas of hemorrhage. In grades II–IV, the dilated small vessels in the lamina propria are lined by reactive-appearing endothelium and infiltrated by mixed cell populations. Multiple representative samples should be obtained and correlated with the endoscopic locations since areas of mucosal destruction and ulceration can be adjacent to large stretches of regenerative mucosa with little or no apoptotic activity. While the grading system is a useful descriptor, it is not intended to reflect severity since all four grades may be associated with extensive involvement and severe symptoms. Grade I covers a wide spectrum of
There is a lack of expert consensus on the minimal diagnostic criterion for gut GVHD because low levels of apoptosis also occur with various medications, including nonsteroidal inflammatory inhibitors, mycophenylate mofetil (MMF), and proton pump inhibitors [44], and with some infectious and/or reactive causes such as cryptosporidiosis, and phosphate enemas [41]. In general, reactive gastropathy does not produce apoptosis. The differential diagnosis of grade II–IV changes includes cytomegalovirus (CMV) enteritis, which produces a range of change from shallow circular ulcers to an inflammatory pseudotumor mass involving multiple layers of the gut (Plate 27.32). The viral inclusions typically involve the endothelium but may also affect the stromal cells and gut epithelium. There is a longstanding controversy as to whether the presence of CMV enteritis (a well known cause of apoptosis in gut biopsies) precludes a diagnosis of GVHD [25]. Studies have addressed this issue using IHC and in situ hybridization for CMV and caspase-3 IH for GVHD [23,45]. In CMV enteritis, apoptosis was largely confined to the cells with immunoreactive CMV antigens, whereas apoptotic cells were much more numerous in GVHD. These studies concur with the NIH chronic GVHD consensus [25] that multiple apoptotic crypt cells seen on H&E stain away from any CMV-infected cells identified by H&E or IHC indicate the copresence of GVHD. The immunosuppressive MMF can produce colitis with focal ulcerations with marked apoptosis and intense acute and chronic inflammation. Some reports of MMF colitis have noted diffuse gross changes as well. In practice, the distinction of MMF colitis from GVHD is often established by improvement in bloody diarrhea after withdrawal of the drug [46]. There are insufficient data on whether MMF can also produce lesions in the upper gut. Antibiotic related pseudomembranous colitis is related to the toxin produced within the colonic lumen by Clostridium species, especially Clostridium difficile. If severe, it can produce ischemic changes with apoptosis. Pseudomembranous colitis is distinguished from GVHD by the characteristic superficial pseudomembraneous cap of neutrophils, mucin, and necrotic cells with or without bacterial rods and by a positive assay for the Clostridium difficile toxin (Plate 27.34). However, occasional patients can have both GVHD and Clostridium difficile infection.
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Transplant-associated microangiopathy
Autologous GVHD
HCT transplant-associated microangiopathy (TAM) refers to a collection of microthrombotic complications, which includes the hemolytic– uremic syndrome [47] and possibly a thrombotic microangiopathy of the colon [48]. It is unclear whether TAM is a discrete clinical and pathologic entity that is distinct from other well-recognized post-transplant complications. Japanese investigators originally described TAM in 16 patients with steroid-refractory diarrhea and later in seven patients who received low-intensity conditioning with umbilical cord donor transplants. Their findings were ulcers, erosion, and diffuse exfoliation from the terminal ileum to the rectum, accompanied by swelling and myxoid change in the subendothelium of the damaged vessels, microthrombi, and fibrinoid necrosis of small arterioles. Many of their patients had concomitant GVHD and/or CMV enteritis. The Japanese investigators linked the genesis of the colonic lesions with exposure to tacrolimus because some patients had marked improvement after withdrawal of the drug [48]. However, colonic TAM should not be equated with tacrolimus toxicity since several viral infections and severe GVHD can produce a histologic picture with endothelial damage identical to TAM. In future studies, it would be useful to determine whether the histologic picture of TAM is caused by tacrolimus toxicity and whether it can be distinguished from that caused by GVHD, because the therapeutic approach involves either escalating or de-escalating the immunosuppressive regimen.
Thirteen percent of autografted recipients, particularly female patients treated for breast cancer or hematologic malignancy, develop a gastrointestinal syndrome that is both clinically and histologically indistinguishable from GVHD occurring in allogeneic recipients (Plate 27.37) [49]. These lesions may persist for several weeks to months following transplantation and rarely can be quite marked. Fortunately, nearly all have durable responses to one or two courses of prednisone. The genesis of this phenomenon is poorly understood. Possible mechanisms include GVHD manifest from maternal microchimerism, and autoreactive T cells resulting from in vitro depletion of selected T-cell subsets in the hemopoietic autograft.
Esophageal GVHD Like most squamous epithelia, that of the esophagus may be involved with GVHD. The diagnosis of acute GVHD presents considerable diagnostic difficulty since a variety of conditions, including acid peptic reflux and infections, can produce mucosal damage. Unless there is a combination of lichenoid interface changes with apoptosis along the basilar portion of the mucosa, the diagnosis of esophageal GVHD should be made with some qualification. Accurate diagnosis requires special stains and often IHC studies for microorganisms. Bacterial, viral, and fungal cultures should be obtained from lesions endoscopically suggestive of infection (Plates 27.35 and 27.36).
Fig. 27.5 Hepatic graft-versus-host disease, day 53. Despite the minimal inflammation within the portal space, the bile ducts (arrow) are reduced to degenerative cytoplasmic masses with a few remaining hyperchromatic nuclei.
Liver The liver is a major target of allogeneic GVHD [8], and occasionally autologous GVHD as well [50]. Liver involvement is heralded by a gradual rise in bilirubin and alkaline phosphatase and aminotransferases enzyme levels. It typically follows acute cutaneous or intestinal GVHD, but can present as the sole manifestation of GVHD. Hepatic GVHD is no longer classified as acute or chronic [25,26]. The histology is largely unrelated to the period when the GVHD arose, but is rather a reflection of the duration of active liver GVHD, the anti-inflammatory effects of immunosuppression, and the anticholestatic effect of ursodeoxycholic acid. In both the experimental and clinical settings, histopathologic and ultrastructural studies demonstrate that the small interlobular bile ducts are the preferential target of the alloimmune reaction (Fig. 27.5, Plate 27.38). In both controlled experimental studies and coded histopathologic studies, the diagnosis is based on the global assessment of characteristic dysmorphic, interlobular bile duct lesions, and cholestasis that is often marked. The histologic interpretation is directly affected by the quality, size, and timing of the sample. Thin-core needle biopsies may be difficult to interpret due to limited sampling of portal areas or crush artifact. The timing of the liver biopsy influences the histologic finding. When liver biopsies to diagnose GVHD are obtained soon after the onset of liver dysfunction, the characteristic bile duct changes may be absent or affect a minority of portal spaces [8].
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The amount and extent of the inflammatory component in liver GVHD is dependent on the degree of immunosuppressive treatment. The portal infiltrate is typically lymphocytic but may include a minor component of loosely scattered neutrophils, eosinophils, monocytes, and plasma cells. Inflammation is typically mild to scant with therapeutic levels of immunosuppressive agents even with extensive bile duct damage (Fig. 27.5). A clinical presentation of liver GVHD with an acute hepatitic onset may develop much later as the solitary manifestation of GVHD following cessation of immunosuppressive therapy or after donor lymphocyte infusion. The hepatic onset is characterized by marked elevation of aminotransferase enzymes with marked periportal inflammation that includes many plasma cells and a lobular hepatitic component with abundant necroinflammatory activity (pyknotic shrunken hepatocytes and acidophilic bodies) (Plates 27.39 and 27.40). The acidophilic body formation reflects the cytokine induction of the Fas/Fas ligand system with the hepatocytes being innocent bystanders. The characteristic dysmorphic interlobular bile ducts have a withered, shrunken appearance with a collapsed lumen, vacuolated cytoplasm, and irregular outlines. Individual or groups of epithelial cells are flattened and often missing nuclei, creating segments of anucleate eosinophilic cytoplasmic syncytia. Remaining nuclei display atypical features with anisonucleosis, hyperchromasia, and dyspolarity. Lymphocytic ductitis is frequently present in the damaged portions of the biliary epithelium, but apoptosis of bile duct epithelium is infrequent. Despite the similarities of GVHD to liver allograft rejection, endothelialitis of central venules is uncommon. With prolonged liver GVHD, bile duct destruction is best appreciated by stains other than H&E (e.g. cytokeratin 7 or 19, trichrome or periodic acid–Schiff [PAS]) (Plate 27.38). GVHD produces a variety of cholestatic changes including canalicular bile plugs, hepatocyte ballooning, and dropout of hepatocytes in the perivenular zone, along with a scattering of lymphocytes and collections of pigment-laden macrophages (Plate 27.40). Rarely, pseudoground-glass PAS, diastase-resistant hepatocytes develop from the accumulation of abnormal glycogen [51,52]. Frequently, there is some degree of ductular reaction, a proliferation of small ductules along the periphery of the portal space, that is common in inflammatory liver diseases. In addition to being a reparative response to duct injury, the proliferated ductules also appear to become a secondary target of liver GVHD because the destructive lymphocytic ductitis and cytologic changes in the ductules are similar to those in the damaged interlobular bile ducts. A particularly striking form of cholangiolar cholestasis with dilated periportal bile-filled ductules occurs in patients with concomitant intestinal GVHD (Fig. 27.6, Plate 27.41). This resembles cholangitis lenta associated with septicemia and results from the coexisting influence of gut GVHD, showering the liver with endotoxin, which in turn stimulates the production of TNF-α and interleukin-6, a promoter of bile ductule proliferation and cholestasis [53]. A consequence of the ductular reaction is the development of stellate fibrosis, periportal fibrous septae which extend out into the liver acinus, and rarely bridging fibrosis (Fig. 27.6) [8]. Yet, even in prolonged refractory liver GVHD with loss of bile ducts, cirrhosis rarely occurs. The few anecdotal case reports of cirrhosis related to GVHD were tainted by inadequate immunosuppression or a previous high prevalence of hepatitis C virus infection [8]. Differential diagnosis The timing, profile, and rate of rise of liver test abnormalities provide good indicators as to the likely diagnostic possibilities (see Chapter 95). The histologic differential diagnosis of liver GVHD includes drug liver injury. Most of the candidate antimicrobial drugs for causing liver injury do not produce the typical clinical or histologic patterns seen with GVHD. If the clinical situation permits, it is prudent to discontinue the
Fig. 27.6 Hepatic graft-versus-host disease, day 56. There is pronounced hepatocellular and cholangiolar cholestasis with portal-to-portal bridging and parenchymal collapse. Cholangioles have proliferated along the limiting plate.
candidate drug rather than trying to exclude it as a cause by histology. A common finding in patients biopsied after HCT is marked hepatic iron overload within both hepatocytes and Kupffer cells. The iron overload, which results from multiple transfusions and/or increased intestinal iron transport, has been associated with elevated aminotransferase levels [8,54]. Infections, especially viral, are an important diagnostic consideration because failure to treat them can lead to liver failure (see Chapter 95). CMV should not be considered in the work-up of severe hepatitis or jaundice since it results in mild anicteric hepatitis whose principal histologic findings in immunosuppressed patients are microabscesses that contain some neutrophils. The only exception is CMV papillitis of the ampulla of Vater leading to biliary obstruction. The life-threatening adenovirus and herpes group viral infections are discussed in the section on infection, below. If the portal spaces are greatly expanded by an infiltrate rich in plasma cells, the differential diagnosis and work-up should include a PTLD (Plates 27.4a and 27.4b) (see Chapter 56), the hepatitic onset of GVHD [55], and, rarely, autoimmune hepatitis [8]. The development of positive tests and/or immunohistologic stains for hepatitis B and C viruses does not exclude the coexistence of GVHD. It may be difficult to distinguish the bile duct changes in cases of severe reactivation of viral hepatitis B or C from prolonged GVHD with a ductular reaction. Nearly all cirrhosis developing after 10 years post HCT is caused by hepatitis C virus. The use of the liver biopsy in monitoring the therapeutic response and prognosis of patients with GVHD is unsettled. To validate the biopsy as a monitor of therapeutic response will require standardization of the biopsy findings in relation to the duration and type of immunosuppressive therapy through linkage to a large multi-institutional database, the use of uniform histologic criteria, and an adequate sample. Although there is no validated histologic grading system for liver GVHD, evidence-based observations of protracted GVHD indicate time to recovery from jaundice is proportionate to the extent of bile duct destruction. Chronic GVHD Since the earlier editions of this text, advances in HCT practice have caused major changes in the presentation and natural history of acute and chronic GVHD (Table 27.1) [24,26]. The clinical manifestations
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[24,26] and certain unique histologic changes [25] listed in Table 27.1 are the minimal criteria that define acute and chronic GVHD. The new clinical classification identifies an overlap syndrome, which is a composite of both acute and chronic features. The classification also includes acute GVHD that is persistent, recurrent or occurs after day 100 without meeting additional diagnostic criteria for chronic GVHD [56]. Since there is no clear histologic dichotomy between acute and chronic GVHD in the liver or gut, the histologic changes specific for chronic GVHD occur in the biopsies from skin and fascia, salivary and lacrimal glands, muscle, and lung. From the pathologist’s viewpoint, chronic GVHD is a multiorgan inflammatory disorder that resembles a mixture of GVHD and several autoimmune diseases. The importance of screening biopsies at day 80–100 of skin or lip in asymptomatic patients still on immunosuppression is controversial. A positive screening biopsy without diagnostic histologic features of chronic GVHD is no longer considered to be chronic GVHD. Likewise, a positive screening oral biopsy unaccompanied by distinctive gross oral features of chronic GVHD is no longer classified as chronic GVHD [25,26]. Accordingly, there is a wide spectrum of utilization of oral and skin biopsies in the diagnosis and management of late acute or chronic GVHD [57]. Biopsies are recommended to confirm active chronic GVHD when alternative diagnoses are entertained, the clinical signs are confined to internal organs or the clinical assessment of activity is obscured by prior changes. In one tertiary care center, 7% of patients sent there for consultation regarding chronic GVHD had been incorrectly diagnosed and treated for active chronic GVHD before referral [58]. Skin Chronic GVHD of the skin has a polymorphous gross appearance potentially involving all regions, including the nails (Plates 27.42–27.48). A detailed glossary of the gross cutaneous manifestations along with a standardized nomenclature and their likely underlying histopathology may prove useful in correlating appearances with the outcome and response to therapy [24,59]. The pathologist should be mindful of several caveats pertinent to the histopathology of chronic GVHD: 1 The histopathology changes over time. Usually, the predominant features of the early manifestations, referred to as lichenoid GVHD (Fig. 27.7), are lymphoplasmacytic inflammation involving the epithelium and tubuloalveolar glands in the eccrine, lacrimal, and aerodigestive tracts (Fig. 27.8). Unlike acute GVHD, chronic GVHD causes extensive destruction of these tubuloalveolar glands and ducts with corresponding clinical sicca syndromes (Fig. 27.8, Plate 27.49). 2 In later stages, referred to as sclerodermoid GVHD, there may be widespread fibrosis, stenosis, obliteration or atrophy of the involved tissues (Fig. 27.9, Plates 27.50 and 27.51). As a result, it may be difficult to distinguish active disease from residual fibrotic damage (Fig. 27.9, Plate 27.52) in the absence of chronic inflammation or serial biopsies showing progression of the fibrosis. However, the initial presentation of cutaneous chronic GVHD may be primarily with changes associated with the later stages of dyspigmentation and dermal sclerosis [59]. 3 Following a flare of chronic GVHD, the histologic changes may be more closely akin to those of acute GVHD. Lichenoid, sclerodermoid, and acute histologic features can coexist. The initial dermatopathologic descriptions of cutaneous chronic GVHD were of a biphasic disorder, initially resembling a combination of widespread lichen planus and lupus profundus, which later progressed to scleroderma-like gross and histologic changes (Figs 27.7 and 27.9, Plates 27.48 and 27.53). Smaller subsets of patients present with localized morphemic lesions or fasciitis (Plate 27.47) [60]. In the current milieu, one may see various combinations of these changes plus superimposed skin lesions that only satisfy criteria for acute GVHD [24,26,58].
Fig. 27.7 Skin, day 314, with the extensive or generalized type of early chronic graft-versus-host disease (GVHD). The so-called lichen planus-like chronic GVHD refers to the acanthotic and hyperkeratotic epidermis that has inflammatory and destructive changes along the dermal–epidermal junction. An eccrine unit at the base of the dermis (arrow) and a follicle deep in the dermis are also being destroyed. The dermal collagen is unaltered.
Full-thickness skin biopsies are essential to fully assess possible changes in the eccrine units, subcutaneous fat, and fascia, and to delineate the degree of dermal fibrosis (Plate 27.50). The specific criteria for the early lichen planus-like stage are the combination of hyperkeratosis, hypergranulosis, and irregular acanthosis with lichenoid basilar layer changes with or without syringitis (inflammation within eccrine units) or lobular panniculitis (inflammation of the subcutaneous fat) (Plate 27.53) [25]. Serial biopsies demonstrate progression of the fibrosis from the papillary dermis downward, consistent with an etiology related to the ongoing inflammation and release of cytokines (Plates 27.50 and 27.52). In contrast to chronic GVHD, the dermal fibrosis in systemic sclerosis (scleroderma) typically proceeds upward from the base of the dermis. Patients with cutaneous sclerodermatous chronic GVHD, like those with progressive systemic sclerosis, have an autoantibody to the platelet-derived growth factor receptor which stimulates the activation of skin fibroblasts in vitro, suggesting a direct role in the cutaneous sclerosis in both conditions [61]. The remodeling of the dermal collagen progresses from straightening of the dermal-epidermal border with loss of the rete ridges and dermal appendages, along with sclerosis of the papillary and adventitial dermis with variable degrees of fibroblastic stroma. If progressive, the reticular dermis becomes thickened by
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Fig. 27.8 Lip biopsy, minor salivary gland. The lymphoplasmacytic inflammatory cells are centered around small ducts. Signs of ductal epithelial cell destruction, irregularity of outline, nuclear stratification and hyperchromatism, and apoptosis (arrows) are similar to graft-versus-host disease changes in small bile ducts. The glandular interstitium is becoming fibrotic and the acini atrophic, resulting in a sicca syndrome.
Fig. 27.10 Lip biopsy, day 376. Fibrotic and destroyed minor salivary gland contains only ectatic ducts. These changes reflect previous damage from chronic graft-versus-host disease (GVHD). Unless they are accompanied by some inflammatory component in other glands or in the mucosa, they should not be taken as a sign of active chronic GVHD.
smudgy collagen bundles. Eventually, the dermal subcutaneous border becomes straightened, and fibrotic septae may form in the subcutaneous fat (Fig. 27.9, Plate 27.50). In patients receiving immunosuppressive treatment, the inflammatory changes may be minimal with evidence of active GVHD limited to epithelial vacuolar degeneration or apoptosis in the basilar layers of the skin and its appendages, oral mucosa or minor salivary glands of the lip. Morpheic GVHD presents as localized dyspigmented macules with variable degrees of induration. A full-thickness biopsy demonstrates nodular fibrous remodeling in the deeper reticular dermis with variable inflammation in the dermis and adjacent subcutaneous fat. The epidermis may have minimal apoptosis or vacuolar changes. Eosinophilic fasciitis of the skin presents with an acute painful deep groove or a rippling cellulite appearance. A deep incisional biopsy shows edema and fibrosis of the fascia and subcutaneous septae with a mixed infiltrate of lymphocytes, histiocytes, and eosinophils [25,60]. A lichen sclerosis atrophicus type of chronic GVHD leads to scarring and stricture when it affects the female genitalia. Histologically, there is epidermal or mucosal atrophy with basal vacuolar changes, a band-like infiltrate associated with small numbers of apoptotic cells, and a subepidermal zone of pale homogenized collagen. Several additional forms of cutaneous chronic GVHD include eruptive angiomas, vitiligo, hyperpigmentation, destructive nail dystrophies, and ichthyosiform change (Plates 27.46 and 27.48) [59]. Oral
Fig. 27.9 Late sclerodermatous chronic graft-versus-host disease (GVHD), day 910. The epidermis is atrophic. The dermal collagen is diffusely sclerotic with homogenization of the collagen, and the dermis–subcutis border is straightened. The dermis below the entrapped eccrine unit (arrow) represents acquired fibrous tissue resulting from chronic GVHD.
Oral chronic GVHD has mucosal changes similar to the skin with lichenoid interface inflammation, exocytosis, and apoptosis. The assessment of GVHD in minor salivary glands should only focus on lobules that are not completely fibrotic. Active GVHD changes include lymphocytic exocytosis into intralobular ducts and acini, and lymphocytic periductal inflammation with or without plasma cells (Fig. 27.8, Plate 27.49). Fibroblastic periductal and periacinar stroma is indicative of chronic GVHD activity, whereas dense fibrosis indicates only previous damage (Fig. 27.10). Because of residual nonallogeneic baseline inflammatory changes from high-dose conditioning [62], there are institutional variations in how the threshold for minimal activity is defined. One such proposal with more stringent criteria requires more than three apoptotic cells in the mucosa and a greater than 10% loss of acinar tissue or ductal
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epithelial necrosis [63]. The same criteria for oral biopsies also apply to the esophageal, vulvar, conjunctival, and lacrimal biopsies. Other sites involved in chronic GVHD Several infrequent manifestations associated with chronic GVHD resemble autoimmune disorders, including vitiligo, myositis (Plate 27.54), serositis, arthritis, synovitis, myasthenia gravis, and bullous pemphigoid. Rackley et al. published a series of 12 patients with a literature review of cardiac manifestations. Striking findings include an obliterative coronary artery vasculopathy resembling that of cardiac allograft rejection (Plate 27.55) and myocarditis, associated with bradycardia and, in some instances, sudden death [64]. A nephrotic syndrome is associated with membranous nephropathy and minimal change podocytopathy [65]. Kamble et al. reported on two cases of vasculitic neuropathy with a review of the literature of central nervous system lesions associated with chronic GVHD [66]. The etiologies of these changes have not been resolved. After thorough effort to eliminate an infectious component, the etiology could include some combination of late toxicity, alloreactivity, and immune dysregulation (autoimmunity).
Fig. 27.11 Lung, cytomegalovirus (CMV) pneumonia, day 68. Several large cells contain Cowdry type A nuclear inclusions (large arrows) as well as six to 12 smaller cytoplasmic inclusions (small arrows) diagnostic for CMV infection. (Hematoxylin and eosin.)
Infection Infection is a frequent complication of HCT and must be evaluated promptly and extensively in tissue and cytology preparations. The pathologist must be prepared to identify unexpected microorganisms, not just those suggested on the basis of nonspecific clinical findings. For example, although a nodular pulmonary infiltrate may indeed be the result of fungal infection, other treatable causes include nocardiosis, legionellosis, toxoplasmosis (Plate 27.56), and CMV, as well as other viral pneumonias. It is obvious, but bears repeating, that identifying microorganisms requires looking specifically for them. This must be done quickly in immunocompromised patients if effective therapeutic drug levels are to be established. Since pneumonia can progress with devastating speed in HCT patients, we immediately process, stain, and study lung tissue and lavaged cells at any hour of any day. The preliminary stains include Wright–Giemsa, Papanicolau, H&E, methenamine silver, PAS, modified Gimenez for Legionella, and Kinyoun for mycobacteria. Fite and Warthin-Starry stains as well as a large number of IHC antibodies and in situ hybridization probes for organisms are used as needed. The pathologist does a disservice to the patient, as well as to clinicians, by studying only H&E and silver-stained sections and reporting that there is no evidence of infection. Allograft recipients who received high-dose conditioning and have GVHD are at significant risk for reactivation of latent viruses, especially members of the herpesvirus group and adenovirus [67–69]. We now rarely see CMV or herpes simplex in bronchoalveolar lavages or tissue biopsies because seropositive patients receive prophylactic antiviral therapy, and patients who develop antigenemia or PCR positivity, are treated pre-emptively. However, treatment does not eradicate these viruses, so late infection may develop unnoticed after treatment is stopped. The incidence of late CMV pneumonia (Fig. 27.11) occurring at a median of approximately 24 weeks post transplant is increasing [70]. HCT recipients are at high risk for acquiring potentially fatal respiratory virus infections when the community prevalence of agents such as respiratory syncytial virus (Plate 27.57) is high. Occasionally, viral infection of a single organ will produce an ambiguous clinical presentation best resolved by prompt biopsy and rapid tissue evaluation. For example, severe abdominal pain may suggest impending bowel infarction from gut GVHD but may actually represent acute herpes simplex virus, varicella zoster virus (Plate 27.58) or adenovirus hepatitis (Plate 27.59), which can be diagnosed within a few hours
Fig. 27.12 Kidney, adenovirus (ADV) nephritis, day 77. Tubules lined by degenerating epithelial cells are strongly reactive for ADV antigen by indirect immunoperoxidase staining using a monoclonal antibody to the adenovirus hexon protein (Chemicon International, Inc., Temecula, CA, USA). ADV species 11 was isolated in culture. Possible ADV nephritis should be considered in patients with hematuria and positive urine culture [71].
by histologic studies of a transjugular liver biopsy. Similarly, hematuria accompanied by costovertebral angle tenderness may be secondary to adenovirus nephritis (Fig. 27.12), which can be diagnosed by renal biopsy [71]. Invasive mold infection develops in approximately 15% of patients undergoing either high-dose or reduced-intensity regimens. The most frequent cause is Aspergillus, followed by zygomycete and Fusarium species (Plate 27.60) [72]. The histologic demonstration of fungi in tissue is often crucial to the diagnosis and prompt treatment of invasive infection since culture isolation is slow and insensitive, fungemia is sporadic, and assay results for antigenemia are often inconclusive. Toxoplasma gondii (Plate 27.56), Pneumocystis jirovecii, mycobacteria, Nocardia, and Legionella are also organisms that can be identified
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rapidly by histologic and immunocytochemical staining and then specifically treated [73–75]. Although these organisms are unusual in most patient populations, their rapid identification can save lives. The incidence of toxoplasmosis over a 20-year period in Seattle was 0.3% [74], of nocardiosis over 25 years was 0.2% [75], and of legionellosis over 12 years was 0.6% [73].
Pulmonary complications Pulmonary complications continue to develop in HCT patients and are most frequent in those receiving allogeneic hematopoietic cells. In addition to the major role of infection already described, other mechanisms of injury may include drug toxicity, irradiation damage, GVHD, and malignant infiltration. The resulting histologic alterations are often nonspecific and may include diffuse alveolar damage, alveolar hemorrhage [76], interstitial and alveolar inflammation, alveolar proteinosis, and pulmonary venous occlusion (Plate 27.61) [77,78]. Several of these may coexist in the same patient and interact to cause significant tissue damage and symptoms. Diagnostic evaluation by the pathologist is closely coordinated with the clinical care team. Consideration is given to the underlying major disease and its treatment, possible exposure to infection or toxic damage, prior pulmonary complications, pretransplant conditioning regimen, post-transplant interval, degree of immunosuppression, whether GVHD is present, results of surveillance cultures and molecular tests for systemic infection, evolution of signs and symptoms, and results of imaging studies as well as pulmonary function tests. Typically, the first involvement for the pathologist is the immediate analysis of bronchoalveolar lavage cytospin preparations for possible infection (see the section on infection) or malignancy. Either negative or positive findings for these two complications will often allow clinicians to focus and thereby simplify therapy. Because the diagnostic yield for bronchoalveolar lavage is low, tissue is often needed to analyze histologic patterns and again carry out detailed morphologic, microbiologic, and molecular studies for infection. Transbronchial biopsies can usually be obtained safely and may fortuitously demonstrate infection, but they provide too little tissue to confidently interpret and categorize the histologic changes. Ideally, the patient is sufficiently stable to undergo video-assisted thoracoscopic surgery. This provides tissue which the pathologist can immediately dissect under sterile conditions so that focal lesions can be cultured for bacteria, fungi and viruses, imprints and postoperative frozen sections can be studied using special stains (see the section on infection), and the most informative areas can be submitted for fixation and permanent sections. Historically, pulmonary complications have developed in one-quarter to one-half of allograft recipients and have frequently been fatal. Improvements in antimicrobial prophylaxis and therapy have dramatically decreased the incidence of viral pneumonias, although these can still develop through reactivation of latent infection as well as through community exposure. A recently described paramyxovirus, human metapneumovirus, infects a significant proportion of transplant patients and can cause fatal pneumonia [79]. The exact nature and extent of its role in pneumonias previously classified as idiopathic are under study. Cytopathic tissue alterations diagnostic for human metapneumovirus have not been described, so antibodies for IHC need to be developed.
Interstitial pneumonia syndrome Approximately 5–15% of allogeneic HCT recipients develop the interstitial pneumonia syndrome (IPS) in which there are multilobar infiltrates by X-ray or scan, symptoms and signs of pneumonia with evidence
of abnormal physiology, and absence of active lower respiratory tract infection [80,81]. IPS is a generally severe and often fatal acute process, in contrast to the insidious chronic course of disorders such as usual interstitial pneumonia of Liebow. The histopathology of IPS is variable in pattern as well as severity and may include diffuse alveolar damage with hyaline membranes, interstitial lymphocytic infiltration, increased alveolar macrophages, and pulmonary hemorrhage. These cases probably have a multifactorial etiology, which may include cytokine-induced damage, toxicity from therapy, cell-mediated injury, and occult infection [82,83]. The decreased incidence of IPS after reduced-intensity regimens strongly suggests that conditioning therapy can play a crucial role [84]. Alkylating agents such as cyclophosphamide and busulfan have long been implicated in pulmonary toxicity. There is now increasing evidence that high-dose melphalan [85], as well as tyrosine kinase inhibitors such as dasatinib [86], may also cause lung damage. Acute GVHD is a risk factor for IPS [80,87]. However, IPS has not been firmly linked to an allogeneic reaction in humans, although there is evidence in mice that nonbacterial pneumonia is a combined effect of GVHD and radiation [88].
Pulmonary GVHD The lung is definitely a GVHD target organ. Bronchiolar epithelium is evidently the major target tissue, although cellular damage is often difficult to identify in the epithelial monolayer. There is no well-defined histologic pattern for acute pulmonary GVHD. Alloreactivity may contribute to the nonspecific features of damage and inflammation which characterize the IPS. The significant association of idiopathic bronchiolitis obliterans organizing pneumonia (also designated cryptogenic organizing pneumonia) with acute skin GVHD as well as chronic GVHD of the gut and oral cavity in a large, case-control study [89] indicates that this bronchiolocentric injury pattern with polypoid masses of granulation tissue obstructing the small airways (Fig. 27.13) could represent a transition between acute and chronic phases. In the 49 patients with bronchiolitis obliterans organizing pneumonia, day 108 was the median day of occurrence. It is well established that chronic pulmonary GVH activity is a major contributor to bronchiolar inflammation culminating in bronchiolitis obliterans. This relationship is supported by published reports of many patients with bronchiolitis obliterans (Fig. 27.14) and chronic GVHD [90], the strong association of airflow obstruction with chronic GVHD, and the symptomatic response to immunosuppressive therapy [82,83]. Because the clinical syndrome of obstructive pulmonary function in a GVHD patient with clear chest imaging studies is distinctive, a strong presumptive diagnosis of pulmonary involvement is usually established without obtaining a biopsy. However, lack of biopsy tissue has limited our understanding of this complication. In some of the few available biopsies, there has been evidence of differing stages of bronchiolar inflammation and fibrosis. In some bronchioles, lymphocytes infiltrate the mucosa and are associated with epithelial injury (Plate 27.62). Other airways show a presumably later stage of lymphocytic bronchiolitis in which there is constrictive narrowing of the lumen (Fig. 27.14) along with a few examples of complete fibrous effacement of the lumen (Plate 27.63). Stains for elastin such as the Verhoeff–Van Gieson are helpful in demonstrating the scarred bronchioles. An intriguing study of the expression of 15 genes in the innate immunity pathway suggests that a loss-of-function mutation in the bactericidal/permeability-increasing gene significantly increases the risk of developing airflow obstruction in human HCT patients [91]. Bronchiolitis obliterans is strongly associated with lung allograft rejection in both humans and animals [92,93], indicating that not only the morphology, but also the mechanisms of the two
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Fig. 27.14 Bronchiolitis obliterans with constrictive subepithelial fibrosis at 5 years post allograft transplantation. The patient had developed obstructive pulmonary dysfunction. This biopsy confirmed that chronic pulmonary graftversus-host disease had developed following a history of skin and gastrointestinal involvement. The arrows indicate smooth muscle bundles. There is also subintimal thickening of the accompanying artery.
Summary
Fig. 27.13 Lung, idiopathic bronchiolitis obliterans organizing pneumonia (BOOP; cryptogenic organizing pneumonia, COP), day 120 following marrow allograft transplantation. The patient developed dyspnea and a cough following tapering of corticosteroid therapy for chronic graft-versus-host disease (GVHD). A chest X-ray showed multifocal alveolar opacities clinically suggestive of fungal or viral pneumonia. The biopsy demonstrated areas of consolidation in which bronchioles were blocked by granulation tissue (arrow) which also extended into nearby alveoli and was associated with interstitial and alveolar infiltration by mononuclear inflammatory cells. In HCT recipients, BOOP is associated with GVHD in the skin, oral mucosa, and gut [88].
processes may be similar. There is vascular inflammatory involvement in lung rejection which may also be present in GVHD. Secondary infection may play a major role in chronic GVHD because patients have important deficiencies of immunoglobulins [94].
Among the future challenges in the pathobiology of GVHD is understanding why and how stem cells are targeted. The immunohistologyguided laser capture of the CK15 basal stem cells, coupled with real-time reverse transcription PCR, has demonstrated the heterogeneity of the basal cells regarding their pro- or antiapoptotic gene expression [18]. Future studies can be directed at events that influence this expression. Immunofluorescence studies on formalin-fixed tissues allow identification of the lymphoid subclasses in tissues and their composition in GVHD lesions. The role of the endothelium as a possible target of GVHD [95,96] and the microvascular changes associated with cutaneous sclerosis have not been well studied in clinical material. Multiparameter flow cytometry coupled with sorting for specific subpopulations of lymphocytes and dendritic cells aids in unraveling the complex immunobiology of GVHD. The influence of genes that are involved in the innate immunity pathway on gut and pulmonary GVHD and infection may provide intriguing clues into susceptibility to these complications [91]. Lastly, improved utilization of biopsies in the management of GVHD should be aided by the standardization of sampling and histologic reporting systems coupled with linkage to a database which includes the type and duration of treatment given before the biopsy.
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3. Wood BL, Borowitz MJ. The flow cytometric evaluation of hematopoietic neoplasia. In: McPherson RA, Pincus MR, editors. Henry’s Clinical Diagnosis and Management by Laboratory Methods. Philadelphia: WB Saunders; 2006. pp. 599–614. 4. Chang C, Storer BE, Scott BL et al. Hematopoietic cell transplantation in patients with myelodysplastic syndrome or acute myeloid leukemia arising from myelodysplastic syndrome: similar outcomes in patients with de novo disease and disease fol-
lowing prior therapy or antecedent hematologic disorders. Blood 2007; 110: 1379–87. 5. Tefferi A. JAK2 mutations in polycythemia vera – molecular mechanisms and clinical applications. N Engl J Med 2007; 356: 444–5. 6. Soll E, Massumoto C, Clift RA et al. Relevance of marrow fibrosis in bone marrow transplantation: a retrospective analysis of engraftment. Blood 1995; 86: 4667–73. 7. Sale GE, Deeg HJ, Porter BA. Regression of myelofibrosis and osteosclerosis following
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hematopoietic cell transplantation assessed by magnetic resonance imaging and histologic grading. Biol Blood Marrow Transplant 2006; 12: 1285–94. Shulman HM, McDonald GB. Hepatic complications of hematopoietic cell transplantation. In: Gershwin ME, Vierling JM, Manns MP, editors. Liver Immunology: Principles and Practice, 2nd edn. Totowa, NJ: Humana Press; 2007. pp. 409–21. Al Sobhi E, Zahrani Z, Zevallos E, Zuraiki A. Imatinib-induced immune hepatitis: case report and literature review. Hematology 2007; 12: 49–53. Hogan WJ, Maris M, Storer B et al. Hepatic injury after nonmyeloablative conditioning followed by allogeneic hematopoietic cell transplantation: a study of 193 patients. Blood 2004; 103: 78–84. Zutter MM, Martin PJ, Sale GE et al. Epstein–Barr virus lymphoproliferation after bone marrow transplantation. Blood 1988; 72: 520–9. Limaye AP, Huang ML, Atienza EE, Ferrenberg JM, Corey L. Detection of Epstein–Barr virus DNA in sera from transplant recipients with lymphoproliferative disorders. J Clin Microbiol 1999; 37: 1113–16. Sasadeusz J, Kelly H, Szer J, Schwarer AP, Mitchell H, Grigg A. Abnormal cervical cytology in bone marrow transplant recipients. Bone Marrow Transplant 2001; 28: 393–7. DeLeve LD, Shulman HM, McDonald GB. Toxic injury to hepatic sinusoids: sinusoidal obstruction syndrome (veno-occlusive disease). Semin Liver Dis 2002; 22: 27–42. Terminology of nodular hepatocellular lesions. International Working Party. Hepatology 1995; 22: 983–93. Ferrara JLM, Cooke KR, Deeg HJ, editors. Graftvs-Host Disease, 3rd edn. New York: Marcel Dekker; 2005. Heymer B, Bunjes D, Friedrich W. Clinical and Diagnostic Pathology of Graft-Versus-Host Disease. Berlin: Springer-Verlag; 2002. Zhan Q, Signoretti S, Whitaker-Menezes D, Friedman TM, Korngold R, Murphy GF. Cytokeratin15positive basal epithelial cells targeted in graft-versus-host disease express a constitutive antiapoptotic phenotype. J Invest Dermatol 2007; 127: 106–15. Daneshpouy M, Socie G, Lemann M, Rivet J, Gluckman E, Janin A. Activated eosinophils in upper gastrointestinal tract of patients with graftversus-host disease. Blood 2002; 99: 3033–40. Socie G, Mary JY, Lemann M et al. Prognostic value of apoptotic cells and infiltrating neutrophils in graft-versus-host disease of the gastrointestinal tract in humans: TNF and Fas expression. Blood 2004; 103: 50–7. Rieger K, Loddenkemper C, Maul J et al. Mucosal FOXP3+ regulatory T cells are numerically deficient in acute and chronic GvHD. Blood 2006; 107: 1717–23. Murata H, Janin A, Leboeuf C et al. Donor-derived cells and human graft-versus-host disease of the skin. Blood 2007; 109: 2663–5. Tzankov A, Stifter G, Tschorner I, Gastl G, Mikuz G. Detection of apoptoses in gastro-intestinal graftversus-host disease and cytomegalovirus colitis by a commercially available antibody against caspase3. Pathol Res Pract 2003; 199: 337–40. Schaffer JV. The changing face of graft-versushost disease. Semin Cutan Med Surg 2006; 25: 190–200.
25. Shulman HM, Kleiner D, Lee SJ et al. Histopathologic diagnosis of chronic graft-versus-host disease: National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease. II. Pathology Working Group Report. Biol Blood Marrow Transplant 2006; 12: 31–47. 26. Filipovich AH, Weisdorf D, Pavletic S et al. National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease. I. Diagnosis and staging working group report. Biol Blood Marrow Transplant 2005; 11: 945–56. 27. Deeg HJ. How I treat refractory acute GVHD. Blood 2007; 109: 4119–26. 28. Saliba RM, de Lima M, Giralt S et al. Hyperacute GVHD: risk factors, outcomes, and clinical implications. Blood 2007; 109: 2751–8. 29. Lerner KG, Kao GF, Storb R, Buckner CD, Clift RA, Thomas ED. Histopathology of graft-vs.-host reaction (GvHR) in human recipients of marrow from HL-A-matched sibling donors. Transplant Proc 1974; 6: 367–71. 30. Kohler S, Hendrickson MR, Chao NJ, Smoller BR. Value of skin biopsies in assessing prognosis and progression of acute graft-versus-host disease. Am J Surg Pathol 1997; 21: 988–96. 31. Kuykendall TD, Smoller BR. Lack of specificity in skin biopsy specimens to assess for acute graftversus-host disease in initial 3 weeks after bonemarrow transplantation. J Am Acad Dermatol 2003; 49: 1081–5. 32. Sale GE, Lerner KG, Barker EA, Shulman HM, Thomas ED. The skin biopsy in the diagnosis of acute graft-versus-host disease in man. Am J Pathol 1977; 89: 621–35. 33. Horn TD, Redd JV, Karp JE, Beschorner WE, Burke PJ, Hood AF. Cutaneous eruptions of lymphocyte recovery. Arch Dermatol 1989; 125: 1512–17. 34. Oyama Y, Cohen B, Traynor A, Brush M, Rodriguez J, Burt RK. Engraftment syndrome: a common cause for rash and fever following autologous hematopoietic stem cell transplantation for multiple sclerosis. Bone Marrow Transplant 2002; 29: 81–5. 35. Baack BR, Burgdorf WH. Chemotherapy-induced acral erythema. J Am Acad Dermatol 1991; 24: 457–61. 36. Marra DE, McKee PH, Nghiem P. Tissue eosinophils and the perils of using skin biopsy specimens to distinguish between drug hypersensitivity and cutaneous graft-versus-host disease. J Am Acad Dermatol 2004; 51: 543–6. 37. Firoz BF, Lee SJ, Nghiem P, Qureshi AA. Role of skin biopsy to confirm suspected acute graft-vshost disease: results of decision analysis. Arch Dermatol 2006; 142: 175–82. 38. Cox GJ, Matsui SM, Lo RS et al. Etiology and outcome of diarrhea after marrow transplantation: a prospective study. Gastroenterology 1994; 107: 1398–407. 39. Thompson B, Salzman D, Steinhauer J, Lazenby AJ, Wilcox CM. Prospective endoscopic evaluation for gastrointestinal graft-versus-host disease: determination of the best diagnostic approach. Bone Marrow Transplant 2006; 38: 371–6. 40. Nydegger A, Catto-Smith AG, Tiedemann K, Hardikar W. Diagnosis of gastrointestinal graft-versushost disease – is rectal biopsy enough? Pediatr Blood Cancer 2007; 48: 561–6.
41. Washington K, Bentley RC, Green A, Olson J, Treem WR, Krigman HR. Gastric graft-versus-host disease: a blinded histologic study. Am J Surg Pathol 1997; 21: 1037–46. 42. Ponec RJ, Hackman RC, McDonald GB. Endoscopic and histologic diagnosis of intestinal graftversus-host disease after marrow transplantation. Gastrointest Endosc 1999; 49: 612–21. 43. Xin W, Greenson JK. The clinical significance of focally enhanced gastritis. Am J Surg Pathol 2004; 28: 1347–51. 44. Welch DC, Wirth PS, Goldenring JR, Ness E, Jagasia M, Washington K. Gastric graft-versushost disease revisited: does proton pump inhibitor therapy affect endoscopic gastric biopsy interpretation? Am J Surg Pathol 2006; 30: 444–9. 45. Hackman RC, Wolford JL, Gleaves CA et al. Recognition and rapid diagnosis of upper gastrointestinal cytomegalovirus infection in marrow transplant recipients. A comparison of seven virologic methods. Transplantation 1994; 57: 231– 7. 46. Papadimitriou JC, Cangro CB, Lustberg A et al. Histologic features of mycophenolate mofetilrelated colitis: a graft-versus-host disease-like pattern. Int J Surg Pathol 2003; 11: 295–302. 47. Ho VT, Cutler C, Carter S et al. Blood and marrow transplant clinical trials network toxicity committee consensus summary: thrombotic microangiopathy after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2005; 11: 571–5. 48. Nishida T, Hamaguchi M, Hirabayashi N et al. Intestinal thrombotic microangiopathy after allogeneic bone marrow transplantation: a clinical imitator of acute enteric graft-versus-host disease. Bone Marrow Transplant 2004; 33: 1143–50. 49. Holmberg L, Kikuchi K, Gooley TA et al. Gastrointestinal graft-versus-host disease in recipients of autologous hematopoietic stem cells: incidence, risk factors, and outcome. Biol Blood Marrow Transplant 2006; 12: 226–34. 50. Saunders MD, Shulman HM, Murakami CS, Chauncey TR, Bensinger WI, McDonald GB. Bile duct apoptosis and cholestasis resembling acute graft-versus-host disease after autologous hematopoietic cell transplantation. Am J Surg Pathol 2000; 24: 1004–8. 51. Lefkowitch JH, Lobritto SJ, Brown RS, Jr. et al. Ground-glass, polyglucosan-like hepatocellular inclusions: a “new” diagnostic entity. Gastroenterology 2006; 131: 713–18. 52. Wisell J, Boitnott J, Haas M et al. Glycogen pseudoground glass change in hepatocytes. Am J Surg Pathol 2006; 30: 1085–90. 53. Chand N, Sanyal AJ. Sepsis-induced cholestasis. Hepatology 2007; 45: 230–41. 54. Kamble RT, Selby GB, Mims M, Kharfan-Dabaja MA, Ozer H, George JN. Iron overload manifesting as apparent exacerbation of hepatic graft-versushost disease after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2006; 12: 506–10. 55. Strasser SI, Shulman HM, Flowers ME et al. Chronic graft-versus-host disease of the liver: presentation as an acute hepatitis. Hepatology 2000; 32: 1265–71. 56. Bridge AT, Nelson RP, Jr., Schwartz JE, Mirowski GW, Billings SD. Histological evaluation of acute mucocutaneous graft-versus-host disease in nonmyeloablative hematologic stem cell transplants with an observation predicting an increased risk of
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Joyce C. Niland & Paul Frankel
Biostatistical Methods in Hematopoietic Cell Transplantation
Introduction With the tremendous strides and newly emerging science within the field of hematopoietic cell transplantation (HCT), advances in study design, biostatistical methodology, and biomedical informatics are required to keep pace. Clinical trial design and analysis methodology have been evolving over the past decade [1], providing the additional tools needed to obtain valid results and advances in evidence-based medicine based on HCT studies. This chapter provides an overview of the study cycle for new treatment discoveries, from observational studies through the phases of clinical trials as applied to HCT. The principles for designing such studies and the fundamentals regarding study conduct and monitoring are described. Subtleties and potential pitfalls of various study designs and analytic approaches are described, along with the requirements for full and complete reporting of study results. Special issues that must be considered during the conduct of human clinical trials are discussed, including appropriate monitoring through independent data and safety boards, along with international standards for the conduct and reporting of human research. With the post-genome era of biologic research underway, the clinical trial database, design, conduct, and analytic tools required to manage the huge amounts of genomic data must be considered. In addition, new methods report on dozens of immunologic parameters that are particularly relevant for HCT. Our greatest challenge will be understanding the meaning of these data, and applying this new understanding to develop targeted interventions and approaches to treatment. Because of the farreaching impact of genomics and related large-scale data on clinical research, for each section of this chapter the potential influence of these data on the study cycle is discussed. The need for evolving advanced biomedical informatics tools to support study design, data management, and analysis of these data is addressed as well. Throughout the chapter, formulas and calculations are avoided in favor of emphasizing the underlying concepts and theories of biostatistical methodology. It is hoped that this approach will provide physicians, fellows, medical students, and health-care professionals involved in HCT research with a basic understanding of, and guide to, appropriate approaches in study design, conduct, statistical analysis, and data computerization. Frequent references to related biostatistical/clinical trials articles and textbooks are included to promote better understanding. For
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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those seeking a more detailed treatment of the history, rationale, and methodology underlying clinical trials, the texts by Meinert and Tonascia [2], Pocock [3], and Friedman et al. [4] provide these underpinnings. For a comprehensive overview of clinical trial informatics applied to the field of oncology, the reader is referred to the text by Silva et al. [5].
The study cycle Figure 28.1 provides an overview of the typical study cycle for new treatment discoveries. While rational drug design through targeted efforts is becoming more common, it is still the case that often initial insights into the potential value of certain therapeutic approaches are gleaned through observational studies. Preclinical (animal and laboratory) studies also play a pivotal role in research hypotheses and in preparation for clinical trials. Often, both observational and preclinical investigations are conducted prior to launching experimentation via clinical trials, that is, planned interventional studies intended to ethically lead to an evaluation of the experimental therapy in people. The final phase of the study cycle is to observe the use of “successful” treatments stemming from the clinical trial results, now offered to the entire patient population, where extended follow-up of a larger patient population may demonstrate unintended and unforeseen consequences of therapy. The application of evidence-based medical approaches to the general population may lead to observational studies that cycle back into the next phase of research to advance medical discoveries. Often, disparate or diminished results will be observed when new treatments are first applied as the standard of care, as the careful selection process applied during clinical trial conduct is no longer in effect, and the patients being treated post clinical trials represent the gamut of stages, phases, and demographics. This field of study, known as outcomes research, will not be covered in this chapter, as this topic is discussed in detail in Chapter 32. The following sections focus on two of the key designs for observational studies, a frequent starting point of the study cycle.
Observational study designs Table 28.1, adapted from Green and Byar [6], suggests that there is a hierarchy of study designs, from the least convincing (anecdotal case reports) to the most convincing (the confirmed randomized control clinical trial). The key differential between observational studies and clinical trials is that the former do not involve any type of intervention with the subjects under study, while clinical trials by definition are experimental in nature and involve an investigator-controlled interven-
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Biological hypothesis Hematopoiesis RBC
Observational studies
BFU-E
CFU-E
Monocytes Macrophage
CFU-M
Sel f Renewal
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Outcomes research
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Fig. 28.1 The study cycle of new treatment discoveries.
Table 28.1 Hierarchy of strength of evidence concerning efficacy of treatment 1. 2. 3. 4. 5. 6. 7. 8.
Confirmed randomized controlled clinical trial Single randomized controlled clinical trial Case series with historical controls Retrospective case-control study Analyses of existing databases Series with literature controls Case series without controls Anecdotal case reports
Adapted with permission from Green and Byar [6].
tion. Much can be learned from conducting well-designed observational studies, and they may serve as a valuable precursor to the next novel treatment approach to be tested in clinical trials, particularly at the point when a randomized trial in HCT is not ethical or practical. While true experiments (i.e. clinical trials) allow an investigator to establish causal associations more conclusively, observational studies can provide major contributions to the understanding of disease processes. In general, observational studies examine the association between the outcome of interest (the “dependent” variable) and the patient’s background, treatment or pretreatment exposures, genetics, laboratory value or health-related behavior (the antecedent or “independent” variable). One difficulty with observational studies is that patients usually differ naturally in other characteristics beyond the specific factors under study. For example, people in various occupations differ not only with respect to their exposures to occupational hazards, but also with respect to their prior and current lifestyles. Such differences may come about by one or more mechanism such as self-selection, education level, fitness for a particular occupation or hiring practices. These potential influences on the outcome under study are known as “confounding factors.” While a robust study design will anticipate and measure confounding factors, many such factors are difficult, costly or impossible to measure fully. In this instance, the role of the primary factor under investigation is more difficult to demonstrate, and the conclusions will require caveats. In addition to such confounders, observational studies that examine
Clinical trials
survival endpoints can be misleading if follow-up is not standardized. For example, if standard follow-up is planned for 5 years, and passive follow-up occurs subsequently, the survival estimates will be unreliable, as passive follow-up gathers more information on the more at-risk patients. If HCT patients are compared with nontransplant patients, and follow-up is not uniform, this will also impact the validity of any conclusions. Researchers should recognize that the statistical analysis of observational studies can often be more difficult than prospective clinical trials. When observational studies are designed and analyzed carefully, one may be able to control some of the biases that are inherent in patient selection or outcome assessment. Two major types of design approach for observational studies, prospective and retrospective, can be applied in the area of HCT research. Each approach has attendant features, analytic methods, and advantages or disadvantages, as described in the subsequent sections. Prospective cohort studies In a prospective cohort design, groups or “cohorts” of subjects are selected based on an observed common exposure to some naturally occurring factor. For example, patients who have a matched sibling donor and receive an allogeneic HCT may be compared with patients with a similar disease status for whom no donor is available. Even through such a natural group assignment process there could be inherent selection bias between the two groups. As an example, donor availability is likely to correlate with family size, which in turn may correlate with the educational and economic levels of the study subjects. The decision to take a patient to transplant is not dictated solely by the availability of the donor; for example, a physician could withhold HCT because of the worsened clinical condition of a patient. Matching on as many potentially confounding factors as possible will help to reduce some of the bias. (However, care must then be taken to take the matching into account when analyzing the data via the appropriate statistical techniques.) There is also potential bias related to the length of time that a patient waits for an allogeneic HCT procedure. Transplant recipients may be at lower risk than all patients intended to go to HCT, as the recipients have not only responded to induction chemotherapy, but also survived relapse
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free for long enough to identify an appropriate donor. This “waiting time” to transplant causes considerable bias if comparing with nontransplant patients as the transplant group excludes those who would have undergone HCT had they survived long enough. To properly analyze data of this nature, patients should be followed from diagnosis, and in some settings it may prove useful to employ time-varying covariates in the model, via techniques such as the one described by Mantel and Byar [7]. An advantage of the prospective cohort design is that it permits the calculation of incidence rates among those “exposed” and those “not exposed” to the antecedent factor of interest, providing a direct estimate of the true relative risk (RR). With this study design, one also can calculate the difference in incidence rates between groups, known as the attributable risk. A primary disadvantage of prospective cohort studies is that they usually are quite expensive and require a long follow-up period to observe a sufficient number of outcome events. Just as with a controlled clinical trial, hypotheses for the observed difference of clinical importance between the groups, the alpha (type I) error that is allowable, and the desired power must be clearly established at the outset of the study, so that the requisite sample size can be calculated. Retrospective case-control studies When the outcome of interest is relatively rare, a retrospective casecontrol study may be the best design for evaluating potential factors associated with an outcome of interest [8]. In a case-control study, the cases are defined as subjects with a particular type and stage of disease. Controls who are free from the disease under study are chosen, matched to the cases based on certain known confounding factors such as age and gender. Care must be taken not to overmatch cases and controls on more than a few key factors, due to the increased difficulty of identifying appropriate matched controls, the increased complexity of the analyses, and the resultant inability to study any of the matching factors with respect to association with the outcome. A drawback of the retrospective case-control design is that incidence rates cannot be directly estimated, as no appropriate denominators are available (populations at risk). Instead, an odds ratio is calculated to estimate the RR between cases and controls, quantifying the odds of having the disease when the factor of interest is present versus when it is absent. The odds ratio is an accurate estimate of the true RR if, and only if, three assumptions hold true: (1) the controls are representative of the general population; (2) the assembled cases are representative of all cases; and (3) the frequency of the disease in the population is rare. Like the prospective cohort study design, case-control studies are subject to uncontrolled variation and potential biases. If a case-control study yields a modest difference in outcome between treatments, this must be interpreted cautiously, as it is difficult to conclude whether a true treatment effect actually exists, or whether the results are confounded by other factors. Case-control studies can be strengthened through several means, including larger sample sizes, adjustment or matching for potential confounding factors, careful testing of model assumptions, and adjustment for multiple comparisons. Systematic reviews and meta-analysis Reviews and meta-analyses of prior published studies will be considered here in the context of observational studies. While both systematic reviews and formal meta-analysis can be useful [9], we will focus on the more rigorous application of meta-analysis. The use of metaanalysis is most commonly employed when a large collection of studies can be said to have, in some manner, addressed the same question, and yet no single study has definitively answered the question, perhaps due
to small sample sizes. A typical analysis will include all relevant studies found in MEDLINE in a predefined time period with predefined study eligibility criteria, and will also search the Cochrane Database of randomized controlled studies. An exhaustive search of the literature is required to avoid selection bias, requiring reviews of studies referenced in the primary publications, but that is not sufficient. Additional effort must be expended to seek out negative trials that went unpublished, due to publication bias favoring positive results. While it may not be possible to eliminate all sources of bias, the recent trend requiring studies to be prospectively registered (http://www.clinicaltrials.gov) to qualify for publication in top journals is improving the landscape for meta-analysis. Recent examples of meta-analysis [10] concluded a positive effect for prophylactic antifungal therapy in patients undergoing allogeneic HCT and high-risk acute leukemia patients. Another recent meta-analysis outside of the transplant setting attracted widespread attention for reporting that high-dose vitamin E increases all-cause mortality [11]. Both studies have merits and limitations. The first paper included a conclusion based on combining allogeneic transplant studies with nontransplant studies, which is problematic in that prophylactic use in allogeneic transplants was already established as the standard of care. The vitamin E paper was criticized for a similar problem, that is, combining various forms of α-tocopherol, but it was also criticized for referring to αtocopherol as being synonymous with vitamin E, when it is simply one member of the vitamin E family. Meta-analysis requires various assumptions, resulting in potentially faulty conclusions when these assumptions do not hold. Nevertheless, both papers have significant merit that could impact clinical care. First, the antifungal paper provides a summary of the result of the one study on prophylactic antifungals in autologous HCT, and presents a valid subset analysis that shows that fungal-related mortality and the incidence of invasive fungal infections were reduced in nontransplant leukemia patients, with a marginally significant impact on all-cause mortality. Also, the second meta-analysis paper makes a good case for avoiding high-dose DL-α-tocopherol acetate, although this was not the primary conclusion of the paper as stated by the authors. Quality of life and cost-effectiveness studies Two burgeoning areas of observational study over the past several years include the study of quality of life (QOL) and cost-effectiveness. Many clinical trials now incorporate these endpoints into the protocol to corroborate the success of new interventions [12], particularly as the margin for improvement decreases as more successful treatment options become routinely available. A literature review has shown that citations for QOL studies grew from fewer than 30 in 1977 to more than 1000 some 20 years later [13]. Analysis of QOL associated with clinical trials may involve multiple scales assessed repeatedly over time [14], raising the challenge of appropriate statistical handling of multiple comparisons. To reduce the number of hypotheses and facilitate the interpretation of trial results for the primary question, “Does the overall QOL differ between treatment arms?,” summary measures collapsing over the multiple QOL items and scales often are used. With multiple QOL assessments over time, missing data can strongly influence the choice of methodology and analytic strategy. Methods have been developed to incorporate QOL into survival analysis, such as the Q-TWiST (Quality-Adjusted Time Without Symptoms of Disease or Toxicity of Treatment) method for quality-adjusted survival analysis using the Cox proportional hazards model [15]. The QTWiST model assumes that patients progress through a series of health states that differ in QOL. The Kaplan–Meier product limit estimate is
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used to calculate the mean duration of each state, and quality-adjusted survival is estimated given a set of covariate values. (Kaplan–Meier/Cox models are described in more detail later in this chapter.) The results can be useful for investigating how prognostic factors might affect treatment benefits in terms of QOL. Further discussion of QOL and costeffectiveness studies is provided in Chapter 32. Impact of genetic data on observational studies Through the mapping and sequencing of the human genome, it has become possible to explore how genotypic differences lead to phenotypic variation, and to begin to understand the genetically influenced mechanisms underlying human disease [16]. Initial exploratory studies involve “data mining” of existing genetic databanks to look for correlations and begin to generate hypotheses. This initial data mining should be “unsupervised” in nature, employing methods that allow nonmathematical biologists to browse and manipulate the vast data already available in genetic databases such as GenBank and the Online Mendelian Inheritance in Man (OMIM) database. Investigators are beginning to use automated sequencing and analysis of DNA as an inexpensive screening of multiple loci for polymorphisms. Sharing of data across all research groups worldwide will be required to expedite discoveries of critical genetic patterns. As we move into the next phase of attempting to relate genetic information to clinical phenotypic data, DNA databanks oriented toward future hypotheses not framed at the outset will need to be created. Such databanks will consist of repositories of identified specimens, and will be able to serve many different “parent” clinical studies in future. To examine genotype–phenotype correlations, it will be critical that the genetic data are linked to repositories of detailed biologic, medical history, and treatment data on the same individuals. The standard endpoints used in clinical trials (e.g. efficacy and safety) will give way to less-clear outcomes such as “classification” and “correlation.” Epidemiologic trials will be increasingly important to tease out environmental versus genetic contributions to disease, and familial history data will become critical. This collection and exploration of complex phenotypic, intervention, and environmental data may prove to be a more daunting information management feat than the amassing of the genetic information, comprised of data elements with a very simple structure by comparison. Guidelines for the use of genomics in clinical research (and in clinical practice) will be required in the future, using an interdisciplinary approach involving clinical investigators, geneticists, biostatisticians, and bioinformaticists. All the newly available sources of information highlight that one key to innovation from observational studies is to observe without necessarily having a clear understanding of what might turn out to be important years later. The importance of observing unintended consequences and unforeseen predictors of outcome is often underestimated, but fortunately, with modern computerized laboratories, increased computerized data standards, growing efforts and advances in data mining, and the rigorous follow-up demanded in the HCT patient population, observational studies and data exploration of clinical data repositories are improving, and are often the leading motivation for future clinical trials for diseases where HCT is the current treatment of choice.
Clinical trial design in HCT research A clinical trial involves a true experimental design in which the investigator controls the intervention. The National Institutes of Health (NIH) defines a clinical trial as a prospective biomedical or behavioral research study of human subjects that is designed to answer specific questions about new interventions (drugs, treatments, devices or new ways of
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using known interventions). Clinical trials are used to determine whether the new interventions are safe and effective. The field of oncology is well suited to the application of clinical trials [17]. Outcomes generally are well defined and measurable, and the participation, compliance, and follow-up rates tend to be better in the face of such serious illness than they would be for less life-threatening diseases. Precursors of modern clinical trials date as far back as informal human experiments conducted in the 1700s, and modern trial methods began to appear in the medical literature in the 1930s. It is generally agreed that the earliest trial using today’s formal methods, such as patient selection and treatment assignment, was the UK Medical Research Council trial of streptomycin for the treatment of tuberculosis, designed by Austin Bradford Hill [18], in which two measures of success (endpoints) were defined as survival and improvement on chest X-rays, as interpreted by a radiologist who was unaware of (i.e. masked to) the type of treatment that the patients had received. There are five phases of clinical trial in the development of a new therapeutic intervention, including phase 0, the newest trial classification from the US Food and Drug Administration (FDA): • Phase 0: Does the therapy hit its target? (“Is the therapy biologically active?”) • Phase I: Determine the appropriate dose. (“How much therapy can be given safely?”) • Phase II: Demonstrate effectiveness. (“Does it work in a group of similar patients?”) • Phase III: Compare against the standard. (“Is it better than what we can offer now?”) • Phase IV: Evaluate the effects post marketing. (“Will the effect hold in the general population?”) A number of legal requirements must be in place before conducting a clinical trial, including review of the protocol by an independent body, determination that the participant’s risk–benefit ratio is reasonable, and a procedure for obtaining the participant’s informed consent prior to enrollment on the trial. (Note that “participant” has become the favored term for individuals enrolled on a clinical trial, rather than patients or subjects, emphasizing consideration of the enrollee’s human rights and expectation of an acceptable QOL, as well as acknowledging that some trials, such as vaccine development trials, enroll healthy individuals.) An in-depth treatment on human subject protections and regulatory requirements is beyond the scope of the current chapter, but a comprehensive overview can be found in the text by Amdur and Bankert [19]. Obviously, the fundamental core of a clinical trial must be based on the scientific question to be answered. However, the design also must incorporate an ethical concern for the participants, and a wise use of available financial, therapeutic, and human resources. The trials must be designed such that the results will ultimately be persuasive to the intended audience(s), including the healthcare and patient populations, and, when applicable, the regulatory bodies responsible for approval of new drugs/devices. The study design also must provide appropriate statistical control against potentially erroneous conclusions. By their nature, clinical trials tend to include lower-risk patients within a certain age group and with particular clinical characteristics. In the real world, patients are less well defined and do not fit the inclusion and exclusion criteria of the trial, such that the trial results will apply only to a proportion of all the patients with the same disease. As can be seen from Fig. 28.2, at least three major errors can occur within a clinical trial, leading to potentially spurious or misleading results. A type I error involves an incorrect conclusion of a statistically significant result when none is actually present. A type II error reflects an inability to detect a significant result when it truly is present. Bias is the third type of error, occurring when the data do not actually measure
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the intended results, but rather are influenced systematically by other existing factors. While type I errors can be diminished through the correct analytic approaches, avoidance of type II errors and bias must occur up front through the appropriate study design, sample size, and robust definition and collection of endpoints. Readers of published studies may wonder why several trials conducted to examine the same therapeutic regimen and endpoints in similar patients with a given disease entity often draw differing conclusions. Variation in clinical trial results can be explained by several factors that must be considered to ensure valid intertrial comparisons. These include, but are not limited to: the specific participant selection criteria; exact response criteria; interobserver variability in assessing the response; dosage modifications proscribed by the protocol; participant compliance with dosing rules; completeness of reporting of the results; and overall sample size of the trial. Prior to contrasting results across clinical trials, the astute reader should assess the comparability of the studies with respect to these critical points (provided that the report is complete enough to allow such an assessment, another frequent downfall in published studies). The primary tool to help control variability of these components within a clinical trial is strict specification of a detailed protocol, and measurement of protocol adherence during the conduct of the trial. The typical components to be specified when authoring a clinical trial protocol are shown in Table 28.2. Some items apply more in certain phases of clinical trials; for example, randomization typically occurs in phase
III trials (although randomized phase I and II designs also are possible). All of these protocol sections at least should be considered for inclusion when authoring a clinical trial protocol, and most are mandatory to create a valid protocol document. Table 28.3 provides a synopsis of the core elements of the first four phases of clinical trials typically conducted at research institutions. The table summarizes the primary aim, sample to be studied, general procedure, and pertinent notes relevant to each phase of study, as discussed in more detail in the following sections. Phase 0 clinical trials While the term “phase 0” is not new, and there is some confusion due to its use in chronobiology studies, this term is meant to reflect early phase I clinical trials. Generally, the goal is simply to evaluate whether the proposed targeted agent or therapy shows potential based on evidence that it “hits” the target. These studies are a subject of ongoing debate, but in 2006 the National Cancer Institute started its phase 0 program, based on FDA guidelines [20,21]. The first National Cancer Institute-sponsored phase 0 study investigated whether or not ABT-888 inhibited poly(ADP-ribose) polymerase (PARP) in the clinic. Two cohorts of three patients each received a single dose at 10 or 25 mg respectively. For the three patients in the second cohort, PARP was
Table 28.2 Key components for inclusion in a clinical trial protocol
Type I error
Type II error
Bias
Incorrectly concluding result is significant
Missing a truly significant result
Not truly measuring what you intend to measure
Must be controlled through design
Can be controlled through analysis
Fig. 28.2 Possible errors in clinical trials.
• • • • • • • • • • •
Objectives Background Hypotheses Drug information Staging criteria Patient eligibility Descriptive/stratification factors Randomization scheme Treatment plan Dosage modifications Toxicities monitored
• • • • • • • •
Study parameters Endpoint definitions Criteria for evaluation Special instructions Statistical considerations Registration guidelines Data submission schedule Inclusion of women, minority groups, and children • Ethical and regulatory considerations • Pathology review • References
Table 28.3 Core elements of phase 0–III clinical trials Phase 0 trials
Primary aim: Sample: Procedure: Notes:
Determine whether the new drug (or a new combination of drugs) is biologically active A small number of subjects (typically 3–15) to better understand the intended effect Observe biologic effects on single administration studies, at minimal doses, that could not be attributed to chance Helps demonstrate sufficient potential to pursue further study
Phase I trials
Primary aim: Sample: Procedure: Notes:
Identify the safe range and maximum tolerated dose for a new drug (or a new combination of drugs) Volunteers with advanced cancer no longer responding to standard therapy, or homogeneous subsets of high-risk patients Gradually increase the dose in successive groups of patients until “significant” toxicity occurs Response is rarely seen and is not a primary aim of a phase I trial
Phase II trials
Primary aim: Sample: Procedure: Notes:
Determine whether new drug has sufficient biologic activity to warrant further investigation Patients with a given type of cancer who have measurable disease Stop after first stage of accrual if insufficient response; otherwise continue to full accrual Biologic activity does not necessarily imply therapeutic benefit, e.g. if drug has serious side-effects
Phase III trials
Primary aim: Sample: Procedure: Notes:
Evaluate in a randomized trial the therapeutic role of a new drug with known biologic activity versus a standard Patients with a given type of cancer randomized to new versus standard Test for differences between groups in major endpoints reflecting benefit Usually expensive and requires a large number of patients
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inhibited in the tumors by 92%, 99%, and 100%, which demonstrated that oral ABT-888 is bioactive and hits its target, as it would not be reasonable to consider such a change in PARP due to chance alone. More generally, phase 0 studies are intended to observe biologic effects on single administration studies (at minimal doses) that could not be attributed to chance alone, demonstrating sufficient potential to pursue further studies. The details of the statistical considerations of such small sample calculations are described in Kummar et al. [20], but, like most statistical methods, it is based simply on detecting an effect that would not reasonably be ascribed to chance alone. Theoretically, even a single patient can be sufficient where the observation could never have happened spontaneously. Nevertheless, most phase 0 studies use between three and 15 subjects to better understand the intended effect. Discussions regarding the risk–benefit ratio and related ethics of phase 0 studies are often contentious. In HCT, there are a variety of situations where phase 0 studies could apply, although they are most often called “pilot” studies due to the fact that often subtherapeutic doses are not given. For patients with acquired immune deficiency syndrome (AIDS) lymphoma, for example, lentivirus-transfected hematopoietic stem cells are genetically modified to code for multiple RNA decoys (small interfering RNA) to combat the AIDS virus (search http://clinicaltrials.coh.org/, keyword AIDS or 04047). While autologous transplant for lymphoma is a common standard second-line therapy, in this setting it has the added benefit of creating an environment that permits genetically modified stem cells to proliferate with limited competition from unmodified stem cells. Five patients are planned for the study, which will demonstrate whether there is successful engraftment of the genetically modified stem cells, and whether they retain their anti-human immunodeficiency virus activity. Another phase 0-type study in HCT involved testing for the persistence of adoptively transfected CD20-specific CD8+ T-cell clones. One unique opportunity afforded to the HCT environment is that genetically modified stem cells, or donor immunized stem cells (e.g. for cytomegalovirus), have an opportunity to proliferate and become the dominate stem cell phenotype. Such initial studies could be classified as phase 0, although some may prefer the term “pilot” as the FDA guidelines for phase 0 include lack of therapeutic intent and limited toxicity exposure (microdoses when possible), which is not particularly fitting for these HCT studies. Phase I clinical trials A phase I clinical trial is conducted to test a new intervention in a small group of people (e.g. < 80) to evaluate the toxicity profile, determine a safe dosage range, and begin to look at possible efficacy. This phase of trial is used to describe which organ systems are affected; to evaluate the extent, duration, and reversibility of toxicities; to observe any possible antitumor activity; and to better understand the pharmacokinetics and pharmacodynamics. Because of this emphasis on determining toxicities, some investigators see an ethical dilemma in enrolling patients in a phase I trial, as clinical benefit (e.g. response) is not necessarily the primary objective. However, while the probability of therapeutic benefit of a phase I intervention is unknown, especially in single-agent first-inhuman studies, the possibility of clinical benefit does exist, and the patient eligibility and physician referral often are predicated upon there being no better option than the phase I therapy. As such, a phase I trial often is the only means of continuing active therapy with the hope that the treatment will benefit some of the individual patients, even during this early testing. Standard phase I trial design For the typical phase I trial in solid tumors, the study sample consists of a heterogeneous group of volunteers with any type of advanced cancer
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that is no longer responsive to standard therapy. In contrast, phase I trials in diseases targeted for HCT often are performed on a homogeneous subset of high-risk patients who share the same disease type. Phase I trials for acute leukemia often are more aggressive with respect to hematologic toxicities, as it is generally accepted that antileukemic activity will be accompanied by myelosuppression. If the mortality in a subset of patients is quite high using an existing therapy, the potential gain may be great, even with accompanying high levels of toxicity. The recent success of new antileukemia drugs encourages patients refractory to standard therapy to try phase I agents, and patients have experienced a complete remission on a phase I study and proceeded to HCT, even in the more difficult to treat leukemias, as Kirschbaum et al. reported at the American Society of Hematology Conference in 2006 [22]. While less complex in terms of statistical analysis when compared with randomized phase III studies, phase I trials still require rigorous “statistical thinking” in terms of design and decision-making. Requirements for entry into a phase I study typically include: (1) a minimum estimated life expectancy of 8–12 weeks; (2) a reasonably good performance status (e.g. minimum Karnofsky Performance Status score of 80); (3) the cessation of all prior experimental therapy for the last 2–6 weeks; and (4) adequate major organ function to allow normal metabolism of the new agent. In addition, with molecularly or immunologically targeted therapies, phase I trials usually are restricted to patients with the specific target. Many new HCT therapies under investigation for the first time have specific molecular targets, such that a primary endpoint is the ability to shut down a specific enzyme or pathway. This is particularly the case in the testing of new biologic agents, where once a dose that saturates a target is reached, what happens at higher doses may not be of interest unless there are alternative pathways involved. Although response rates are examined within a phase I trial, because these trials generally consist of very small sample sizes, lack of response should not necessarily deter the investigator from proceeding to a phase II trial for estimation of therapeutic effect. Conversely, responses observed during the phase I trial should not be used as a substitute for performing a full phase II or phase III study. Dose-escalating phase I designs. For traditional interventions, the phase I trial will involve a dose-escalation scheme to determine the maximum tolerated dose (MTD) as a primary endpoint. This primary aim is carried out under the assumption that a larger dose or more aggressive regimen is more likely to provide therapeutic benefit to the patient, but will also increase the risk of adverse events. Because adverse events occurring at high doses may be irreversible and life-threatening, the MTD is a relative concept that weighs an acceptable level of toxicity against potential clinical gain. Establishment of the MTD is required not only for a new individual drug, but also for any new drug combination, as the cumulative effect of combining therapeutics with known toxicities becomes a critical unknown that must be established. The study plan must avoid overaccruing patients to subtherapeutic doses that are unlikely to be beneficial, while avoiding overexposure of patients to unacceptably high toxicity levels [23]. A dose-escalating study design should specify the starting dose, steps in the dose escalation, number of patients to be tested at each dose level, and criteria for moving to the next level and for specifying the estimated MTD. The dose range selected must be considered relatively safe yet likely to encompass the MTD. This requires a predefined level of toxicity considered to be unacceptable, referred to as a dose-limiting toxicity (DLT). A safe starting dose is selected based on preclinical pharmacology, animal toxicity or use in other types of patient [24]. For first-in-human use, one-tenth of the mouse equivalent dose that is lethal to 10% of nontumor-bearing animals (MELD 10) is often used, although arriving at a precise estimate can be difficult [25].
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Table 28.4 Examples of phase I dose-escalation schema
Enter 3 patients
Modified Fibonacci scheme*
Unit step size scheme†
Constant step size scheme‡
30 mg 60 mg 100 mg 150 mg 200 mg
30 mg 60 mg 90 mg 120 mg 150 mg
30 mg 40 mg 50 mg 60 mg 70 mg
For comparison, all three dose escalation schema examples were set at a starting dose of 30 mg. * Escalation factors: 100%, 67%, 50%, 33%, 33%, . . . † Escalation factors are set equal to the starting dose, e.g. 30 mg in this example. ‡ Escalation factors are set equal to an arbitrary constant, e.g. 10 mg in this example.
Grade III+ toxicity?
No
Step up 1 dose level No
Yes Any grade IV+
2/3 grade III
1/3 grade III
Add 3 more patients
Grade III+ toxicity? Yes
MTD = next lower dose
No 6 pts at MTD?
Yes
Accrue 6 pts
Phase I closed
Fig. 28.3 Traditional phase I trial schema. MTD, maximum tolerated dose; pts, patients.
The most common study procedure in a dose-escalating phase I trial is to increase the dose gradually in successive groups of three patients, until the predetermined level of unacceptable toxicity is observed. Typically, a dose ladder is prespecified, often the “modified Fibonacci scheme,” in which a sequence of incremental doses is established as the dose-escalation ladder. Escalation proceeds until the MTD is reached. Alternative dose-escalation schema include the unit step size scheme, in which a constant step size equal to the initial dose is applied, and the constant step size scheme, in which each dose is increased by a constant fixed increment over the previous dose (Table 28.4). Designs where the step size depends on the non-DLT toxicities are often used in first-inhuman phase I studies, but are also used more generally [26]. While the sample size necessary to progress to the MTD cannot be anticipated within a phase I plan, in our experience the typical number of levels ranges from three to 15, with fewer levels when testing a new combination of well-known drugs, and more levels for first-in-human use. A typical sample size might end up being 24, but studies with two patients (e.g. unacceptable toxicity at a dose where the blood concentration was below the level of effect seen in preclinical studies) and 60 patients have been conducted. In HCT trials, the study regimen often will have been used in other types of patient, so that the types of toxicities and a crude estimate of the MTD are available for planning purposes. However, patterns of toxicity can change markedly from one type of patient to another, such that caution is still required. A dose-escalation procedure commonly in use is shown in Fig. 28.3. Three patients are accrued to the first dose level. If no DLT is observed in those three patients (after one complete course plus any predesignated waiting period to observe drug-related toxicity), the dose is escalated to the next level specified by the protocol. If a single patient experiences DLT, three additional patients are treated at the same dose level; the dose may continue to be escalated only if no additional DLT is observed in the additional patients treated at that level. If a second DLT is detected, de-escalation ensues, with the initial estimate of the MTD being the preceding dose level. The trial is considered to be complete (MTD determined) when six patients have been accrued at the estimated MTD dose level, with the occurrence of at most one DLT out of six. In non-HCT studies, DLT is usually defined as treatment-related nonhematologic grade III or IV toxicity, with a few exceptions, and no hematologic DLT. Hematologic DLTs in a leukemia trial often can only be detected in the absence of blasts, and defining hematologic DLTs often requires a discussion of duration and results of a bone marrow aspiration. In HCT studies, DLTs are considerably more difficult to define, and no consensus has emerged. The most common rules today are still grade 3 toxicities by the Bearman Scale from 1988 [27].
One criticism of phase I trials is that “the recommended dose has no interpretation as an estimate of the dose which yields a specified rate of severe side effects” [28]. With the usual phase I escalation schema, only six patients are observed at the estimated MTD, and no measure of statistical precision (standard error or confidence interval) can be calculated. This is particularly troubling when the target is a dose with a specified amount of severe toxicity (the DLT rate), in an attempt to achieve a favorable risk–benefit ratio. Statisticians have examined the operating characteristics of phase I designs to try to improve upon the rather ad hoc design described above. Storer [29] provides an introduction to work in this area, showing how poorly the usual designs perform with respect to providing a reliable estimate of the MTD. He proposes some simple variations in the designs that may perform better, and methods for obtaining confidence limits for the MTD. The article by Korn et al. [30] provides a discussion of Storer’s approach along with other alternatives, and evaluates their performance via computer simulations. Ivanova et al. [31] have described an up-and-down design for the sequential allocation of subjects to dose levels, yielding a local estimate of the toxicity probability calculated from all previous responses. One point that helps mute criticism of the standard design is that generally there is no consensus regarding the target DLT rate. Most new phase I designs have rarely been implemented, partly due to concerns such as length of time to complete the trial, potential for excessive toxicity, and complexity of updating the models for dose escalation throughout the trial without an automated system in the clinic. As more institutions adopt electronic medical records, the capability of and access to computers at the point of care may change this last situation. Before implementation in the clinic, computer simulation studies are carried out to compare the performance of the proposed designs. Such comparisons traditionally use the performance criteria of proportion of patients treated at the recommended MTD (and the number above and below), and the average number of patients required to complete the trial, and base these simulations on a variety of assumptions regarding the true dose–toxicity profile. In future simulation studies, investigators may also prefer to incorporate the actual time to trial completion as a performance measure as well, as it is unethical to spend excessive time examining an unpromising regimen before moving on to the next therapeutic approach. Clarke et al. [32] have investigated a new phase I approach intended to minimize the time to trial completion by allowing new patients to enter the trial without waiting for toxicity evaluation on the full prior cohort of patients, yet conditioned on all available toxicity information to that point.
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In HCT studies, there often are additional considerations to the phase I study besides concerns about DLTs. Specifically, some agents tested in HCT studies are designed to help reduce graft rejection. In such a setting, there is, hopefully, a therapeutic window below which the dose is too low to prevent rejection, and above which unacceptable toxicity occurs. Designs in this situation usually rely on extensive simulations to obtain rules that result in acceptable operating characteristics [33]. Regardless of the particular study design, uncertainty in estimating the MTD requires investigators to be cautious and monitor toxicities in a phase II trial very carefully, being prepared to adjust the phase II treatment dose as additional information accumulates. The phase I study result is a critical first step, for when the phase II treatment dose is modified mid-trial, the statisticians and the investigators or the data safety and monitoring board may decide that the study is essentially a “do-over,” and restart the patient count from the modification. This is less common in a phase III setting, when such modifications are generally minor. Phase II clinical trials Following establishment of the safety and toxicity profiles of a new intervention, a phase II clinical trial is conducted to determine whether the new therapy has sufficient evidence of biologic activity to warrant further investigation. Phase II trials are performed not only for antitumor activity, but also to prevent relapse, for prophylaxis and treatment of graft-versus-host disease (GVHD), for treatment of symptoms of other therapies (e.g. pain), for prophylaxis against and treatment of specific infections or for a combination endpoint (such as protocol-defined “clinical benefit”). The primary components of a phase II trial are summarized in Table 28.3. Standard phase II trial design Phase II trials typically are conducted in those patients who are most likely to respond favorably to a new regimen, so that potential activity of the new regimen is not missed. Therefore, participants with maximum performance status and a minimum amount of prior therapy should be selected whenever ethically possible. In addition, all patients in a phase II trial should be evaluable for the primary endpoint, which should be objective. Subjective methods of assessing treatment effect make the outcome of the phase II study more problematical and less convincing. One approach to phase II trials is the single-group design, with no control group. Like the phase I study, the single-group phase II trial is subject to the problems of any uncontrolled clinical trial. However, these studies are intended as low-cost screens for choosing safe doses and for eliminating treatments that do not have sufficient promise for further study. Similar to the phase I study, the uncontrolled phase II group study design depends on careful definition of the eligibility criteria, and uniform application of these criteria in admitting patients to the study throughout the accrual period. It is also extremely important to define clearly within the study protocol the response endpoint and its method of measurement. The number of patients to be studied in a phase II trial depends on the specified degree of disease activity required for the new treatment to be of therapeutic value, specified as the percentage of patients expected to respond to the treatment. The study should be large enough to furnish information that will rule out with high probability (e.g. 80–90%) a treatment that does not have this desired degree of activity, taking into account possible random variation in the limited sequence of patients studied. The simplest approach is to compute an upper confidence limit, and if this limit is less than the desired activity level, the treatment is rejected as of little interest. If the upper limit is greater than the desired level, investigation will proceed to determine just how active the regimen
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Table 28.5 Number of patients required for the phase II trial of an agent for given levels of therapeutic effectiveness and type II error Therapeutic effectiveness (%) Rejection error (beta) 5% 10%
5
10
15
20
25
30
35
40
45
50
59 45
29 22
19 15
14 11
11 9
9 7
7 6
6 5
6 4
5 4
Reproduced with permission from Gehan and Schneiderman [36].
might be, via a phase III trial. Of course, other factors might preclude further study, such as additional evidence of unacceptable toxicities or a higher MTD than indicated by the phase I study results. Statistical methods for computing the upper (one-sided) confidence limit are found in most elementary statistical texts, such as in Chapter 5 of Glantz [34] or Chapter 14 of Pagano and Gauvreau [35]. Commonly, a single-sample, two-stage design is employed in phase II trials, thereby allowing early termination of the study after the first accrual phase if the new regimen does not appear to be promising with some level of specified confidence. Table 28.5, from Gehan and Schneiderman [36], shows the number of patients required in the first stage of accrual if one desires either 95% confidence (rejection error or beta = 5%) or 90% confidence (beta = 10%) for efficacy levels ranging from 5% to 50%. The new treatment is rejected as not sufficiently active if no responses are seen among “n” patients treated. For example, if the study produced no responses among the first 11 patients, one would have 95% confidence in rejecting the new treatment as being active in 25% of patients. If one or more responses are seen among the first set of patients, accrual is continued into the second stage, until a large enough study group is reached to estimate the response rate with a prespecified level of precision that depends on the final sample size. As an example, with 35 subjects entered after concluding both stages of accrual, the response rate could be estimated with a maximum standard error of approximately 8% (95% confidence interval width of approximately 16%). The typical phase II study uses a binary endpoint – response or no response. Response could be defined to include only complete response, or to include both complete plus partial responses, as the outcome of interest. However, the counts themselves usually are based on quantitative measurements (e.g. estimates of the amount of reduction in tumor volume or counts of cutaneous lesions in herpes zoster). Generating waterfall plots can often provide additional information [37]. Variants of the phase II trial design Important work has been done on designs that control both risks of error (alpha and beta) and achieve significant savings in the number of patients required. Simon provides a thorough review of different approaches to phase II trials [38]. He has developed an optimized two-stage design with a smaller sample size in situations where the lack of efficacy becomes clear early in the accumulation of the data, allowing the possibility of earlier stopping at the end of the first stage of accrual [39]. Simon concludes that two-stage designs with a target sample size of 35–50 and a substantial probability of early termination usually are appropriate. Fleming [40] offers other commonly used procedures for two-stage designs. Since the relationship between type I and II errors, interim stopping points, and sample size are not always obvious or monotonic, we often explore around the “edges” of these designs to seek preferred designs, calculating the operating characteristics of alternative
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designs. Three-stage designs also are employed, especially when a quick “bail-out” option is needed for early lack of evidence [41]. Storer has proposed that designs for control of both alpha and beta risks be expanded to consider three possible decision outcomes: response rate unacceptable, response rate acceptable, and an intermediate region in which further study is required before a decision can be made [42]. Several statisticians have done work on combining decision-making risks with estimation, where the precision of the estimates is based on confidence limits. For example, Chang and O’Brien [43] have created a method and offer a computer program for obtaining confidence limits for the response rates from the results of multistage phase II studies. Another area of interest is Bayesian methods, based on the quantitative expression of prior beliefs about the treatment to be tested, and using this quantitative information and a measure of its precision in the study design and analysis. Phase II trials are a likely arena for using this approach, and Thall and Simon [44] provide a useful introduction to this topic. In a two-stage phase II trial, participant accrual typically must be suspended while the results of the first-stage accrual are examined to determine whether or not to continue into the second-stage accrual. This temporary suspension has drawbacks in that the momentum for patient accrual is lost, and the overall time to completion of the study is lengthened. In addition, there may be logistical problems in reactivating the trial if the data show that it is warranted to proceed to the second stage, particularly in a multicentered study setting. Herndon [45] has proposed an alternative “hybrid” design that allows accrual to continue while the results from the first stage of accrual are being examined to determine whether the study should continue to the next stage. Other variations on the standard phase II design include adding a phase II treatment arm to a phase III clinical trial [46], randomized phase II trials (or “active control phase III trials”) to select the most promising of several new agents [47], “calibrated” phase II trials, in which a subgroup of patients are randomized to a standard treatment to verify that the population under study is capable of responding to an active treatment [48], and randomized screening trials, often used when comparing a new agent combined with a known regimen versus the known regimen alone [49]. In these designs, one does not enroll typical phase III sample sizes. Typically, either the measure of statistical significance is reduced (e.g. p < 0.2), or a selection decision design is used, where the chance of choosing an inferior regimen is kept small. As a result, these randomized phase II studies can enroll conventional phase II sample sizes to each arm, and early stopping rules can apply both jointly and independently to the two arms. Often historical controls and phase II studies are suggested in lieu of random concurrent controls, that is, comparing patients treated earlier at the same institution(s) using a therapy that is different from the new therapy under consideration, or using historical control results from the literature. While it has been argued that historical controls are convincing in certain situations [50,51], most consider such comparisons to be suggestive of benefit only, rather than providing a valid, reliable basis for evaluation of a new therapy. A few medical advances have been so clear cut that historical controls would suffice (e.g. the discovery of penicillin or, more recently, the approval of PS-341 in second-line treatment for multiple myeloma); however, such quantum leaps in clinical discovery are rare. Phase II studies are also often used for decisionmaking prior to phase III studies or when phase III studies are unlikely, as is often the case in the transplant setting or in rare diseases such as T-cell chronic lymphocytic leukemia [52]. Accepting inferior decisionmaking as a long-term approach has drawbacks, and advances in oncology are more often observed through careful scientific evaluation through clinical experiments, and the phase III clinical trial is the accepted “gold standard” for establishing a benefit of a new therapy.
Phase III clinical trials After the efficacy of a new HCT drug or regimen has been established in a single group of participants through early therapeutic trials, generally the new therapy is tested against a standard (or a placebo, such as a placebo plus HCT versus HCT plus new agent) within a phase III trial. As shown in Table 28.3, in this phase of trial participants are assigned to therapeutic strategies by random, or probabilistic, assignment, to determine which therapy is superior. The phase III clinical trial requires an extremely detailed protocol to serve as a guide to the organization and conduct of the study, and to provide a plan for analysis of the results, allowing replication and confirmation of the study results. The protocol must specify the eligibility criteria for deciding whether the patient qualifies for the clinical trial, based on the scientific question asked. Hypothesis testing is used to determine whether any differences observed are significantly greater than would be seen by chance alone. The scientific credibility of a properly designed phase III study meets the international guidelines generally required as proof of the efficacy and safety of new drugs and therapeutic devices as the final step in approval for marketing. The International Conference on Harmonization (ICH) Technical Requirements for Registration of Pharmaceuticals for Human Use were developed by the FDA and the comparable regulatory bodies in Japan and the European Union, resulting in publication of the ICH Tripartite Guideline for General Considerations for Clinical Trials [53]. The ICH E9 guideline Statistical Principles for Clinical Trials was adopted by the Committee for Proprietary Medicinal Products in 1998. The principles within this document are scientifically sound, and are being followed by the three ICH regions, although at the current time some regions may be applying these guidelines more stringently than others [54]. To prevent delays in receiving marketing authorizations for new drugs, it is strongly recommended that the statistical guidelines provided in ICH E9 are consulted, and that planned statistical analysis strategies are modified to reflect these principles when designing drug development studies [55]. Standard phase III trial design The primary rationale for randomization within a phase III trial is to assure that the study arms can be compared in an unbiased fashion. Although the need to evaluate competing therapeutic strategies on comparable groups has been acknowledged for many years [56], the difficulty of assuring that groups of patients treated in different manners are truly comparable often has been underappreciated. The randomized trial balances treatment groups statistically, not only with respect to known and recorded factors, but also with respect to unknown patient factors. While the randomized groups will not be identical, any observed differences in therapeutic outcomes between them can be evaluated relative to differences expected through random chance alone. The group comparison is based on probability theory, and provides a measure of the statistical precision of the treatment difference estimate, accounting for random variation. This precision is measured by the standard error of the estimate, the confidence interval, and/or the p-value, as described in introductory statistical texts [34,35]. In addition to utilizing randomization to minimize patient variability that might confound the evaluation of treatment effects, HCT protocols also often attempt to minimize the variability in supportive treatments. However, strict requirements to a protocol-specified supportive care regimen may greatly impair participation among centers and accrual within centers, such that allowing for a more general supportive care regimen within wider guidelines may be necessary in HCT studies. The phase III study plan is based on information stemming from the preceding early phase studies, and calls for accrual and follow-up of
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participants over a specified period of time. All participants are observed until the occurrence of a specified endpoint has occurred, such as death, progression of target disease or relapse, or to the administratively defined time for analysis at the end of the predefined observation period. HCT studies that include courses of nontransplant treatment as an integral part of the strategy must enroll and follow patients from the time that nontransplant treatment is begun. If the pretransplant treatment strategy differs between the two arms, care must be taken to ensure that patients are enrolled and followed from a similar time point for both arms. Randomization should occur immediately after ascertaining eligibility, as undue delay between randomization and transplantation can lead to high patient drop out, complicating valid interpretation of the trial results. Randomization process. The randomization procedures in a phase III clinical trial should be clearly described and justified in the protocol. To obtain valid trial results, it is crucial that the research team has no knowledge of the assignment at the time of decision for study entry for any given study participant, otherwise the advantage of the random assignment may be lost due to selection bias. A typical procedure is to screen the patient for eligibility based on already available information (disease, routine laboratory work, etc.), obtain patient consent, then perform any study specific eligibility tests (e.g. multiple-update gated acquisition scan), and then register the patient and obtain a random assignment to one of the treatment groups. These assignments are made according to a series of random numbers, with the sequence being computer generated in advance or as the patients are entered into the study. It is desirable to minimize heterogeneity in the participant group to avoid imbalances in patient characteristics. However, the benefits of a homogeneous study population must be balanced against the need to accrue sufficient numbers of patients in a timely manner, and the need to have the study sample reflect the population in which the treatment eventually will be used, to be able to generalize the study results. The randomization schedule often is balanced at successive points in accrual, by ensuring that the patients within a “block” (e.g. every six patients) are evenly assigned to the randomization groups as accrual progresses at each site. To protect the research team from being able to anticipate treatment group assignment, the block sizes can be varied, and block size information should not be specified in the protocol. If considerable variation in prognosis among the study patients is anticipated, more of the higher-risk patients may be assigned randomly to one treatment group and more lower-risk patients to the other group, making it difficult to detect a true treatment difference due to the unfair comparison. Heterogeneity among patients can be addressed in the design phase by using “stratified randomization,” in which patients are divided into risk groups (strata) based on a small number of known prognostic variables, such as age (younger/older) and remission status at transplant (first complete remission/beyond first remission). Randomization is carried out within each age/transplant status group. Because the number of strata grows rapidly with each new characteristic, stratification should be confined to only a few proven prognostic variables. For example, if there are four stratification factors, each of which takes on one of two possible values (e.g. male or female, disease stage III or IV, age young or old, and previous radiation therapy or not), this results in 16 possible strata. Therefore, designs that stratify on more than three factors generally are unnecessary and unwise. For additional prognostic variables, covariate adjustment can help account for imbalance in treatment arms. For the primary objective, these covariates should be specified a priori as part of the protocol. If the study is to be carried out at several sites (clinics, hospitals, and study centers), the variability among centers in transplant trials may be
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quite considerable, even for tightly written protocols. In the case of such multicentered HCT trials, stratified randomization should be used to generate separate randomization assignments for each site, to ensure that the treatment arms are statistically balanced within each site. In their seminal 1976 paper on clinical trials design, Peto et al. recommended that, unless the trial size is quite small, there is generally little need for prerandomization stratification, other than for treatment center in the case of a multicenter trial [57]. Another approach to obtaining balance on participant covariates is to use Efron’s “biased coin” design or variations on this idea [58,59]. An example would be to balance a study adaptively on gender and age as new patients are accrued to the study, instead of randomizing within each of four gender–age strata. With this method, as each new participant is enrolled in the study, the degree of balance between the two treatment arms is examined separately for gender and by age. Randomization proceeds with a skewed probability favoring assignment to the treatment group that reduces any existing imbalance in prognostic factors between the two arms. One caution is that applying correct analytic methods within adaptive randomization can be more complex, as discussed by Lachin et al. [60] and Simon [61]. Study sample size. Planning the size of the study is crucial to determine the number of participants required to acquire sufficient information for comparing the treatment groups. While controlled trials tend to reduce the inherent bias in the data through the study design, extremely small sample sizes may limit the ability to avoid bias fully. Most treatments have relatively minor effects on disease, and it has become appreciated that trials can be heavily influenced by chance [62]. Establishing the reality of small differences between treatments often requires a very large sample size, making such studies quite expensive. Recruitment may have to occur from many hospitals, or even across several countries, making control of data quality quite difficult. Attaining a sufficient sample size for a phase III HCT trial requiring a human leukocyte antigen-identical sibling donor can be particularly problematic, as only about one-third of otherwise eligible transplant candidates have such a donor. For trials studying interventions designed to prevent later complications, for example trials to prevent chronic GVHD, relapse or late organ failure, early attrition from transplant-related mortality decreases the effective sample size available (and could bias the analysis of the longer-term endpoint if not handled appropriately). In calculating the desired sample size, several parameters must be specified by the investigator, based on prior data, past experience, and clinical relevance. The investigator must specify whether the statistical evaluation of the hypothesis is to test for no difference between the two arms, or to prespecify a desired direction of the test, for example that the new treatment is better than the standard treatment. These are known as two-sided and one-sided alternative hypotheses, respectively, with the two-sided approach being the more conservative method preferred by most peer-reviewed journals (unless strong biologic evidence or the use of a placebo comparison arm dictates that the direction of the test should be one-sided). The investigator also must determine the difference between treatment arms that is clinically important, generally based on his or her clinical experience and what the community has accepted as worthy of a change in practice in similar patients. The alpha error rate for the null hypothesis of no difference must be specified in advance, that is, the probability of declaring that the two treatments differ when they are really equivalent, with the apparent difference being due to random variation. (A traditional alpha error level is 5%.) The beta error rate also must be declared in calculating the study sample size, representing the probability of missing the specified clinically important treatment difference (often set at 20% or below, such that the power of the test [1 − beta] is 80% or higher).
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While this overview of the phases of clinical trials may make it seem relatively straightforward to move from phase I to phase II, and phase II to phase III, often this is not the case. Evidence for safety and dosing may be based on an accumulation of results from several trials, and, when appropriate and expedient, phases I and II may be combined in a single trial, particularly in the face of limited patient resources. At times, it may become necessary to study more than one intervention in the same patient, such as enrolling the same individual in both an experimental transplant trial and the follow-on trial of a new GVHD prophylaxis. In such instances, very careful planning of entry criteria, measurement of potential confounding factors, and adjustment for the primary treatment arm must be considered in the conduct and analysis of these companion trials.
Impact of genetic data on clinical trials design The genomics era has led to a rapidly improving understanding of germline polymorphisms and mutations, along with tumor genetics. Many specific oncogenes, tumor suppressor genes, genes related to DNA repair, and other genetic abnormalities found in cancer have been characterized for a variety of tumor types [63]. Germline mutations and heritable polymorphisms that increase the risk of cancer are better understood, and other polymorphisms are known to impact the metabolism of various drugs. Knowledge of these molecular markers can be exploited within clinical trials being conducted for the detection and treatment of cancer. Four avenues of expanding genomic research that impact the design of future clinical trials are made possible through the availability of the human genome mapping. First, genetic testing will provide earlier detection of the genetic risk of disease, and improved sensitivity and specificity in the diagnosis of disease. With the growing availability of genetic markers for presymptomatic conditions in hematologic malignancies, early intervention and prevention protocols will become more feasible and prevalent. Eligibility will be developed that includes disease specificity as determined by genetic testing. In addition, genetic testing may be used to refine detection of disease as the primary endpoint of a clinical trial [64]. For example, detection of minimal residual disease (MRD) can be approached through the detection of the unusual expression of a gene in leukemia cells, such as the aberrant expression of the WT1 gene in acute myeloid leukemia (AML), or the presence of very subtle genetic lesions, such as the detection of ras point mutations in AML [65,66]. However, it can be technically difficult to discriminate the rare aberrant expression or mutated allele from normal background expression or wild-type alleles, such that using genetic targets as a marker of MRD generally suffers from less sensitivity and specificity compared with the detection of translocations or gene rearrangements. Because of this concern, qualitative polymerase chain reaction (PCR) assays require controls to detect false-positive results from contamination or a suboptimal assay, false-negative results due to reaction failure, and/or contamination of the assay. The second new scientific area of pharmacogenomics is advancing greatly our knowledge of the human’s ability to metabolize, tolerate or respond to drugs based on the individual’s genetic make-up [16]. In this field, large-scale genomic technologies such as gene sequencing, statistical genetics, and gene expression analysis are systematically applied to speed the discovery of drug-response markers, whether they act at the level of the drug target, drug metabolism or disease pathways. Pharmacogenomics offers the prospect of predicting an individual’s susceptibility to side-effects when undergoing a new therapy, and to individualize treatment selection based on his or her genetic profile [67]. For further information, see Chapter 12.
Clinical trials are already using genetic prescreening and treatment “run-in” periods as part of the study eligibility, to ensure that the participants can metabolize and tolerate the drug, to individually calibrate the most potentially efficacious dose, and/or to stratify subjects by their genetic ability to respond to or tolerate the study drug. Defining patient populations genetically could improve greatly the outcomes, safety, and efficacy profiles achieved within therapeutic oncology research. As an example, a phase I study at City of Hope National Medical Center combining fixed-dose irinotecan with two other agents being escalated states that UGT1A1 7/7 patients are ineligible. As a note of warning, such laboratory-defined polymorphisms only scratch the surface of human genetic variability, so, when feasible, phenomenologic eligibility criteria (e.g. fasting bilirubin levels) may still need to be employed. The third emerging scientific area, pharmacogenetics, involves the development of new drugs based on information about genes and gene targets. Knowledge of molecular characteristics of certain cancers has made it possible to identify patients who could benefit from therapies that target those features [63]. For example, in the majority of patients with chronic myeloid leukemia (CML), leukemic cells have a “telltale” chromosomal abnormality involving “swapping” of genetic material between chromosomes 9 and 22, resulting in the development and proliferation of the leukemic cells. Effective pharmacologic intervention may be designed to target and specifically to inhibit this faulty enzyme. Based on a randomized phase III trial of 1106 patients, in December 2002 the FDA granted accelerated approval of a drug involving a genetic target, imatinib mesylate (Gleevec), for the initial treatment of patients newly diagnosed with Philadelphia (Ph)+ CML. The approval was granted in a record time of just under 6 months from receipt of the application, and was based on a statistically significant decrease in the risk of disease progression detected at the first interim analysis. In the fourth scientific avenue, clinical trials exploring novel gene therapies provide exciting possibilities for repairing or replacing defective genes, or inserting therapeutic genes, impacting study design and conduct. Gene therapy is predicated on the ability to clone and manipulate genes (see also Chapters 10 and 11) [63]. For some diseases, introducing a functional homolog of the defective gene that produces even small amounts of the missing gene product can have beneficial effects, making these diseases strong candidates for gene therapy. An example of a pilot study in gene therapy is a five-patient study at City of Hope to assess the feasibility and safety of cellular immunotherapy utilizing ex vivo-expanded autologous CD8+ T-cell clones genetically modified to express the interleukin-13 zetakine chimeric immunoreceptor in patients with advanced glioma. With the availability of genomic data, more exploratory and “datamining” studies will be conducted to help design the subsequent clinical trials, to find correlations, and to identify the constellation of genes that may determine the toxicity and efficacy of new therapies to be tested within a trial. Both randomized clinical trials and prospective observational studies that include storage of genetic tissue will provide opportunities to gain insights into the genetic basis of variation in response to treatments [68]. Ideally, all future clinical trials will include the banking of genetic samples, to continue to build extensive databases linking clinical and genetic data for future exploratory “data mining” and the discovery of new patterns and predictors. Future clinical practice may require that all trial results be coupled with genetic information to help determine treatment choices by genotype. By identifying those patients most likely to respond to novel drugs, it will be easier to demonstrate efficacy and safety in phase III studies, leading to smaller, more effective clinical trials with corresponding cost savings. In addition, this focused approach will be more ethical as it is more likely that a suitable efficacious drug will be administered to the right patients in a safe manner.
Biostatistical Methods in Hematopoietic Cell Transplantation
While these new trials will be faster to complete, they will be more complex to analyze, as genetic information will be collected and correlated with clinical results within the trial plan. Trials will involve gene-based eligibility criteria and stratification by genetic risk/response data. The rapid pace of development of new therapies for experimentation will put rising pressure on standardization within the entire clinical trials arena, to support the requirement for even more efficient flexible study design tools. Already by the end of 2002, among 360 active breast cancer and CML trials identified via the NIH-sponsored website http:// clinicaltrials.gov, 61 (17%) included a genetic component. Of the 249 breast cancer trials, 26 were using hairy-related 2 (HER2) to characterize disease, four included testing for breast cancer 1/2 (BRCA1/BRCA2) for eligibility and stratification purposes, and three included microarray expression data for exploratory analyses. Among the 111 CML trials, 35 were utilizing the presence of the Ph chromosome as either an eligibility or a stratification factor.
Clinical trial conduct Single- versus multicenter studies While phase I and small phase II HCT trials usually can be completed within a single institution, randomized phase III clinical trials frequently require collaboration among several or many sites to achieve the targeted sample size. National cooperative groups such as the Southwest Oncology Group and the HCT Clinical Trials Network have been established to conduct randomized trials that may require thousands of patients to attain sufficient power. Alternatively, several transplant centers may join forces to achieve sufficient sample sizes to conduct trials within a smaller consortium setting, such as the City of Hope National Medical Center–Fred Hutchinson Cancer Research Center–Stanford University Medical Center consortium for the conduct of regional HCT trials. National and international registries such as the International Bone Marrow Transplant Registry are conducting large-scale observational studies based on the results that are routinely reported to them from hundreds of HCT centers. Each of these multicentered approaches creates new challenges and obstacles that must be conquered to conduct a valid study. One central location must be established as the Data Coordinating Center (DCC), responsible for final protocol documentation and dissemination, case report form development, database programming, and data storage. Data quality assurance, protocol adherence monitoring, and statistical analysis must also be addressed. Central registration of patients, review of eligibility and incoming data records by trained staff, insuring audit procedures for review of data records against the original source data, and ensuring appropriate handling of Institutional Review Board (IRB) approvals, amendments, and severe adverse events at all sites are responsibilities of the DCC in a multicenter model. In both single- and multi-center studies, an effective, convenient, and accurate mechanism for data capture and transmission needs to be developed and distributed to all participating sites. Implementation of a system for data collection ranges from paper-based data collection forms to online data entry systems. Mailing or faxing of paper forms for central data entry at the DCC has been the more traditional mode to date. However, electronic data transfer from distributed sites of data entry has been available since the 1980s [69], and is becoming more commonplace. In this case, a secure mode of data transfer with redundancy checking and audit trails must be established. At City of Hope, we have over 10 years of experience with directing a national DCC for cancer research that has established online data capture via the Internet using web-based forms [70]. Numerous additional considerations arise when transmitting data via the Internet, such as ensuring security of the data via encryption, and fault tolerance of the online system.
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Data and Safety Monitoring Boards Data and Safety Monitoring Boards (DSMBs) play an essential role in protecting the safety of cancer clinical trial participants, and in assuring the integrity of the research being conducted [71]. DSMBs serve a datamonitoring function above and beyond that traditionally served by IRBs, and as such are particularly important for studies that involve blinded or masked data, and cooperative clinical trials involving several centers. Generally, the DSMB monitors ongoing phase III trials; however, their purview also may include earlier-phase studies. DSMB members are charged with monitoring the accumulating trial data at regular intervals and making recommendations regarding appropriate protocol and operational changes based on the data. The DSMB helps ensure that trials do not continue beyond the point when the objective(s) have been met, and a clinically meaningful answer of importance to the scientific community and the public has been obtained. However, the decision to continue or to stop a trial, or to modify the protocol in a major way, rarely is straightforward. Such deliberations require considerable judgment and attention to numerous factors, to protect the safety of the research participants while ensuring the highest quality scientific research. The size and composition of a DSMB study panel are driven by the nature of the study involved, but it generally consists of five to eight members whose expertise encompasses the scientific area under study, ethics, regulatory affairs, and biostatistics. The committee should be multidisciplinary in nature, and made up of individuals who are free of any significant conflict of interest [72]. Once the DSMB membership has been established and charged with its responsibilities, the DSMB Chair, coordinator, and biostatistician typically agree to a basic format and schedule for the presentation of ongoing data, so that DSMB members will receive adequate reports within a sufficient time frame for the decision-making process. The study Principal Investigator may participate as an ex officio member of the DSMB; however, for phase III trials the data should remain “blinded” to the Principal Investigator during all discussions of the interim results. Although exceptions can be considered by the DSMB chair on a case-by-case basis, this principle is particularly important if the Principal Investigator is involved with patient care during the course of the study. The DCC will provide interim results to the DSMB on study progress, safety, and relative efficacy of the treatment arms, subject to restrictions of the protocol design. Methods such as group sequential designs satisfy the valid objectives of interim monitoring, while avoiding undesirable consequences such as increased alpha error [71]. After each meeting, the DSMB members will make a recommendation regarding the continuation of the trial(s) under review. Critical issues that should be incorporated in this assessment include increased morbidity/mortality related to the study intervention; severe or increased adverse reactions; unsatisfactory performance of the DCC or study sites; suspicion of fraud; inability to complete the study in a timely manner due to lack of patient enrollment; failure to comply satisfactorily with recruitment criteria; and any other issues concerning possible important protocol deviations or suggested changes. Depending on the findings and circumstances, actions to be taken by the DSMB include suggestions for increasing patient recruitment, extending the period of recruitment, stopping recruitment because of an inadequate rate of accrual, modifying the protocol because of safety or accrual issues, discontinuing the protocol because of poor protocol compliance, or terminating the study early following a planned interim analysis with highly significant results. An interim analysis that strongly suggests that the protocol cannot be successfully completed, for example if the initial study design estimates are found to be invalid or seriously inaccurate, also can lead to the recommendation for early study termina-
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tion. Other issues of concern in monitoring trial conduct include the problems of premature publication, and exaggerated estimation in trials that are stopped early [73].
Monogenic 11%
Impact of genetic data on clinical trials conduct The inclusion of genomic data in clinical trials will influence future organizational structures to provide the optimal infrastructure for management and analysis of the complex merging of genetic and clinical data. The application of industrial processing and quality improvement techniques may emerge more prominently to assist in decreasing the attendant possibility for errors with such complex data [74]. Clearly, the addition of genomic data to clinical trials raises new ethical questions regarding confidentiality, particularly in light of the enactment of the Health Insurance Portability and Accountability Act. In the past, there was relatively unrestricted access by third parties for secondary uses of data, and inadequate data anonymization, neither of which is tolerated under the Act’s regulations that became effective in April 2003. There are significant technologic and policy issues that must be addressed in protecting participant confidentiality and security of the data. Concerns about subjects’ rights, informed consent, privacy, and ownership of genetic material require strict attention in the development of DNA banks [16]. The biggest challenges to data access likely will stem from the complex organizational, social, political, and ethical issues that must be resolved to allow linkage of clinical and DNA information. Genetic data are more reliable, persistent, and specific than ordinary identifiers such as name, social security number, etc. As genetic research proceeds, such data may come to be highly predictive of current and future health status, making the data extremely sensitive in nature. It is likely that a separate consent process and/or wording for genetic tissue analysis will be required in the future. It may be necessary to move the consenting process earlier in the participant recruitment activities when genetic testing is a key component of eligibility screening for a clinical trial. Within the clinical trial setting, some test results may be noninterpretable with respect to the impact on health or treatment decisions, and should remain within the realm of the research team and database, at least until such interpretation becomes available. Safeguards will be critical when genetic data are incorporated into routine clinical practice as well. Privacy policies and procedures will be required to minimize the risk of inappropriate disclosure of private information for linked samples. The HIPAA mandates both high standards of data privacy, as well as the extent of patient access to their own data, and extends to clinical data generated by researchers who also are clinicians providing patient care. The use of genetic manipulations to create novel interventions dictates the need for greatly increased monitoring of protocol conduct, decision support for patient treatment, identification of adverse events in real time, and regulatory reporting of severe adverse events (SAEs). Figure 28.4 shows the number and type of gene transfer trials that were ongoing in 2002, with cancer being the predominant focal area and encompassing 62% of all such trials. As the number of gene transfer trials has grown, so has the number of SAE reports, from 285 reported from 1988–99, to 3263 reported in the period 1999–2002. In an attempt to enhance the safety of such highly experimental trials, the NIH has developed a system for the automated reporting of SAEs stemming from gene transfer trials, called the Genetic Modification Clinical Research Information System (GeMCRIS). This system is intended to facilitate the evaluation and analysis of safety information across all gene transfer clinical trials, and to provide database reports that will inform diverse user groups, including IRBs, bioethics committees, DSMBs, investigators, and research participants themselves. Secu-
Marking 8%
CVD 8%
Other 3%
Infectious disease 8%
Cancer 62%
Fig. 28.4 Gene transfer trials by type of application. CVD, cardiovascular disease.
rity features are in place to protect trade secrets and confidential commercial information contained in the database. GeMCRIS is web based and could provide the basis for continuous review of studies involving humans, even being suggested for extension to nongene-transfer trials in the future. It is hoped that the use of such systems will optimize patient safety, while providing useful data to inform the design and conduct of both ongoing and future trials. The critical nature of such SAE reporting was highlighted by the closure of a French trial studying human gene transfer as a possible treatment for X-linked severe combined immunodeficiency, after enrolling 11 patients. The strategy of the experiment was to correct the early block in T-cell and natural killer lymphocyte differentiation by transducing the subjects’ CD34+ cells ex vivo with a defective Moloney murine leukemia retroviral vector. This vector contains the common γ gene for the cytokine receptors responsible for the delivery of growth, survival, and differentiation signals to the early lymphoid progenitors. While nine of the 11 subjects experienced significant restoration of their immune system following the gene transfer intervention, two of the nine also experienced SAEs that appeared to be directly related to the experimental therapy. Both children developed a T-cell leukemia that appeared to be due to insertional mutagenesis after receiving the gene transfer product. Calls for tightening of government oversight of gene therapy research in humans also were heard following the death of an 18-yearold participant in a gene therapy experiment at the University of Pennsylvania involving transferring DNA to treat a genetic defect that reduces liver function. However, as of this writing, this single death is the only one directly attributable to gene therapy research. It is hoped that future government oversight will not restrict genetic research unreasonably, as it holds such great promise for the eradication of life-threatening disease.
Database management for clinical research The choice of software tools for research data management depends on the complexity of the study design and the volume and types of data to
Biostatistical Methods in Hematopoietic Cell Transplantation
be collected. Options range from simple spreadsheets such as Excel for a very small straightforward study, to an Access database for a study of intermediate size and complexity, to MS SQL Server, SAS or Oracle databases for larger, more complex data collection efforts. For any approach chosen, however, some basic principles of database design hold true. Each entity (e.g. person) in the database requires a unique identifying code. More than one type of datum should not be mixed within the same data field, for example, entering both the value for weight and the code for kilograms as one data point within the field. Continuous values should always follow the same format and convention for data entry (e.g. white blood cells × 103), and categorical data should be entered using invariant coding schemes (e.g. M for male, F for female). Deviations from these basic rules make the ultimate data analysis and interpretation of the database difficult if not impossible without a major reworking of the entire dataset. Options for statistical analysis software also range widely, depending on the complexity of the required analyses and the expertise of the individual doing the programming. Data entered into an Excel spreadsheet can be manipulated directly within this system for simple statistics such as frequencies and means. Anything more complex than this is best run in an actual statistical analysis package, with the simplest being menu-driven systems such as Systat, and the more complex but powerful choice for biomedical research typically being S-Plus, R or, more typically, the SAS statistical package. Typically, few physicians have the time or inclination to learn to program these latter packages adequately, and would be better off consulting a biostatistician for analyses at this level of sophistication. In the future, the health-care institution within which clinical trials are being conducted is likely to be fully networked, facilitating the deployment of electronic data capture and decision-support tools, and allowing health-care providers and biomedical researchers ready access to electronic information for health care that could be used for biomedical research activities [75]. However, the real challenge in integrating data from electronic clinical care systems into a clinical trials data system has to do with the information access, data flow, coding schema (or lack of coded data in the case of free text information), compatibility of the rules and timing for data collection, comparability of field definitions, political and organizational boundaries, data security, and confidentiality. In large part because of all these challenges, the current clinical trials process is coordinated and monitored largely offline rather than dynamically, requiring duplicate data entry and resulting in inconsistencies, increased errors, and delays in reporting and analysis. Documentation of protocols is nonstandard, and there is no consistent means of determining eligibility for them. The health-care data network will need to be able to translate to and from any electronic medical record system to allow data to be collected at the point of use in a structured form to facilitate clinical trials and outcomes analysis. Further, unifying information “architecture” needs to be developed for cancer clinical trials, so that data can be exchanged across systems and institutions in a seamless fashion, based on protocols that are written in a standard format and structure. At City of Hope National Medical Center, we have developed a webbased system that delivers protocol information dynamically from our clinical trials database [76], and are working with the Association of American Cancer Institutes to achieve virtual exchange of protocol information nationwide. The National Cancer Institute’s Center for Biomedical Informatics also is working to harmonize the informatics approaches to cancer center clinical trials information management across the nation’s cancer centers through the caBIG initiative [77].
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Impact of genetic data on clinical trials information systems The rapid pace of future genetic discoveries and trials will result in extremely demanding data integration requirements. Much more flexible and responsive clinical trial data management systems will be needed to incorporate the high throughput of genomic data, while establishing the correct identifiers and appropriate key structure to be able to merge these data with incoming correlative clinical information. In addition to data from DNA or other genetic tissue from the participants in the dataset, a high-quality, large-sample clinical dataset with well-characterized participant and longitudinal follow-up for effects of treatments will be needed, both to store analyzable data for the clinical trials, and to provide the databanks for exploratory mining for future discoveries and new hypothesis generation [16]. A new generation of professionals with both biomedical and computer science skills will be required to provide advanced biomedical informatics and biostatistical expertise to build and use the critical tools for data retrieval, storage, classification retrieval, and correlative analysis capabilities for complex clinical and genetic data. With the advent of clinical trials that incorporate genomic components, the need for electronic data integration standards across systems and institutions becomes even greater. To facilitate sharing and pooling of genetic results worldwide, standards must be adopted for the coding and exchange of such data, and genetic protocol results will need to be published in a sharable electronic form, for merging into existing resources such as GenBank and OMIM.
Analysis of clinical research data Increasingly more sophisticated statistical techniques are required for HCT research, along with the application of sound basic biostatistical principles. The protocol needs to clearly define the primary and secondary endpoints to be analyzed, and the statistical plan for conducting the analyses. Certain biologic measurements may have more than one usage, and our understanding of the meaning of these endpoints may be evolving over time with new discoveries, such that definitions need to address how the information will be applied within the specific protocol. For example, the study of MRD generally aims to understand the biology and clinical significance of leukemia that persists in patients who are in complete pathologic remission [64]. Following marrow transplantation, the detection of MRD usually has been associated with subsequent relapse in childhood acute lymphoblastic leukemia, t(15;17)+ AML, and CML. However, MRD also has been detected in patients enjoying longterm remission. Therefore, the study of MRD has evolved from the objective of identifying patients at high risk for relapse, to explaining how an abnormality associated with leukemia can persist for years in an otherwise “cured” patient. This section on statistical analysis first covers some of the general principles and guidelines to be observed in the analysis of clinical research data, and then discusses more specific forms of analysis that frequently are applicable to HCT data. The intent-to-treat principle Biases and misinterpretations can be caused by careless use of the definitions of censoring in the statistical analysis, such as assuming that losses to follow-up due to withdrawal from the study are not related to differential risk of the endpoint. More obvious risks of bias can arise through the common practice of eliminating patients from the analysis after entry into the study by declaring them to be “inevaluable” for the primary study endpoint. The argument is that if the purpose is to deter-
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mine whether a new treatment is effective in forestalling or preventing disease, a patient who fails to go through the assigned cycle of treatment (e.g. because of failure to take the full course of drugs) should not be regarded as contributing valid information on the question. While the concept itself is valid, it is clear that deleting patients who are unwilling or unable to take the assigned regimen provides only one picture of the difference between treatment efficacies. Including in the comparison all patients thought to be candidates for the treatments at the time of randomization can provide quite a different view, and answer quite a different question. The inclusion of all patients in the analysis is called the “intent-totreat” approach, for obvious reasons. Both approaches are reasonable, but the plans and logic should appear in the protocol. Generally, the FDA will want an intent-to-treat analysis because the treatment difference expected in practice, taking valid account of drop-outs in actual practice, is most relevant in approving a new drug for the market.
Subgroup analyses Another common pathway to faulty conclusions lies in searching for possible treatment differences by analyzing multiple subgroups of patients, with the idea that although the new treatment may not be efficacious for all patients who are believed to be reasonable candidates for the study, it may be efficacious for certain subgroups (e.g. women versus men, younger persons versus more frail older persons, or patients with less serious disease versus more advanced disease). Planning such searches and the specific analyses at the writing of the protocol is wise. Unplanned searches at the end of the study will increase the possibility of uncovering differences that are due to random variation alone, and hence risk erroneously concluding that the treatment is effective in some subgroup. If such searches are planned beforehand, the size of the study can be enlarged to minimize the chance of such errors in looking at subgroups. Investigators should be clear in reporting the results of clinical trials to point out the degree to which conclusions may have arisen through unplanned searches for subgroup treatment differences, and readers should be aware of the possible dangers of undisclosed subgroup analysis.
Interim analyses The protocol should clearly state the plan for accruing patients to the trial, the target time and number of patients to be accrued, and the plan for stopping the trial and carrying out the target analysis. However, it is common to analyze the data at some preliminary points in the accrual and follow-up of the patients. Interim evaluations are conducted to protect patients against unexpected excessive or severe toxicities, and to allow earlier termination of the trial if that seems wise, either for unforeseen difficulties in execution of the trial, or for data suggesting a large and persuasive advantage in efficacy for the experimental treatment in a randomized study. Here it is important to protect both the patient and the study against overinterpretation of what may be chance variations in early data (see the section on DSMBs and also a comment in the next section, on statistical guidelines for early stopping of clinical trials). Group sequential methods call for analyzing accumulating responses in groups as they become available. The strategy is to identify a priori stopping boundaries at each interim analysis time point that preserve the desired type I and type II statistical errors. An adaptive design is one in which the data accumulating in a trial are examined periodically – or even continually – with the goal of modifying the trial’s design depending on what the data show about the unknown hypotheses. Among the
possible modifications are stopping early, extending the trial beyond its original sample size if some of the assumptions in the original design are not being met, reducing the power, dropping arms or doses, and adding arms or doses. Although adaptive designs can be conducted using the frequentist approach, in classical models flexibility is penalized and adaptation is difficult to effect, while the Bayesian approach appears more flexible, although most still require that the frequentist operating characteristics at least be simulated to provide adequate control of type I error. Berry gives a general introduction to the Bayesian approach and describes its role in clinical trials [78,79]. Returning to the example of the accelerated FDA approval of Gleevec, the protocol-specified analysis was to have occurred at 5 years. However, an interim analysis conducted after a median follow-up time of 14 months revealed a statistically significantly lower time to treatment failure (i.e. disease progression) in the Gleevec arm over the arm receiving interferon and cytarabine. It is important to note that because FDA approval for use of Gleevec in CML patients was based on an early interim analysis, the long-term effects of treatment with Gleevec were largely unknown initially.
Equivalency testing While the majority of HCT trials are interested in demonstrating a significant difference in efficacy, equivalency studies also are being reported more often in HCT, to demonstrate that there is a nonsignificant difference in efficacy. In such a setting, the classical test of significance can be inappropriately applied, with the danger of incorrectly accepting the null hypothesis of no difference solely because of inadequate sample size. If one wishes to use significance testing, the appropriate “null” hypothesis is that the standard therapy is more effective than the experimental therapy by at least some specified amount [80,81]. Since correct interpretation of the significance tests relies heavily on sample size, and statistical significance is often overemphasized as a binary decision rule, some investigators conduct equivalency trials using confidence intervals [82]. This approach is more intuitively appealing, and allows for sequential monitoring using repeat confidence intervals. It is impossible to demonstrate that a new treatment is exactly equivalent to a standard treatment. Therefore, to examine therapeutic equivalence, a maximum “acceptable” difference (delta) in the effectiveness of two treatments must be specified in advance. With a fatal outcome (e.g. overall survival), the new treatment can only be considered equivalent to the standard if no more than a very small decline in efficacy is allowed; with less toxic outcomes, larger differences may be clinically acceptable. Other parameters that must be specified are the confidence level (alpha) for the upper limit of the true difference between the new and the standard treatment, and the probability (1 − beta) that the confidence limit for the true difference will not exceed the specified value of delta [82]. Because the acceptable difference between two equivalent treatments usually is small (particularly in contrast to the larger differences desired when demonstrating superior therapeutic efficacy), the required sample sizes tend to be large. Whenever the common response rate is greater than the quantity (1 + delta)/2, sample sizes for equivalency testing are lower than those required if the same trial were to be (inappropriately) conducted using the traditional significance testing approach. Frequently in HCT studies, if the response rate is too low (e.g. less than 50%) investigators are more likely to conduct efficacy trials in an attempt to identify better treatments. It is only when response rates are satisfactorily high or toxicity is severe and the intervention is primarily intended to lower toxicity, that equivalency tests become of paramount importance.
Biostatistical Methods in Hematopoietic Cell Transplantation
Analysis of recurrent states The statistical discussions so far apply with events that occur once during follow-up, for example the risk of first progression after HCT, death, or first occurrence of GVHD. However, often there is interest in not only the occurrence of a state (e.g. GVHD, pulmonary infection or hospitalization for any reason), but also how long it persists and how often it recurs. Describing such data for a group of transplant patients followed over time, with censoring, and comparing groups of patients, present statistical challenges. One measure of the importance of the state of interest (e.g. GVHD) is the proportion of surviving patients, uncensored by specific definition in the protocol, who are presently in that state at each time post transplant. This can be estimated by the prevalence curve [83], taken from Storb et al. [84] and shown in Fig. 28.5, which provides an estimate of the prevalence of chronic GVHD among disease-free surviving patients who received prednisone and in those that did not. The figure also indicates that chronic GVHD is higher among the patients who had earlier developed acute GVHD. However, as the investigators point out, one must be cautious in drawing causal conclusions, as the groups compared are defined by events that could be linked causally to the occurrence of chronic GVHD in ways that might obscure or complicate the interpretation. Examples would be the relationship between survival and the use of prednisone, or the preceding occurrence of acute GVHD and its concomitant implications for prednisone treatment and survival. As another technique for states that may recur and are sequential in nature, the probability that a patient is in a given state can be modeled by Markov or semi-Markov multistate models. These models have been
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used by Klein et al. [75] and Keiding et al. [85] to study the antileukemic effect of GVHD on relapse and death in remission following transplantation. Surrogate endpoints Cancer clinical trials often experience a problem in recruiting patients to studies, and/or sustaining the length of follow-up necessary to accumulate definitive endpoints, such as overall survival. In addition, subjects usually branch to alternative therapies upon progression, increasing patient variability, and new therapies arise, limiting conclusions. Studies of AIDS are even more difficult, as subjects are known to jump from one experimental study to another before disease progression. This prompts the statistical considerations of earlier endpoints as substitute or “surrogate” markers of later fatality, with the idea of drawing reliable conclusions about comparative risks of death from earlier results. Disease progression in cancer and in HCT has been used as a surrogate marker for length of life, but with some reservations about the reliability in translating progression comparisons to valid prediction of differences in mortality and RR. As more is learned about early markers in HCT, the interest in time-efficacy in research becomes more acute, and interest in the use, of early markers as surrogates to guide research grows. Two useful papers in this area, one by Prentice [86] that deals with statistical aspects of the definition and operational use of surrogate markers, and another by Ellenberg and Hamilton [87] on surrogate markers in cancer, provide good introductions to these concepts and related applications. Analysis of time to an event
1.0
Grade O Acute GVHD
Prednisone
0.5
No Prednisone 0 Grade I–IV Acute GVHD
1.0
Prednisone 0.5
No Prednisone 0
0
1
2
Years after Marrow Graft
Fig. 28.5 Prevalence curves for active chronic graft-versus-host disease. (Reproduced from [84], with permission.)
3
While most phase I and II studies focus on a binary end point, such as toxicity or response to therapy, phase III studies (and some phase II transplant studies) typically concentrate on longer-term events and the time to those events, such as time to progression of disease or survival time after transplant. The data for a given patient will include the time from HCT to occurrence of the event or to the time of data analysis, if the patient is treated successfully. Because the times of transplant vary over the study accrual period, each patient will have his or her own data point consisting of the time from HCT (or study entry) to event or end of the study at time of analysis. There also will be information as to whether or not the patient experienced the event of interest (e.g. relapse or death) by the time of analysis. The patient who avoids the event by the time of analysis is said to have a “censored” data point, as all that is known for that patient is that the event has not happened thus far. This form of censoring is called “administrative” censoring, because the incomplete information on the patient is due to the administration of the study, rather than to any irregularities in the data collection. More information will not be known about the censored patients unless the study is extended by further follow-up of all surviving patients and is reanalyzed. When allowing additional follow-up, it is, as noted earlier, critical to avoid passive follow-up due to the bias created. In comparing the results for two groups containing censored data points, standard methods for comparing means, such as the two-sample t-test, should not be used, as they do not take into account the censored aspect of the data. Methods for handling censored data are covered in several statistical texts [34,35], and texts that describe methods that are more advanced yet intended for the clinical scientist rather than the statistician also are available [88–90]. Often an extended time of follow-up is required to observe sufficient endpoints to allow a statistically precise determination of the relative superiority of one or the other treatment. Long periods of study will necessarily mean that some patients will be lost to follow-up due to other
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than “administrative” censoring, for example moving away from the study site or decision of the patient to leave the study for personal reasons. These losses to follow-up will also result in censored data points, and are usually treated in the same way as administratively censored data points in the analysis. However, losses to follow-up for reasons other than administrative censoring can be due to reasons that build biases into the study and the analysis. If the patients tend to drop out of one of the treatment arms for reasons that might be associated with the treatment itself and/or with the risks of the endpoint of interest (e.g. due to excessive toxicity combined with patient refusal for follow-up), an analysis that treats this as administrative censoring has these biases built into it. The analysis may then be misleading, particularly if such biased losses to follow-up occur relatively frequently. On the other hand, administrative censoring should not create a similar possibility of bias, as the patients are blindly and randomly assigned to the treatments over time. This form of censoring is considered to be “uninformative,” meaning that the reasons for censoring should not be expected to be related to the risk of event if the patient were to stay in the study. Kaplan–Meier survival analysis The familiar Kaplan–Meier graph is used for describing the typical censored clinical trial dataset, plotting estimates of the probability of “surviving” to time “t” post transplant (i.e. the probability of not experiencing the endpoint, e.g. progression, from transplant to time t). The graph indicates the times of death by dropping down a fraction of its value at each time of death (the event of interest). The surviving (censored) data points typically are denoted by hash marks on the survival function. In HCT studies, the survival pattern differs from more common survival distributions, with a high early mortality rate followed by a plateau in mortality after transplantation. Unfortunately, interest is often highest exactly where the data are least informative – at the end of the curve, where few patients are still at risk. This uncertainty should be quantified by reporting either pointwise confidence intervals or confidence bands. Standard errors can be computed for the Kaplan–Meier curve at any time point, and the estimated survival curve graphed with a plus and minus distance (confidence limits) around the curve at a few time points.
interval-censored data, and Kim et al. [95] for doubly-censored data. Even when technically incorrect, most HCT and cancer trials report survival analysis as if the event in question (e.g. progression or relapse) occurred on the last day of the interval. Fortunately, randomization protects against the influence of this approximation. In nonrandomized studies, the maximum influence is based on the surveillance interval. As long as clinically relevant differences are much larger than the surveillance window, the influence is limited, and as a result this is generally a bigger problem for nontransplant studies. Weighted Kaplan–Meier estimator for matched data. Studies on the comparison of transplantation with respect to standard therapy present a number of statistical challenges: they usually are not randomized, often are retrospective (based on registry data), and the treatment assignment is time dependent (waiting time to transplant). These challenges motivate the use of matching on a set of known prognostic factors and the waiting time to transplant, as an approach to identify a set of “controls” from the group of nontransplanted patients. Usually, the latter is a larger group than the transplanted one, and thus is likely to have more than one control for many of the cases. When a variable number of patients treated with conventional therapy matches each transplanted patient, the standard estimating and testing procedures need to be modified in order to account for the fact that matched data are highly stratified, with strata containing a few, possibly censored, observations. A weighted version of the Kaplan–Meier estimator, which accounts for a variable proportion of matching, has been proposed and its statistical properties studied [96]. The problem of the comparison of the survival experience in the two treatment groups was addressed, and two tests, based on the distance between the survival estimates calculated at a prefixed time point, were examined through simulations, the aim being that of obtaining an unbiased estimate of the effect of HCT. The procedures proposed were applied to data collected from an Italian study whose aim was the evaluation of HCT compared with intensive chemotherapy for pediatric patients with acute lymphoblastic leukemia. Although the focus was on a retrospective observational study, the methods proposed also are applicable to prospective studies. The log-rank test
Analysis of multivariate interval-censored survival data. Multivariate failure time data are observed in biomedical studies when subjects are followed for the occurrence of multiple events, such as recurrent infections or GVHD. In some studies, the multivariate events can be interval censored [91]. Interval-censored survival times occur when the outcomes are not directly observable but are detected from periodic clinical examinations or laboratory tests, such as periodic PCR testing for positivity for the Ph chromosome, or cytogenetic testing for signs of disease relapse. In interval censoring, the exact times of events are not known since the events could have happened at any time during the interval between the last visit when the subject was determined to be negative for the outcome and the first positive visit. Also, the timing and width of the interval often differ across subjects. Other examples of multivariate interval-censored data are from time to multiple tumor recurrences, which are usually diagnosed from periodic clinical examinations. If all study subjects were monitored according to the same examination schedule and no appointments were missed, this type of data could be treated as multivariate grouped survival data and analyzed using the approach of Guo and Lin [92], a grouped-data version of Wei et al.’s method [93]. However, the approach is not applicable to the more common situation when there are missed visits. In the proposed approach, the marginal distributions are based on a proportional hazards model for discrete survival data that has been utilized by Finkelstein [94] for singly
The analysis for comparing two curves that represent two treatment groups in a phase III study is usually carried out by a log-rank test of statistical significance (sometimes called the Cox actuarial or proportional rank analysis), and an RR is calculated, providing an estimate of the risk of failure in one group compared with the risk of failure in the other. This single number is based on the assumption that the RR between the two groups does not change over time. Several texts describe these calculations in detail [34,35,88–90]. If the RR is greater than 1, the data favor the control group; if it is less than 1, the experimental group is favored. The 95% confidence interval for the RR is preferred to simply the RR and the log-rank p-value. In the accelerated FDA approval of Gleevec mentioned earlier, the risk of progression was compared by the log-rank test. The RR was calculated to be 0.183, with a 95% confidence interval of 0.117–0.285, and a p-value much less than 0.001. The results from such a log-rank analysis might be represented by the layout as found in Table 28.6. The
Table 28.6 Results of log-rank analysis Treatment group
Relative risk
95% confidence interval
p-value
Gleevec
0.183
(0.117, 0.285)
15%). • Use: hat, long sleeve shirts and pants if outside for long period of time. • Avoid contact with people with respiratory illness or other transmissible diseases. • Autologous and teenage patients should be discussed separately. Discuss with your physician when restrictions may apply to your child’s care after one year post transplant. Reproduced with permission from the Fred Hutchinson Cancer Research Center Long-term Follow-up Program.
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Long-term recovery (Table 31.14) Nurses, under the guidance of the transplant center medical team, can successfully act as liaison between the referring physician and the transplant physicians. They can collect important and pertinent information
to triage transplant-related problems and, after discussion with the transplant physicians, relay possible solutions to the team providing care at home. One organized approach is to conduct long-term follow-up rounds on a regular basis to discuss nonurgent problems. Issues most frequently requiring the expertise of the transplant team are the diagnosis and treat-
Table 31.14 Long-term recovery Assessment Fatigue Pain Functional status Complete blood count Kidney function Liver function Magnesium level Drug levels Chronic GVHD symptoms (allogeneic patients): • Skin: erythema, dryness, macular or urticarial rash and pruritis, hyperpigmentation, vitiligo, mottling, lichenoid plaques, hyperkeratosis, exfoliation scleroderma or morphia • Mouth: dryness, mucositis, erythema, lichenoid changes, striae, tightness around the mouth, sensitivity to hot, cold or spicy foods, gingivitis • Eyes: dryness, grittiness, blurring, excessive tearing, photophobia and pain • Liver: elevated liver function tests not attributable to other causes and sometimes jaundice • Gastrointestinal system: anorexia, nausea, vomiting, diarrhea, dysphagia, malabsorption, weight loss • Lungs: cough, wheezing, dyspnea on exertion, history of recurrent bronchiolitis or sinusitis, bronchiolitis obliterans • Nails: ridging, onychodystrophy, onycholysis • Hair: premature graying of scalp hair, eyelashes, eyebrows, thinning of scalp hair, alopecia, decreased body hair • Vagina: dryness, dyspareunia, stricture or stenosis, erythema, atrophy or lichenoid changes not induced by ovarian failure or other causes • Myofascia: stiffness and tightness with restriction of movement, sometimes with swelling, pain, cramping, erythema, and induration, most commonly affecting the forearms, wrists and hands, ankles, legs, and feet, contractures • Muscles: proximal muscle weakness, cramping • Skeletal: arthralgia of large proximal girdle joints and sometimes smaller joints • Serosal: unexplained effusions involving the pleural, pericardial or peritoneal cavities • Thrombocytopenia, eosinophilia, hypogammaglobulinemia • Energy level: unusual fatigue Medication compliance Nutritional needs Symptoms of infection Hormonal issues Neurological symptoms Sexual dysfunction Teaching Symptoms to be reported: skin changes, nausea, anorexia, weight loss, diarrhea, dysphagia, dry or sensitive mouth, dry, gritty eyes or excessive tearing, coughing, wheezing or shortness of breath, thinning of scalp hair, ridged nails, loss of range of motion or stiffness, vaginal symptoms When to report fever How to access medical help after hours Review of infection prevention Importance of medication compliance Importance of reporting abnormal lesions or lumps, bowel changes or abnormal vaginal bleeding Importance of regular exercise Care coordination Health-care provider appointments Medication administration Laboratory draws Return visit to transplant center at 1-year anniversary of transplant and thereafter as needed Vaccinations after the first year Allogeneic patients: • Plans for work-up of chronic GVHD symptoms based on assessment • Plans for treatment of chronic GVHD GVHD, graft-versus-host disease.
Nursing Role in Hematopoietic Cell Transplantation
ment of chronic GVHD, pulmonary complications, gastrointestinal symptoms, engraftment issues, treatment of serious and life-threatening infections, booster immunization, recurrent disease, and the development of secondary malignancies [33]. The need for the transplant team to act as a resource may persist from a few months to several years. While patients who live at a distance from the transplant center benefit from periodic follow-up visits to the transplant center, the general oncologist or hematologist at home can provide the routine follow-up care. Timely and consistent communication between the transplant centers and community practices is essential for safe continuity of care [34]. Transplant nurses can also provide ongoing support in the outlying communities by offering educational programs and by sending out educational material when the need is recognized. Facilitation of support groups is another way for transplant nurses to provide ongoing education and support for local patients in the long-term recovery phase. If the transplant center is involved in ongoing research, the research nurse plays an integral role in continuing with the collection of data and following research protocols. In some research centers, patient health questionnaires are regularly mailed to post-transplant patients to update demographics, quality-of-life information and survival documentation. Research nurses can triage problem issues that are identified from the questionnaires.
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The role of the nurses working with the HCT patients during longterm recovery requires patience and perseverance when considering the recurring nature of chronic GVHD and other long-term issues. As in other phases of HCT nursing, the work is rewarding and challenging, and it allows for the establishment of long-term and trusting relationships. While nurses working in the office setting may find the problems of this population to be daunting at times, the eventual recovery and return of patients to a satisfying and full life is a reward experienced by those who are fortunate enough to work with these patients.
Nursing practice issues in hematopoietic stem cell transplant Various nursing roles are needed to successfully care for transplant patients. The organization of nursing roles usually parallels that of the center in which the program resides and is based on that institution’s nursing philosophy and care delivery model. In the ambulatory setting, nursing models must complement the medical care delivery model for successful care. For example, primary care in the outpatient setting is often structured by having a specific RN work with a specific provider caring for one group or team of patients. See Table 31.15 for descriptions of the various nursing roles.
Table 31.15 Nursing role descriptions in hematopoietic stem cell transplantation Nursing title
Role description
Inpatient staff RN
Cares for patients requiring acute care. Includes assessment, patient teaching, and carrying out the medical care plan. Inpatient nurses may care for patients on a designated transplant unit or on an oncology unit
ICU RN
Cares for patients requiring critical care. Includes assessment, patient teaching, and carrying out the medical care plan. ICU nurses may care for patients in an ICU incorporated into the transplant unit or in a main hospital ICU
Nurse coordinator
Coordinates pretransplant preparations including donor identification. Initiates patient education regarding transplant process. May continue as case manager during patients’ transplant course. This position is required of National Marrow Donor Program designated centers
Discharge planning RN
Assists team in planning and coordinating patients’ discharge from hospital to ambulatory setting. Provides patient and caregiver education required to manage care at home
Outpatient clinic RN
Cares for patients requiring ambulatory care. Includes assessment, triage, care coordination, patient teaching, and carrying out the medical care plan
Outpatient infusion RN
Cares for patients receiving infusions and transfusions. In many centers the stem cell infusion occurs in the outpatient infusion unit
Home infusion RN
Cares for the patients receiving infusions in the home setting. Educates patients and care providers to administer intravenous solutions in the home
Apheresis RNs
Cares for patients and donors undergoing apheresis procedures
Long-term follow-up RNs
Cares for patients after the acute phase of transplant. Provides education and triage for ongoing complications or late effects of transplant
Research RNs
Partner with the principal investigator to implement research protocols. Coordinates research activities with primary care team
Nurse manager
Responsible for nursing care delivered in a specific unit or clinic. Ensures appropriate staffing levels for the safe delivery of care
Clinical nurse specialist
Responsible for advancing transplant nursing practice by promoting evidence-based practice, conducting nursing research, creating nursing care guidelines, and evaluating outcomes of nursing care at the population level
Nurse educator
Responsible for orientation and continuing nursing education for transplant nurses
Nurse practitioner/physician’s assistant
Provides medical care under the direction of the attending physician. Performs procedures such as lumbar punctures and bone marrow biopsies
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Achieving and maintaining an expert nursing staff is a constant challenge for institutions with HCT programs. Initial orientation of a nurse with oncology experience generally takes 6–8 weeks. After this time, the nurse can be counted in staffing patterns but is not considered fully competent until he or she has completed their first year. The nurse-topatient ratio is also an issue that must be considered when planning a HCT unit. Most inpatient units staff on a one nurse to two or three patient ratio. This is usually a much lower ratio than on other oncology units and may need to be justified to hospital administrators. Patients develop strong feelings of trust and confidence in specific nurses, and facilitating work schedules to maximize these relationships can be a challenge for nurse managers. Continuing education must be built into HCT nurses’ work schedules. This education includes updates and refreshers on nurse practice topics as well as education on new medical protocols. It is also important for the nurses to remain up to date with the results of medical research. Each unit should identify topics of high risk and high complexity that qualify for annual competency review and testing. Examples on an inpatient unit include “Administration of high-dose chemotherapy” and “Administration of stem cells.” Examples in an outpatient clinic include “Steroidinduced diabetes teaching” and “Donor screening.” It is important for HCT nursing leaders to have a proactive strategy to prevent “burnout” in their staff. These nurses are at high risk for experiencing job-related stress, moral distress, and compassion fatigue as they work in fast-paced units with acutely ill patients with uncertain outcomes. Flexible scheduling and supporting time off requests assist many nurses to keep their resiliency to continue to work with these patients. It is also important that the nurses have time to share their experiences with each other as they may not be understood by other support people in their lives, such as family members or even other health-care professionals. The need for HCT nurses to network on a national level had been identified since the 1980s, when many transplant units began. Nurses in
the initial transplant centers organized national conferences, including conferences in Seattle, Minneapolis, and Omaha. The conferences were very well attended but quite a stress on the resources of the individual centers. In 1989, the Oncology Nursing Society acknowledged the unique needs of several subspecialties and created “Special Interest Groups.” The Blood and Marrow Stem Cell Transplant (BMSCT) Special Interest Group of the Oncology Nursing Society is one of the largest. In 1998, the Center for International Bone Marrow Transplant Research/American Society for Blood and Marrow Transplantation agreed to have a concurrent nursing conference with their medical conference sponsored by the BMSCT Special Interest Group. This conference has been planned by this group for over a decade, and has grown from a half day to 3 full days.
Summary Expert nursing care is essential to the success of a HCT program. Dr E. Donnall Thomas, founding Medical Director of the Fred Hutchinson Cancer Research Center’s Transplant Program and recipient of the 1990 Nobel Prize in Physiology and Medicine, called transplant nurses “his secret weapons” [35]. Nurses who are drawn to this subspecialty within oncology, and are successful in mastering the required skills, are rewarded by intense, intimate, and long-term relationships with the patients during this time of crisis in their lives. There is great satisfaction in knowing that they have made a positive difference in the lives of these patients.
Acknowledgment The authors would like to acknowledge Judy Campbell and Juanita Madison for their contributions to the original chapter published in the third edition of this text.
References 1. Ford R, McDonald J, Mitchell-Supplee K, Jagels B. Marrow transplant and peripheral blood stem cell transplantation. In: McCorkle R, Grant M, Frank-Stromborg M, Baird S, editors. Cancer Nursing: A Comprehensive Textbook. Philadelphia: WB Saunders; 1996. pp. 504–30. 2. Woltz P, Castro K, Park BJ. Care of patients undergoing extracorporeal photopheresis to treat chronic graft-versus-host disease: review of the evidence. Clin J Oncol Nurs 2006; 10: 795–802. 3. Franco T, Ford RC. Models of care delivery for hematopoietic stem cell transplant patients. In: Ezzone S, Schmit-Pokorny K, editors. Blood and Marrow Stem Cell Transplantation: Principles, Practice, and Nursing Insights, 3rd edn. Boston: Jones & Bartlett; 2007. pp. 423–9. 4. Kelley CH, Randolph S. The role of the homecare nurse throughout the continuum of blood cell transplantation. J Intraven Nurs 1998; 21: 361–6. 5. Holmes W, Kapustay PM, Walker F, Williams L, Ezzone S, editors. Peripheral Blood Stem Cell Transplantation: Recommendations for Nursing Education and Practice. Pittsburgh, PA: Oncology Nursing Press; 1997. pp. 1–44. 6. Nelson JP. The blood cell transplant program. Semin Oncol Nurs 1997; 13: 208–15. 7. Grant M, Cooke L, Bhatia S, Forman SJ. Discharge and unscheduled readmissions of adult patients undergoing hematopoietic stem cell transplanta-
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Nursing Role in Hematopoietic Cell Transplantation 21. Crawford SW. Critical care and respiratory failure. In: Thomas ED, Blume KG, Forman SJ, editors. Hematopoietic Cell Transplantation, 2nd edn. Malden, MA: Blackwell Science; 1999. pp. 712– 22. 22. Horak DA, Forman SJ. Critical care of the hematopoietic stem cell patient. Crit Care Clin 2001; 17: 671–95. 23. Saria MG, Gosselin-Acomb TK. Hematopoietic stem cell transplantation: implications for critical care nurses. Clin J Oncol Nurs 2007; 11: 53–62. 24. Plunkett P. Ethical issues in transplantation. In: Whedon MB, Wujcik D, editors. Marrow and Blood Stem Cell Transplantation: Principles, Practice, and Nursing Insights, 2nd edn. Boston, MA: Jones & Bartlett; 1997. pp. 506–23. 25. Hagen SA, Craig DM, Martin PL et al. Mechanically ventilated pediatric stem cell transplant recipients: effect of cord blood transplant and organ dysfunction on outcome. Pediatr Crit Care Med 2003; 4: 206–13. 26. Afessa B, Tefferi A, Dunn WF, Litzow MR, Peters SG. Intensive care unit support and acute physiol-
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David S. Snyder
Ethical Issues in Hematopoietic Cell Transplantation
Introduction Many of the ethical issues raised in hematopoietic cell transplantation (HCT) are common to those involved in other forms of medical therapy utilizing advanced technologies. These issues are informed by the traditional principles of patient autonomy and the importance of informed consent and confidentiality; justice, that is, the fair allocation of limited resources; beneficence and nonmaleficence; and fidelity or nonabandonment when the goals of treatment shift from cure to supportive care. However, certain unique features of HCT compound the complexity of these ethical issues, including: the young age of most patients; the fact that most of the diseases are life-threatening and often fatal within a short time in the absence of an HCT, yet are potentially curable with this treatment; the fact that a living donor is required who is often a minor; the ability to select potential donors for an affected sibling by utilizing preimplantation genetic diagnosis; the great financial expense of the procedure and controversies about reimbursement; and the fact that survivors of HCT may be cured of their primary malignant disease but may face other serious complications, such as sterility, chronic graftversus-host disease (GVHD), psychologic distress, sexual dysfunction, cognitive impairments, and occupational disability. In this chapter, some of the ethical issues in HCT are reviewed under the following general headings: who is eligible for HCT, informed consent for the patient, informed consent for the donor, alternative sources of stem cells, HCT for nonmalignant diseases, and end-of-life issues.
Who is eligible for HCT? Case scenario 1 A 49-year-old man with stage II multiple myeloma has a human leukocyte antigen (HLA)-identical sibling. He is eligible for a protocol utilizing tandem autologous HCT followed by a reduced-intensity regimen allogeneic HCT. His physicians feel this approach represents his best chance for long-term, disease-free survival and possible cure of his myeloma. His insurance plan considers this treatment plan experimental, and will only authorize a single autologous HCT. Case scenario 2 A 32-year-old man with chronic myeloid leukemia (CML) has no sibling donors available for allogeneic HCT. Although his company’s insurance Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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plan covers the costs of procurement and the actual transplant for a matched unrelated donor HCT, it will not pay for the search costs, which the patient cannot afford. Although the company sponsors a donor drive in the name of this patient, he cannot access any of the donors who might be identified. These two scenarios, which are based on actual cases, help to highlight some of the difficult issues involved in determining who undergoes an HCT procedure. This process involves both medical decision making based on clinical and protocol-determined eligibility criteria and thirdparty payer decision making. Each of these processes is ethically complex. First, the medical decision making: in the interest of justice, it is imperative that transplant teams develop standardized sets of criteria by which patients are selected for HCT. Some of these criteria are straightforward, such as the patient’s diagnosis and stage of disease, age, and adequacy of major organ function. Other criteria are more subjective and often relate to psychosocial issues that may influence the patient’s ability either to tolerate the HCT procedure or to comply with the long-term follow-up regimen. Multidisciplinary teams are needed that include not only physicians, but also nurses, psychologists, and social workers. It is important to avoid arbitrary and inconsistent decision making in deciding when not to offer HCT to a patient. For example, an HCT center may decide that a young patient with acute leukemia who has Down’s syndrome is not an acceptable candidate because of expected psychosocial management problems that might compromise the outcome. In one survey of 58 pediatric HCT centers, 16 leukemic children with Down’s syndrome had been transplanted at 10 centers. This was only about 20–25% of the predicted number based on incidence data. In fact, the outcomes were no different than those expected from a similar cohort of patients with acute leukemia who did not have Down’s syndrome. The author cautioned against physician bias toward these patients [1]. In an accompanying editorial, physicians were advised to use a utilitarian approach, that is, to recommend against HCT for children with Down’s syndrome only if data show poorer outcomes for them. Many HCT teams are concerned that patients with a history of active substance abuse may be poor candidates because of expectations of higher complication rates and poor compliance in their long-term care. A retrospective study from one transplant center identified 17 of 468 HCT patients as lifetime substance abusers (alcohol in 71%, marijuana in 30%, and opiates in 30%). When these 17 patients were paired with matched control subjects, a significant difference in survival probability was seen, 60% versus 10% (p = 0.0022) [2]. More data may be needed to substantiate these findings, but they do support the current practice at many HCT centers of excluding such patients. A survey of 597 HCT professionals (physicians, social workers, and nurses) was conducted in which 17 case vignettes were posed that
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highlighted a range of psychosocial issues that could potentially impact the long-term outcome of the transplant procedure [3]. The issues included suicidal ideation, use of addictive illicit drugs, a history of noncompliance, and others. At least 10% of responders indicated they would not proceed in all 17 vignettes, but there was a lack of unanimity for most of the cases in terms of recommendations to proceed or not. The decision making seemed contingent on perceptions of the severity and currency of the psychosocial issue and the ability to manage the patient’s potential noncompliance. A challenging case was recently published in which the issue of whether an incarcerated minor with relapsed acute leukemia should be offered an HCT, and who should pay for it [4]. This type of case highlights the sharp conflicts between a physician’s obligation to provide optimal care versus social justice. Is it cruel and unusual punishment to deny a prisoner a potentially life-saving treatment when the crime was not judged to be severe enough to justify the death penalty? Is it right for the state to pay for such a procedure when other law-abiding citizens without insurance or other resources are denied this type of treatment? There is a need for Federal and statespecific guidelines to establish when it is appropriate medically and socially for prisoners to receive technologically advanced and expensive healthcare. Decision making regarding third-party payer reimbursement is seen as a conflict over fairness and justice. Physicians as patient advocates are often in conflict with insurers who argue that HCT for certain diagnoses and disease stages is experimental and unproven. Physicians often believe that it is unfair to have patients accepted or denied access based on geography, their position in society or their ability to pay when a therapy is investigational, high cost or both. In analyzing patterns of utilization in four states, Mitchell et al. found that black patients, those enrolled in health maintenance organizations, those covered by Medicaid, and self-pay patients were less likely to receive HCT when admitted for either leukemia or lymphoma [5]. Insurers believe that it is unfair, and uneconomical, to be pressured by physicians and patients to pay for care that is not standard therapy [6]. Uniform and fair methodologies are needed to bridge these two views, rather than relying on costly litigation [7]. One approach is taxonomic, with a treatment modality labeled as experimental or standard. Thirdparty payers would cover experimental therapy if it met certain criteria, such as a phase III trial approved by the appropriate government agency, for example the Food and Drug Administration, and if the treatment were likely to benefit the patient. The treatment should be medically necessary, safe, and effective. The therapy must be as beneficial as any established alternative, and the improvement must be attainable outside of the investigational setting [8]. It is necessary to balance the premature dissemination of poorly studied, toxic, and expensive therapies to desperate patients by accepting limitations to HCT on the one hand, and by accepting that the patients’ best interests may be served by an experimental therapy on the other. Legal authorities caution that, although insurers may deny autologous transplant for a patient with cancer because the treatment is experimental, the more relevant question may be, “Is it the best option available for that particular patient with that particular disease?” The controversy in the recent past related to autologous HCT for breast cancer exemplifies these dilemmas. Welch and Mogielnicki highlighted some of the lessons that could be gleaned from this experience, including: (1) it is premature to discuss the cost-effectiveness of an intervention when its clinical effectiveness is unknown; (2) the National Institutes of Health should have an important role in determining what is experimental therapy; (3) public officials should not mandate coverage in the absence of clear data; and (4) the news media watchdog role should be extended to health care [9].
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An alternative approach is to reach a consensus based on available data of efficacy, duration of expected benefit, and the quality of wellbeing after the treatment [10]. Panels of expert physicians and insurers could review available data on a regular basis and develop grids that designated which diagnoses and disease stages would be covered, based on outcome analyses. Different categories could be considered, such as (1) diseases for which HCT is curative and may be the only curative therapy available, for example CML; (2) diseases for which HCT may not be curative but definitely prolongs survival, for example autologous transplant for CML, and possibly breast cancer and multiple myeloma; and (3) diseases for which the benefits of HCT are not yet known, for example autologous transplant for autoimmune diseases such as multiple sclerosis. One model is the Oregon Medicaid system, which adopted such an approach and covered HCT for all major hematologic malignancies including chronic lymphocytic leukemia, but did not cover chemotherapy for metastatic renal cell carcinoma. Such an approach is followed in California for Medi-Cal patients and by Blue Cross/Blue Shield. Another component of this approach is to restrict payments to “centers of excellence” that have demonstrated track records in their field. One of the most difficult challenges that the medical profession and the public at large face is how to assure that progress in developing new therapies will continue in a managed care system designed to minimize health-care expenses and to reimburse only for medically necessary and proven treatments. Who is going to pay for ongoing research that will lead to the breakthroughs of the future? One could argue that third-party payers have an ethical obligation to participate in this process by helping to underwrite the expenses involved in clinical research, and thereby to help establish which new treatment modalities are in fact effective and superior to established methods. Such new therapies may in fact be cheaper in the long run if they lead to definitive cures for patients and obviate the need for years of expensive, supportive chronic care.
Informed consent for patients undergoing HCT Case scenario 3 An 18-year-old woman with relapsed acute myelogenous leukemia (AML) has an HLA-matched brother available for allogeneic HCT. She has expressed an interest and willingness to undergo a transplant. However, during the informed consent process, she refuses to listen to any discussion about possible risks and complications from HCT. Can her doctors assume that she has given her consent and proceed to HCT? Respect for patient autonomy is reflected most critically in the informed consent process, in which patients are informed of their diagnosis, prognosis, potential benefits and risks of the proposed therapy, and alternative treatments available. Although this statement is true for any type of medical intervention, in the setting of HCT there are extra complexities, including the facts that many patients are minors, that almost all patients being considered for HCT have diseases that will likely be fatal without a transplant and are potentially curable with the procedure, and that patients who survive their original disease and the early transplant mortality are at risk for development of a wide variety of chronic complications that may impair their quality of life in many ways. After a patient has been told that he or she has a fatal disease and that a potentially curative procedure is available, how much does the patient really hear and/or retain about the potential risks and complications? How accurate are the available data about details of mortality and morbidity – including risk of sinusoidal obstructive syndrome (SOS), acute and chronic GVHD, cytomegalovirus and other infections, chronic radiation effects, avascular necrosis, sterility, the risk of second malignancies, and quality of life after transplant – and how many of these data
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Chapter 32
are revealed to prospective HCT patients? If patients choose not to hear all of these specifics and make choices solely on the options of life and death, is this really informed consent? Must we insist on inflicting them with these truths? Can patients exercise their autonomy by choosing not to be fully informed? One study compared the process of informed consent as viewed by physicians or nurses in comparison to the view of patients [11]. For the HCT nurses, it was most important that patients know about the sideeffects and complications of the procedure. The physicians wanted the patients to know about the diagnosis, therapeutic options, and outcomes of treatment. The physicians’ views matched the patients’ most closely, as they made decisions based mainly on outcome and life or death. In another study of adult and pediatric patients and their physicians, the three main reasons that patients and their parents chose HCT were: (1) trust in their physician; (2) fear that their illness would get worse without the transplant; and (3) belief that the HCT would be a cure. Most of the adult patients remembered that complications could occur, but they could recall fewer than half of those mentioned. Most of the patients believed they had been given adequate information and that it was not too technical, although the physicians thought the information conveyed was in fact too technical and excessive. Most of the patients believed that their physician wanted them to undergo the BMT, and the patients made their decision based on that advice. One HCT nursing director suggested that the role of the HCT nurse in this process may be to ensure that patients have been completely informed, although the nurse must realize that patients may not be able to comprehend the complex details of treatment, and that the intensity of the choice between life and death may overshadow the risks of potentially life-threatening complications. The nurse can function as a patient advocate; which may create conflicts with the physician at times, and the nurse must not take away hope from the patient [11]. Lee et al. analyzed the degree of concordance or discordance between 313 patients and their physicians in estimating the chances of success after HCT [12]. There was significant concordance between patients and physicians when the outcomes were likely to be favorable, but when patients had more advanced disease, they tended to fail to recognize the higher risks of relapse and death. The authors speculated that physicians may tend to minimize the risks in their discussions with patients who have advanced disease. Divulging more information about these risks may not change the patients’ decisions to move forward with the HCT, but it may promote better psychologic adjustment after HCT. Andrykowski et al. focused on the question of whether patients had “returned to normal” following HCT, and examined differences between their expectations and actual outcomes, and the impact these differences might have on their sense of wellbeing [13]. They studied 172 diseasefree survivors from five HCT centers and found that only a minority felt they were back to normal. Before HCT, only 19% of patients had not expected to return to normal, and 47% anticipated they would. Following HCT, 32% (possibly up to 52%) stated they had not returned to normal. The discordance between pre-HCT expectations and their current functional status was associated with greater current psychologic stress. Despite this discrepancy, the survivors’ evaluation of their decision to pursue HCT was generally quite positive. The investigators asked why there would be such differences between pre-HCT expectations and actual outcomes. Some patients may hear the word “cure” pre HCT, and therefore expect that they will be “as good as new” after the transplant. Patients may not have been adequately informed of potential sequelae at the time of the HCT. Some patients may underestimate the risk of bad things happening to them, the socalled optimistic bias. This may be an adaptive mechanism to deny threats and to be unrealistically optimistic in the face of stress or adversity. In the short term, this adaptive mechanism may be beneficial, but
in the long run, it may promote psychologic distress because of unrealistic expectations. These findings suggest that providing details of risks and benefits during the informed consent process may have little impact on the patients’ decision making. Only a minuscule number of patients actually turn down HCT if it is offered, mainly because they view HCT as their sole chance of disease cure and survival. These studies suggest, however, that, following HCT, periodic discussions of possible complications and outcomes should continue to take place between patients and their physicians to help optimize the patients’ psychologic wellbeing. The issue of involving children in medical decision making is important since many candidates for HCT are minors. Most investigators recognize that children’s capacity for decision making and autonomy develops over time. The parents or guardians must give consent for their minor child, but the wishes and concerns of the child should be taken into account [14]. The American Academy of Pediatricians states that one “should not exclude children and adolescents from decision-making without persuasive reasons” [15]. It is generally accepted that, starting at age 7 or 8, children are capable of giving assent to any proposed intervention, and that their assent should be required to proceed. The assumption is made that the parents will act as surrogate decision makers for their children and make choices based on the best interests of the child. The courts may need to become involved if parents do not seem to be following this approach, for example if a parent is a Jehovah’s Witness and refuses blood transfusion for the child.
Informed consent for donors of hematopoietic cells Case scenario 4 A mother leaves the country with her 16-year-old son who is HLA matched with his 22-year-old brother who has acute leukemia, because she fears that if her younger son donates stem cells then he will die or get leukemia too. She feels she is going to lose one son and does not want to lose the other. The older son dies from refractory leukemia without HCT. Case scenario 5 In performing HLA typing on members of a large family because a relative has leukemia, it becomes evident that one of the siblings has a different father from the rest of the children. The source of cells for an allogeneic transplant is usually a living donor who may be closely related to the patient, who may be a minor, or who may be an unrelated volunteer. In each of these situations, the autonomy of the donor must be respected, as reflected in an appropriate informed consent process. Donors must be informed of the specifics of the procedure that they may undergo, especially the potential risks involved. The process must be free of coercion and must respect the confidentiality of the donor and the family when it is a family donor in question. Cultural diversity issues may complicate the search for a potential donor if unusual or irrational beliefs are brought to bear. Special and important considerations are raised when the donor is a minor. It is generally accepted that the risks involved in being a marrow donor are very small. Estimates of the incidence of life-threatening or incapacitating complications are about 0.29% for adult donors at several centers, and 0.4% in a pediatric population from a single institution [16]. A potential benefit of the donation process is that pre-existing medical problems that require intervention may be detected in the donor. In this study from Seattle, 206 medical problems were observed in the 1549 donor evaluations, with hypertension, obesity, and cardiac problems the most common (see Chapter 42) [17].
Ethical Issues in Hematopoietic Cell Transplantation
Many collection centers are using granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood cells (PBCs) from matched siblings or unrelated donors as their preferred source of cells [18–20]. Many of the risks associated with marrow harvests are minimized or prevented, such as complications from general anesthesia, the pain from multiple iliac crest punctures, local hematomas, and/or infection at the aspiration sites. The use of G-CSF for this purpose is considered to be generally safe except for donors who have certain underlying medical conditions such as hemoglobinopathies, autoimmune disease, or coronary artery disease. In a randomized study comparing PBC to bone marrow donations, the donors found the PBC collections to be the less burdensome and preferred method [21]. In a study of sibling donors for patients enrolled in a randomized trial comparing PBCs with bone marrow cells, Rowley et al. found that the intensity and nature of pain was very similar for the two groups, although all the PBC donors returned to normal function by 14 days compared with 80% of the bone marrow cell donors [22]. It is still not settled whether there is any overall advantage to the recipient from one source of cells over the other. Donor preference may be the deciding factor if there is no clear advantage between the two cell sources. In reviewing the experiences and attitudes of the first 20 volunteer unrelated donors at the University of Minnesota, Stroncek et al. found no serious physical or emotional after effects [23]. Nine of the 20 donors reported that a friend or family member had discouraged them. Nineteen said they would donate again, and 17 would advise others to donate. One donor had orthostatic hypotension for 1 day, and all donors received one or two units of autologous blood. The benefits of being a marrow donor for a family member or an unrelated recipient are a matter of debate. At one extreme, it is argued that there are no benefits to the donor, and therefore even the small risk of complications becomes more significant. Others state strongly that there are remarkable benefits to the donor of a psychologic nature, especially when donating to a family member, such as the satisfaction of helping to save a loved one’s life [24]. On balance, the very low risks associated with marrow donation and the fact that marrow is a renewable resource like blood, and that significant benefits are derived by the donor, make it ethically appropriate to encourage PBC and marrow donation. Of course, it is imperative that any coercion of potential donors be avoided. To that end, the HCT center should keep results of HLA typing confidential (or not even initiate testing) until all family members have had the opportunity to discuss privately their desire to proceed [11]. Another argument for strict confidentiality related to HLA typing results is that previously hidden facts about paternity may be brought to light to an unsuspecting family. Revelation of such information could have devastating effects on the entire family unit. The psychologic state of the donor must be respected. If the outcome is poor, especially if the patient fails because of GVHD, there will be a natural tendency for donors to blame themselves for the patient’s death. Appropriate counseling before and after the HCT can help lessen this burden for the donor. In light of these concerns, it has become the legal standard that no court can force anyone to undergo medical testing or a procedure that is intended only to benefit another individual. Confidentiality and noncoercion are especially important issues in relation to unrelated donors. The National Marrow Donor Program has very explicit policies and procedures designed to respect potential donors’ autonomy by requiring informed consent at every step of the process, including the time at which a donor is first listed in the registry; before collection of blood for confirmatory typing, infectious disease markers, and research samples; prior to notification of the transplant
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center that a donor is willing to proceed to donation; and before general anesthesia [25]. The donor’s privacy is of utmost importance to avoid any extraneous influences or pressures as the donor decides whether or not to donate. The identity of the donor is known only to the individual donor center, and many precautions are put in place to ensure this. Generally, the recipient and donor are allowed to contact each other directly only by mutual consent 1 year after the HCT. The National Marrow Donor Program considers these measures to be critical in promoting increased participation in the program by altruistic individuals. The World Marrow Donor Association reviewed the ethical issues for unrelated donors, focusing on respect for the donors’ autonomy, wellbeing, and needs. They stressed the importance of noncoercion and adequate informed consent, especially when requests for donation of a second cellular product are being considered [26]. The process by which decisions are made to use minors as marrow donors is somewhat controversial [27–29]. Some authors have questioned the manner in which informed consent is obtained to collect marrow from minors and have asked whether parents can truly give informed consent. The need to involve the minor in the consent process by seeking his or her assent is a widely accepted concept, especially for children over the age of 7 or 8 years. Delany has questioned whether it is really legal to harvest marrow from a minor and has labeled the process “altruism by proxy” [29]. One legal concept permits a medical procedure only if it serves the best interests of the child who undergoes it. If there is disagreement between caregivers, it may be necessary to go to court. A second concept argues that parents can give valid consent to treatments that are “not against the interests of the child.” This approach was developed to allow blood drawing from children whose legitimacy or parentage was at issue. Some have argued that this principle is relevant to any medical intervention carried out on children purely for other people’s benefit. Delany argues that marrow donation is not in the best interest of the donor, and that it may be against the child’s interests, especially if the potential donor is too young to have established an emotional bond with the intended recipient. She also contends that the parents are not well suited to give informed consent for the donor due to conflict of interests related to the sick child, and that an informal tribunal or other forum independent of the parents and medical advisors should be responsible for approving each proposed donation. This procedure would be analogous to the strategy developed by the Law Commission in England to safeguard mentally incapacitated adults from being inappropriately volunteered for marrow extraction. Month disagreed strongly with this interpretation of the issues [29]. She believed that the positive aspects for the child donor are considerable and include the saving of a sibling’s life and the benefit of many years of a whole family not burdened by the psychologic trauma of the death of a child. Further, since HCT is an accepted therapeutic option that can be lifesaving, and often offers the best cure rate, is there really any reason a morally competent sibling would not want to donate? The risks of donation are minimal, especially when compared with the risks of not donating marrow, that is, the death of a sibling. Savulescu argued that although the bone marrow donation is not in the donor’s medical interests, it may be in his or her overall interest [29]. To save the life of a loved one is one of the most important things one can do. Even for donors who are too young to understand at the time, the potential for a future sense of achievement and the love and gratitude of the saved sibling is important. There are benefits to the family unit as well. Parents should be the ones to give informed consent after they are educated about the risks and benefits of the donation, just as parents often make decisions with conflicting interests of their children, but with a commitment to the overall good of the family.
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An unusually difficult case was recently published involving a 15-yearold boy with relapsed AML who was incarcerated for sexually assaulting his 9-year-old sister [4]. The conflicts involved in the decision making about whether or not to offer the boy HCT were discussed in a preceding section of this chapter. The 9-year-old sister was the only HLA-matched relative available. Should she be asked to donate stem cells to her brother? Is there a reasonable alternative source of stem cells available for the patient, for example a matched unrelated donor? Is the mother the right person to give informed consent on her behalf? Should there be a legal guardian appointed to protect the interests of the sister? Would the potential harm to her, psychologic and physical, outweigh the benefits? Although these are questions relevant to almost every case involving a minor donor, the conflicts were clearly more acute in this case. The formal process for approving the use of a minor for marrow donation varies between countries and between centers within the same country. A survey of 52 HCT centers in Europe and the United States found that some centers had an ethics committee decide the validity of the therapeutic proposal [28]. In France, there is a law that requires a committee of three experts to assess the psychologic and medical consequences of donation of the organ by a minor, and notes that “the refusal of the minor donor to accept the removal shall always be respected.” In the United States, a Washington State court ruling stated that the advantages to the donor are undoubtedly greater than the risks, and defers to the parents to give informed consent. Some institutions require an authority outside the family and hospital, for example a “tutelary judge” or commission of experts, to make the decision. A questionnaire was sent to 70 HCT centers in North America by pediatricians at the MD Anderson Cancer Center to poll the HCT teams on their approaches to marrow donations by minors [30]. The impetus for sending the questionnaire was a case brought to the MD Anderson pediatricians in which they were asked to collect marrow from a young child to be donated to the child’s parent. There was a significant difference in size between the child and parent, and three HLA mismatches. The parent had relapsed AML. Ethical concerns were raised by hospital staff members. As a result, the MD Anderson Center developed a process in which the minor donor and surrogate are interviewed by a social worker, pediatrician, and anesthesiologist, none of whom are involved in the care of the recipient. If any concerns are raised, the case is referred to the ethics committee for consultation. Fifty-six (80%) of the centers responded to the questionnaire. There was consensus in endorsing the validity of parental consent. Most centers would use donors as young as 6 months old and would use them more than once if needed. The centers were willing to use minor donors for patients on experimental protocols, and the projected outcome of the HCT did not affect the decision to use the minor as a donor. Issues raised included the nontherapeutic nature of the donation, the vulnerability of the minor, and the potential conflict of interest when a parent acts as a surrogate for both the donor and a sick family member. Even greater conflict was seen if a parent was the intended recipient of the marrow, and most agreed that the other parent or both should be involved in the informed consent procedure. The majority of the centers and physicians had policies or practices of using the parents as the surrogate; a minority preferred to use a parent plus a child advocate or child protection agency. If there were disagreements between the parents, eight centers would cancel the HCT, and the rest would either seek consultation with the ethics committee, use an unrelated donor, refer the parents for another opinion or refer the decision to a child protection agency. When asked if the use of a child intentionally conceived as a marrow donor were ethical, most of the physicians answered that they would be willing to use such a donor. They would consider using umbilical cord blood (UCB) in that case as being safer for the donor.
The participation of pediatric donors in clinical research raises additional issues beyond standard clinical practice [31]. When can Institutional Review Boards (IRB) approve such activities? The IRB must focus on whether the proposed research procedure is different for the donor compared with a standard stem cell collection. If the proposed research adds or replaces procedures to the standard clinical donation, and thereby increases the risks to the donor, such a protocol must be approved by the Secretary of Health and Human Services with the advice of a panel of experts (category 46.407 of the Federal Regulations). Some have argued that minors who are asked to donate G-CSFprimed PBC should be considered to be research subjects, since the data about long-term risks in this setting are not well known. The parents would be the best persons to decide for their child donors, but IRB approval would be required [16,32].
Alternative sources of hematopoietic cells Case scenario 6 A couple decides to reverse a vasectomy to create a new baby to be a possible donor for their teenage daughter with CML who has no family or unrelated donors. HLA-matched siblings are usually considered to be the primary source of cells for HCT. For the 70% of patients who lack such a donor, alternative sources of stem cells include matched related donors other than siblings, unrelated volunteer donors, UCB cells, and possibly autologous stem cells in the appropriate clinical setting. In this section, the ethical issues raised by three potential sources of stem cells are discussed, namely, UCB [14,33–35], “children conceived to give life” [36], and xenografts from baboons or other primates. Since the first UCB HCT was performed in 1988, about 6000 such transplants have been carried out worldwide [37]. There are over 170,000 UCB units available in 37 banks in 21 countries [38]. The advantages of cord blood over adult marrow include: the lower risk of carrying viruses; a reduction in procurement time from the 4–6 months needed for an unrelated marrow donor, to 1–2 weeks; and the fact that cord blood cells are potentially usable across greater HLA disparities. The major disadvantages are the limited number of cells available, the fact that one cannot go back to the donor for more cells if needed, and the risk of transmitting a genetic disease not previously diagnosed in the donor’s family. Marshall raised several ethical issues related to cord blood cell collections and cord blood banks, starting with consent and privacy [39]. Is informed consent required for a product that is otherwise discarded as waste? Is the mother (or parents) the appropriate one(s) to give consent? Is consent required to conduct a battery of tests to detect possible infectious or genetic diseases, or both? Should donors, that is, the parents, be informed of the results of such tests? Who owns the cord blood? Obviously the donor himself cannot give informed consent, yet an extensive medical profile about the donor may be generated, raising concerns about invasion of privacy. Should consent be obtained for follow-up tests that are not consented to directly? What should be done with abnormal results from such tests? Should banks maintain medical files and genetic data, but in such a way that names and identifiers are delinked from the donated sample [40]? What are the obligations to inform the family if evidence of human immune deficiency virus or an abnormal gene is found? Some authors argue that the infant donors should be the first to benefit from use of their own cord blood cells [41]. Records should be kept of diseases detected and parents informed. Therapy should be offered for treatable diseases. Commercialization of cord blood cells for therapy should include a financial benefit for infants; for example, royalties from
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each donation should be placed in a national trust fund for research and treatment of children with serious diseases. Kurtzberg et al. argued that cord blood that was previously discarded material should be a public resource with no financial gain to any party [42]. Regulation of donor banks by an agency such as the Food and Drug Administration is needed. For example, there are companies that try to persuade parents that they should store the cells as insurance against future needs, when in fact the chances are remote that the donor would ever need or want to use the cord blood cells. There is a pressing need to protect the confidentiality of the information collected, to notify parents and children of the test results, and to assure equitable access to cord blood samples. For the health of the mother and baby, the collection process should be restricted to full-term, uncomplicated pregnancies, and should not interfere with good obstetrical practice at delivery. The American Academy of Pediatrics has addressed the issue of cord blood banking and issued a set of guidelines to help physicians regarding types and quality of blood banks (for-profit versus not-for-profit), ethical and operational standards including informed consent policies, financial disclosures, and conflict-of-interest policies [43]. Their three main recommendations are: (1) the storage of cord blood in private banks for later personal or family use should be discouraged unless there is a full sibling in the family with a known malignant or genetic condition that might benefit from cord blood transplantation; (2) cord blood donations should be encouraged when stored in a bank available for public use; and (3) private storage of cord blood as “biologic insurance” should be discouraged. Considerable attention and debate were generated in response to the actual case outlined above as case scenario 6. The practice of “conceiving a child to give life” is not a rare event [44]. Some question whether a good outcome for the recipient lessens the ethical dilemmas posed. It was argued that such a practice does not impose harm on persons or relationships within the family, and does not show lack of respect for the child conceived [11]. Others raised their concerns about performing prenatal testing to diagnose the genetic disease affecting the intended recipient and for HLA typing, and the potential for aborting in the case of a mismatch. One hematologist’s view of the concept is that such a child is conceived not to replace the sick sibling, but to give life to the sibling who would not survive without a transplant [44]. The benefits of this approach include the fact that the cord blood is disposable material, and that no anesthesia or blood transfusions are needed for the donor. From the recipient’s perspective, the transplant can be performed earlier since a marrow transplant could require a delay of 6 months or more. Alby described three characteristics of pregnancies conceived for HCT: (1) families in that situation face the trauma of the impending death of the sick child; (2) the pregnancy outcome with respect to HLA matching and the HCT results are both uncertain; and (3) biology has intruded into the family dynamics [36]. A family may attempt a cure at any cost, especially in an emergent situation such as acute leukemia. A child may be viewed subconsciously as a replacement child, but should never be reduced to the role of a therapeutic tool. In terms of family dynamics, a child may be viewed as bad or good based on HLA matching. Many are concerned that such a child is being conceived only for transplantation. Yet a child is always born for something: to maintain the integrity of a couple, to gratify the parents’ need for a family, to repair their vulnerability. All parents must go beyond the idealized view of their children and accept that they have an identity of their own, and all children have some symbolic role in the family fantasy. In that light, is there really anything wrong with conceiving a child to donate cells for the purpose of saving the life of a sibling? There are risks for children born for HCT, of both a physical and a psychologic nature. The physical risks are less from cord blood collec-
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tion than from marrow harvesting. Such a child would be born into a psychologically stressed family dealing with the emotional and financial trauma of caring for a sick child. In case of the death of the recipient, the new child is exposed to parental depression, identification with the deceased sibling, loss of identity if the parents cannot accept the death, and shared guilt with the parents. When the procedure is successful, the donor is seen as the savior, and both the child and the family must find a normal way of life again. Advances in in vitro fertilization techniques have opened the door to preimplantation genetic diagnosis (PGD). Candidate embryos can be screened in vitro for HLA matching [45] and for various genetic traits [46,47] as the basis for selecting which embryos to implant. HLA typing of embryos to select a potential stem cell donor for an afflicted relative has been proposed as a legitimate means of securing a donor for a patient that is in need of a life-saving transplant. The ethics of this approach have been reviewed, and the conclusions were that using PGD to choose a stem cell donor is unlikely to cause harm to anyone, is likely to be beneficial to some, is a reasonable use of limited health resources, and should be permitted in countries where PGD is already allowed [48]. Morgan et al. also concluded that the use of assisted reproductive techniques to create a stem cell donor can be considered to be ethically acceptable [49]. As a recent case in point, in the United Kingdom, the Human Fertilisation and Embryology Authority gave permission for PGD to be utilized to select an unaffected embryo to serve as a future hematopoietic cell donor for a sibling with thalassemia. The technique has been used in the United States since 2000 [50]. Pennings et al. argued that PGD-guided selection of embryos for this purpose is morally defensible on the condition that the procedure to be performed on the future child is acceptable for an existing child, and that the “instrumentalization” of the donor child does not demonstrate disrespect for the child’s autonomy and intrinsic worth [51]. Devolder considered the risks and benefits of utilizing preimplantation HLA typing for selecting embryos to be used as donors of stem cells for sick siblings and concluded that it would be unethical not to allow this procedure, since there is no indication of harm to the donor child, and some lives would be saved [52]. She argued further that it should be up to the parents to decide whether their current or future children can act as a donor for a loved one, and that the pool of potential recipients should not be restricted to siblings or other family members, but should be expanded to include anyone whom the couple loves. The ability to create multipotential stem cells in vitro from pluripotential embryonic stem cells is a subject of wide public debate. Legal and ethical distinctions have been drawn between human reproductive cloning and nuclear transplantation for the production of stem cells for therapeutic applications. Only the latter will be discussed here as a potential source of hematopoietic cells for use in HCT in the future. Many scientists, ethicists, government bodies, and medical organizations in the United States, Canada, and Europe have advocated for the legalization of research for the purpose of therapeutic cloning [53–58]. The potential to utilize such cells to treat a variety of medical conditions appears to be great, although significant technical hurdles are yet to be overcome before there can be clinical applications. Many serious ethical issues must be considered including the protection of human dignity, the source of unfertilized eggs and/or embryos to be used for research, informed consent from potential donors, the privacy of donors, and financial incentives to donate eggs and/or embryos. The utilization of an unusual source of stem cells generated considerable media attention when investigators attempted to reconstitute the immune system of a patient with acquired immune deficiency syndrome (AIDS) using stem cells collected from a baboon [59]. A number of scientific and safety issues are raised, including the risks of the baboon cells attacking the human host, the unknown ability of the baboon cells
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to mount an immune response against pathogens to which the AIDS patient is exposed, the concern that the patient will be made sicker by the conditioning regimen, and the question of whether the patient will have to fight off baboon viruses. Ethically, there are concerns about how to protect the rights of the first patients treated this way. How does one select the first patients for procedures that are not likely to benefit them, that is, the so-called “patient-pioneer” or “human guinea pig”? It is critical that the patient understand how unlikely it is that he or she will benefit, and that the experiments be done in a rigorous way so that specialists in the field can learn from the experience. There are fears of transmission of animal pathogens to humans, perhaps necessitating germ-free colonies of donor animals. Pigs are being used for solid organ transplants to humans, perhaps as a bridge to tide the patient over until a human organ becomes available. Barker proposed caution in xenotransplantation and a postponement of solid organ xenotransplants until the issue of informed consent to the infectious disease risks is adequately addressed in an open public policy process [60].
Transplantation for nonmalignant diseases Most HCT procedures are performed to treat malignant diseases for which HCT often represents the only curative option. Many nonmalignant diseases are also treated by HCT, especially in the pediatric patient population, and include primary marrow failure such as severe aplastic anemia and a variety of congenital immune deficiency syndromes. The balance of risk and benefit for patients with these diseases is clearly in favor of HCT since the diseases are often rapidly fatal, and curative alternative options are generally not available. The balance may not be so clearly defined for a variety of diseases that affect marrow-derived cells, which may be a source of significant morbidity for affected patients and possibly shortened life span, but are not considered to be rapidly fatal. Further, the data that demonstrate the long-term benefit from HCT for these conditions may be inconclusive or even nonexistent. Examples of this category of diseases would include hemoglobinopathies such as sickle cell disease, certain metabolic storage diseases, autoimmune diseases such as multiple sclerosis or rheumatoid arthritis, and acquired immune deficiency diseases, specifically AIDS. How does one select the right patient to be treated by a procedure that carries significant risk of morbidity and possibly early mortality? How can one demonstrate the efficacy of HCT in diseases that usually progress over years rather than weeks or months? How does a parent decide to consent to an HCT for a child with such a disease? This discussion focuses on hemoglobinopathies as a paradigm for these issues since there has already been a broad experience in performing transplants for thalassemia [61], and a body of literature about the ethical dilemmas involved is available. These issues were discussed at an international meeting on HCT in children held in March 1994 [33]. In regard to hemoglobinopathies, the difficult questions are whom to treat and when, and whether to offer HCT as a curative procedure to all affected children as early as possible. Some participants favored HCT for all patients with sickle cell disease who have an HLA-matched sibling in early childhood, given the high risk of morbidity in adult life. Others argued that it should be reserved for those who show clinical indicators of poor prognosis, as adopted by the ongoing national trial to recruit 30–60 children over a 5-year period. For thalassemia, the clinical course is more variable. Some proposed that HCT should be offered before any morbidity has occurred, while others would reserve it for children whose iron overload was well controlled and who were without liver disease. Giardini discussed the ethical issues of HCT for thalassemia in an editorial in 1995 [62]. He argued in favor of HCT since the risk of mortality from disease-related complications remains high even with
desferrioxamine chelation. The success of standard therapy depends on compliance of the patient and a health-care system that is able to carry it out. The cost of this effort is about $32,000 per patient per year in developing countries, and about $60,000 per year for adults in the United States. These patients require the input of specialists, including cardiologists, hepatologists, psychologists, and endocrinologists. In comparison, an HCT for hemoglobinopathies in 1991 cost about $173,250 in the United States and about half that in Europe. Giardini argued that it is ethically and economically appropriate to recommend HCT for thalassemia, especially given the progress in dealing with GVHD and cytomegalovirus, the availability of newer antibiotics, and so forth. Estimated outcomes for patients with class of risk I are 3% probability of death, 4% of rejection, and 93% of disease-free survival. For patients in class of risk II and III with organ damage from iron overload, the mortality from HCT is higher, but their expected survival with standard therapy is much lower. Although other experimental approaches show promise, such as artificial hemoglobin, new oral iron chelators, gene therapy, and agents to stimulate fetal hemoglobin, the fact is that currently HCT offers the only chance of cure of the disease. Kodish and associates examined the issue of parents’ decision making concerning HCT for children who were affected with sickle cell disease [63]. A questionnaire was presented to parents with hypothetical chances of cure versus mortality to see how they balanced these two outcomes. In the study of 67 parents, 54% were willing to accept some risk of short-term mortality. Thirty-seven percent would accept at least a 15% mortality risk in the short term, and 12% were willing to accept a 50% or more risk. Sixteen (24%) of the 67 parents would not accept HCT even if there was a 0% mortality risk. Differences were found between the group of parents who would accept some risk and those who would not. Parents who were highschool graduates, who were employed outside of the home or who had more than one child with sickle cell disease were more willing to take the risks. Of note, parents were more willing to take risks for girls than for boys. The parents’ decisions were not related to the clinical severity of their children’s illness, unlike the way the doctors might make such a decision. For parents with more than one child, their willingness may reflect their knowledge of the disease, or the burden of caring for children with sickle cell disease. Clearly, the concerns and values of the parents must be considered along with weighing the medical issues of risks and benefits. The difficult issue is what criteria to use to select patients to undergo HCT for sickle cell disease. As one model, the IRB at the University of Chicago in 1988 allowed two groups of children to be treated with HCT: children who already had had a stroke and therefore were receiving monthly transfusions to prevent the next stroke, and those with recurrent painful crises who had required hospitalization for at least 60% of the preceding year. Recent advances in HCT, such as the use of peripheral blood hematopoietic cells from allogeneic donors and reduced-intensity conditioning regimens, have been utilized in the treatment of hemoglobinopathies [64,65]. These advances help reduce the risks of short- and long-term toxicities for either the donor or the recipient, and thus create a more favorable risk–benefit ratio. Parents of minors with hemoglobinopathies will need to be informed about these innovations to help them make an appropriate decision for their children who may be candidates for HCT.
End-of-life issues Case scenario 7 A 50-year-old physician with CML is dying from SOS and GVHD with liver and respiratory failure early after HCT. He has no written advanced
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directive. He made his second wife promise that she would not let him stay on life support for more than a week if he had no hope of recovery. She is devoutly Catholic, but feels she made a commitment to her husband to withdraw life support. His adult children from his first marriage, who are atheists, say their father would never agree to withdrawal of life support, that he is ardently opposed to euthanasia, and that to him withdrawal of life support is no different from euthanasia. Respect for patient autonomy requires caregivers to determine the wishes of their patient with respect to resuscitation and life support. Advanced directives, such as a Power of Attorney for Health Care, are extremely effective vehicles to facilitate discussions between patients and their doctors, for patients to convey their wishes, and to name a surrogate to speak for them when they become incapacitated. Yet very few patients execute such documents or discuss these matters with their doctor. Such discussions about end-of-life issues are difficult for most physicians, nurses, and patients. They are even more difficult in the context of HCT, where the patients are generally young and, although they have potentially fatal diseases, often have a significant chance for cure with this treatment. During the informed consent process that leads up to the HCT, physicians are obligated to discuss the risks and complications of the planned therapy, including the chance of early mortality. As discussed above in the section on informed consent for patients, when faced with life and death choices, patients often block out the details of possible complications in the decision-making process. Physicians have trouble raising end-of-life issues in the context of proposing a potentially lifesaving therapy, in part for fear that patients will take away a negative message about their prognosis [66]. The fact remains that up to 15–20% of patients may succumb to early allogeneic transplant-related mortality. HCT patients often decompensate rapidly and unpredictably, for example from severe SOS or GVHD. There may not be an opportunity to discuss end-of-life issues when the patient is in respiratory failure on the way to the intensive care unit. The quality of patient care would be much enhanced if such discussions and appropriate documentation of patients’ wishes were carried out well before this point, and were then acted upon as dictated by the patient or the surrogate. Many of these patients will be maintained by intensive life support for extended periods of time, despite generally poor chances of recovery. At some point in the course of the HCT procedure, the goals of therapy must shift from cure and aggressive interventions to palliation and supportive care for a dying patient. Rigorous attention to pain control is essential throughout the HCT process and certainly during the period of dying. The principles of fidelity and nonabandonment require caregivers to make it clear to patients and their families that they will not be abandoned as the goals of treatment shift from cure to caring for the dying patient. Although it may be appropriate to withhold interventions that are medically futile, how is this to be determined? How much of our limited health-care resources should be expended providing what is ultimately futile medical care? How great an emotional cost do families have to pay? These are difficult questions that need to be addressed at both the macrolevel of society and the microlevel of individual patients and families. It is the patient (or surrogate) who is best able to understand and decide on the issues of his or her ability to pay for a treatment and the possibility of financial destitution of a family by continuing futile care. The surrogate must utilize substituted judgment to relay the decisions that the patient would have made if he or she had the capacity. In cases in which no surrogate is legally designated, an ethics committee may need to be involved to consider quality of life issues and death with dignity. To explore the variables involved in instituting do-not-resuscitate (DNR) orders for HCT patients, a retrospective review of 40 patients who died on the Seattle Veterans Administration Medical Center HCT
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unit was carried out [67]. This center has a DNR policy that requires physicians to document the discussion held with the patient/surrogate, and for the patient/surrogate to sign a DNR consent. Patients with fatal diseases often suspend their feelings about terminal illness in hopes of a cure through HCT. The dying trajectory of these patients is often sudden and unpredictable. The health-care providers often focus on aggressive medical management when these situations arise, and, as a result, the topic of death is avoided. The process of executing DNR orders was believed to facilitate the transition from aggressive care to supportive care in critically ill HCT patients. Of 42 deaths that occurred on the HCT unit during a 5-year period, six patients had a resuscitation attempt and 36 had DNR orders (two records were not available for review). The DNR consent was signed 26% of the time by the patients themselves, 32% by spouses, 24% by mothers, and 18% by other family members. The two groups of patients did not vary in terms of age, diagnosis or type of HCT. The non-DNR group developed life-threatening complications earlier in their course, whereas multisystem organ failure was the common factor among the DNR patients. The death of non-DNR patients may not have been anticipated because the complications that developed were emergent and were considered reversible. These conditions may have precluded discussions regarding DNR status. For the DNR patients, this designation occurred close to the time of death: 32% on the same day as death, 82% less than 5 days before death, and 18% 5–14 days prior to death. In 11 studies of HCT patients who required mechanical ventilation, the rates of survival to discharge from the hospital ranged from 0% to 11.1%, with a mean of 4.7% [68]. It would be clinically useful to define a subgroup of patients whose chance of survival is so low that both patients/surrogates and physicians can agree that intensive care can no longer fulfill the original goals of the HCT. Criteria to identify patients who are destined to die after HCT are needed to help reduce emotional and financial expenditures of families and institutions. At the Fred Hutchinson Cancer Research Center from 1980 to 1992, 25% of all HCT patients were ventilated. Patients who were more than 20 years old, had disease in relapse, and had grafts that were not HLA identical had a 50% chance of needing ventilation. Only 6.1% survived. To identify predictors of death in mechanically ventilated HCT patients, Rubenfeld and Crawford conducted a nested case-control study comparing all 53 survivors of ventilatory support with 106 matched control subjects who did not survive [68]. These patients were selected from the 865 individuals who were mechanically ventilated for at least 24 hours during that 12year period. Survivors were defined as those who were alive for 30 days after extubation and who were discharged from the hospital. Survival was statistically associated with younger age, lower score on the APACHE (Acute Physiology, Age, Chronic Health Evaluation) III scale, and shorter time from HCT to intubation. Of the 106 control subjects who died, 82% died while on the ventilator, and the remaining 18% died in the hospital a median of 18 days after extubation. Of the 53 survivors, median survival after extubation was 634 days (range 54 days to 12 years). Eighteen (34%) survived for less than 6 months and five (9%) for less than 1 year; 30 (57%) lived for more than 1 year. No patient who was intubated for more than 149 days survived. The survival rates changed from 5% to 16% over the most recent 5 years, which was not explained by changes in the age of patients, the rate or timing of intubation or the percentage of allogeneic transplants that were not HLA identical. The improved survival may be due to better antimicrobials and the use of cytokines. There were no survivors among the 398 patients who had lung injury and either required more than 4 hours of vasopressors or had sustained hepatic and renal failure. Using these three criteria, an accurate prediction of death could be made in the first 4 days for more than half of the patients who did not survive. Of the 60% of patients who developed any
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two of these three risk factors, half had done so by day 2, and 90% by day 4. On average, these risk factors developed six ventilator days and nine hospital days before death. If life support had been withdrawn on the first day that two out of three risk factors were met, and if death had followed swiftly, more than 7300 hospital days and 4800 ventilator days could have been avoided for the 812 patients who died. A multi-institutional study confirmed these observations in adult HCT patients, for whom the combination of mechanical ventilation and hepatic and renal dysfunction was associated with a probability of death of 98–100% [69]. A scoring system for pediatric patients undergoing allogeneic HCT called the Oncologic Pediatric Risk of Mortality (OPRISM) may be useful in predicting fatal events, and thus help parents decide to establish a supportive care strategy for their children [70]. Although most physicians and ethicists may agree that futile, inappropriate or unreasonable medical care should not be provided even if requested, how are these terms to be defined, and by whom? In cases in which the patient/surrogate continues to request treatments that the caregivers consider futile, the physician is not ethically required to provide that care, but is obligated based on the principle of nonabandonment to
facilitate the transfer of the patient to another physician who will. Consultation with the institutional ethics committee may be helpful in these most difficult situations.
Closing remarks The indications for HCT have expanded over the years to encompass a wide range of diagnoses and disease stages, including patients with early stages of hematologic malignancies considered to be at high risk for relapse, solid tumors, congenital disorders, and autoimmune diseases. Potential sources of stem cells have also expanded to include autologous or allogeneic cells from bone marrow, peripheral blood or cord blood from related or unrelated HLA-matched donors. It is imperative that the rights of both patients and donors be respected in step with these technologic advances. The ethical principle of justice requires caregivers and insurers to provide potentially life-saving yet high-risk procedures to HCT candidates in an open and equitable manner. Patient autonomy, nonmaleficence, and nonabandonment are guiding principles in making decisions about end-of-life care.
References 1. Arenson EB Jr, Forbe MD. Bone marrow transplantation for acute leukemia and Down syndrome: report of a successful case and results of a national survey. J Pediatr 1989; 114: 69–72. 2. Chang G, Antin JH, Orav EJ, Randall U, McGarigle C, Behr HM. Substance abuse and bone marrow transplant. Am J Drug Alcohol Abuse 1997; 23: 301–8. 3. Foster LW, McLellan LJ, Rybicki LA, Dabney J, Welsh E, Bolwell BJ. Allogeneic BMT and patient eligibility based on psychosocial criteria: a survey of BMT professionals. Bone Marrow Transplant 2006; 37: 223–8. 4. Opel DJ, Diekema DS. The case of A.R.: the ethics of sibling donor bone marrow transplantation revisited. J Clin Ethics 2006; 17: 207–19. 5. Mitchell JM, Meehan KR, Kong J, Schulman K. Access to bone marrow transplantation for leukemia and lymphoma: the role of sociodemographic factors. J Clin Oncol 1997; 15: 2644–51. 6. Vaughan WP, Purtilo RB, Butler CD, Armitage JO. Symposium: Ethical and financial issues in autologous marrow transplantation: a symposium sponsored by the University of Nebraska Medical Center. Ann Intern Med 1986; 105: 134–5. 7. Faber-Langendoen K. Ethical issues in the allocation and reimbursement of bone marrow transplantation. Leukemia 1993; 7: 1117–21. 8. Beatty PG. Bone marrow transplantation for the treatment of hematologic disease: Status in 1994. Exp Hematol 1995; 23: 277–88. 9. Welch HG, Mogielnicki J. Presumed benefit: lessons from the American experience with marrow transplantation for breast cancer. Br Med J 2002; 324: 1088–92. 10. Tamura T, Matsuzaki M, Harada H, Ogawa K, Mohri H, Okubo T. Upregulation of interferon-a receptor expression in hydroxyurea treated leukemia cell lines. J Invest Med 1997; 45: 160–7. 11. Downs S. Ethical issues in bone marrow transplantation. Semin Oncol Nurs 1994; 10: 58–63. 12. Lee SJ, Fairclough D, Antin JH, Weeks JC. Discrepancies between patient and physician estimates for the success of stem cell transplantation. J Am Med Assoc 2001; 285: 1034–8.
13. Andrykowski MA, Brady MJ, Greiner CB et al. ‘Returning to normal’ following bone marrow transplantation: outcomes, expectations and informed consent. Bone Marrow Transplant 1995; 15: 573–81. 14. Massimo L. Ethical problems in bone marrow transplantation in children. Bone Marrow Transplant 1996; 18(Suppl 2): 8–12. 15. Harrison C, Kenny NP, Sidarous M, Rowell M. Bioethics for clinicians. 9. Involving children in medical decisions. Can Med Assoc J 1997; 156: 825–8. 16. Pulsipher MS, Nagler A, Iannone R, Nelson RM. Weighing the risks of G-CSF administration, leukopheresis, and standard marrow harvest: ethical and safety considerations for normal pediatric hematopoietic cell donors. Pediatr Blood Cancer 2006; 46: 422–33. 17. Buckner CD, Petersen FB, Bolonesi BA. Bone marrow donors. In: Forman SJ, Blume KG, Thomas ED, editors. Bone Marrow Transplantation. Boston: Blackwell Scientific Publications; 1994. pp. 259– 69. 18. Anderlini P, Przepiorka D, Lauppe J et al. Collection of peripheral blood stem cells from normal donors 60 years of age or older. Br J Haematol 1997; 97: 485–7. 19. Bishop MR, Tarantolo SR, Jackson JD et al. Allogeneic-blood stem-cell collection following mobilization with low-dose granulocyte colonystimulating factor. J Clin Oncol 1997; 15: 1601–7. 20. Pavletic ZS, Bishop MR, Tarantolo SR et al. Hematopoietic recovery after allogeneic blood stem-cell transplantation compared with bone marrow transplantation in patients with hematologic malignancies. J Clin Oncol 1997; 15: 1608–16. 21. Heldal D, Brinch L, Tjonnfjord G et al. Donation of stem cells from blood or bone marrow: results of a randomised study of safety and complaints. Bone Marrow Transplant 2002; 29: 479–86. 22. Rowley SD, Donaldson G, Lilleby K, Bensinger WI, Appelbaum FR. Experiences of donors enrolled in a randomized study of allogeneic bone marrow or peripheral blood stem cell transplantation. Blood 2001; 97: 2541–8.
23. Stroncek D, Strand R, Scott E et al. Attitudes and physical condition of unrelated bone marrow donors immediately after donation. Transfusion 1989; 29: 317–22. 24. Anonymous. Ethics of organ transplantation from living donors. Transplant Proc 1992; 24: 2236–7. 25. Anonymous. National Marrow Donor Program® Donor & Patient Confidentiality Guidelines. Minneapolis. 2003. pp. 1–4. NMDPnetwork.nmdp. org. 26. Bakken R, van Walraven AM, Egeland T. Donor commitment and patient needs. Bone Marrow Transplant 2003; 33: 225–30. 27. Curran WJ. Beyond the best interests of a child – bone marrow transplantation among half-siblings. N Engl J Med 1991; 324: 1818–19. 28. Burgio GR, Nespoli L, Varrasi G, Buzzi F. Bone marrow transplantation in children: between therapeutic and medico-legal problems. Bone Marrow Transplant 1989; 4(Suppl 4): 34–7. 29. Delany L, Month S, Savulescu J, Browert P, Palmer S. Altruism by proxy: volunteering children for bone marrow donation. Br Med J 1996; 312: 240– 3. 30. Chan K-W, Gajewski JL, Supkis D, Pentz R, Champlin R, Bleyer WA. Use of minors as bone marrow donors: current attitude and management. J Pediatr 1996; 128: 644–8. 31. Peerzada JM, Wendler D. Hematopoietic stem cell transplant research with pediatric donors: when can institutional review boards approve it? Transplantation 2006; 81: 1616–20. 32. Pentz RD. Healthy sibling donation of G-CSF primed stem cells: a call for research. Pediatr Blood Cancer 2006; 46: 407–8. 33. Roberts I. Bone marrow transplantation in children: current results and controversies. Bone Marrow Transplant 1994; 14: 197–9. 34. Gluckman E, Eurocord Network Organisation. Ethical and legal aspects of placental/cord blood banking and transplant. Hematol J 2000; 1: 67– 9. 35. Dame L, Sugarman J. Blood money: ethical and legal implications of treating cord blood as property. J Pediatr Hematol Oncol 2001; 23: 409–10.
Ethical Issues in Hematopoietic Cell Transplantation 36. Alby N. The child conceived to give life. Bone Marrow Transplant 1992; 9(Suppl 1): 95–6. 37. Samuel GN, Kerridge IH, Vowels M, Trickett A, Chapman J, Dobbins T. Ethnicity, equity and public benefit: a critical evaluation of public umbilical cord blood banking in Australia. Bone Marrow Transplant 2007; 40: 729–34. 38. Duarte RF, Pamphilon D, Cornish J et al. Topical issues in unrelated donor haematopoietic stem cell transplants: a report from a workshop convened by the Anthony Nolan Trust in London – 2005. Bone Marrow Transplant 2006; 37: 901–8. 39. Marshall E. Clinical promise, ethical quandary. Science 1996; 271: 586–8. 40. Rubinstein P, Stevens CE, Adamson JW, Migliaccio G. Umbilical cord blood cells: informed consent. Bone Marrow Transplant 1995; 15: 160. 41. Ammann AJ. Placental-blood transplantation. N Engl J Med 1997; 336: 68–70. 42. Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996; 335: 157–66. 43. Section on Hematology/Oncology and Section on Allergy/Immunology. Cord blood banking for potential future transplantation. Pediatrics 2007; 119: 165–70. 44. Schaison GS. The child conceived to give life. The point of view of the hematologist. Bone Marrow Transplant 1992; 9(Suppl 1): 93–4. 45. Verlinsky Y, Rechitsky S, Sharapova T, Morris R, Taranissi M, Kuliev A. Preimplantation HLA testing. J Am Med Assoc 2004; 291: 2079–85. 46. Grewal SS, Kahn JP, MacMillan ML, Ramsay NKC, Wagner JE. Successful hematopoietic stem cell transplantation for Fanconi anemia from an unaffected HLA-genotype-identical sibling selected using preimplantation genetic diagnosis. Blood 2004; 103: 1147–51. 47. Van de Velde H, Georgiou I, De Rycke M et al. Novel universal approach for preimplantation genetic diagnosis of {beta}-thalassaemia in combi-
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33
Richard P. McQuellon & Michael Andrykowski
Psychosocial Issues in Hematopoietic Cell Transplantation
Introduction Hematopoietic cell transplantation (HCT) is an aggressive, dynamic, and evolving medical procedure. It is used in the treatment of a variety of life-threatening diseases, often as a high-risk, best choice for patients with few treatment alternatives. Consequently, HCT is associated with the potential for a wide variety of serious physical, psychological, and social complications. In this chapter, we discuss the origin, recognition, and management of the psychological and social complications associated with HCT. We use the term “psychosocial” to refer to issues that are psychological and/or social in nature. Similarly, we use the term “HCT” to refer to a class of medical procedures that involve the transplantation of hematopoietic stem cells (HSCs) derived from peripheral blood, cord blood or bone marrow. For the most part, the literature examining psychosocial issues in HCT does not distinguish among stem cells of different origin. A set of generic psychosocial issues is germane regardless of the origin of stem cells or the underlying disease. We direct the major part of our discussion to psychosocial issues pertinent to adult HCT recipients. We also provide some discussion of psychosocial issues pertinent to donors and caregivers. The recognition and study of psychosocial issues associated with HCT has a relatively long history [1–3]. As HCT has gradually evolved as a medical procedure, some of the psychosocial issues associated with it have evolved as well. There have been several recent trends in HCT that can affect how recipients and their families experience the transplant, and consequently what type of psychosocial issues emerge. First, peripheral blood rather than bone marrow is now the main source of stem cell support. This eliminates the need for the bone marrow harvest and its concomitant general anesthesia requirements. Stem cells can now be harvested in a series of one to three sessions in an apheresis unit lasting approximately 1–3 hours. Before stem cell collection, stem cell donors are likely to be primed with growth factors in order to increase the concentration of HSCs in the peripheral blood. Second, older individuals are now being transplanted with greater frequency. This increases the likelihood of physical morbidity for recipients. It also increases the possibility that older caregivers may be called upon to provide supportive care to their loved one at a time when they may need considerable health care themselves. Third, the use of reducedintensity conditioning regimens is becoming more common. These Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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reduced-intensity regimens produce less physical morbidity and thus may often be done on an outpatient basis, resulting in less isolation of the patient and less exposure to the hospital environment. Fourth, with the advent of new testing methods, better matching in the allogeneic transplant setting is possible. This has the potential to reduce the incidence of graft-versus-host disease (GVHD) and the distress and psychosocial problems that accompany this serious and often life-threatening complication. Finally, much more information on potential clinical outcomes associated with HCT is available to potential HCT recipients and their families. For example, a prospective HCT recipient with lymphoma can now go to the website for the Center for International Blood and Marrow Transplant Research (CIBMTR; http://www.cibmtr.org/) and view Kaplan–Meier survival curves relevant to their specific disease status. There they would see the following: “Among 2,292 patients receiving autotransplants for follicular lymphoma between 1996 and 2004, 3-year probabilities of survival were 73% ± 1.5% and 52% ± 7% for chemosensitive and chemo-resistant disease, respectively.” This greater access to information regarding clinical outcomes may be either frightening or comforting to potential recipients and their families depending on their understanding of their disease and overall outlook. Another recent trend in HCT is the increased emphasis on post-transplant survivorship monitoring [4]. Greater emphasis is now placed on periodic and systematic monitoring of a range of clinical outcomes in recipients. This is likely to identify psychosocial problems that may be present and consequently improve the chances they will be addressed. Several important studies of recovery following HCT conducted with relatively large cohorts of recipients and caregivers have identified key potential problem areas and pointed to potential areas for psychosocial intervention [5,6]. Also, an important survey of 600 HCT survivors was conducted in 2006 by the Bone Marrow Transplant Information Network (http://BMTinfonet.org). When asked to identify the most significant issues facing them following transplantation, 73% of respondents endorsed emotional/psychological health as a problem. The results of the survey reflect a reality for a significant number of recipients: HCT is highly stressful, and psychosocial issues remain a primary concern for the majority of recipients for a long period of time after transplantation.
Psychosocial issues in HCT: recipients Two major sources of distress can be identified for HCT recipients during the course of treatment and recovery. First, HCT is often used after other conventional treatment options have failed in diseases that
Psychosocial Issues in Hematopoietic Cell Transplantation
are virtually always life-threatening. Furthermore, HCT itself is associated with significant risk of mortality: treatment may actually hasten a patient’s death relative to what might have been expected had conventional treatment, or no treatment, been employed. The context of HCT is one of uncertain and yet ever present life-threat that serves as a significant source of stress for recipients, family caregivers, donors, and medical staff. In its simplest form, the potential HCT recipient is faced with this decision: high-risk treatment with a chance of cure or some alternative course of action with no chance of cure. A second source of distress in the HCT setting is the aggressiveness of the procedure. High-dose or reduced-intensity chemotherapy regimens with or without localized and/or total body irradiation produce acute side-effects (e.g. nausea, mucositis, hair loss, and fatigue) and long-term effects (e.g. chronic GVHD, secondary malignancies, etc.). In the best of circumstances, most HCT recipients typically experience an array of taxing physical stressors within the context of the psychological and social stress associated with medical uncertainty and life-threatening circumstances. Even when the transplant is a success and recipients resume their life outside of a closely monitored medical environment, the need for ongoing surveillance for recurrent disease is a periodic reminder of an ever-present awareness: they have entered a high-stakes medical scene and will remain there for the rest of their lives. Stages of HCT Over the course of the HCT process, recipients are confronted with an often highly predictable sequence of treatment-related events. The word “stage” refers to a variable time period that is characterized by specific tasks and physical and psychosocial stressors that recipients and their caregivers must negotiate. In the broader cancer survivorship literature, the time period from diagnosis through the long term has been described as the “seasons of survival” [7]. Descriptions of the number and nature of stages of HCT have varied [2,8]. We will organize our discussion of psychosocial issues confronting the HCT recipient around five relatively distinct stages: (1) the decision to undergo HCT; (2) pre-HCT preparation; (3) post-HCT hospitalization; (4) hospital discharge and early recovery; and (5) long-term recovery (Table 33.1). These stages represent a general map of the process and not an invariant sequence of events and stressors facing all recipients. Some psychosocial issues that appear during one stage of HCT may surface again at a later time. For example, the prospect of a shortened life span due to a life-threatening illness suggests patients may struggle with an intense awareness of their mortality at the time of their decision to undergo HCT [9]. Such concerns may fade into the background at the time of hospital discharge and early recovery, only to re-emerge as significant concerns during long-term recovery. Many of the physical stressors can be directly related to anxiety, depressive symptoms, and distress that may appear periodically during each stage of treatment and recovery. Stage 1: the decision to undergo HCT The decision process leading to HCT is usually highly stressful. Few individuals realize that the very fact they are contemplating HCT places them in a category where a natural life span may be less than likely. At best, recipients can expect a host of toxic short- and long-term sideeffects that typically exceed those associated with conventional therapeutic options. At worst, the recipient may die during the course of HCT, experiencing an earlier death relative to what might have occurred had other conventional or palliative therapeutic options been selected. Despite the evolution of the medical context of HCT, there has been little interest in how these recent changes may affect the decision matrix or the decision process more generally. Most of the extant research on
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HCT-related decision making has focused upon recipients’ provision of informed consent. This is a rather limited perspective on HCT-related decision making, however. The formal consent process is only the final step in an often protracted decision-making process. Earlier steps in the decision sequence include the decision to consider HCT as a treatment option and to undergo formal medical evaluation for HCT (i.e. medical referral for HCT). Medical referral for treatment. The decision to undergo HCT begins with its consideration as a treatment option. In most cases, the possibility of HCT is raised initially by the medical oncologist managing the patient’s care. In other instances, patients may raise the prospect of HCT with their physicians on their own. Regardless of the source of referral, up-to-date knowledge regarding HCT is critical at this juncture. This is particularly true when HCT is viewed as the only viable hope for disease cure. In these instances, patients are likely to quickly embrace HCT as their only alternative, disregarding the alternative of no further curative treatment. In effect, a psychological commitment to HCT may be made at the time this option is first raised and considered [10], perhaps before the patient possesses any substantial knowledge of the risks and benefits associated with HCT. This commitment may be immutable by the time the patient arrives at the transplant center for initial evaluation. The referring physician therefore plays a critical role in ensuring that the decision to undergo HCT is truly an “informed” decision. Selection and screening of candidates. The period of time leading up to HCT poses many psychosocial stressors. We have identified the pattern of some of these stressors in Table 33.1. The isolation procedures and physical hazards (e.g. infections and severe toxicity) associated with HCT have been assumed to have the potential to create great distress in prospective patients. Consequently, the American Society of Blood and Marrow Transplantation included a psychiatric/psychosocial assessment as part of the guidelines for treatment established in 1995 [11]. The intent of this assessment is to obtain information about a number of areas of functioning, including current psychological distress, past or present psychiatric history, current social support, coping style, history of medical noncompliance, and past or current problems with alcohol or substance abuse. While psychosocial considerations, such as a history of alcohol abuse or medical noncompliance, may be used to screen out candidates for solid organ transplantation [12], rejection of an HCT candidate strictly on the basis of psychosocial considerations is rare. Psychosocial information obtained during the pre-HCT evaluation is most often used to anticipate problems in caring for the patient and to develop an individualized plan of care over the course of HCT. For example, to facilitate care planning, Molassiotis developed a brief scale to prospectively identify patients prone to emotional difficulties during post-HCT hospitalization [13]. Standardized approaches to the pre-HCT psychosocial evaluation have been described, including the Psychosocial Levels System [14], the Transplant Evaluation Rating Scale (TERS) [15], and the Psychosocial Assessment of Candidates for Transplantation Scale (PACT) [16]. All three approaches focus, for the most part, upon the content areas indicated above. The PACT and the TERS yield comparable information [17], while the TERS appears to possess somewhat better psychometric properties than its precursor, the Psychosocial Levels System [18]. There is a need for more research in this area of screening and selection of HCT candidates. For the most part, the clinical utility of standardized approaches to pre-HCT psychosocial evaluation for selecting appropriate candidates for HCT remains to be empirically established. While it might be anticipated that inadequate social support, a history of treatment nonadherence or substance abuse problems would be associated with poorer post-HCT outcomes, this linkage has not been firmly
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Table 33.1 Themes, tasks and psychosocial issues associated with five stages of hematopoietic cell transplantation (HCT) Stage
Issue
*1. The decision to undergo HCT
Theme: active decision making • Confronting mortality and the possibility of death • Managing the uncertainty of treatment outcome • Considering alternative treatments • Financial considerations/insurance limitations • Psychosocial evaluation • Informed consent process • Symptoms of anxiety, depression, and distress
2. Pre-HCT preparation
Theme: aggressive treatment • Managing acute treatment side-effects • Adapting to isolation and hospital routine • Adopting the patient role • Confronting unfamiliar procedures and treatment (e.g. total body irradiation) • Separation from family and friends • Adjusting to altered body image (e.g. hair loss, weight loss, and Hickman catheter placement)
3. Post-HCT hospitalization
Theme: watchful waiting • Waiting for engraftment • Heightened physical and emotional vulnerability • Contending with the boredom of isolation • Maintaining morale and hope • Dealing with life-threatening complications • Encountering acute psychologic distress • Managing discouragement
4. Hospital discharge and early recovery
Theme: transition from intense medical surveillance • Managing the loss of daily psychosocial support of the medical team and allied health-care professionals (e.g. pastoral care) • Contending with the stress of frequent medical appointments, readmissions, and setbacks • Reintegration into valued social roles (e.g. parent, spouse or social companion) • Managing unexpected sequelae (e.g. profound fatigue) • Adapting to potential frustration, depressive symptoms, and anger • Complying with self-care guidelines and daily medicine regimen
5. Long-term recovery
Theme: establishing the new normal life • Re-establishing primary identity and relinquishing the patient role • Recovery of valued roles in the community, at work, and at home • Adjust to losses associated with transplantation, e.g. fertility • Return to employment • Accepting the possibility and reality of long-term effects (e.g. cataracts or second malignancies)
* Many of these issues continue over the course of the five stages. For example, confronting mortality and the prospect of death may be revisited more or less intensely each time the patient returns to the transplant center for ongoing evaluation and follow-up over the course of many years. Also, anxiety, depressive symptoms, and distress may manifest themselves over the course of the entire HCT process. We place these symptoms in stage 1, but they might just as easily emerge in any stage.
established. However, several studies suggest an association between psychosocial status at the time of HCT and important post-HCT outcomes such as survival [19–22] and risk for medical complications [21,23]. While there has been no solid link between pre-HCT psychosocial variables and post-HCT survival [24–28], this remains a provocative area of research. Hoodin and Weber conducted a systematic review of the literature and identified 15 methodologically sound studies that highlight psychosocial factors affecting survival after HCT [29]. Based on their review, they cautiously suggest that survival after HCT is not affected substan-
tially by depressed mood (in contrast to Loberiza et al., noted above), psychopathology or social support. However, longer survival may be related to less anxious preoccupation, higher fighting spirit, and better quality of life (QOL) ratings before transplantation. The authors concluded that this is an important area of inquiry, and that the literature is not sufficiently developed to provide strong evidence for a relationship between psychological variables and survival following HCT. Whatever impact psychosocial factors have on survival, they may be weak relative to patient characteristics and clinical factors such as histologic subtypes [28].
Psychosocial Issues in Hematopoietic Cell Transplantation
Provision of informed consent. Informed consent occurs in a face-toface meeting between the transplant physician and the recipient and caregivers either before and/or at the time of pre-HCT hospitalization. At this meeting, the multipage consent forms are gone over in more or less detail, depending on the preference of the recipient. Some recipients want to go over every word; others prefer to gloss over the document. In any case, the end product is a witnessed, legal document signed by the patient and physician. In theory, the consent process allows patients to make an informed and voluntary decision regarding whether to proceed with HCT. In practice, however, this ideal is unlikely to be realized [1,10]. There are several reasons for this, including the nature and amount of information required to make an informed decision, the stressful context within which an informed decision is made, and the reluctance of potential recipients to actively consider the risks associated with HCT. It is not uncommon for potential HCT recipients to come into the initial consultation with limited knowledge of their disease and their treatment options. More importantly, they may have little understanding of the morbidity and mortality associated with transplantation as well as survival rates without a transplant. Stiff et al. studied recipients’ understanding of their disease and treatment plan at the time of the initial HCT consultation in 99 HCT candidates [30]. Participants were assessed both before and after a 3-hour multidisciplinary team consultation that was held before transplantation. Prior to the consultation, about three-fourths of these potential recipients reported adequate information about their disease, yet a large percentage of patients lacked knowledge about their 1-year prognosis with or without any therapy. After the consultation, two-thirds of the patients reported they had obtained enough information to make an informed decision about whether or not to undergo HCT. This was compared with only 23.2% prior to this consultation. While the consultation clearly increased the number of individuals who believed they knew enough to make an informed treatment decision, the fact that one-third of respondents did not believe they had enough information to make an informed decision indicates that a substantial number of HCT recipients may not be making informed decisions to undergo this treatment. Significant elevations in psychological distress have been observed in adult HCT recipients within the 48 hours after provision of informed consent [31]. Most consent forms contain exhaustive descriptions and listings of the medical risks associated with HCT, sufficient to frighten even the most psychologically stable adult or parent. Furthermore, research has documented that many recipients are considerably distressed in the days prior to HCT [32–37]. Distress can compromise the consent process by inhibiting communication with health-care providers, limiting rational consideration of the risks and benefits of HCT, and inhibiting comprehension or memory for information communicated during the important consent discussions. Indeed, research suggests that distressed individuals may be more likely to agree to undergo experimental therapies [38]. HCT is being used increasingly as a “first-line” therapy for some medical conditions, making the treatment decision even more complex and distressing for all concerned. While HCT may increase the likelihood of cure or prolonged remission relative to other therapeutic options, these other options (e.g. conventional oral or intravenous chemotherapy) may be associated with less morbidity and virtually no short-term mortality risk. An example of this dilemma is seen in the treatment of chronic myeloid leukemia (CML). Conventional therapy for CML, while fairly benign, is not curative, and without further treatment the disease will likely ultimately accelerate. Patients with a suitable donor may be advised to consider undergoing HCT before their disease worsens. Given the increased morbidity and mortality associated with HCT, an optimal strategy might be to postpone HCT as long as possible.
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However, if CML enters an accelerated phase, risks for HCT-related morbidity and mortality increase relative to what the risks would have been if HCT had been implemented during the chronic, stable phase of the disease. This decision is now even more complicated with the recent success of the oral chemotherapeutic agent imatinib mesylate (Gleevec) for the treatment of CML. This agent has produced prolonged remissions in many CML patients and may even produce cure. The decision of whether and when to undergo HCT fundamentally involves a trade-off between the increased toxicity associated with HCT and the increased potential for disease cure, or at least long-term, disease-free survival. QOL considerations must be balanced against quantity of life considerations. There is evidence that patients are willing to risk substantial toxicity and increased risk of mortality for increased survival even if the outcome of treatment is uncertain [39,40]. Based on the premise that the HCT decision is based upon patients’ knowledge of HCT and their preferences for certain mortality and morbidity (e.g. QOL) outcomes, Sebban et al. developed a bedside decision board to assist CML patients and their physicians in deciding between HCT and other more conservative management options for CML [41]. They tested the decision board with 42 healthy hospital personnel and not HCT recipients. Satisfaction with treatment preference was higher for those exposed to the decision board compared with those presented with an abbreviated version of the decision board. Such decision tools could be a welcome addition to the pre-HCT decision context but require testing and evaluation with actual prospective HCT recipients before they can be advocated for routine use. Stage 2: pre-HCT preparation The second stage of HCT begins with the start of the preparative regimen, consisting of either high-dose or reduced-intensity chemotherapy, often in combination with total body irradiation. Typically, the recipient is hospitalized during this period, although it has become more common for patients to receive their pre-HCT preparative regimen on an outpatient basis. While side-effects vary as a function of the type and intensity of the preparative regimen, they are often severe, even with aggressive supportive care, and may continue to be experienced after preparative therapy has been completed. Critical psychosocial issues during the pre-HCT preparation phase center on: (1) adaptation to hospitalization, including becoming accustomed to the hospital routine and being confined to a single hospital room and the immediate surroundings; (2) learning proper infection control procedures (I would suggest not being too specific as these vary from center to center); and (3) maintenance of the recipient’s psychological strength and coping capacity. Simple hospital routines such as frequent vital sign monitoring in the middle of the night can be annoying. Pain and nausea are typically controlled through liberal use of antiemetic and analgesic medications, all of which can alter the recipient’s psychological status. Cognitive-behavioral therapies can also be used as adjunctive therapy for symptom control, especially with patients who are averse to antiemetic and analgesic medications. In a pair of well-designed studies, Syrjala et al. examined the utility of several cognitive-behavioral interventions (e.g. hypnosis, relaxation and imagery, and cognitive-behavioral coping skills training) for controlling pain and nausea associated with pre-HCT preparative therapy [42,43]. While hypnosis and coping skills training appeared to have little impact upon nausea and vomiting [42], all three interventions reduced pain associated with mucositis. Results suggest that hypnosis can significantly add to the effects obtained with standard analgesic medications. Widespread incorporation of cognitive-behavioral therapies into the management of HCT recipients may be difficult since implementation of these therapies is typically time-consuming and costly. However, more abbreviated cognitive-behavioral interventions could be developed
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and could be effective. For example, some evidence suggests a singlesession psychoeducational intervention designed to enhance coping with treatment side-effects can be effective in reducing nausea, fatigue, and anxiety in autologous HCT recipients [44]. As administration of the pre-HCT preparative regimen occurs either in the hospital or in an outpatient clinic setting close to the transplant center, this period of time can be used to plan for the management of recipients’ psychosocial needs as they will likely emerge during postHCT recovery [45]. If not completed already, a detailed psychosocial evaluation can be performed at this time. Identification of potential risk factors for psychosocial difficulties post HCT is key to their prevention and management. A proactive approach can be helpful as it establishes a relationship between the HCT recipient and a mental health-care professional (e.g. social worker, counselor, psychologist or psychiatrist) who might later help manage any emerging psychosocial difficulties. Elements of this proactive approach include identification of coping resources and deficits, discussion of psychosocial difficulties that might be anticipated, and training in behavioral skills to improve symptom management and facilitate coping. Some research has focused upon distress experienced during this preHCT phase of the transplant process. Trask and colleagues assessed psychological distress in 50 HCT recipients (both allogeneic and autologous) prior to transplantation [36]. Fifty percent and 51% of recipients reported clinically significant levels of emotional stress and anxiety, respectively. Twenty percent reported clinically significant levels of depression. Andorsky et al. assessed pre-HCT physical and mental functioning using the Medical Outcomes Scale Short Form 36 (SF-36) in 320 HCT recipients (allogeneic and autologous) [46]. Results indicated that mean mental functioning scores prior to HCT were no different from those of the general population. Of course, mean group scores can obscure the fact that a significant proportion of respondents might be experiencing significant distress. Nevertheless, this recent study suggests many current HCT recipients, perhaps a majority, may approach their transplant with reasonably good mental functioning. Stage 3: post-HCT hospitalization Upon completion of cytotoxic preparative therapy, infusion of hematopoietic cells takes place. Most recipients experience this brief infusion of stem cells as anticlimactic. However, completion of this infusion of stem cells highlights a phase of the HCT process characterized by profound physical vulnerability. During the weeks after stem cell infusion, recipients are monitored daily for evidence of stem cell engraftment and immune system reconstitution. Many recipients request printed copies of their daily laboratory results and diligently focus on key milestones indicating engraftment. Indeed, the mood shifts of recipients and family members often fluctuate daily depending heavily on the up or down movement of their “counts.” Once the side-effects of preparative therapy have resolved, and if no serious infectious complications have arisen, the recipient enters a period of hopeful waiting. Weakness and fatigue are common at this time, although the recipient may actually feel relatively good. Should serious medical complications develop, however, this quiescent state quickly ends. Given the life-threatening nature of many medical complications during this early post-HCT period, recipients often react to medical setbacks with considerable distress. A lengthy hospitalization, often characterized by a series of vexing medical complications, can be profoundly demoralizing, especially if a tentative discharge date had been set, only later to be postponed. Some recipients respond to a prolonged hospitalization with lethargy and decreased motivation, others with anger and agitation. In extremely rare cases, a recipient may attempt to leave the hospital against medical advice. At these times, the psychosocial team can play a critical, if not lifesaving, role by helping the
recipient to better cope with the stresses, disappointments, and lack of control characteristic of this post-HCT stage of the transplant process. The primary psychosocial issues during this early period of post-HCT recovery center on the promotion of continued effective coping with the physical and psychological stresses associated with HCT. In addition to anxiety and depressive reactions [47], recipients may experience neurocognitive symptoms or neurologic complications [48–50]. Episodes of delirium may occur and may last several hours to several days. Symptoms are diverse and can include alterations in level of consciousness, altered sensory or perceptual function (e.g. hallucinations), impairments in memory, concentration, and higher cognitive processes, mood swings, impaired judgment, and sleep disturbance [51]. HCT recipients differ in the amount of time they are required to spend in a dedicated hospital “transplant unit” before and after HCT. The point along this inpatient–outpatient continuum that characterizes a recipient’s experience is critical to understanding the particular psychosocial difficulties they may encounter. The more HCT is experienced as a lengthy inpatient procedure, the more likely recipients are to evidence psychosocial difficulties associated with prolonged confinement in an institutional setting (e.g. boredom, loss of control, loss of identity, social and physical isolation, interrupted sleep, etc.). These are all mitigated when HCT is an outpatient procedure. Other difficulties may be substituted, however. For example, while hospitalization and strict infection control procedures can be stressful, the immediate availability of supportive care from the medical team can be enormously reassuring to the recipient. Assistance with an emergency medical crisis is immediately available for recipients while they are hospitalized; such assistance may not be as readily available in the outpatient setting. This small difference can have a profoundly disconcerting impact on some recipients. Some recipients might even prefer the psychological security afforded by hospitalization on a specialized unit in lieu of the freedoms associated with a more outpatient-based HCT procedure. The stressors associated with HCT can bring out both the best and the worst in patients and their families. The so-called “difficult” patient or family member (someone who is angry, demanding, and often uncooperative with the HCT team) can pose many challenges. Certain principles underlie the successful management of such individuals, including frequent communication among medical team members, the formulation of a reasonable treatment care plan, and the consistent implementation of this treatment plan by all members of the team. Multidisciplinary team meetings can be very helpful in planning and ensuring a consistent course of treatment for challenging patients. Ongoing, effective communication with the difficult patient or family member is critical, along with continued attempts to identify, convey understanding of, and address the fears and anxieties likely underlying their difficult behavior. Despite the presence of multiple and significant stressors during the post-HCT hospitalization and the difficulty in conducting psychosocial research during this stage, there is some relevant research on this time period. Prieto and colleagues studied both the physical and psychological status of 220 inpatients receiving HCT [52]. Patients were assessed at hospital admission, on the day of their transplant, and 7 and 14 days after HCT. Anxiety was highest at hospital admission and decreased thereafter. The proportion of recipients who qualified as a “case” of anxiety as measured by the Hospital Anxiety and Depression Scale declined from 22.7% at the initial assessment to 8% 14 days after HCT. In contrast, the percentage of recipients qualifying as a “case” of depression increased from a low of 11.4% at the initial assessment to 21% 7 days after transplant. The authors suggest their figures might underestimate the prevalence of anxiety and depression symptoms because of the use of psychopharmacologic treatments during hospitalization. Zittoun et al. assessed QOL as well as depression and anxiety symptoms during
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hospitalization in 179 HCT recipients [53]. Recipients were assessed 10, 20, and 30 days after completion of their pre-transplant preparative regimen. At each assessment, approximately one-fourth to one-third of respondents reported clinically significant levels of depression and anxiety. The results of these studies suggest that significant psychological distress during the post-HCT hospitalization period is fairly common. Consequently, periodic monitoring of distress during hospitalization is recommended with short-term counseling and/or psychopharmacologic intervention utilized if needed. However, there are significant challenges to accurate assessment of mood and psychosocial status during this stage of the HCT process. In particular, accurate recognition of depression at this time can be problematic. Recipients’ eating and sleep–wake cycles are almost always disrupted to some degree, and fatigue and lack of energy are common side-effects of intensive HCT preparative regimens. A lack of energy may also be the result of anemia, sleep disturbance, and/or nutritional deficits. Persistent fear and worry can also be a significant energy drain. Lethargic recipients might be mislabeled as depressed, or, even worse, truly depressed recipients might be mislabeled as simply being fatigued due to the variety of lethargy-inducing factors potentially present in the HCT setting. Health-care providers must be open to considering the variety of underlying explanations for the significantly depressed or fatigued HCT recipient, carefully weigh the evidence supporting different competing explanations, and ultimately prescribe the appropriate course of treatment. Stage 4: hospital discharge and early recovery When engraftment has occurred, blood counts are near normal, and recipients are medically stable, they are ready to leave the HCT unit. HCT recipients may also be moved to a less restrictive setting such as a “step-down” unit in the hospital or lodgings close to the HCT center for a period of time after discharge from the hospital transplant unit. This intermediate move allows for continued careful monitoring of those recipients whose homes are distant from the transplant center. Frequent medical follow-up on an outpatient basis is the norm during the first several weeks and months post HCT. The period after treatment ends brings new challenges [54]. Hospital discharge and return home mark the beginning of an often long process of convalescence, recovery, and life reintegration. Critical psychosocial issues associated with the early recovery period include coping with any anxiety generated by the recipient’s gradual weaning from the intensive medical care available in the transplant unit, coping with the often profound physical debilitation experienced by recipients at this time, and adjusting to the need to vigilantly practice a regimen of self-care behaviors designed to minimize the risk of infection. While hospital discharge and a return home is an eagerly anticipated milestone, it can be highly distressing. Many recipients report anxiety at the realization they will no longer be under the constant surveillance and care of medical staff. Fears that life-threatening difficulties might emerge when appropriate medical assistance is not easily available can temper any excitement surrounding a return home or a move to temporary lodging outside the hospital. Frequent clinic follow-up is needed during the early recovery stage. Such follow-up necessitates either remaining close to the transplant center or regularly commuting many hours from the recipient’s home, which may be quite geographically distant from the center. Coping with physical debilitation is a significant challenge for nearly all patients in the early recovery stage. It is not uncommon for recipients to report generalized weakness and fatigue during the 6–12 months after HCT [37,55]. These symptoms can drastically interfere with resumption of routine activities characteristic of the recipient’s premorbid lifestyle. How recipients react to these symptoms and their functional limitations
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can be influenced by expectations for post-HCT recovery. While most recipients experience some physical limitations, the anticipated timeline for post-HCT recovery varies considerably [56]. Recipients and family members are often overly optimistic in their expectations. Many hope for a quick return to a healthy, fully functioning, “normal” life. Frustration, depression, and anger can result when these expectations are not met [10]. Most HCT recipients experience significant physical deconditioning as a result of their lengthy hospitalization, yet they are rarely enrolled in any kind of systematic program of physical rehabilitation. This is likely due to many factors including geographic distance between the transplant center and the recipient’s home. The few studies that examine the benefits of exercise programs for HCT recipients show great promise [57,58]. Routine screening following HCT for psychosocial distress is uncommon in spite of the fact that the procedure is physically and emotionally demanding. One study evaluated 80 autologous (44%) and allogeneic (56%) transplant patients prior to admission, at the first clinic visit after hospital discharge, and at 100 days post transplant [59]. A total of 44% of patients had symptoms of either depression, anxiety or post-traumatic stress disorder after transplantation. The measurement of pretransplant distress was associated with detection of distress after transplantation, suggesting that pretransplant assessment is predictive of post-transplant functioning. An important finding of the study was that there was an association between self-reported distress and noncompliance with medication following transplantation. This suggests the obvious to the clinician: distressed patients may be less likely to comply with the rigorous regimen of medications following transplantation. Promotion of appropriate self-care behavior is also a significant issue during the early recovery stage. Good self-care behavior is intended to reduce the risk of infection and includes both behavioral restrictions (e.g. avoid crowds) and the prescription of specific activities (e.g. engage in some form of physical exercise). Patients may be required to take up to 30 pills each day at varying schedules, an assignment which is taxing for even the most diligent. Failure to adhere to this self-care regimen can result in very serious medical complications and even death. Despite the importance of appropriate self-care after HCT, little research has examined this issue. Our own experience suggests adherence to self-care guidelines can be a significant problem after HCT in both adult and pediatric settings. We have known of one male HCT recipient who acquired a mortal wound infection after shaving with a forbidden straight razor and one patient who could not stop smoking and contracted a fatal fungal infection. While these are notable examples of nonadherence, less flagrant violations of self-care guidelines are common and can result in equally disastrous consequences. In this regard, there is some indication in the literature that screening might identify those at risk for nonadherence of the strict medication regimens [59].
Stage 5: long-term recovery Successful recovery after HCT is characterized by waning of the physical side-effects of treatment, decreased symptoms of disease, a return to premorbid levels of psychosocial and physical functioning, and a resumption of valued life roles. This process usually begins 3–6 months after HCT and can continue for up to 2 years or more [56]. Syrjala and colleagues have illustrated the recovery trends in patients with treatment-related distress and depression versus no depression, respectively, in a sample of allogeneic and autologous transplant recipients (Figs 33.1 and 33.2). It is critical to recognize, however, the wide variability in the “the trajectory of recovery” following HCT [37]. Unfortunately, some recipients never fully recover, either physically and/or psychosocially, while for others recovery requires a lengthy journey, a journey which may not end in the same place it began.
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Fig. 33.1 Mean scores for treatment related distress comparing autologous versus allogeneic transplant recipients over time. HCT, hematopoietic cell transplantation.
Fig. 33.2 Respondents with physical limitations over time, according to the presence or absence of depressive symptoms at baseline.
Physical and psychosocial late effects of HCT. One of the benchmarks often used by HCT recipients to gauge the progress of their long-term recovery is the extent to which they perceive they have “returned to normal” [60]. Clinical experience and empirical research both suggest that most disease-free HCT recipients will experience the sense that they have “returned to normal” within the first several years following transplant [10,37], although the recovery process may take as long as 3–5 years [56]. What is “normal,” of course, is highly dependent upon each recipient’s unique perspective. While acknowledging that they
have “returned to normal,” it is not uncommon for even disease-free HCT recipients to say that their life will never be the same. Even with the return of what they might consider a normal lifestyle, few recipients are left unchanged by their HCT. Recipients may evidence a variety of late physical effects, such as pulmonary problems, cataracts, sterility, endocrine dysfunction, chronic GVHD, and weakness/fatigue (with possible sexual dysfunction). Even in the absence of such late physical effects, most recipients experience some interruption in normal developmental tasks, for example attending school and developing peer group relationships in the case of pediatric recipients, or maintaining a career and establishing satisfactory marital and family relationships in the case of adult recipients. Recipients may also evidence a variety of psychosocial late effects such as anxiety, depression, strained interpersonal relationships or even post-traumatic stress disorder [61]. These psychosocial late effects may persist for months or years after HCT or they may first emerge several years after HCT. In all instances, the presence of significant psychosocial late effects exact a significant toll on a recipient’s QOL [32]. GVHD in its acute or chronic form can precipitate many psychosocial stressors for patients [62]. These can include additional medical appointments and accompanying stressors for caregivers, as well as symptoms of depression and anxiety. The extent to which GVHD is a psychosocial stressor seems intuitively obvious, yet its impact on mental health has yet to be sufficiently documented. There are some conflicting data in the literature regarding to what extent either acute or chronic GVHD affects mental health. In one study, the patients’ experience of acute or chronic GVHD in the 12 months following transplantation was not reflected in worse scores on the Mental Component Subscale or Physical Component Subscale of the SF-12 [62]. Clinical observation suggests that both acute and chronic GVHD could precipitate intense anxiety and/or depressive symptoms, and/or discouragement in patients seeking to return to normal following HCT. Indeed, we have counseled patients contending with bouts of chronic GVHD several years post transplantation whose functional activities and work life have been severely impaired by this complication. Hjermstad and colleagues studied health-related QOL, fatigue, anxiety, and depression in HCT recipients 3–5 years after transplantation [63]. A total of 248 patients (n = 61 allogeneic HCT, n = 69 autologous HCT, n = 118 conventional chemotherapy) were included in the study. Importantly, both the transplant and conventional chemotherapy patients reported more fatigue than population norm values after 3 years. No differences were found between the groups in terms of anxiety and depression. When comparing across groups, anxiety and depression were less of a problem than fatigue. The authors concluded with a strong recommendation for regular follow-up care for patients even after 3 years post transplant, with an emphasis on assessment of functional status and fatigue. Despite the presence of physical and psychosocial late effects, the majority of long-term HCT survivors report relatively satisfactory global QOL. For example, Broers et al. described how 90% of their sample of HCT recipients reported good-to-excellent QOL 3 years post-HCT [64], despite the fact that about 25% continued to experience significant functional limitations even 3 years after transplantation. Thus, recipients often acknowledge having quite good QOL while at the same time experiencing the continued presence of significant physical or psychosocial difficulties. This apparent paradox can be at least partially explained by some evidence that suggests patients with a chronic disease may alter their perspective on QOL, establishing a lower ceiling of acceptability and redefining what constitutes “good” QOL. This apparent paradox might also be explained by consideration of the potential of the transplant experience to exert a positive impact upon recipients’ lives. It is very important to note that the long-term impact
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of HCT is not uniformly negative. Recipients can and often do experience positive psychosocial sequelae of their transplant experience [65]. These positive psychosocial “late effects” are often subsumed under the label of “post-traumatic growth” and can include improved self-esteem, perception of a new meaning in life, redirected and re-established life priorities, enhanced capacity for compassion, improved family and social relationships, and renewed spiritual faith. Interestingly, HCT survivors’ reports of such “benefits” of the transplant experience often coexist with reports of significant physical and psychosocial difficulties. Andrykowski et al. compared HCT survivors with matched healthy controls on a variety of indices of physical and psychosocial functioning [65]. Relative to healthy controls, HCT survivors reported poorer physical, psychological, and social functioning. Conversely, however, HCT survivors also reported significantly more psychological and interpersonal growth, with the size of these statistically significant group differences also suggesting clinical importance. Hence, long-term post-HCT recovery can involve more than simply adjustment to the potential negative physical and psychosocial late effects of HCT and the quest to re-establish the premorbid status quo. Rather, long-term recovery can involve attempts to derive meaning from the HCT experience and identify benefits that may have paradoxically come from it. In the end, successful long-term recovery may involve the establishment of a fundamentally altered life equilibrium, a new normal if you will, one that incorporates both the negative as well as the positive impacts of the HCT experience. Management of late effects of HCT. Given that many HCT survivors report good QOL, derive benefit from their transplant experience, and view themselves as having returned to normal, some degree of complacency with regard to the management of the late effects of HCT is understandable. However, it may be misleading to adopt an overly rosy view of the long-term recovery of HCT recipients. These reports are probably best viewed as a testimony to the resilience of the human spirit when confronted by difficulties, rather than being a reflection of a survivor’s objective physical and psychosocial status. The fact remains that research has repeatedly demonstrated that many survivors continue to report a variety of clinically important physical and psychosocial difficulties long after transplant. Recognition of psychosocial late effects when they occur is best achieved by routine and periodic monitoring in HCT survivors. The CIBMTR, the European Group for Blood and Marrow Transplantation, and the American Society for Blood and Marrow Transplantation have developed screening guidelines for long-term HCT survivors [66]. These guidelines recommend a clinical psychosocial assessment conducted by a mental health professional at 6 months and 1 year following transplant and annually thereafter. An assessment of depressive symptoms, psychological distress, and sexual functioning is specifically recommended, as well as inquiry into family functioning and the psychosocial status and adjustment of the spouse or other primary caregiver. Guidelines also recommend inquiry into dietary and exercise habits, tobacco and alcohol use, and substance abuse. In addition to their impact upon general physical health, these health behaviors may be linked to the experience of distress and hence may be useful indicators of psychosocial difficulties. Management of the psychosocial late effects of HCT may involve some appropriate combination of pharmacotherapy, traditional counseling and psychotherapy, cognitive-behavioral intervention, and complementary and alternative medicine. Unfortunately, there have been very few efforts to carefully evaluate the impact of these interventions during the long-term recovery phase. Consequently, the specific evidence base for these interventions in the HCT setting is virtually nonexistent. However, there is a much stronger and growing evidence base regarding
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the impact of psychosocial or psychoeducational interventions on psychosocial adjustment in cancer survivors in general [67]. While the HCT survivor may evidence some unique needs or concerns relative to the larger population of cancer survivors, all share this common experience: they are survivors of a life-threatening and QOL-threatening illness and treatment process. As a result, interventions shown to be effective in minimizing distress and enhancing psychosocial adjustment in cancer survivors in general might be expected to evidence similar positive effects with HCT survivors. There is a critical need at the present time to build an evidence base to support efforts at management of the psychosocial late effects of HCT and the fostering of optimal psychosocial adjustment in HCT. There is a small but growing body of research examining the impact of physical activity and exercise interventions on both physical and psychosocial outcomes. In general, participation by HCT recipients in structured programs of exercise has been associated with better QOL, less distress, and less fatigue [57,58]. Finally, in approaching the management of psychosocial difficulties in HCT survivors, we suggest a perspective that views long-term recovery from HCT as a process and not a discrete event. Research has suggested recovery may be characterized by gradual passage through a series of psychosocial transitions [68]. These transitions are characterized by the changing physical and psychosocial concerns of primary importance to the HCT recipient [37,69]. Early in the course of postHCT recovery, recipients are likely to be most concerned about surviving the transplant and achieving a measure of medical stability. Psychosocial concerns are few at this time. As months and years pass, however, recipients begin to focus more upon psychosocial issues revolving around sexuality, occupational adjustment, social relationships, fears of recurrence, and coping with chronic physical limitations. Ultimately, most recipients achieve a level of psychosocial equilibrium and move on with their life. This view of recovery as a process has clear clinical implications. Specifically, staff should be aware that psychosocial concerns are likely to change over time as recipients gradually adapt to one set of concerns only to have others emerge and become salient. Early in the course of recovery, recipients might show no interest in addressing issues of sexual or occupational functioning. Later in the course of recovery, however, they may have great concern regarding deficits in these areas. As a result, clinical efforts to assist recipients’ recovery must focus on appropriate areas of concern at the appropriate time in the course of recovery [69].
Psychosocial issues in HCT: hematopoietic cell donors While the HCT donor is “matched” to the recipient with regard to human leukocyte antigen typing, the donor may be either a related family member or an unrelated volunteer donor. Until the mid 1980s, HCT donors were almost exclusively drawn from the ranks of human leukocyte antigen-matched relatives, and donation of bone marrow was the norm. In the last two decades, however, the number of unrelated volunteer donors has increased, as has the harvesting of hematopoietic cells from peripheral blood. In the United States, most unrelated volunteer donors are identified from a registry maintained by the National Marrow Donor Program [70]. Several important psychosocial issues arise in the context of hematopoietic cell donation. These issues revolve around identification of the short- and long-term physical and psychological outcomes associated with donation. While some parallels can be drawn between hematopoietic cell donation and both blood and living solid organ donation, these comparisons are imperfect. Significant differences exist among these three types of donation with respect to the risks associated with donation,
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the prior relationship between the donor and recipient, and the donor’s knowledge and direct experience of the medical outcomes experienced by the recipient of their donation. These differences are so great as to preclude simple translation of what is known about blood and solid organ donation to the donation of hematopoietic cells. For example, hematopoietic cell donation requires a much higher level of commitment and health risk than that associated with blood donation, but far less commitment and health risk than that associated with living solid organ donation. Thus, only minimal comparison can be made among these types of donation, and the uniqueness of hematopoietic cell donation must always be kept in mind. A number of studies have examined physical outcomes, primarily pain and physical disability, following the donation of hematopoietic cells from either bone marrow or peripheral blood [71–73]. Serious physical complications after either type of stem cell donation are rare. However, some localized pain and discomfort is typically associated with the harvesting of bone marrow [73,74]. Some pain and discomfort are also typically reported in conjunction with the administration of granulocyte colony-stimulating factor preparatory to harvesting of hematopoietic cells from peripheral blood [72,73]. Despite the pain and discomfort that accompanies harvesting procedures, most donors return to full normal activity within a few days [71,72]. Studies which have compared the two hematopoietic cell harvesting procedures have generally found that bone marrow donation is associated with somewhat greater pain and discomfort and/or a more gradual return to full normal functioning [71,72,74–77]. How patients experience the differences between the two harvesting procedures from a psychosocial perspective, that is, anxiety before, depressive symptoms after, etc., has not been studied. It is unlikely that one would be recommended over the other based on psychosocial criteria alone. In addition to the acute physical impact of hematopoietic cell donation, research has examined the long-term psychological and social outcomes associated with hematopoietic cell donation. Because of the obvious difference in the donor–recipient relationship between related and unrelated hematopoietic cell donors, the research has typically examined outcomes separately for these two groups. Several studies have examined post-donation outcomes in related hematopoietic cell donors [78,79]. In general, related donors tend to report little emotional distress associated with marrow donation and little change in their relationship with the recipient. Typically, the experience of related marrow donation is generally positive, and few if any regrets regarding donation are expressed. Reports of increased self-esteem, happiness, and life satisfaction are common among donors. However, a minority of donors do report negative experiences (e.g. estrangement from the recipient). A recent review of pediatric sibling hematopoietic cell donation suggests that distress following donation might be more prevalent in pediatric, as opposed to adult, hematopoietic cell donors [80]. More research in this area is merited, particularly research in the pediatric donor setting and research focusing upon identification of risk factors for poor long-term donor adjustment in both children and adults. Most of what is known about long-term psychosocial outcomes experienced by unrelated hematopoietic cell donors comes from a comprehensive study of several hundred adult, unrelated hematopoietic cell donors recruited through the National Marrow Donor Program [81,82]. Donors were assessed at several time points, including after provision of informed consent for donation as well as 1–2 weeks and 1 year post donation. Results suggested that a sizable minority of hematopoietic cell donors experienced donation as stressful and inconvenient when queried 1–2 weeks following donation, with 12% admitting to a fair degree of worry about their health [83]. When queried 1 year following donation, donors were generally quite positive about their donation. One year post donation, 87% of donors stated they believed their donation experience
was “very worthwhile,” while 91% indicated they would be willing to donate again. Some evidence suggested donors with longer marrow collection times (perhaps experiencing more acute pain during the harvesting procedure) and those experiencing lower back pain or difficulty walking following donation viewed their experience as more stressful and experienced less positive psychosocial outcomes. Psychosocial outcomes in related and unrelated HSC donors have been directly compared in a single study [84]. A total of 77 donors (41 unrelated and 36 related) completed a series of pre- and post-donation assessments. Related donors experienced more depressive symptoms both prior to and following donation, and they also reported more moderate and severe pain than unrelated donors following donation. Demographic differences and differences in intraoperative events during stem cell harvest were thought to be an unlikely explanation for these results. The authors suggested that differences in the motivations and pressures that underlie the act of donation might contribute to the greater morbidity in the related donor group. To what extent do the donor’s long-term psychosocial outcomes vary as a function of the actual transplant outcome? In the large study of unrelated donors described above, Butterworth et al. found that the death of the recipient rarely produced guilty feelings or feelings of personal responsibility [83]. Rather, grief was an almost universal response to the recipient’s death and was often surprisingly intense given the recipient was an unrelated stranger. Of course, these data describe the experience of unrelated donors. Given the existence of a prior donor–recipient relationship and the likelihood the donor may be attendant to the recipient’s death, more negative reactions, perhaps involving guilt and anger in addition to profound grief, might be expected among related donors. However, only two studies have examined related donor reactions following the death of the hematopoietic cell recipient. In a small study of 23 related donors, bereaved donors reported more depressive symptoms than nonbereaved donors 6 months after marrow donation [78]. However, an earlier and somewhat larger study found that bereaved and nonbereaved, related donors did not evidence any major differences in adjustment and attitudes as a function of the medical outcome of the transplant [84]. All in all, the available data suggest the negative impact of a recipient’s death on the long-term adjustment of both related and unrelated donors might be less than expected, with donors of both types experiencing few, if any, long term-negative outcomes. In summary, the available research suggests that serious physical and psychosocial complications are uncommon following hematopoietic cell donation. This is true for both related and unrelated donors, and for harvesting of stem cells from both bone marrow and peripheral blood. However, this does not imply that hematopoietic cell donation is a completely benign experience. A degree of acute pain and reduced functional status as well as negative psychological and social outcomes have been reported by some donors after their donation. Consequently, at least some monitoring of both the short- and long-term physical and psychosocial reactions of hematopoietic cell donors is warranted. Much of the research on donor reactions is at least a decade old at this time. As the HCT setting is continually evolving, it is important that research evolves as well to capture any changing trends in donor reactions to their donation experience. Current research priorities include early identification of donors at greatest risk for negative responses to donation, as well as the development of brief, supportive psychosocial interventions to minimize any negative post-donation reactions. This section has focused, for the most part, upon the adult hematopoietic cell donor. It is not uncommon for children to be called upon to serve as hematopoietic cell donors for a seriously ill sibling or even parent. In rare cases, children are conceived with the intention that they might serve as a hematopoietic cell donor for an ill sibling [85]. Pediatric hematopoietic cell donation raises important ethical and legal issues
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[86,87], and the psychosocial consequences of such remain to be adequately identified [88]. Clearly, this is an area in need of more research and informed ethical and legal deliberation.
Psychosocial issues in HCT: informal caregivers Caregivers are often the unsung heroes in the transplant process. Without them, the recipient would face a lonely, harrowing inpatient stay, and the healthcare providers would have less help in coaching, guiding, and motivating the recipient throughout the long treatment and recovery process. By informal caregivers we mean family members, friends, and acquaintances of the HCT recipient, who often play a crucial role in the transplantation process. They are typically expected to provide daily companionship, furnish emotional support, assist in the recipient’s performance of daily tasks and responsibilities, and even help with the provision of medical care. At the same time, they must make provision to either suspend their daily roles or continue them, a great challenge and burden for some. The need to perform in this caregiver role may extend for many months and even years beyond the actual time of transplantation. With recent trends for an earlier hospital discharge of HCT recipients, informal caregivers are asked to carry an increasingly heavy and prolonged caregiving burden. Almost without exception, caregivers perform their demanding role with healing care and grace. Caregivers that stay with their loved one during hospitalization for HCT are exposed to many of the same psychosocial stressors as recipients themselves. While informal caregivers do not experience the acute physical demands of transplantation, they are susceptible to the understandable worries and fears associated with their loved one’s treatment. Caregivers can also experience geographic dislocation, a radical change in normal daily activities, poor sleep, isolation from other family members and friends, financial stress, feelings of helplessness and inadequacy in the midst of the recipient’s suffering, and considerable outof-pocket expenses [89]. Despite their own considerable needs, HCT caregivers typically receive little care and support as the focus of care and concern is typically centered on the HCT recipient. Unlike the HCT recipient, however, caregivers are not permitted to assume the “sick role.” Assumption of the sick role gives the HCT recipient implicit permission to depend on caregivers for help with even the most basic of functional activities and to suspend their regular responsibilities. Caregivers, however, are subjected to increased expectations associated with the caregiver role. These expectations typically involve serving as a primary source of physical care and emotional support for the HCT recipient. Both the recipient and medical staff can hold these expectations. In addition, caregivers can also place heavy expectations upon themselves, asserting the need to “be strong” for the recipient. Being strong in this context often involves ignoring their own physical and psychological needs in lieu of a total focus upon those of the HCT recipient. In short, it is not easy being a caregiver in the HCT setting! Several general issues have been the focus of caregiver research in the HCT setting in a small but growing body of research. First, researchers have tried to identify the “needs” of HCT caregivers, broadly defined. Second, attempts have been made to document the nature, extent, trajectory, and consequences of the distress and burden experienced by HCT caregivers. Finally, attempts have been made to compare caregiver burden and distress associated with HCT treated in an inpatient setting versus HCT treated in a more outpatient setting. Only a few studies have attempted to directly assess the needs of HCT caregivers. This is surprising given that a careful explication of the nature and timing of needs experienced by caregivers is a necessary precursor to any effective clinical attempt to address these needs. In a study of 58 mostly spouse caregivers, the most prominent needs were related to information, particularly regarding home care after discharge
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and diagnosis, fear, and leisure activity deficits [90]. While the additional needs of HCT caregivers can be inferred from other research on caregiver stress and burden (described below), there is a clear need for additional research to directly document the spectrum of informational, psychological support, and practical needs of HCT caregivers. Such need assessments should focus on the entire trajectory of HCT treatment and recovery as it is likely that the specific needs of caregivers evolve and change over time. One strategy for identifying the nature and trajectory of the HCT caregiving experience is to contrast it to the status and experience of the HCT recipient. Comparison of the HCT caregiver with healthy controls or normative data also places the status of the HCT caregiver in an appropriate context. In a well-designed prospective and longitudinal study, Langer et al. studied 131 spouse caregiver–recipient dyads in the HCT setting from pre transplant through 24 months post HCT [91]. Comparison data were also collected from a sample of nonmedical healthy controls. While negative affect declined over time among both caregivers and recipients, caregivers reported more depression and anxiety relative to recipients and controls. Caregivers and recipients reported similar levels of marital satisfaction prior to transplant. However, caregivers reported lower levels of marital satisfaction relative to the recipient at 6 and 12 months post transplant. Bishop et al. compared current health-related QOL and post-traumatic growth in 177 spouse/partner caregivers and HCT recipient dyads and 133 recipient-matched, healthy controls [6]. Recipients were roughly 2–19 (mean 6.7) years post transplant. Caregivers and healthy controls reported better physical health than HCT recipients. However, both partners and recipients reported more depressive symptoms and sleep and sexual problems than controls. Caregivers reported less social support, dyadic satisfaction, and spiritual well-being and more loneliness than both recipients and healthy controls. Furthermore, caregivers reported less post-traumatic growth than HCT recipients. In short, while reporting better physical health status, HCT caregivers reported equal or significantly worse psychological and social status relative to HCT recipients. Recent trends toward shorter transplant-related hospital stays have resulted in a greater portion of HCT care furnished in the outpatient, ambulatory setting. While significant cost savings have been achieved without compromising clinical outcomes, identification of how this trend may affect the HCT caregiver is important [92]. It is assumed that caregiver burden increases with shorter inpatient hospital stays and a consequent shifting of greater responsibility for care to the outpatient setting. Thus, a third important research issue has been the extent of caregiver burden and distress associated with HCT in the inpatient setting relative to that associated with HCT in the outpatient setting. Grimm et al. compared the emotional responses and needs of 26 HCT caregivers from an inpatient setting with those of 17 caregivers from an inpatient/outpatient setting [93]. Caregivers were monitored from prior to transplant through 12 months post transplant. In general, few differences were found in caregiver distress between the inpatient and inpatient/outpatient settings. Surprisingly, some evidence suggested the inpatient/outpatient setting might be associated with less caregiver mood disturbance. Given the small sample size and the lack of random assignment to inpatient or inpatient/outpatient settings, these results are not definitive. However, this research does reinforce the importance of considering caregiver outcomes, in addition to cost and recipient outcomes, when evaluating the relative merits of different models of HCT care delivery. This is particularly true as the presence of an appropriate caregiver is a requisite for ambulatory, outpatient-oriented modes of HCT care delivery [92]. In summary, the presence of a committed caregiver during any inpatient hospitalization and throughout the long process of recovery is
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common and is an invaluable asset to the HCT recipient and professional staff on the unit. In fact, some preliminary data suggest the presence of a caregiver during HCT hospitalization is linked to better survival during the first year post HCT [94]. However, despite the clear value of the caregiver to the HCT recipient, the available research suggests the caregiving experience in the HCT setting can be physically, financially, emotionally, and socially stressful. Importantly, the negative impact of the HCT caregiving experience may linger for years and may equal or even exceed that of the HCT recipient experience, with fewer compensatory reports of psychological growth in caregivers relative to recipients. Given this, it is very surprising that we were unable to locate any research focused upon the provision of care to the HCT caregiver. This is an important area for future research.
Psychosocial issues in HCT: professional caregivers Provision of comprehensive, high-quality care to HCT recipients requires well-trained nursing, medical, and allied health-care professionals to work together under highly stressful conditions. All HCT inpatient units need to have the capacity for intensive care of patients; they are designed to provide for a wide range of patient needs. Due to the fragile nature of the immunocompromised patient, the medical status of HCT recipients is often highly volatile. This potential for rapid and profound shifts in the HCT recipient’s medical status requires that the health-care staff remain ever vigilant while managing an increasingly complex array of machines and medications. Alongside these acute life-threats are the mundane tasks such as daily walking and mouth hygiene which are required to avoid serious medical complications such as infection and pneumonia. Often, the bedside nurse must be the motivating force, encouraging compliance to the daily tasks of living, which can be overwhelming to a patient who is profoundly fatigued, nauseous, vomiting, and diarrhetic. This role can be especially burdensome with a patient who is not motivated to follow the daily treatment regimen. Furthermore, the health-care staff must often do their work while under close scrutiny from worried and vigilant family members who are present at the recipient’s bedside for extended periods of time. Some family members record all medication given and every entry to the hospital room of any health-care provider. Finally, despite the stresses posed by the difficult nature of their work and the difficult nature of the HCT setting, medical staff must respond appropriately and compassionately to the expressed and often unexpressed needs and concerns of the recipient and their family. This is clearly a challenging task. A very small body of research has examined professional caregivers in the HCT setting and attempted to document the stressful nature of their work. Molassiotis et al. surveyed 129 nurses and 26 doctors from 16 HCT centers [95]. One-half of their respondents reported emotional exhaustion, and 80% reported feelings of low personal accomplishment. While overt depression appeared to be less common than might be observed in the general population, signs of clinically significant anxiety were seen in more than 10% of respondents. Specific sources of distress that were reported by these professional caregivers included having to work with dying patients on a regular basis, coping with the overwhelming responsibilities of their role in the HCT setting, adjusting to rapid advances in HCT technology, and coping with the often excessive demands of patients and family members. The picture was not quite as bleak in a study of burnout and job satisfaction in HCT nursing staff [96]. Encouragingly, these investigators found burnout was low and job satisfaction was high among the nurses they studied. However, while most study respondents indicated they derived a sense of personal accomplishment from their work, the investigators reported that about 25% of study respondents appeared to evidence some worrisome symptoms of anxiety.
The psychological impact of the transplant outcome on medical staff should not be understated. The stakes are very high in the HCT setting. These high stakes, coupled with the need for medical staff to engage in frequent, lengthy, and often emotionally intense interactions with recipients and their family members, create a fertile environment for distress. It is not uncommon for members of the HCT team to become deeply attached to patients and family members. The consequences of this deep attachment range across the spectrum of human emotional experience, from deep feelings of sadness and distress when a patient expires, to joy and celebration when a patient “beats the odds” and is rewarded with extended life. More effort needs to be devoted to examining and understanding the emotional labor involved in the provision of care in the HCT setting and the development, implementation, and maintenance of support programs for the professional caregivers in the HCT setting. In sum, the work of professional caregivers in the HCT unit is highly demanding and stressful. The HCT unit requires highly flexible personnel who can respond quickly and appropriately to a variety of medical circumstances and emergencies. Their response needs to be both technically sound and sensitive to the emotional, social, and spiritual needs of HCT recipients and their family members. The difficulty in responding in this way increases when patients are critically ill and staffing is short. Given the extraordinary day-to-day pressures under which HCT units typically function, it is truly remarkable how often these units function at a very high level and indeed often serve as models of workplace cohesion.
Conclusion HCT is not a monolithic procedure, and broad and sweeping generalizations about the experience of recipients, donors, caregivers, and medical staff are necessarily limited. In fact, the description of the inpatient experience prior to HSC infusion runs from “It was like rolling off a log” to “It was the hardest thing I have ever done.” However, the core of the HCT experience is similar for nearly all recipients. Fundamentally, it is a life-threatening treatment for a life-threatening disease that can produce a range of physical and psychosocial morbidities. Based on the relevant literature and clinical experience, we have attempted to highlight and summarize some of the important psychosocial issues that arise over the course of transplantation and that are germane to recipients, donors, and caregivers in the HCT. In preparing this chapter, we noted a continued growth in the literature addressing psychosocial issues associated with HCT. However, this growth has been uneven. Most of the extant research has focused upon the adult HCT recipient. There is a clear need for more psychosocial research germane to the pediatric HCT setting, as well as more research examining the psychosocial needs of informal caregivers. In addition, there is a need for studies that identify risk factors associated with the difficulties encountered by recipients over the trajectory of diagnosis, treatment, and long-term recovery, as well as research that evaluates interventions to prevent or minimize psychosocial problems. While the focus of these intervention studies should be on the HCT recipient, attention should also be directed toward the development and evaluation of interventions to minimize distress and enhance psychosocial adjustment in the caregivers of these HCT recipients. The psychosocial care of recipients, donors, and caregivers remains a challenging task for all health providers from the early stages of decision making through long-term survivorship. This task will be enhanced by the recommended screening and preventive practices guidelines for long-term survivors established in 2006 [4]. These guidelines will draw attention to the biological, psychological, social, and emotional needs of recipients throughout their seasons of survivorship [7].
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Karen L. Syrjala & Samantha Burns Artherholt
Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients
Introduction Knowledge of health-related quality of life (QOL) during and after hematopoietic cell transplantation (HCT) has expanded rapidly in the past decade, although it always lags behind major changes in transplantation eligibility and methods. Cross-sectional and longitudinal surveys have described QOL in patients from pretransplant through at least 10 years post-transplant [1–13]. As a result, much is known about QOL outcomes over time for autologous and allogeneic myeloablative transplant recipients within and across diagnoses. The central consistent findings of these studies are that: (1) physical function returns to pretransplant level for approximately 75% of survivors by 1 year; (2) by 3 years, 80–90% of survivors are back to full-time work; and (3) the majority of patients navigate the challenges of HCT with good psychologic health. Notwithstanding these generally positive outcomes, a majority of patients will have residual problems in specific domains of QOL such as energy, sexual function, fertility, and musculoskeletal symptoms. Excellent measures and methods for evaluating quality of life have been validated with HCT patients. Population-normed instruments are available for comparison with HCT-related outcomes. Computer-adapted measures are being tested to facilitate routine, rapid assessment and immediately accessible results, enhancing the potential usability of QOL assessments in clinical care. Progress has been made in previously underconsidered groups impacted by HCT. Research has described pediatric outcomes, caregiver impacts, and cognitive deficits during and following treatment. Studies have begun to examine QOL in transplant recipients receiving reducedintensity conditioning regimens. However, a dearth of research remains in two areas: clinical trials to improve QOL outcomes, and QOL outcomes research following relapse post-HCT. In addition, research is lacking in documenting outcomes for families of patients who die. This chapter reviews what is known about QOL during and after HCT, and considers measurement issues necessary to understand when interpreting results or doing research that includes QOL outcomes. Other chapters discuss the application of QOL within outcomes research, in the psychosocial domain, and with sexual problems (see Chapters 29, 33, and 35).
Definition of health-related QOL Most QOL researchers would concur that “health-related QOL refers to the extent to which one’s usual or expected physical, emotional and Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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social wellbeing are affected by a medical condition or its treatment” [14]. Elements essential to any definition of QOL are multidimensionality and the patient’s perspective. With time, QOL has grown to encompass not only subjective evaluation, but also those behaviors that may be reported by patients but go beyond subjective experience to include function and activity reports. While medical outcome studies often define outcomes in terms of peak toxicity or time to an event such as relapse or death, QOL is usually described in relation to nontransplant controls or as change from pretransplant status. A central tenet is that QOL is relative to the current situation and the expectations of patients within the circumstances in which they find themselves. As an example, patients with major toxicities during transplant can report good psychosocial QOL and satisfaction with QOL because they believe they are doing as well as possible in a difficult situation, and they have the support they need from people important to them [6]. Despite this subjectivity, QOL correlates with medical, functional, and observational outcomes [15–17]. An important recent transition in outcomes research is the concept of patient-reported outcome (PRO). This concept has been instituted by the Food and Drug Administration for medical product development and labeling [18]. PRO measures include reports of disease symptoms, treatment adverse effects, functional status, or overall wellbeing. A useful guideline for determining appropriateness of a PRO is offered by Lipscomb et al.: “the concept measured by the PRO instrument should be relevant and specific to the population, condition, and treatment to yield clinically valid and useful information” [19]. This definition is somewhat narrower than that of QOL and linked more explicitly to medical outcomes. However, the term “PRO” is being broadly accepted as covering aspects of health status evaluation that come directly from those individuals being evaluated, without modification or interpretation by another observer. It seems likely that, over time, PRO will replace QOL as a term and focus of clinical trial endpoints, given their importance to medical treatment and potentially reimbursement decisions. However, components of QOL not directly linked to medical decision making, such as spirituality or social function outcomes, will continue to be of interest to patients and families preparing for treatment, recovery, and long-term survival needs.
Dimensions Concepts of QOL continue to adhere broadly to a three-level “pyramid” first described by Spilker [20]. He tops the pyramid with global assessment of wellbeing. The middle level includes multidimensional assessment of the physical, psychologic, economic, spiritual, and social domains. The base details subcomponents of each domain; for example, work and symptoms are aspects of the physical domain. Other
Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients
subcomponents include social and recreational activities, sexual function, relationships with the medical team, religion, perspective on life, personal control, and components of health and treatment that contribute to stress or worry for the patient. Ferrell and colleagues [21] interviewed 119 adult bone marrow transplant survivors at least 100 days post-transplant to determine what QOL meant to these patients. They categorized the many positives and negatives noted by survivors into themes and then into four domains matching those of Spilker: physical, psychologic, social and spiritual/existential wellbeing. Consistent with these definitions, the multidimensional QOL measures used in most HCT studies include physical, mental, and social function domains, in some cases with the addition of items specifically relevant to HCT questions of interest (Fig. 34.1). Table 34.1 lists measures commonly used to assess QOL components in HCT. Smith and colleagues [22] found that patients perceived health status and QOL as distinct from each other. Physical function weighed more heavily in assessments of health status, while emotional health weighed more heavily in judgments of QOL. Social function was not prominently considered in either global rating. Consistent with this emphasis on health outcomes, medical studies incorporating a QOL assessment have successfully used the shortened version of the Functional Assessment of Cancer Therapy – BMT Module (FACT-BMT) with only the physical wellbeing, functional wellbeing, and transplant-specific items. This abbreviated measure is called the Trial Outcome Index [23,24]. Researchers are attending to understanding resilience as they document that most patients with cancer and receiving HCT do very well in the psychologic domain. Four concepts being examined in detail are spirituality, optimism, benefit finding and “post-traumatic growth” [5,25–28]. In essence, the ability to find positive meaning beyond the diagnosis and to perceive gains as well as losses in the disease and transplant process predict better long-term adaptation. Most often gains are reported as greater appreciation for life, closer interpersonal relationships, reprioritizing “what really matters,” feeling closer to God, inner strength, and a sense of peace and thankfulness [5,21,29,30]. While these concepts do not appear in most health-related QOL measurement, they are usually noted as important outcomes by patients, clinicians, and researchers examining qualitative outcomes of HCT [28,31]. As measured by most scales, spirituality includes finding connection and meaning in life as well as practicing organized religion [29,32]. A challenge for QOL experts has been to determine a definition of spiri-
tuality distinct from religion and still specific enough to be measured rather than being all-inclusive of peaceful, positive feelings. Although research has reported relationships between spirituality and mortality, as well as other health outcomes, reviews conclude that most studies finding relationships between spirituality and health outcomes are methodologically flawed [29,30]. A survey of 1422 individuals from the general population in the United States reports that those who describe themselves as both spiritual and religious have less psychologic distress than those who are religious and not spiritual. However, associations to health status have not been adequately tested because significant sociodemographic differences cannot be controlled between these groups [32]. Benefit finding, optimism, and growth have been associated with improved outcomes in studies that have solid methodology [25–27], although long-term benefits to survival and QOL remain unclear [33]. When designing an assessment plan, the relevance of specific QOL dimensions to the cohorts and outcomes being examined merits careful consideration. For example, a study assessing African-American elderly and the health-related QOL relevancy of items found that spirituality was more relevant for these males and females than were many of the usual QOL content items [34]. As another example, if investigators are examining the QOL impacts of an antiviral medication, they will likely want to assess symptoms, and possibly physical and cognitive function, but evaluation of emotional or social function may not be warranted. In summary, QOL is always evaluated from the patient’s perspective. It is rated in part by internal comparisons with how much worse things could be. Thus, patients with poor physical health may report good psychologic or overall QOL. Increasingly, studies are measuring optimism and benefit finding as outcomes that may predict both mental health and medical outcomes. While many dimensions of QOL can be considered for evaluation, physical and mental components are usually included. In some cases, study hypotheses may focus on the PRO of symptoms and function, in which case mental health may not be salient.
Phases of HCT Transplantation is a dynamic and individual experience [31]. While fairly consistent patterns can be described, a patient will have his or her own trajectory of illness and recovery depending on both medical and
Physical Physical abilities or deficits Fatigue/stamina Musculoskeletal problems/mobility Other physical symptoms Sexual function Fertility Work capacity
Fig. 34.1 The principal dimensions of quality of life, and elements that have been evaluated within each of these dimensions.
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Social
Mental
Social support Economic and health resources Access to information Relationships Recreational activity Caregiver effects
Depression Distress/worry Post-traumatic stress Cognitive function Coping style/optimism Self-efficacy/perceived control Positive growth
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Table 34.1 Selected quality of life measures used in hematopoietic cell transplantation (HCT)
HCT specific
Minutes to complete
Number of items
Scores
Yes
12
30
9 Subscales, no total score
Yes
10
27 + 23 37
No No
4 5 5
23 27 36
5 Subscales plus BMT module (TOI includes 3 subscales) overall score 4 Subscales, 8 subscales, total score
Symptoms MDASI (MD Anderson Symptom Inventory) MSAS (Memorial Symptom Assessment Scale – Short Form)
Adaptable No
3 5
13 32
Total score 4 Subscales, total score
Mental health, emotional wellbeing BSI (Brief Symptom Inventory-Short Form) CESD (Center for Epidemiologic Studies – Depression) CTXD (Cancer and Treatment Distress) HADS (Hospital Anxiety and Depression Scale)
No No Yes Yes
4 5 3 4
18 20 18 14
3 Dimensions, global severity Total score 5 Subscales, mean total 2 Scores
Coping style Brief COPE
No
5–10
28
14 Subscales in 3 groups; can select from subscales
Social function MOS Social Support Survey (Medical Outcomes Study)
No
4
19
4 Subscales, overall index
No
3
12
Total score
Yes
10
40
9 Subscales, overall score, medical impact score
No
10
35
4 Subscales, total score
Relevant measures tested in HCT* Multidimensional QOL EORTC-QLQ-C30 (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire – C30) FACT-BMT (Functional Assessment of Cancer Therapy – BMT) or abbreviated Trial Outcome Index (TOI) PedsQL (Pediatric Quality of Life Inventory) plus Cancer Module SF-36 (Short Form 36 Health Survey)
Spiritual, existential wellbeing FACIT-SP (Functional Assessment of Chronic Illness Therapy – Spirituality and Wellbeing Scale) Sexuality SFQ (Sexual Function Questionnaire)
8 Subscales plus physical and mental components, no total score
Caregiver and family impact Caregiver QOL – Cancer
* Updated measure information is available from websites for many of the measures listed. Those noted below can be located with a web search of the name or with the web address listed. If not listed, no website was found and the author needs to be contacted. • EORTC-QLQ-C30 (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire – C30) http://www.eortc.be/home/qol/ExplQLQ-C30.htm • FACT-BMT (Functional Assessment of Cancer Therapy – BMT Module) http://www.facit.org/about/welcome.aspx • PedsQL (Pediatric Quality of Life Inventory and Cancer Module) http://www.pedsql.org/ • SF-36 (Health Survey Short Form 36) http://www.sf-36.org/ • MDASI (MD Anderson Symptom Inventory) http://www.mdanderson.org/departments/prg/dindex.cfm?pn=0ee78204-6646-11d5-812400508b603a14 • MSAS (Memorial Symptom Assessment Scale – Short Form), contact Victor T. Chang
[email protected] • BSI (Brief Symptom Inventory) http://www.pearsonassessments.com/tests/bsi.htm • CESD (Center for Epidemiologic Studies – Depression), no restricted access, numerous websites provide measure and scoring • CTXD (Cancer and Treatment Distress, HCT-specific measure) http://www.fhcrc.org/science/clinical/biobehavioral/projects/ • HADS (Hospital Anxiety and Depression Scale) http://www.gl-assessment.co.uk/health_and_psychology/resources/hospital_anxiety_scale/hospital_anxiety_scale.asp • Brief COPE http://www.psy.miami.edu/faculty/ccarver/CCscales.html • MOS Social Support Survey http://rand.org/health/surveys_tools/mos/mos_socialsupport.html • FACIT-SP (Functional Assessment of Chronic Illness Therapy) http://www.facit.org/about/welcome.aspx • SFQ (Sexual Function Questionnaire) http://www.fhcrc.org/science/clinical/biobehavioral/projects/
Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients
psychosocial factors [35]. There are two methodologic concerns in past research that contribute some instability to results across studies. One is that much of the research is cross-sectional, clustering patients with widely varying times since transplant and diverse diseases or treatments. Another is the range of assessment instruments used to evaluate QOL outcomes, which prevents ready comparison across studies. Nonetheless, outcomes are more similar than different, lending confidence to the robustness of QOL results. The psychosocial components of the phases of HCT are only summarized here; a detailed review can be found in Chapter 33. Pretransplant Patients arrive at transplant with differing diseases, histories of treatment, levels of physical function, and psychologic, social, informational, and financial resources. Psychosocial predictors of outcome have received the most attention, although data indicate that pretreatment physical QOL also predicts transplant outcomes [1,6,12,36]. Distress, in particular, predicts an acute transplant symptom course, especially for pain and distress [7,36–38]. Psychosocial needs are greatest just prior to transplant if judged by the percentage of patients with clinically significant anxiety or depression at different phases of treatment and recovery [1,6,36]. The conclusion from the review of numerous studies is that pretransplant physical condition and psychologic status predict later physical and psychologic adaptation in addition to survival [1,6,9,10,39– 42]. These replicated findings support guidelines calling for routine assessments of psychosocial function [43,44]. Acute treatment During the immediate post-transplant weeks, assessments find that survival is the axis around which other aspects of QOL are evaluated. In consequence, patients attribute their experiences to physical rather than psychologic causes during this phase [6,36]. Patient focus is dominated by mouth pain, nausea, fatigue, hair loss, infection risks, adhering to treatment requirements, and efforts to maintain self-care. As would be expected, patients with acute graft-versus-host disease (GVHD) have measurable declines in QOL compared with those not experiencing acute GVHD [23,45]. As hospitalization has shortened and treatments for acute toxicities have improved, investigations have increasingly moved from a focus on acute QOL to recovery and long-term outcomes. Recovery Initial studies focused recovery from HCT on the first year, by which time physical function is known to return to pre-HCT levels for most survivors. Recent research has documented that, for allogeneic transplant recipients, recovery of QOL is a longer process, requiring 3 and even sometimes 5 years [1]. During recovery, QOL issues arise that may have received minimal attention during the acute transplant phase, such as work, family relations, infertility, and sexual function [1,35,46]. Multiple studies report that the following risk factors predict a slower or poorer return of function: physical health pretransplant, depression, lack of social support, lower education level, female sex, and medical complications during recovery, in particular chronic GVHD [1,6,7,9,10]. Older adults have more difficulties with medical recovery from highdose treatment and are less likely to return to work or social activities [3,7,47–49]. Risk of chronic GVHD or other medical sequelae increases with age, and active treatment for complications is associated with poorer QOL during recovery. On the other hand, younger women have been reported to have poorer performance status and more psychosocial concerns following HCT [35]. In all, while medical risks vary signifi-
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cantly with age, QOL is more noteworthy for a lack of differences based on age than for any remarkable distinctions based on age. While survivors have slower recovery in the years following transplantation if they struggle with chronic GVHD, or have pulmonary disease, infection or other medical complications [7,13,47,50,51], once these medical complications resolve, QOL improves to levels equivalent to those of patients who never had them [3,23,45,51]. Similarly, patients receiving allogeneic transplants may have more physical and emotional difficulties in the first year, but by 12 months, physical and psychologic difficulties do not differ between types of transplant [1,3]. Return to work justifiably receives a good deal of attention in recovery and long-term function reports. While 30–60% of survivors begin to return to work by the end of their first year post-transplant, return to full-time work continues to occur for patients into the third year [1,12,52,53]. Fortunately, by 3 years post-transplant, studies agree that 80–90% of survivors have returned to full-time work or school [1,7,12,54–57]. Interestingly, although the pace is slower, overall rates of return to work for HCT recipients are similar to those in patients with other cancers [57]. It appears that rates of return to work may differ to some extent by the country in which a patient lives, perhaps as a result of the different economic incentives or rehabilitation programs available [40,58–60]. Long-term function Longitudinal and cross-sectional studies find that, by 5 years, a large majority of adult survivors of HCT are functioning well in their return to “normal” life in the domains of physical, psychologic, social, existential, and overall subjective QOL [2,4,5,12,55,61,62]. However, specific deficits remain at rates higher than those of the general population. Chronic GVHD continues for some patients, and is an important predictor of poorer overall health and QOL [11]. Long-term survivors report more accumulated health problems, musculoskeletal dysfunction, and more limitations in sexuality and social function than case-matched controls [2,5,63]. Results are inconsistent in terms of whether HCT survivors have higher rates of psychologic dysfunction, although studies agree that a majority do well [2,5]. Survivors also report more difficulty obtaining life and health insurance coverage (although actual rates of coverage are comparable to rates in control groups) [2,55]. Sexual dysfunction is one of the most consistent long-term deficits found across studies and time points after treatment. Both men and women have lower rates of sexual activity and satisfaction after HCT than the general population, with women’s sexual dysfunction one of the most frequent and persistent long-term QOL problems [4,40,64–71]. Chapter 35 details these sexual function issues following HCT. Fatigue Fatigue is the most persistent symptom beyond the first year of recovery [45,48,56,72,73]. While fatigue during acute treatment usually refers to tiredness, fatigue in survivors is often described as lack of stamina and weakness. Many biologic mechanisms have been postulated to explain persistent fatigue following HCT or other cancer treatments. Considered among the potential causes are the effects of interleukins and interferons, anemia, hypothyroidism, metabolic abnormalities, infection, treatmentrelated hormone and immune suppression, total body irradiation (TBI), sleep disruption, lack of physical activity, depression, and the need for many medications [74]. Knobel et al. [75] examined fatigue in autologous survivors 3 years or longer post-transplant. While men did not differ from the general population, women reported significant problems with fatigue. Neither disease factors nor time since treatment predicted level of fatigue. For women, those treated in first remission reported more fatigue than
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women transplanted at more advanced stages of disease. Since biologic explanations were not detectable, this finding may reflect the relativity of subjective report. Women with less prior treatment may have noticed a greater contrast in their energy than women who came to transplant fatigued from prior treatment. Gonadal dysfunction also was not associated with post-transplant fatigue. Follicle-stimulating hormone and luteinizing hormone levels did not predict fatigue level, nor did estradiol or testosterone levels in male or female survivors. Likewise, hemoglobin and thyroid levels were unrelated to reported fatigue. Immune markers tested, including interleukin-6 and soluble tumor necrosis factor receptors p55 and p75, did not predict fatigue either. On the other hand, interleukins, interferons, depression, and numerous medications have been associated with fatigue in other non-HCT cancer studies [14,76]. Although common pathways continue to be investigated, successful treatments for fatigue have included psychostimulants, antidepressants, erythropoietin, and exercise [14,77,78]. Regardless of etiology, more than one researcher has demonstrated an improvement in fatigue with exercise interventions [78–80]. Cognitive function Cognitive performance is an area of great individual variability pretransplant. Studies of adults indicate that 20–56% enter transplant with cognitive deficits that could interfere with function [81–84]. To date, there is no indication that adult cognitive abilities decline more rapidly after HCT than with nontransplanted adults [85]. We completed a prospective, longitudinal study of neuropsychologic function in 142 adult allogeneic HCT survivors. Declines in functioning were evident from pretransplant to 80 days post-transplant across all cognitive and motor skills tested. By 1 year, function returned to pretransplant capabilities in information-processing speed, verbal fluency, and verbal memory. However, problems remained with motor strength and dexterity. Previous chemotherapy was associated with increased risk of impairment in verbal fluency and memory [83]. Similar findings have been reported in other studies, with deficits mainly in the areas of psychomotor speed, executive function, and information-processing speed [86–88]. Interestingly, cognitive complaints may be largely unreflective of cognitive performance, and QOL is more related to perceived deficits than actual impairment [86]. Further research is needed on risk factors for specific impairments, as no consensus has been reached regarding specific treatment-related risks [88,89]. Several investigators have described cognitive disorders in patients taking calcineurin inhibitors or glucocorticoids for GVHD. For the most part, these have been case reports. A number of mechanisms have been postulated as the cause, and unless stroke or other permanent brain events occur, reports have indicated that these effects resolve with discontinuation of the drug [90–92]. Longer-term neuropsychologic effects have been described [93]. Abnormalities in neurologic and magnetic resonance imaging exams from 8 months to 5 years after transplant were greater for patients with chronic GVHD, corticosteroid or cyclosporine use. Meanwhile, long-term cyclosporine use and age increased the risk for neuropsychologic impairment. Disease, donor status, and conditioning regimen have been unrelated to long-term neuropsychologic results [83,93,94]. Patients who experience acute delirium during the treatment phase of HCT may be at elevated risk for depression, anxiety, and fatigue at 30 days, and worse mental health, more anxiety, fatigue, and distress at 80 days post-HCT [95]. These patients also had worse executive and frontal lobe functioning, attention, and processing speed at 80 days than HCT recipients who did not have a delirium episode. In children, prospective studies to 3 years after HCT have found no decline in cognitive performance for those over the age of 5 at transplant [96,97]. However, patients under the age of 3 are at increased risk of
intellectual decline over time post-transplant [96,98]. Long-term outcomes do not differ based on TBI versus chemotherapy-only conditioning regimens [96,99,100]. Disparate findings, with researchers reporting cognitive deficits in one-third to two-thirds of survivors, may be attributable to inclusion requirements of the samples (autologous versus allogeneic, age range, or history of prior treatment). Alternatively, some disparity may result from differences in tests selected, from time points at which testing was done or from differences in HCT treatment at the sites where patients were examined. In considering neuropsychologic function before and after transplant, it is important to recognize that performance is susceptible to disruption from virtually any source. Similar to fatigue, cognitive dysfunction is a common endpoint of many physiologic and mental strains. As etiologies are further examined, causes will likely differ between patients, treatments, and time points. Late effects As late effects are more closely studied, it is clear that medical risks continue at a higher rate for long-term survivors than for nontransplanted adults and children [11,40,47,53,63,101]. It is also certain that infertility concerns impact a substantial minority of both male and female survivors [102]. However, it is not similarly clear whether there are other QOL late-onset complications among survivors. Medical late effects such as cardiac failure or second cancers are distinct events that can occur long after treatment has been completed. In contrast, physical and psychologic problems can wax and wane following treatment, and the prevalence of problems ranges widely across studies. Thus, it is challenging to discover whether QOL deficits accumulate after transplant more rapidly than would be expected in an aging population. We would speculate, however, that survivors remain vulnerable to late QOL complications as a response to increased medical risks. Chapter 105 presents a review of late medical risks. Relapse Little progress has been made in understanding the QOL of patients who relapse. Issues of family, social network, and spiritual needs become vital to patient outcomes as their physical health is jeopardized. The symptoms and course of QOL, whether proceeding to death or returning to remission, remain largely unexplored, in part because patients are often censored from studies when they relapse. This is changing as more patients live with active disease or move between relapse, second transplant, and remission status following HCT. In summary, the physiologic, and psychologic phases and impacts of HCT have been well defined. Common problems post-transplant include fatigue, cognitive difficulties, sexual dysfunction, social limitations, and efforts to manage continuing uncertainty and fear. Late medical effects can be expected to have related QOL impacts.
Risk factors and diversity in HCT outcomes Risk factors for some QOL outcomes have been identified. Differences in medical course and short-term outcomes have been documented for autologous and allogeneic stem cell recipients, for patients with different diseases or pretransplant histories, and for patients who receive TBI versus those who receive chemotherapy-only regimens. Another area of diversity is the wide range of patients and others affected by transplant who respond within their own developmental phase of life or within their roles: adult or child patients, caregivers who are parents of children or spouses of adults, and males or females whether as patients or as caregivers.
Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients
Conditioning regimen Few sustained differences in QOL have been confirmed relative to the use of TBI or chemotherapy only as a conditioning regimen. Research indicates that TBI is the best predictor of neuropsychologic deficits at 80 days post-transplant, but differences are no longer measurable after 1 year [47,103]. Two medical differences that could influence QOL are a greater incidence of cataracts in patients receiving TBI, and a greater likelihood of alopecia in patients receiving busulfan with cyclophosphamide [53]. Few studies document other differences predicted by conditioning regimen within the domains of QOL. Clinical reports and early toxicity data suggest that nonmyeloablative or reduced-intensity conditioning recipients will differ from myeloablative recipients in their course of morbidity and function [104,105]. Since populations receiving less-intensive conditioning have generally not been eligible for high-dose conditioning, it is difficult to compare QOL and function across the two groups. Nonetheless, comparisons have been somewhat informative. One prospective longitudinal study compared the QOL of patients receiving reduced-intensity conditioning and myeloablative HCT, and found that both groups returned to baseline QOL by 2 years post-transplant [106]. Compared with autologous transplant recipients, patients receiving reduced-intensity conditioning transplants report more anxiety but overall better QOL in the first year post-transplant [45]. A continued study of the long-term outcomes of patients receiving reduced-intensity conditioning transplants is underway. Chapter 71 discusses the differences in eligibility as well as treatment for reduced-intensity conditioning HCT recipients. Autologous, allogeneic HCT Type of donor has been compared directly in numerous studies examining risk factors for QOL outcomes. Few studies have found measurable differences in psychologic QOL over time between autologous, allogeneic, and unrelated transplant recipients, despite their medical and treatment differences [1,49]. Some studies have reported that autologous HCT recipients have better QOL in all domains than allogeneic transplant recipients [73,107], while others have found that allogeneic HCT recipients report less impairment [56,108]. Still others have found that any discrepancy in QOL based on transplant type disappears by 2 years post-transplant [3]. Despite the discrepant findings, it appears that transplant recoveries for autologous versus allogeneic HCT recipients are more similar than different, particularly in the psychologic domains. Male or female Little attention has focused explicitly on gender effects in QOL outcomes. Nonetheless, nearly all research documents some gender effects within QOL surveys or interventions. In reviewing these outcomes, it becomes rapidly apparent that female recovery is more complex than male recovery, at least based on self-reports [1,7,10,47–49,109]. Norms for males and females on psychologic tests are always separated because of the well-established fact that women endorse more symptoms than men given the same levels of health. Women report more fatigue and generally report higher levels of distress, even when using standardized scores within gender normed measures [1,110]. Two additional consistent findings across studies are that women have more sexual problems and are slower to return to work [1,47,64]. In summary, risk factors tend to be relatively stronger during the acute phase of transplantation, with few aspects of treatment, disease or individual characteristics distinguishing long-term QOL in survivors. The major treatment factors influencing QOL are the use of reduced-intensity conditioning regimens and immunosuppression post-HCT, particularly
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for calcineurin inhibitors. Females seem to have greater difficulty during and following HCT across numerous studies and outcomes. On the other hand, age and type of donor have had less consistent influence on outcomes. Allogeneic transplant and older age seem to be risk factors primarily for acute differences in QOL.
Pediatric QOL Both parents and children have documented elevated distress during acute treatment, with declining distress in the 6 months following HCT [111,112]. Risk factors for high levels of distress and low QOL include receiving a transplant from an unrelated donor, increasing age, and lower socioeconomic status [113,114]. By 1 year post-transplant, both mothers and children report good QOL [112,114], and most long-term survivors lead independent, productive lives in adulthood [115,116]. Nonetheless survivors of pediatric HCT are noted to have higher anxiety as well as feelings of sensitivity and vulnerability when compared with survivors of pediatric bone cancers [8]. Other researchers have reported that pediatric survivors do better than their peers in psychosocial domains [117]. The rate of successful return to school (85–95%) looks similar to rates of return to work in adult survivors [118]. In sum, results testify to the resilience of children not unlike the resilience of adults. The restriction to these overall positive outcomes is that it has not been possible yet to track many survivors past their twenties. Cohorts of mature adult survivors of pediatric HCT are not yet large enough to evaluate their QOL.
Caregivers and parents of pediatric transplant recipients Medical staff and patients could not manage HCT and recovery without the presence of a family caregiver or multiple caregivers. As central a role as families play, research on family caregivers is only recently making headway in evaluating QOL needs. QOL in caregivers both before and after HCT is low compared with norms and that of their patient counterparts [119–122]. Spouses are the principal source of support for most adults undergoing transplant, and patients relying on spouses for caregiving report better QOL than those relying on others (such as friends or children) [119]. Caregivers report more distress than patients do during the acute transplant period [121,123,124]. However, research also indicates that the stressful impact of supporting a loved one through HCT can last years beyond the active caregiving phase [125]. Parents of pediatric HCT patients report significant distress in the course of their child’s treatment. Although some studies report that mothers of pediatric HCT patients suffer the greatest trauma in the course of their child’s treatment [126], other researchers find that mothers and fathers both report significant distress symptoms [127,128]. Younger mothers and those experiencing more depression or anxiety at the time of HCT are more likely to continue to be distressed 18 months later [126]. Other caregivers manage remarkably well from early evidence, despite their levels of acute distress.
Interventions to improve QOL outcomes Clinical trials to improve QOL have been surprisingly infrequent in HCT populations. During acute treatment, several studies have demonstrated that pain and distress are improved with hypnosis or other psychosocial care [38,129–131]. Several clinical trials have demonstrated that fatigue is reduced with exercise programs [78–80,132,133]. In reviewing the limited randomized controlled trial research, it seems that interventions have had modest effects, while the cost-effectiveness can be questioned
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[58,134]. As next steps, researchers need to define which QOL deficits benefit from intervention, in which high-risk patients, and at what points in time.
Measuring QOL What to measure and how to measure it is challenging when QOL is an HCT outcome. While PRO data can be rich and informative, inadequate appreciation of data collection requirements necessary for high-quality data and the importance of minimizing nonrandom missing data have limited the clinical value of many early QOL studies. In selecting measures of QOL in HCT recipients, the ideal is to use measures with established norms, reliability, and validity in HCT cohorts. In addition, an availability of broader population norms is valuable to permit a comparison of HCT results with those of other groups. However, the use of generic measures tested across broad populations has a downside as well. There is an inherent trade-off in benefits of generic versus specific QOL measures. HCT-specific measures may be more precise and informative but are then only relevant to an HCT-specific problem. Another challenge is the changing nature of what is relevant to measure based on the dramatic changes in QOL over the phases of HCT. Finally, there is the added value and potential reduction of missing data when using online or multiple electronic methods for data collection. In QOL measurement, a “response shift” is expected as patient conditions change medically. Patients naturally reprioritize concerns, and self-report is, by human nature, relative to expectation rather than being tied to medical status. In this process, health may decline, but many patients still report good psychologic function [6,135]. Another factor that can shift response is the changing relevance of assessment dimensions. While physical symptoms may be the central focus during acute treatment, focus shifts in long-term follow-up to functional abilities and emotional adaptation [35,46]. This is a major reason why excellent measures in one circumstance may be of less value at a different time. If no patients work in the 3 months after HCT, there is no gain from asking about work, and to do so would seem out of touch with patient experiences. However, after the first year, not asking about work would be a major oversight. Generic measures There are three QOL measures routinely selected when providing a “generic” assessment of HCT outcomes: the FACT-BMT, the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire – C30 (EORTC QLQ-C30), and the Short Form 36 Health Survey (SF-36), which is not a cancer-specific measure. One or another of these is used in most cancer QOL studies. Each of these well-standardized, reliable, and valid measures has some questions in common with the others and asks some questions that are not represented in the others. Yet each of them has been used effectively in HCT research. The FACT-BMT asks questions specific to the treatment of cancer and the HCT process, and focuses its emotional wellbeing content on concerns specific to the disease and treatment. It does not evaluate physical abilities per se. As such, it is quite disease- and treatment-oriented and may be less relevant during recovery and follow-up. The “BMT module” of added questions contains items that elaborate the more common HCT symptoms and concerns [136]. The EORTC QLQ-C30, in contrast, has a more explicit focus on functional abilities, what a patient is able to do physically, and specific symptoms, and a rather brief psychologic review. The HCT module, the EORTC QLQ-HDC29, measures side-effects and HCT-specific issues. Psychometric studies of this new module are ongoing [137]. The SF-36, meanwhile, is a health-related QOL measure not specific to disease
[138]. Its evaluation of physical ability corresponds most closely to that of the EORTC QLQ-C30 [139]. It provides a brief assessment of psychologic function, and it has minimal emphasis on physical symptoms. Given its general health orientation across populations and lack of symptom evaluation, the SF-36 is appropriate for use in the years posttransplant as issues become less HCT symptom-specific. Research has documented that these QOL measures are correlated but do not share enough variance to be considered equivalent [139]. Investigators find that the instruments have analogous global health items (correlation r = 0.82; 67% of the variance is shared). But the physical and psychologic dimensions of the EORTC QLQ-C30 and FACT share only limited variance. Virtually no variance is shared between the EORTC QLQ-C30 and FACT on scales of social wellbeing or cognitive function [139]. With its focus on treatment-related issues, the FACT covers the areas of greatest influence on patients during acute treatment, whereas the EORTC QLQ-C30 provides assessments that may be relevant through recovery. Each of these measures has normative information on use with HCT. Each has translations in multiple languages, and all are appropriate to capture issues faced by HCT patients. These scales have particular value for their abilities to provide a normative reference for a sample or an individual patient. For comparison, Table 34.2 lists the subscales for these three QOL measures. Figure 34.2 shows the capacity of the SF-36 to distinguish the function of 10-year HCT survivors and matched controls. Numerous other measures exist that are relevant to HCT but are not as widely used. Hence comparative data are not as readily available. The Sickness Impact Profile (SIP) [140], with 12 subscales, effectively tracks decline and recovery in health-related QOL before and after HCT [12], but is not cancer specific and is no longer widely used. Other scales have relevance in specific circumstances but do not have the extent of testing in HCT to provide normative information. Still, these multidimensional measures of QOL might be of relevance depending on the goals of assessment. The Cancer Rehabilitation Evaluation System – Short Form (CARES-SF), the Functional Living Index-Cancer (FLIC), and the Memorial Symptom Assessment Scale – Short Form (MSAS-SF) are some notable examples [108,141,142]. For a detailed consideration of measures, the reader is referred to books on this topic or articles comparing measures [143,144].
Mental composite t score Role emotional Social function Mental health Vitality -Physical composite t score Physical function Role physical Bodily pain General health 40
Controls Survivors
P = .01
P = .007 P = .002 P = .06 P = .02 P = .02 P = .03 P = .004 P = .10 P = .001
50
60 70 80 Mean score
90
100
Fig. 34.2 Health-related quality of life standardized mean scores and standard errors of the means on eight domains and two composite scales (physical and mental) on the Short Form 36 Health Survey (SF-36) for 10year survivors compared with case-matched controls. (Reproduced from the American Society of Clinical Oncology and Syrjala et al. [2] with permission.)
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Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients Table 34.2 Subscales of the three most widely used global health-related quality of life measures
Subscales
Summary scores Number of items
EORTC-QLQ-C30
FACT-BMT
SF-36
PedsQL plus Cancer
Physical functioning Emotional functioning Social functioning Role functioning Cognitive functioning Global quality of life Pain Nausea/vomiting Fatigue EORTC QLQ-HDC29 No – only subscales
Physical wellbeing Functional wellbeing Emotional wellbeing Social/family wellbeing BMT scale
General health Physical function Role – physical Body pain Vitality (fatigue) Mental health Role – emotional Social function
Physical function Emotional function Social function School function Cancer module
Total score
Total scale score
30 in core 29 added in EORTC QLQ-HDC29
27 in core 23 added in BMT scale
Physical composite t-score Mental health composite t-score 36
23 in core 27 in cancer module
See text for abbreviations.
Specific measures
0.6
Since generic measures are unlikely to have the sensitivity to capture the details of main interest in a clinical trial, measures specific to anticipated outcomes may need to be added to a generic tool. Considering symptom and function outcomes helps to clarify this point. Patients can be assessed for general physical function with a measure such as the EORTC QLQ-C30 or SF-36, but if an intervention is targeted explicitly to reducing fatigue, these scales may not have the sensitivity to capture outcome changes [75]. A specific fatigue measure will provide greater sensitivity to differences in outcomes. These focused forms will also provide investigators with more detail on where changes are occurring: is sleep or stamina improved; or does the patient feel more alert? A construct central to cancer QOL and HCT specifically is the concept of disease- and treatment-specific distress: what are the worries, stresses or fears specific to a patient having a transplant that contribute to how he or she is doing? General cancer distress measures are advocated for routine clinical and research use by the National Comprehensive Cancer Network guidelines [145]. Studies find that HCT-specific distress is a better predictor of pain, nausea, and stress responses during acute treatment than is depression or other general mood indicators [37,38], while depression is a better predictor of long-term outcomes [1]. Figure 34.3 shows the mean level of transplant-specific distress in patients over time in comparison to their general depression scores. The difference in these two measures visually demonstrates why a generic measure may not always be specific or sensitive enough to capture selective information.
0.4
Mean z scores
-0.4 -0.6 -0.8 -1.0 -1.2
Depression Distress Physical Limits
-1.4 5 s ar
ye
When a patient is unable to provide a self-report of QOL, a proxy responder may be the only option. This proxy is often a nurse or caregiver, or the parent of a young child. Research on the validity of proxy responses
-0.2
s ar ye
Proxy or parent
0.0
3
The gold standard for QOL assessment is a patient completing written or computer assessment with a staff person who is available to answer questions and to assure that the patient is competent and responding as instructed, as well as to review responses and correct skipped or double responses while the patient is present. However, often the choice is between no information or mailed, phone or proxy responses with someone answering for the patient.
0.2
ar ye 1 ys da 90 -tx e Pr
Alternative QOL measurement strategies
P < .001, each time course differed from the others
Fig. 34.3 Trajectories of physical limits, depression, and distress from before hematopoietic cell transplantation (HCT) to 5 years after HCT. Scores are transformed to z-scores to be on the same scale. Physical function and depressive symptoms recovered by 1 year and on average were stable thereafter. Distress related to disease and treatment differed from depression over time and declined gradually over the 5 years following treatment. Patients were 319 adult, male and female recipients of allogeneic or autologous HCT who had been treated with myeloablative therapy, with nonrelapsing survivors followed to 5 years. (Reproduced from JAMA and Syrjala and Zaza [164] with permission.)
indicates that, on average, parents and other proxies differ equally in responses when compared with patient reports [146,147]. Proxies, including parents, tend to underestimate patients’ symptoms. In contrast, they tend to rate global QOL and mental health lower than patient self-reports [146]. Fortunately, proxies are more accurate when they are rating concrete, observable events. Another finding from this research is that clinicians and parents underestimate the QOL information a child or patient with limited communication ability can still transmit. Of interest, children’s self-reports of their own health status significantly match physician ratings, while parents’ ratings do not [15]. When evaluating pediatric QOL, there are several assessments to consider [15]. The Pediatric Quality of Life Inventory (PedsQL) includes
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both a patient report and a parent report evaluating QOL for pediatric cancer patients on dimensions parallel to the FACT or EORTC QLQC30 [147–150]. Multiple versions suited to the developmental ages of children are comparable. A companion Cancer Module provides treatment-specific assessment of symptoms [151]. Both of these measures have well-established reliability and validity, although it is not clear that they have been used with HCT patients. In contrast, a relatively new measure is the Child Health Ratings Inventory (CHRIs), with its DiseaseSpecific Impairment Inventory-HSCT (DSII-HSCT; a transplantspecific module). Preliminary studies in HCT cohorts indicate good reliability, and moderate correlations between child and parent reports of QOL [152]. Given the large number of measures translated for age and language, it should be possible for the majority of patients to complete a general QOL measure appropriate to their needs if they can read. Alternatively, most measures can be administered orally, including by telephone if necessary. Administration by computer, mail or telephone interview The shortening of QOL measures, the increased attention to symptoms, and the necessary brevity of physician time have led researchers to explore the use of automated assessment systems, with some success. Testing of QOL assessment across modalities with HCT or other cancer patients substantiates the transferability of written measures to a touchscreen or other bedside computer [153–156]. Measures confirmed to be equally reliable and valid with written, mailed, and computer formats include the EORTC QLQ-C30, the SF-36, the FACT, and Memorial Symptom Assessment Scale [153–156]. Completeness and quality of data are improved with the computer administration of measures [154]. Software can immediately catch skipped items, eliminate double responses, and reduce burden by intentional skip patterns using item response rules. In contrast, at least one error occurred in 44% of paper response forms. As important, patients generally prefer the electronic response version, and it takes somewhat less time to complete. When onsite assessment is not an option, web and telephone methods are also effective. Bush et al. [4] demonstrated the feasibility, high compliance and satisfaction of a web-based system for collecting QOL data at home in the year following transplant. An automated telephone symptom monitoring for HCT recipients has been successfully tested and compared with written reporting [157]. A computerized system (Interactive Voice Response) can be set to contact patients at home at any designated day or time selected by the patient. If the patient does not respond, the system will call after set periods as often as programmed. The technology can be programmed to download information to a physician or nurse if critical set points are reached. In a comparison of Interactive Voice Response versus paper and pen, HCT patients responded at the same rate as on paper and reported the same symptom severity. In addition, 98% found the system easy to use [157].
Clinical utility of QOL data After a major review of oncology studies, a National Cancer Institute working group concluded that QOL assessment in cancer trials is fea-
sible, has scientific quality, can yield interpretable findings, and brings added value to cancer care decision making, especially when there is a substantial QOL difference but no survival difference between competing therapies [158]. Although the collection of QOL data is frequent in research settings, the frequency of its use in clinical settings is much lower [159,160]. Lee et al. [160] found that most HCT physicians consider providing QOL data to be part of their responsibility when informing patients of the risks of treatment. However, a majority of physicians do not believe that this QOL information influences patients’ decisions on whether to undergo HCT. Only 53% of physicians surveyed report using published QOL data to inform their practice. More effort is needed to translate QOL findings into usable information for clinical practice. One important component of usability is consensus on what constitutes a “clinically meaningful difference,” also called a “minimum important difference.” Increasingly, accepted strategies consider a meaningful difference to be a change in scores or a difference between groups of onethird to one-half a standard deviation of the observed scores [161,162]. Monitoring and discussion of QOL with HCT recipients has the potential to increase patient involvement in care, reassure patients, and improve patient satisfaction. Use of QOL assessments can improve physician–patient communication and enhance patient QOL outcomes [163].
Conclusion QOL before, during, and after HCT has been well described. Studies agree that most patients regain high levels of physical and psychologic QOL by 3 years following transplantation. At the same time, a subset of 5–20% of patients will have medical problems, such as chronic GVHD, that impinge long-term on QOL. Even for patients who return to full-time work and normal social activities, and those who have good psychologic and physical function, specific areas of difficulty have been identified. Recognized long-term problems include fatigue, cognitive deficits, musculoskeletal symptoms, and sexual dysfunction for both males and females. The impact on QOL of medical late effects is receiving more attention but remains an area deserving of further investigation. To date, few clinical trials have been published that improve QOL during either acute treatment or long-term recovery. By nature, QOL shifts with the situation a patient is in, reflecting not only observable abilities, but also expectations for the immediate circumstance. As a result, very ill patients can report good QOL in some areas. In assessing QOL, many options have been tested with HCT patients and work well. A generic QOL measure provides valuable normative information that permits a comparison of scores with patients of comparable age, gender, and/or disease. At the same time, specific, focused measures such as those for fatigue or sexual function may be needed to provide adequate sensitivity when defining the nature of a target problem or to detect clinical trial differences. Fortunately, there are many effective examples of QOL evaluation with HCT patients, at all ages and phases of treatment and survivorship. Finally, a remaining challenge is to determine a method that facilitates use of QOL data in clinical decision making.
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D. Kathryn Tierney
Sexuality Following Hematopoietic Cell Transplantation: An Important Health-related Quality of Life Issue
Introduction For more than two decades, investigators have explored the healthrelated quality of life (HRQOL) of hematopoietic cell transplant (HCT) recipients. Findings from these studies indicate that the majority of HCT recipients report good-to-excellent HRQOL [1–7], and have identified both positive and negative sequelae that influence HRQOL. Positive sequelae noted following HCT include a heightened appreciation for life, improved interpersonal relationships, personal growth, changes in priorities, and enriched spirituality [8–11]. Sequelae that negatively influence the HRQOL of HCT recipients are well documented and include fatigue or decreased physical strength [12–14], lack of or perceived lack of social support [3,12], cognitive changes [3], inability to resume previous role responsibilities [13,15], chronic graft-versus-host disease (GVHD) [12,13], recurrent infections [13], emotional distress [3,13], insomnia [3,6,13,14], and alterations in sexual health [3,4,12,13]. Investigators have an opportunity to improve the HRQOL of HCT recipients by exploring strategies to minimize the negative sequelae and enhance the positive ones. Alterations in the sexual health of HCT recipients are an important HRQOL issue and the focus of this chapter. First, the concept of sexuality will be explored, followed by what is known about sexual health in HCT recipients, including the types of alteration, their etiologies, and the impact of these alterations. Assessment of sexual health and possible interventions to improve sexual functioning and decrease sexual dissatisfaction will then be described. Finally, areas for future research will be explored.
The concept of sexuality In order to fully appreciate the impact of the diagnosis and treatment of cancer on an individual’s sexuality, one first has to consider a broad view of sexuality. Sexuality encompasses much more than sexual activity. Sexuality includes the view a person holds of him- or herself as a sexual being. Gender roles and what it means to be a man or a woman are components of sexuality [16]. Body image, self-confidence, and self-esteem all influence one’s sexuality and its expression. Sexuality is shaped by age, developmental stage, culture, sexual beliefs, expectations, sexual preferences, past intimate relationships and experiences,
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
and the sexual partner. Sexuality is expressed not only in sexual activity, but also in appearance, attitudes, values, roles, and relationships [16,17]. Life-threatening illness does not eliminate the need of individuals for intimacy, affection, and emotional connection to others. The World Health Organization views sexuality as an integral component of the human experience, which can enrich and enhance personality, communication, and love [18]. The American Society of Clinical Oncology and the Institute of Medicine are working to raise awareness of the needs of cancer survivors, and calling for the development of survivorship care plans that address psychosocial needs, including sexuality [19]. Standards for the scope of nursing practice published by the Oncology Nursing Society and the American Nurses Association mandate that nurses assess and intervene to treat sexual dysfunction [20]. Cancer survivors have identified altered sexual health as a distressing consequence of the diagnosis and treatment of cancer that negatively influences HRQOL. One study found that two variables, altered sexuality and family distress, exerted the most negative effect on measures of social wellbeing in long-term cancer survivors [21]. Cancer patients ranked loss of sexual feelings as the sixth most severe side-effect of chemotherapy treatment, preceded by effects on loved ones, loss of hair, fatigue, altered role responsibilities, and effects on social activities, respectively [22]. Altered sexual health following HCT has been reported by 22–70% of recipients [23,24], and for many these changes persist for years following treatment [3,25,26]. HCT recipients report that alterations in sexual health negatively affected their HRQOL [3,4,12,13]. Changes in sexual health following treatment for a life-threatening malignancy are complex. Sexual health may be altered as a direct result of physiologic changes due to the cancer or treatment. In addition to physiologic changes, alterations in sexual health may result from psychosocial variables such as anxiety, depression, altered relationships, and the uncertainty that accompanies the diagnosis and treatment of cancer. An individual’s sexuality is often expressed within the context of a relationship with another individual. Therefore, altered sexual health in one member of the couple will affect the sexual health of the other. The sexual partner may also experience depression, anxiety, and uncertainty, further contributing to alterations in sexual health. The American Psychiatric Association, in the Diagnostic and Statistical Manual of Mental Disorders, collectively describes sexual dysfunctions as a group of disorders that may affect one or more phases of the sexual response cycle [27]. These disorders are characterized by changes, physiologic or psychologic, that adversely influence sexual functioning, leading to psychologic distress or stress within relationships. Diagnoses for disorders of the sexual response cycle are shown in Table 35.1.
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Table 35.1. Diagnoses of disorders of the sexual response cycle. Hypoactive sexual desire disorder Female sexual arousal disorder Male erectile disorder Orgasmic disorders Sexual pain disorders, including dyspareunia and vaginismus Data from [27].
These diagnoses are based on the four phases of the sexual response cycle [28,29]. The first phase, desire, is the interest in sexual activity and includes sexual fantasy and thoughts. The concept of desire has biologic, behavioral, and cognitive components and is poorly understood [30,31]. Desire is strongly linked to the second phase of the sexual response cycle, arousal [31]. The subjective experience of excitement and pleasure, and the associated physiologic changes, characterize the arousal phase. In men, arousal leads to an erection due to a complex sequence of neurovascular and cellular physiologic changes [32,33]. Arousal in women is associated with vasocongestion leading to vaginal lubrication and swelling [31]. Orgasm, the third phase, is a period of intense sexual pleasure and is accompanied by the release of sexual tension and rhythmic muscular contractions of the perineal tissues and reproductive organs. Emission and ejaculation are the two phases of orgasm in men. Relaxation and an evaluation of the sexual experience are noted in the final phase of the sexual response cycle, resolution. The phases are not necessarily linear but are strongly interconnected. It must be emphasized that sexuality is a much broader concept than the sexual response cycle; however, the sexual response cycle offers a standardized approach to assessing and diagnosing alterations in sexual health.
Altered sexual health in HCT recipients There has been some work specifically investigating the alterations of sexual health experienced by HCT recipients. One noteworthy study evaluated the sexual health of HCT recipients longitudinally, reporting that, in both genders, the incidence and types of alteration in sexual health increased from before HCT to the last assessment 3 years post HCT [26]. However, the majority of information on the altered sexual health of HCT recipients is derived from studies focusing on the multidimensional construct of HRQOL and not specifically designed to evaluate sexual health. These HRQOL investigations have highlighted the incidence and the types of altered sexual health faced by HCT recipients. Numerous HRQOL studies have reported diminished sexual satisfaction following HCT [4,5,12,34–36], and other studies have reported sexual dysfunction [13,24,26,37]. Altered sexual health is often cited as negatively impacting HRQOL of HCT recipients [3,4,12,13]. A summary of findings from investigations specifically designed to explore the sexual health of HCT recipients is shown in Table 35.2.
Etiology of altered sexual health in HCT recipients Alterations in sexual health may be due to a number of biologic, physiologic, psychologic, and social variables, and the complex interactions among these variables. Consequently, the etiology of altered sexual health will rarely be limited to a single cause. In HCT recipients, alterations in sexual health may be due to the malignancy, the diagnosis, therapy antecedent to HCT, the preparative regimen utilized for transplant, and the complications and treatment of complications following HCT. In male HCT recipients, reported alterations in sexual health include hypoactive sexual desire disorder, erectile dysfunction (ED),
ejaculatory dysfunction, hormonal changes, and infertility. Alterations in the sexual health of female HCT recipients include hypoactive sexual desire disorder, premature ovarian failure (POF) and menopause, arousal disorders, dyspareunia, and infertility. Biologic and physiologic variables in men The effects of the high-dose preparatory regimen on the hypothalamicpituitary-gonadal axis in men are well documented. Damage to the gonads from the preparative regimen leads to a loss of the secretion of the sex steroids, testosterone and inhibin, leading to an absence of negative feedback to the hypothalamus and pituitary, resulting in elevated levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The hypothalamus secretes gonadotropin-releasing hormone (GnRH) into the circulation, stimulating the release of the gonadotropins, FSH and LH, from the pituitary. FSH and LH control gonadal function, resulting in the production of gametes and the synthesis of sex steroids. The Leydig cells in the testicles, under the influence of LH, start androgen synthesis and secretion. FSH initiates spermatogenesis, which is then maintained by testosterone [38,39]. Negative feedback to the hypothalamus and pituitary occurs when testosterone and inhibin levels rise, inhibiting the secretion of GnRH and the gonadotropins [40]. Both radiation and chemotherapy damage gonadal tissue, leading to decreased secretion of sex steroids and thus a loss of the negative feedback inhibition at the level of the hypothalamus and pituitary. The result is the characteristic findings of elevated levels of FSH and LH indicative of infertility [38]. The degree of gonadal damage will depend on age, the dose of radiation and chemotherapy, and the type of chemotherapy administered [38,41]. Irradiation directly to the testes in doses of 12–15 cGy produces irreversible azoospermia [39]. Radiation and chemotherapy, particularly alkylating agents, result in a dose-dependent depletion of the germinal epithelium lining the seminiferous tubules of the testes, and this depletion results in azoospermia, testicular atrophy, and infertility [41,42]. Features of germinal cell depletion consist of small testicular size and reduced testicular volume [41]. Endocrine values in men with germinal cell depletion include elevated FSH (25–90 mIU/mL), elevated LH (8–25 mIU/mL), low-to-low-normal testosterone (200–700 ng/100 mL), decreased testosterone production rate (3.5 mg/day), and decreased free testosterone (8.6 ng/100 mL) [41]. Testosterone is strongly correlated with sexual desire and arousal [43]. Levels of testosterone in male HCT recipients often remain within normal limits as the Leydig cells are relatively resistant to the effects of chemotherapy and radiation [39,44]. However, subtle damage to the Leydig cells, evidenced by a reduction in the amount of testosterone produced and a decrease in free testosterone level, may be found on more extensive hormonal evaluation [45]. Men over the age of 45 may be at increased risk of testosterone insufficiency [46]. Elevated prolactin levels have been found in some men following HCT and may indicate damage to the hypothalamus [42,47]. Hyperprolactinemia has been associated with infertility, ED and decreased libido [43,47–49]. Radiation damage to the thyroid may result in hypothyroidism or subclinical hypothyroidism [42,50], and levels of free testosterone and bioavailable testosterone are reduced in men with hypothyroidism [51]. ED is defined as the inability to achieve or maintain an erection sufficient for satisfactory sexual intercourse. The etiology of ED may be hormonal, vascular, neurologic, secondary to medications, psychologic or a combination of these factors [49]. Cavernosal arterial insufficiency has been reported as a cause of ED in men following HCT, with the risk being higher in those men receiving total body irradiation compared with those receiving chemotherapy only [52]. Pelvic irradiation can cause penile arterial insufficiency contributing to ED in some men [42,49,52,53].
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Table 35.2. Investigations of altered sexual health following hematopoietic cell transplantation (HCT). Study
Measurement(s)
Findings
n = 51 men Mean age 33 (range 19–49) years Mean time from HCT 58 (range 6–154) months [47]
Investigator-designed questionnaire on sexual function Hospital Anxiety and Depression Survey Hormonal assessment
15% diminished desire 24% ED 13% delayed ejaculation 9% dry ejaculation 20% premature ejaculation
n = 31 Median age 38 (range 16–56) years Assessed pre and 3 months post HCT [94]
Derogatis Interview for Sexual Functioning
Pre/3 months post-HCT: 36%/48% sexual dysfunction 65%/52% sexual dissatisfaction 36%/40% decreased desire 14%/11% arousal problems 43%/43% orgasm problems 25%/25% ED
n = 29 men Mean age 35 (range 18–61) years Mean 36 (range 7–97) months post HCT [48]
Psychosexual Functioning Questionnaire – male version Psychosocial Adjustment to Illness Scale Hormonal assessment
38% 21% 38% 48%
n = 16 men Mean age 36 (range 20–49) years Median of 3 years (range 7–96 months) post HCT [95]
Investigator-modified Psychosocial Adjustment to Illness Scale Hormonal assessment
25% diminished desire 12% intermittent ED
n = 102 Mean age 37 (SD ± 9.9) years Assessed pre HCT and 1 and 3 years post HCT [26]
Investigator-modified Derogatis Sexual Functioning Inventory Symptom Checklist-90
In women pre/3 years post HCT: 40%/52% arousal disorder 14%/46% orgasm disorder 30%/52% lubrication problems 14%/33% pain In men pre/3 years post HCT: 15%/20% arousal disorder 4%/6% orgasmic disorder 13%/22% getting an erection 13%/16% keeping an erection
n = 168 Median age 33 years Median time from HCT 14 months [68]
Questionnaire on sexual functioning and fertility EORTC QLQ-C30 Patient ranking of perceived changes in sexual relationships
Autologous/allogeneic HCT recipients: 44%/51% decreased desire 48%/57% decreased sexual activity 27%/42% decreased pleasure 29%/45% decreased sexual ability
n = 126 Mean age 27 (range 9–50) years Mean 47 (range 6–149) months post HCT [24]
“Faces” Scale of Life Satisfaction Hormonal assessment when possible
24% ED 13% ejaculation difficulties 22% of all subjects reported diminished sexual satisfaction
n = 64 Median age 39 (range 20–65) years Mean 52 (range 19–89) months post HCT [96]
EORTC QLQ-C30 Leukemia-BMT specific module Questionnaire on sexual functioning and fertility Questionnaire on patient perception of changes in HRQOL
Autologous/allogeneic HCT recipients: 21%/60% diminished desire 30%/68% decreased sexual activity 18%/47% decreased pleasure 29%/53% diminished ability to engage in sexual activity
diminished desire altered body image ED sexual dissatisfaction
ED, erectile dysfunction; EORTC QLQ-C30, European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30; HRQOL, health-related quality of life.
Hypertension is a major contributor to ED in healthy men [49,51] and a common side-effect of calcineurin inhibitors utilized for GVHD prophylaxis and treatment. ED and diminished intensity of orgasm may be caused by chemotherapy agents associated with peripheral neuropathies
administered before HCT or with the preparative regimen [54]. Other lifestyle factors that may contribute to ED include cigarette smoking, alcohol use, and high cholesterol levels [49,55]. The immunosuppressant sirolimus (Rapamune) commonly causes hyperlipidemia.
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Findings of fatigue and decreased physical stamina are often reported following HCT [12–14]. All phases of the sexual response cycle can be disrupted by fatigue and weakness [17,43]. Biologic and physiologic variables in women In women, the effects of the high-dose preparatory regimen on the hypothalamic-pituitary-gonadal axis are understood. Loss of the secretion of estradiol from the ovaries damaged by the preparative regimen leads to an absence of negative feedback to the hypothalamus and pituitary, resulting in elevated levels of FSH and LH. As in men, the hypothalamus secretes GnRH into circulation, stimulating the release of the gonadotropins FSH and LH from the pituitary. FSH initiates growth and maturation of the ovarian follicles, and LH controls ovulation and corpus luteum formation [38]. Negative feedback to the hypothalamus and pituitary occurs when estradiol levels rise, inhibiting the secretion of GnRH, FSH, and LH [40]. Both radiation and chemotherapy damage gonadal tissue, leading to decreased secretion of estradiol and thus a loss of the negative feedback inhibition at the level of the hypothalamus and pituitary. In women, the findings of elevated levels of FSH and LH are indicative of infertility and menopause [38]. The degree of gonadal damage will depend on age, the dose of radiation and chemotherapy, and the type of chemotherapy administered [38,41]. A single high dose of total body irradiation produces ovarian failure in all women [38]. Alkylating agents and radiation therapy have cytotoxic effects on both dividing and resting ovarian cells. Ovarian failure is more likely in women over the age of 25 [41,44]. Serial ultrasound examinations have demonstrated that, almost immediately following the preparative regimen, the ovaries show evidence of structural damage, shrinkage, and loss of follicles [46]. In addition to decreased levels of estradiol and elevated levels of FSH and LH, a small number of women tested had evidence of abnormal androgen function [42]. Radiation damage to the thyroid may result in hypothyroidism or subclinical hypothyroidism [42,50], which can contribute to POF [56]. Estrogen deficiency is associated with a multitude of symptoms, including hot flashes, night sweats, insomnia, mood swings, irritability, depression, vaginal dryness, atrophy and fibrosis, pruritus, urogenital symptoms, changes in cognitive function, changes in appearance, reduced bone density, and cardiovascular disease [44,57,58]. POF results in symptoms of estrogen deficiency that are more pronounced and severe due to the sudden loss of estrogen [23,58]. Table 35.3 summarizes results from investigations of POF in female HCT recipients. Findings from an ongoing longitudinal investigation evaluating the effects of POF on sexuality indicate that many women report symptoms of estrogen deficiency and alterations in sexual health on the first assessment completed prior to HCT [59]. In 48 women evaluated pre HCT, 46% reported hot flashes, with 27% reporting the hot flashes to be moderate-to-severe in intensity. Thirty-five percent reported vaginal dryness during sexual intercourse, and 60% reported diminished libido. Of the 28 women in this sample who reported sexual activity in the preceding month, 36% reported decreased arousal, 29% described decreased lubrication, 25% experienced pain during vaginal penetration, and 47% reported problems with orgasm. These findings indicate that therapy antecedent to HCT contributes to symptoms of POF and alterations in sexual health. In female HCT recipients, the etiology of vaginal atrophy is multifactorial and includes the effects of POF, the preparative regimen, in particular radiation therapy, and chronic GVHD of the vagina. Estrogen deprivation results in the vaginal mucosa becoming thinner with less lubrication, leading to symptoms of itching, dryness, and burning, increased risk of infections, and dyspareunia [58,60]. Additionally, estrogen deficiency affects urogenital tissues, leading to dysuria, urinary frequency, incontinence, and an increased risk of urinary tract infections
[60,61]. Radiation may contribute to vaginal narrowing and stenosis [42]. One investigation reports the incidence of vaginal chronic GVHD as 36% 1 year post HCT, increasing to 49% by 2 years [62]. Symptoms of vaginal chronic GVHD include cystitis, excoriation, ulceration, adhesions, and stenosis [62–64]. Decreased physical stamina and fatigue are commonly reported following HCT [12–14]. Fatigue and weakness can cause disruptions in all phases of the sexual response cycle [17,45]. Psychosocial variables Investigations of HRQOL in HCT recipients report a number of longlasting psychologic issues, which may alter sexual health. Long-term survivors of HCT report depression [8,65], anxiety [8,13,65], decreased self-confidence and self-esteem [12,66], altered body image [46,48,66,67], and distress secondary to infertility [66,68]. A sense of wellbeing, mood, and energy all significantly influence a woman’s sexuality [43]. Body image scores for both male and female HCT recipients were significantly lower compared with a healthy sample [67]. Higher levels of psychologic distress correlated with poor body image, disruption in sexual relationships, and reduced sexual satisfaction in one study [24]. In men, higher levels of psychologic distress pre HCT significantly predicted for sexual dissatisfaction 3 years post HCT [26]. In both genders, the desire, arousal and orgasm phase of the sexual response cycle can be altered by psychologic distress, especially depression and anxiety [30,69]. Psychologic stress, especially anxiety, depression, and relationship stress, can contribute to ED [49]. POF is more than an early transition into menopause as it is associated with a number of psychologic stresses including feelings of lost youth and femininity, and altered body image due to changes in appearance [39,58]. A woman’s sense of femininity and what is means to be a woman may be negatively altered by infertility [16,70]. Similarly, in men, infertility may alter their sense of virility and masculinity [16,70]. Infertility and alterations in gender roles may result in a loss of self-confidence [46,66,67,70]. If a couple has not completed childbearing, infertility may lead to distress within the relationship. Additionally, single individuals may be hesitant to establish new intimate relationships for fear of rejection secondary to their infertility [30,71,72]. Not surprisingly, infertility was more distressing for younger individuals or those who had not completed their families [66,68]. The time from the diagnosis of cancer or life-threatening illness through treatment and recovery following HCT is lengthy, psychologically difficult, and filled with uncertainty. During HCT and the early recovery phase, a caregiver is required who assumes significant responsibility for the care of the HCT recipient. Often this caregiver is the sexual partner. The effects of role changes on intimate relationships are not yet understood. Several recent publications have cited the burdens of caregiving and the significant problems faced by caregivers following HCT. In a longitudinal investigation evaluating marital satisfaction, findings indicate that the spousal caregivers experienced higher levels of depression and anxiety at 6 months and 1 year post HCT than the HCT recipient and a healthy normative population [73]. Additionally, marital satisfaction scores decreased for the caregiver from pre HCT to the 1-year assessment, whereas marital satisfaction scores for the HCT recipient remained stable. The decreased marital satisfaction scores were significantly more pronounced if the caregiver was female. The sexual partner may experience psychologic distress including anxiety, depression, and uncertainty [69,71,74], which can lead to alterations in sexual health. Findings from an ongoing longitudinal investigation indicate that nearly half of the 28 spouses/partners of women preparing for HCT experienced alterations in sexual health [59]. Specifically, seven (25%) indicated slight problems performing sexual
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Table 35.3. Investigations of premature ovarian failure in women post hematopoietic cell transplantation (HCT). Study
Measurement(s)
Findings
n = 15 Mean age 22 (range 17–30) years Assess pre HCT and 3–4 months post HCT [63]
Hormonal assessment Pelvic ultrasound Interview
53% 25% 25% 33% 73% 40% 53%
n = 37 Mean age 30 (range 17–46) years Time from HCT 13–84 months [23]
Gynecologic examination Hormonal assessment
83% vasomotor symptoms (hot flashes, sweating, irritability) 76% vaginal dryness and dysuria 76% dyspareunia 70% sexual problems (decreased desire, altered self-image, anxiety, decreased self-confidence)
n = 36 Mean age 26 (range 14–43) years Mean 4 years (range 8 months to 9 years) post HCT [66]
Questionnaire regarding menopausal symptoms, sense of femininity, sexual difficulties, social activities and coping Interview for effects of hormone therapy on symptoms
61% hot flashes 36% night sweats 81% vaginal dryness Problems in 22 sexually active women: 73% decreased desire 64% orgasm problems 82% problems with sexual intercourse
n = 30 Mean age 37 (range 25–49) years Mean time with ovarian failure 16.6 months (range 6–36 months with an additional subject 4 years and another 9 years) [80]
General and gynecologic examination Complete blood count, liver function tests, lipid panel, haemostasis parameters, hormone levels Medical history Pelvic ultrasound, mammography, and computerized bone mineralometry Investigator-designed questionnaire for menopausal symptoms
90% vasomotor symptoms, hot flashes, night sweats, and palpitations 61% musculoskeletal pain 54% vulvovaginal atrophy with dyspareunia, itching, and burning 54% mood changes with anxiety, depression, irritability, and headache 45% atrophic skin changes 42% urinary tract symptoms 26% weight gain 16% insomnia 16% memory changes Gynecologic examination revealed cervical and uterine atrophy, loss of pubic hair, pale mucous membranes, vaginal dryness, stenosis, and decreased vaginal elasticity
n = 44 Median age at evaluation 30 (range 18–43) years Median 357 (range 261–4628) days post HCT [97]
Physical examination Laboratory studies Gynecologic examination Interview for menopausal symptoms and sexual functioning
Received radiation and chemotherapy: 67% hot flashes, night sweats, insomnia, and mood changes Received chemotherapy only: 38% hot flashes, night sweats, insomnia, and mood changes In 30 sexually active women: 77% vaginal dryness 53% diminished libido and arousal 60% dyspareunia 70% diminished pleasure Gynecologic findings: tissue atrophy, loss of pubic hair, small uterus, introital stenosis, and atrophic vulvovaginitis
n = 74 postpubertal allogeneic HCT recipients Median age 30 years Mean 49 (range 4–118) months) post HCT [61]
Gynecologic examination Hormonal assessment Interview regarding HRT, menopausal symptoms, and sexual activity
78% hot flashes 61% genitourinary symptoms 94% of 52 sexually active women reported difficulties with intercourse Gynecologic findings: vulvovaginal atrophy, loss of pubic hair, and decreased thickness of genital mucosa
HRT, hormone replacement therapy.
hot flashes cystitis symptoms irritability mood changes depression diminished libido dyspareunia
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activities, three (11%) indicated constant problems, and two (7%) indicated being totally unable to perform sexual activities. The couple may find that resuming sexual activity after HCT is difficult due to the long period of forced abstinence. The HCT recipient may be struggling with body image changes and concerns about their sexual functioning. Both the HCT recipient and the spouse/partner may be suffering from depression and anxiety that is interfering with the desire phase of the sexual response cycle. As a recent cancer survivor stated (personal communication), it can take time for couples to find one another again. The psychosocial adjustment of HCT recipients is influenced by the presence or absence of social support [3,7,12]. Social support has been found to facilitate healthy adjustment following HCT, with strong family relationships predictive of better psychosocial adjustment [75]. Decreased social support negatively influenced the psychosocial coping of HCT recipients even years following treatment [3]. If a major source of social support for the HCT recipient is the sexual partner and relationship stress arises due to alterations in sexual health, the recovery process may be jeopardized.
Assessment and interventions for altered sexual health Barriers to addressing issues of sexuality and sexual health include personal discomfort, lack of education and limited time on the part of the healthcare professional [17,53,76], and reluctance of the part of patients to disclose concerns. Strategies to overcome professional barriers include increasing awareness of the types of sexual problem experienced in the population served, gaining experience in assessing sexual health, acquiring knowledge of interventions, and identifying available resources [77]. One approach to beginning an assessment of sexual health is to simply ask patients if they have resumed sexual activity. One report demonstrated that by asking two specific questions during an office visit – “Are you sexually active?” and “Are you having any sexual difficulties or problems at this time?” – the number of sexual concerns revealed increased from 3% who spontaneously disclosed concerns to 16% [78]. Interventions for addressing sexuality begin before HCT, when disclosure of possible alterations in sexual health should be discussed. In addition to stating the risks of infertility, POF, and menopause, describing possible alterations in sexual health is an essential element of informed consent. Information regarding possible alterations in sexual health should be included in the consent form and the educational materials provided by transplant centers. These education materials can serve as a valuable resource to HCT recipients during the recovery process. The educational materials should include information on when it is safe to resume sexual activity, what precautions are needed, when to consult a healthcare professional with concerns, and a list of resources. HCT recipients experience higher levels of psychologic distress when there is a discrepancy between their expectations and the outcomes following HCT [65]. This finding suggests that one intervention for improving post-HCT sexual health and HRQOL is education aimed at minimizing the discrepancy between expectations and possible outcomes. It has been shown that individuals have a remarkable capacity to adapt to changes and re-establish norms [10]. Educating HCT recipients and their spouses/partners regarding possible changes in sexual health facilitates their ability to adapt. Simply informing couples that decreased libido is common for many months following HCT is one place to start. By initiating a discussion of sexual health before HCT, the healthcare provider has accomplished two goals in addition to providing informed
consent. First, the healthcare provider has identified him- or herself as a resource for sexual health concerns. Second, they have validated that sexuality and altered sexual health are legitimate concerns. Ideally, discussions of sexual health will include the sexual partner. Including the sexual partner may facilitate a discussion between the couple, identify the partner’s anxieties, and enlist the partner’s support for coping with changes. The transplant team should identify a group of specialists for referrals to help address alterations in the sexual health of HCT recipients. Several referrals may be needed to address physiologic, biologic, and psychosocial variables that could be contributing to altered sexual health. These resources should include a dedicated and interested gynecologist and urologist, reproductive specialists, and mental health professionals for counseling couples [53]. Grieving lost fertility may occur following HCT, and assessment of the emotional response to infertility should be evaluated. A number of accepted and investigational approaches for fertility preservation include sperm or oocyte cryopreservation, cryopreservation of testicular or ovarian tissue, hormonal suppression of ovarian function, and embryo cryopreservation [39]. Other reproductive techniques that may be attempted in the post-HCT setting include in vitro fertilization and in vitro fertilization by intracytoplasmic sperm injection [39]. A detailed discussion of fertility preservation procedures and success rates is beyond the scope of this chapter, but the reader is referred to reference [39] for a more detailed discussion. Much of the responsibility for fertility preservation before HCT rests with the referring oncologist/hematologist as most patients have already received therapy that may have affected fertility prior to arriving at the transplant center. However, keeping informed of advances in assisted reproductive technologies that may be utilized by HCT recipients and making appropriate referrals are an important responsibility of the transplant team. Alterations in sexual health are nearly always related to a combination of biologic, physiologic, and psychosocial variables. To comprehensively assess alterations in sexual health, the healthcare provider must assess multiple biologic, physiologic, and psychosocial variables and the interactions among these variables. In making the diagnosis of a sexual disorder, considerations include the onset of the symptoms, the situational context, the frequency of occurrence, and the psychologic and physiologic conditions that may be contributory. There has been an increased focus on the sexuality of cancer patients and alterations that arise during and following treatment; however, there is a paucity of studies investigating treatment strategies [17]. Key factors for the successful treatment of any alteration in sexual health will be open communication between the couple and a desire on the part of both for sexual activity. Hypoactive sexual desire disorder Sexual desire is the least well understood and most difficult aspect of altered sexual health to treat. Desire is strongly linked to the second phase of the sexual response cycle, arousal, so individuals with hypoactive sexual desire disorder are likely to experience problems with arousal. Many HRQOL investigations of HCT recipients have demonstrated that hypoactive sexual desire disorder and arousal problems are common. The dynamic relationships between the phases of the sexual response cycle cannot be overemphasized. Women who experience dyspareunia may quickly lose interest in sexual activity. Similarly, in men, loss of desire for sexual activity may be secondary to ED. In these situations, interventions need to be targeted at treating the dyspareunia or ED, and not specifically focused on decreased desire. In many cases, the discussion of sexual difficulties with the HCT recipient and partner can be therapeutic. The information, reassurance
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that treatment options are available, and opportunity to explore alterations in sexual health with a healthcare provider can often effectively address many areas of concern [17,79]. In fact, the most effective intervention may be for the healthcare provider to begin a dialog on issues of sexuality [53]. Fatigue, altered body image, decreased self-confidence, depression, anxiety, and uncertainty are key variables to assess when evaluating hypoactive sexual desire disorder. Depression and anxiety in both the HCT recipient and the sexual partner should be evaluated. Assessment of the relationship with the intimate partner needs to be evaluated for stress, discordance of expectations, and adequacy of communication. When couples were asked if there were any arguments or problems related to alterations in their usual sexual activities before HCT, 24 (86%) of the spouses/partners and 20 (71%) of the women reported no arguments, yet the majority reported alterations in sexual activities [59]. These data raise the question as to whether the alterations in sexual health were not a concern of the couple before HCT or whether they were simply not discussing the sensitive issues surrounding altered sexual health. If depression is identified as a contributing factor for hypoactive sexual desire, antidepressant therapy can be helpful. However, the selection of the antidepressant is critical. Delayed or absent orgasm may occur with selective serotonin reuptake inhibitors, which may necessitate a change in dose or alternative antidepressant [53]. Resources for the recovering HCT recipient and his or her partner include educational materials from the transplant center, support groups, and Internet discussion groups. Additionally, the American Cancer Society publishes two books, one for men and one for women, titled “Sexuality and Cancer,” authored by Leslie R. Schover. Testosterone replacement in men may restore sexual desire and arousal [43]. In male HCT recipients with low-to-low-normal testosterone levels, testosterone replacement therapy was effective in improving sexual desire [52]. The role of testosterone in female sexual desire and arousal is not clearly understood, and the use of testosterone therapy in women is controversial [43]. However, some women may find improvement in sexual desire and arousal with testosterone replacement therapy [43,53,80].
POF, vaginal atrophy and dyspareunia Assessment of women with POF includes medical history, physical examination, gynecologic examination, laboratory analysis, hormonal evaluation, medication profile, and a review of coexisting health problems and degree of fatigue. The hormonal testing should include estradiol, FSH, LH, androgen level, and thyroid function tests. Many symptoms of estrogen deficiency can be ameliorated or minimized with hormone replacement therapy (HRT). HRT is effective in relieving hot flashes, improving sleep, maintaining vaginal elasticity and lubrication, and decreasing changes in the appearance of the skin and changes in breast size. One study found that symptoms of estrogen deficiency were eliminated in 22 of 27 women [66], and two other investigations reported almost immediate relief of symptoms in the majority of women treated [23,61]. In a more recent investigation, HRT effectively relieved hot flashes in 66% of female HCT recipients within 10 days (range of 7–15 days) [80]. The HRT also relieved other symptoms; specifically, insomnia was reduced in 66%, improved psychologic and emotional states were noted in 66%, and 53% had a decrease in vulvovaginal atrophy after 5 (range 4–12) weeks of therapy. On gynecologic examination, the appearance of the genitalia returned to normal following HRT. The authors conclude that HRT should be offered to all women with no medical contraindications soon after HCT.
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There are many gaps in knowledge relating to the use of HRT for the treatment of POF in HCT recipients. There are several studies that suggest that currently prescribed HRT may not be adequate for HCT recipients [24,42,66]. The evidence for questioning the adequacy of currently prescribed HRT is the findings of low estradiol levels despite HRT, the large number of women receiving HRT not having menstrual cycles, and the degree of vaginal dryness found despite HRT. Data from one study revealed that, at 3 years post HCT, 76% of the women were taking HRT yet 52% cited lubrication and arousal problems, 33% experienced dyspareunia, and 46% experienced difficulties with orgasm [26]. Findings from this investigation indicate that when HRT is initiated may be important to maximize benefit. Those women who had not started HRT by 1 year post HCT experienced distressing sexual dissatisfaction at 3 years even though they were all taking HRT by this time. The benefits of HRT in relieving symptoms of estrogen deficiency are clear, but the selection of the HRT for female HCT recipients is challenging and requires additional study. One author recommends low-dose estrogen replacement be administered for the first month and increased to full dose in the second month if the woman has only recently become estrogen deficient [56]. However, if she has been estrogen deficient for 12 or more months, low-dose estrogen replacement should be initiated with a gradual increase to maintenance doses by 6 months to avoid sideeffects. A recommended estrogen dose in young women with POF is the equivalent of 1.25 mg of conjugated equine estrogens [56]. In women with an intact uterus, progestins should be added [56]. Careful selection of the type of HRT is critical as some oral contraceptives may reduce circulating free testosterone by increasing sex hormone-binding globulin levels, resulting in diminished sexual desire [43]. In women with adequate estrogen replacement and complaints of diminished desire, fatigue, and a poor sense of wellbeing, testosterone replacement may be beneficial [56,80]. The use of HRT following physiologic menopause has been an area of debate and controversy for some time. Recently, however, the results of the Women’s Health Initiative Study have reported that the risks of HRT – breast cancer, heart attack, stroke, and blood clots – outweigh the benefits in women following physiologic menopause [81]. The risks of HRT in female HCT recipients may not be the same as the risks of HRT in women following physiologic menopause. For females with POF, HRT is administered to compensate for the premature loss of endogenous hormones [39,56]. Selection of the type of HRT should be guided by the woman’s symptoms and preferences, and designed to prevent future complications such as osteoporosis [80]. In general, women with POF should continue HRT until the age of physiologic menopause [56]. Due to the complexities of HRT and the gaps in knowledge regarding HRT for female HCT recipients, a prudent course of action is to refer women to a gynecologist for a thorough evaluation and individualized approach to HRT. This individualized approach must encompass not only the woman’s symptoms and preferences, but also a broad assessment of factors that may increase the risks associated with HRT. These factors include a family history of cancer and cardiovascular disease, genetic markers, hypertension, and hyperlipidemia, as well as lifestyle issues such as exercise patterns, obesity, diet, and smoking history. Nonhormonal strategies proposed for relieving symptoms of estrogen deficiency include the use of herbs, vitamins, yoga, acupressure, acupuncture, exercise, and diet modifications. Few of these interventions have been rigorously tested. In a recent systematic evidence review, Remifemin, a formulation of the herb black cohosh, has evidence to support its use in the relief of hot flashes [82]. Antidepressants are another nonhormonal strategy that can ameliorate some symptoms of menopause. Hot flashes were reduced by 60% with the use of venlafaxine (Effexor) [83]. Other antidepressants that may be
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helpful include paroxetine (Paxil), fluoxetine (Prozac), and citalopram (Celexa) [84]. Gabapentin (Neurontin), clonidine hydrochloride (Catapres), and bellergal (Bellergal-S) are other nonhormonal agents that may decrease hot flashes [84]. Resources are available to women who want to explore nonhormonal strategies for the relief of menopausal symptoms. Several that have been well received by female HCT recipients include: “The Estrogen Decision,” by Susan Lark, published by Celestial Arts in 1999, and “Natural Menopause,” by Susan Perry and Kate O’Hanlan, published by Perseus Books in 1997. These books provide information on the role of HRT and alternative strategies in alleviating symptoms of menopause. Nonhormonal strategies for managing menopausal symptoms will require a consistent commitment by the woman to maximize potential benefits. Female HCT recipients with vaginal atrophy, narrowing, and dyspareunia may benefit from the consistent use of vaginal lubricants and dilators. Vaginal lubricants should be water soluble to avoid the risk of infections. Vaseline is not water-soluble and therefore should not be used as a lubricant. K-Y Jelly® (Johnson & Johnson, New Brunswick, NJ), vitamin E, Replens® (LDS Consumer Products, Cedar Rapids, IA), and Astroglide (Biofilm Inc., Vista, CA) can all be used as a lubricant prior to sexual intercourse. Replens can be used as a vaginal moisturizer as well as a lubricant. As a vaginal moisturizer, one applicator of Replens can be inserted into the vagina three to four times per week at bedtime. Astroglide has been reported by some users to last longer during sexual activity. These products can be found at the local drug store or via the Internet. Dilators can be obtained from a gynecologist or often through the radiation therapy department. Women should lubricate the dilator and then insert the dilator into the vagina until it is slightly uncomfortable and lay with the dilator in place for 10–15 minutes three or four times per week. As the vaginal tissues begin to stretch, the woman can increase the depth of penetration and gradually increase the size of the dilator. The level of commitment for pursuing these measures in a consistent manner will optimize results. A woman experiencing dyspareunia should assume a position during sexual intercourse that allows her to control both the rate and depth of penetration, enabling her to stop penetration if she becomes uncomfortable. Women worried about lubrication and their response to sexual stimulation can be encouraged to self-stimulate, masturbate, as a way to explore their response without simultaneously being concerned about their partner’s needs [85]. A number of medications may affect sexual functioning in women, including antiandrogens, sedatives, antidepressants, and stimulants [86]. Evaluation of the medication profile and adjustment of medications may improve sexual functioning. Chronic GVHD of the vagina can also contribute to vaginal atrophy, stenosis, and dyspareunia. Epithelial cell damage, mononuclear cell inflammatory infiltration, fibrosis, and atrophy are histologic features of chronic GVHD [87]. Additional features of chronic vaginal GVHD include inflammation, stricture formation, narrowing, and obliterated introitus [64,88]. A comprehensive program for the prevention of vaginal chronic GVHD incorporating patient education, topical estrogen, early initiation of HRT, vaginal dilatation in the absence of sexual activity, and regular gynecologic examination minimized the development of severe GVHD of the vagina [62]. These investigators also outlined treatment for established chronic GVHD of the vagina, which included topical steroids, topical cyclosporine, and vaginal dilatation in women who had evidence of vaginal narrowing. Fifteen of the 28 women treated for chronic GVHD of the vagina had complete resolution of symptoms, eight noted improvement, and in five women the symptoms remained stable. Unlike other reports of chronic GVHD of the vagina [64], no patients in this report required surgical intervention.
Erectile dysfunction Assessment of ED includes a medical history and physical examination, a review of the medication profile, evaluation of coexisting health problems such as hypertension, anemia, hyperlipidemia, and fatigue, and an endocrine evaluation. Hormonal testing should include total testosterone, free (bioavailable) testosterone, sex hormone-binding globulin, FSH, LH, prolactin, and thyroid function [49,52]. Additionally, the transplant team can make a referral to a urologist for a comprehensive evaluation of the functional and structural capacity of the penis [89]. In men, testosterone is strongly linked to both sexual desire and arousal. Testosterone replacement therapy often restores sexual desire and arousability in the subset of men with low testosterone levels, and testosterone replacement therapy may also improve erectile function [43,52]. Testosterone replacement therapy is available as an intramuscular injection, transdermal patches, gel and oral forms. Testosterone enanthate (Delatestryl) and testosterone cypionate (Depo-Testosterone) are two testosterone replacement products available [49,89]. The recommended dose is 100–200 mg intramuscularly every 2–4 weeks [89]. Testosterone replacement is contraindicated in men with a history of prostate cancer, and routine monitoring of prostate-specific antigen levels should be performed at 6-month intervals [90]. Testosterone replacement therapy can contribute to coronary artery disease by its effect of lowering high-density lipoprotein levels [89]. Hyperprolactinemia observed in some male HCT recipients is associated with decreased desire and ED. The use of bromocriptine mesylate (Parlodel) can be effective in lowering prolactin levels [91]. Performance anxiety can lead to ED and subsequently decreased libido. Suggesting self-stimulation, masturbation, to men may improve their confidence in their erectile function in a setting where they do not need to worry about their partner’s needs [85]. Cognitive interference can lead to ED, so encouraging the man to stay very focused on sensations during sexual activity will help minimize negative thoughts and performance concerns. Suggesting that the couple prolong foreplay will help to ensure that the male is aroused and may also improve erectile function. Antihypertensives, anticholinergics, antihistamines, cardiac medications, antidepressants, alcohol, and other recreational drugs can all contribute to ED. A thorough review of the medication profile is an important component of the assessment for the etiology of ED. Reducing doses or trying alternative medications may result in improved sexual functioning. Sildenafil (Viagra) and other phosphodiesterase inhibitors such as vardenafil (Levitra) and tadalafil (Cialis) have made an important contribution to the effective treatment of men with ED. Sildenafil does not improve sexual desire but increases the penile response to sexual stimulation [32,49]. Sildenafil should be taken approximately 1 hour prior to sexual activity in a dose of 20–100 mg orally. The side-effects of sildenafil are generally mild and well tolerated, and include disturbed color vision, headache, flushing, and dyspepsia [32]. Sildenafil is contraindicated in men taking nitrates for angina or hypertension. A number of other strategies for the treatment of ED, such as cavernosal injections, vacuum devises, and surgical interventions, are available, but in general these strategies are not well accepted by men [53]. One group of investigators hypothesized that a combination of testosterone replacement therapy combined with sildenafil would improve erectile functioning in men following HCT [92]. The sample consisted of eight men aged 22–58 years who were 2–24 months post HCT with evidence of hypogonadism, ED, decreased libido, ejaculatory disorders, and documented cavernosal arterial insufficiency. The men were treated with a combination of testosterone replacement therapy given monthly for 6 months and sildenafil taken one to two times per week. Results
Sexuality Following Hematopoietic Cell Transplantation: An Important Health-related Quality of Life Issue
indicated that all eight men had an improvement in erectile function allowing for satisfactory sexual intercourse, seven men had resolution of ejaculatory dysfunction, and all had an improvement in both total and free testosterone levels. The authors concluded that this combination therapy is a safe and effective therapy for ED in HCT recipients and should be evaluated in a larger clinical trial.
Areas for future research Over the past two decades, investigators have explored the HRQOL of HCT recipients and have identified a number of positive and negative sequelae that influence HRQOL. The next step is for investigators interested in improving the HRQOL of HCT recipients to begin to design and test interventions to minimize the negative sequelae, particularly strategies to minimize alterations in sexual health. Throughout this chapter, areas for future study have been raised. Designing and testing an education program to help HCT recipients and their spouses/partners develop realistic expectations of possible changes in health, including alterations in sexual health, may bring expectations and possible outcomes closer together and thereby decrease psychologic distress. Many research questions can be generated regarding the HCT recipient’s caregiver. One question concerns what the issues and concerns of caregivers are during and after HCT. In addition, what strategies can be designed and tested to address caregivers’ concerns? Additional investigation is needed to develop insights into the effects on intimate relationships when the spouse/partner is the caregiver. Investigations of the biologic, physiologic, and psychosocial variables that underlie alterations in sexual health need to continue. The identification of variables that can predict which HCT recipients are at risk for alterations in sexual health is needed. The effects of nonmyeloablative transplantation on fertility and sexual health have not been fully explored. Anxiety and depression are known to be long-term psychologic issues for some HCT recipients. What intervention strategies reduce these negative emotional consequences? Questions regarding HRT therapy for female HCT recipients include: (1) what is the most efficacious combination of hormone replacement?; (2) does testosterone therapy benefit women by improving libido and arousal?; (3) when is the best time to begin HRT?; (4) how well does HRT protect women from the long-term sequelae of POF, such as osteoporosis and cardiovascular disease?; (5) what strategies can improve
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adherence to the use of HRT?; and (6) what nonhormonal strategies are effective in alleviating symptoms of estrogen deficiency? The monitoring and prophylactic measures described earlier for vaginal chronic GVHD were effective in preventing the development of severe GVHD, avoiding the need for surgical intervention [62]. What additional preventative strategies can be employed? Since most HCT recipients during the time of risk for chronic GVHD have returned to their homes and referring physicians, what can the HCT team do to see this program implemented by a woman’s local gynecologist? The understanding that cavernosal arterial insufficiency is a significant cause of ED in HCT recipients led to the development of a treatment strategy that included the use of testosterone replacement therapy plus sildenafil, which was very effective in a small number of male HCT recipients [92]. This intervention strategy needs to be investigated in a larger sample of men suffering from ED post HCT.
Conclusion Sexuality is a broad concept encompassing much more than sexual activity, and remains an important aspect of being human regardless of the diagnosis and treatment of life-threatening illness. The understanding of alterations in sexual health following HCT is not complete. It is clear that the etiology of alterations in sexual health involves biologic, physiologic and psychosocial components, as well as the complex interactions among these variables. While understanding the etiology of alterations in sexual health is not complete, enough knowledge exists to allow healthcare professionals to begin to address this critical aspect of HRQOL. A beginning step is to include an assessment of sexual health as part of routine care following HCT. It has been shown that an open discussion of sexuality with a healthcare professional can be therapeutic and address many areas of concern. Developing a referral network of other healthcare professionals, including gynecologists, urologists, reproductive specialists, and mental health professionals, enables the HCT team to further assist HCT recipients with alterations in sexual health. While the focus of this chapter has been alterations in sexual health following HCT, it must be stated that many HCT recipients report satisfying sexual relationships after transplant [6,24,93]. The challenge for the HCT team is to identify those HCT recipients who are experiencing alterations in sexual health and intervene to improve sexual functioning, decrease sexual dissatisfaction, and thereby improve their HRQOL.
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Susan K. Stewart
Hematopoietic Cell Transplantation: The Patient’s Perspective I still find it almost bizarre that I would be the one to get so sick. I was very physically active. I rode my bike 2,000 miles a year, swam about 100 miles and was active in my children’s lives. Leukemia happens to people you read about in the newspaper during their appeals for donors or money for transplants. It doesn’t strike enormously healthy, happy and vital 39-year-old family men. HCT survivor Mike Eckhardt, 1995
Preparing for transplant Like Mike, most people have never contemplated the notion that they may some day have an illness that requires treatment with hematopoietic cell transplantation (HCT). Regardless of whether a patient is newly diagnosed or previously learned of the illness and has exhausted other therapies, the prospect of HCT is traumatic for both the patient and his or her loved ones. Having lived through this experience myself in 1989, after being diagnosed with acute myelogenous leukemia, I know first hand how difficult it can be to process information under duress, make life or death decisions, and cope emotionally with the HCT experience. In 1990, I created Blood & Marrow Transplant Information Network (BMT InfoNet; www.bmtinfonet.org) to help other families navigate the HCT experience. Through lay-language books about HCT, survivorship symposia, and peer counseling, BMT InfoNet services more than 10,000 patients and their loved ones each year. Their experiences help inform this chapter. Patients are overwhelmed with fear: fear that the transplant will not cure; fear that the transplant might kill; fear of pain; and fear that the side-effects of treatment will be intolerable. Many are also angry and/or depressed over the loss of control over their wellbeing. A disease now controls their body, and the patient must rely on a team of strangers to save his or her life. Preparing a patient for the rigors of HCT against this backdrop of fear, anger, and/or depression is challenging. Some patients are so overwhelmed emotionally that they hear and process little of what a physician tells them. They can process only discrete packets of information, are overwhelmed by details, and prefer to leave much of the decision making in the hands of the physician. At the other end of the spectrum are the patients who attempt to regain control over their body by accumulating as much information as possible about their disease and treatment options. They want complete, detailed information about the risks and benefits of treatment. They will search external sources, such as the Internet, for information. They want to be fully engaged as partners in their care. Since patients’ styles of coping with troubling medical information vary greatly, it is important to ascertain in advance how much information a patient wants, and how best to deliver it. Level of education, cultural biases about discussing illness and death, the degree of trust a
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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patient has in the medical establishment, as well as individual coping styles will define the best method of discussing treatment options with a patient. Asking how much detail the patient wants when discussing medical treatments, and whether he or she prefers to hear it all at once or in small segments, enables a physician to tailor the delivery of information to best suit each individual patient. Despite these differences, there are certain universal elements of communication that are helpful to bear in mind when preparing a patient for transplant.
Put the risks into perspective When a patient meets with the transplant team, a long list of potential treatment-related complications is described. For many, this is the first time they have heard a detailed discussion of the risks associated with HCT. The prospect of developing these complications is frightening for patients, and many leave the consult with the transplant team more depressed than when they arrived. It was like a whirlwind, a dream. One day our child was a normal 15-year-old boy who would live to be 80. The next day we were staring at blackboard diagrams about transplants, and hearing doctors tell us our son might die. It wasn’t real. We didn’t understand. All we could do was hug each other and cry. (Lorraine Boldt, mother of transplant survivor)
Patients need help putting the risks associated with HCT into perspective. Grouping the complications into (a) those that always occur, (b) those that often occur, and (c) those that seldom occur provides patients with a manageable framework for understanding relative risks and coping emotionally with the prospect of complications. Knowing that the risk of developing severe liver damage is small, while the risk of developing mucositis is high, for example, conveys a far more accurate picture of what a patient should expect. Without such a framework, patients may imagine the risk of developing very serious complications to be greater than it is. It is also important to distinguish between those side-effects that are temporary and those that may persist long term. Most people believe they can deal with a problem for a discrete period of time. Knowing in advance what the normal range of duration is for each complication enables patients to better cope with the difficulty when it arises. Without such information, patients can become distressed if a complication persists, and may become concerned that it signals relapse, disease progression or another serious problem.
Hematopoietic Cell Transplantation: The Patient’s Perspective
It is equally important to discuss how complications will be managed. Although a detailed discussion of each therapy is not necessary, patients should know that treatment for each complication exists. This is particularly important when discussing painful complications, since some patients fear pain more than death. Many patients do not automatically assume that pain medication will be available to relieve discomfort. This must be explicitly stated. Leave out this discussion and a patient can assume that he or she will suffer tremendous pain, or that the complication is not treatable, and this can increase the emotional load.
Communication in lay language Although many patients are well educated, most are unfamiliar with the medical terms used routinely by transplant personnel. Terms like “CBC”, “aspirate,” “Hickman,” “bilirubin,” “TPN,” and even “stem cells” are often foreign to people without medical training. Even more commonly used words such as “prognosis” and “remission” may be meaningless to patients who have never used these terms. Most patient advocacy groups, as well as the general print media, strive to use language that can be understood by a person with a fifth- or sixth-grade education. Given that HCT patients are under a great deal of emotional stress and may not be able to process information as easily as the average person, medical procedures and terms should be described as simply as possible. In the absence of a clear explanation, patients may misinterpret the message being conveyed by the physician. Using precise language also improves a patient’s understanding of what he or she is about to experience. For example, when describing a painful procedure, using words such as “hurt” or “pain” does not adequately prepare a patient for the sensation he or she will feel. Terms like “pressure”, “stinging sensation” or “dull ache” convey a more precise idea of the sensation. Precise language will help reassure patients that these sensations are normal when they occur.
Repetition and reinforcement is important The volume of information that HCT patients receive is overwhelming, and few completely digest the information during the first presentation. Patients who are emotionally overwhelmed may shut out complicated or frightening information, and later recall only part or none of what was said. It’s important to repeat information at several points during treatment. A patient who was unable to absorb critical information when it was initially presented may be better prepared to absorb it later after the initial shock about the diagnosis and the difficult treatment he or she is about to undergo wanes. Some patients have found video- or audiotaping the initial consultation with the transplant physician to be helpful. Such a record enables them to double-check their understanding of what was said later when reflecting upon the information. It also helps them catch important details they may have missed during the initial presentation. Suggesting this option to patients can provide them with a valuable tool, and can reassure them that the physician wants them to be as informed as possible about the impending treatment. Suggesting that patients bring a support person to the consultation whose sole purpose is to take notes can also be helpful. Proactively suggesting this as well as taping the conversation will help those who might be too distraught to think of these tools on their own. Encouraging patients to ask questions can also enhance understanding. Some patients come from a cultural background where questioning a physician is considered rude or disrespectful. Inviting them to ask questions to confirm their understanding can give them “permission” to do so. Inviting questions means more than simply saying, “Do you have any questions?” Inviting questions in a way that elicits more than a
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yes/no answer may allow patients to better formulate and state their concerns. For example, a physician might say, “Some patients don’t understand what I mean when I say they will become neutropenic. Do you understand what I mean by neutropenic, or should I explain it more clearly?” Such a question normalizes a confusion the patient may have and affirms the physician’s responsibility to make the explanation as clear as possible. Patients who have questions during treatment sometimes do not feel that they have an adequate opportunity to present them to the physician. Creating an atmosphere that encourages questions from patients is essential. A physician who appears busy or distracted can discourage patient questions without meaning to do so, leaving the patient feeling anxious and uninformed. Sitting down when entering a patient’s hospital room, for example, signals to the patient that the physician has time to focus on the patient’s concerns. Consciously making an effort to engage a patient in conversation, rather than simply delivering news, can create an atmosphere that elicits patient concerns. Suggesting that the patient maintain a notebook of questions that arise when the physician is not present can also help patients organize their thoughts.
Different learning styles It is well accepted by educators that individuals have different styles of processing information and learning. A person who is primarily a visual learner does best when charts or pictures accompany the explanation. Seeing the person with whom he or she is talking, rather than just hearing an explanation, enhances understanding. An untidy environment can interfere with this patient’s ability to absorb information. Auditory learners, on the other hand, do best with verbal explanations. They are more likely to use telephone support systems, such as helplines, than pore over charts or diagrams that explain a problem. They are easily distracted by sounds. Kinesthetic learners learn by doing. They like hands-on explanations and can be distracted by movement. When educating transplant patients, accommodating the variety of learning styles is important. A person who is primarily a visual learner can miss key information if the education plan relies solely on verbal explanations. Patients who are primarily auditory learners can have difficulty absorbing print information. Kinesthetic learners may benefit from a tour of the facilities where procedures will be performed, with an opportunity to handle equipment that will be used during transplant. Although it may be difficult to ascertain each individual’s learning style, providing the patient with a menu of learning tools will maximize the likelihood that he or she will absorb important information. Augmenting verbal explanations of procedures with written publications or audiovisual aids produced by organizations like BMT InfoNet or the National Marrow Donor Program (NMDP; www.marrow.org) enables a patient to choose the information vehicle that best suits his or her needs.
Discussing pain control is important Many patients fear pain even more than death. Yet while all transplant teams inform patients about potential complications, some neglect to discuss the steps that will be taken to manage pain. They assume that patients know that pain medication will be administered as needed. Many patients, however, have had little experience with hospitalization and painful medical procedures, and do not automatically assume that pain relief will be available. Others have undergone painful medical procedures without adequate pain control and have little reason to assume that pain will be managed more effectively by the transplant team.
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Explicitly telling patients that pain control medications will be used to prevent and control pain, and educating them about when to ask for pain relief, can ease their anxiety. Many patients try to be stoic, asking for pain medication only after pain becomes intense. Explaining that pain is easier to manage before it becomes intolerable, normalizing the use of pain medications in the transplant setting, and encouraging patients to request relief before pain becomes unbearable can minimize the discomfort and anxiety a patient experiences. If a patient-controlled analgesia device will be available, letting the patient know that he or she will have some control over the administration of pain medication can make the prospect of pain more manageable. It is equally important to warn patients and caregivers in advance about possible side-effects of pain medications. Most are unprepared for the change in mental status that accompanies the use of some medication, and both patients and caregivers can become alarmed when this occurs. Patients can see or hear things that are not real and talk nonsensically. Caregivers who observe this without understanding that this is a side-effect of the medication, and patients who later realize they have been hallucinating, can become emotionally distraught. Assuring both patient and caregiver that these drug reactions are within the range of normal and are temporary can alleviate unnecessary anxiety. Fear of addiction discourages some patients from asking for pain relief. Patients, as well as many physicians, do not realize that drug addiction is a psychological phenomenon, whereas drug dependence is a physical phenomenon. Although a patient may develop a physical dependence on a drug, he or she can be weaned from the drug by tapering the dosage. An addiction, however, requires psychological interventions to address the underlying cause of the addiction, as well as tapering or discontinuation of the drug. Patients who have never before had a drug addiction are unlikely to develop an addiction to pain medications administered as part of HCT [1].
Discuss psychological difficulties One important area of patient education that is often overlooked is a discussion of the psychological difficulties that patients and family members encounter during transplant. Patients may be told that the transplant will be a “difficult” or “stressful” experience, but these words fail to describe or normalize the type and depth of emotions the patient and his or her family are likely to experience. If not given this information, a patient becoming angry or depressed during treatment and recovery may assume that there is something wrong – that he or she is a weakling or is not coping as well with the stress as most patients. After all, the patient reasons, if these emotional difficulties were common among HCT patients, the medical team would have said so in advance, just as they warned of the other medical complications. Some become angry with themselves for not coping well, which exacerbates their emotional distress. As difficult as the transplant experience is for patients, it can be even more difficult for the family member/caregiver who is keenly aware of the patient’s progress or lack thereof. The person upon whom the caregiver normally relies for support – the partner – may be the patient, who is ill equipped to deal with anyone’s emotional load besides his or her own. Usual support networks, like family members or friends, may fail to comprehend the severity of the situation and be unprepared to address the caregiver’s needs. It is important that the health-care team be explicit with both the patient and caregiver about the range of emotions they may experience during and after transplantation. Anger about the disease, resentment over the loss of control of the patient’s wellbeing, fear about medical procedures or possible treatment failure, and depression about the slow pace of progress are typical emotions patients and caregivers face. The
patient may express distress by lashing out at the medical team or a loved one. Assuring both patient and caregiver that these emotions are normal, and explicitly telling them what types of assistance are available to help them cope, maximizes the likelihood that they will seek and accept help, should they need it. Patients need to understand that HCT is an extraordinary experience and that they can expect to need more than their everyday coping tools to manage the stress. Normalizing the use of antianxiety or antidepressant medications in this circumstance, and assuring the patient that, in the absence of a history of substance abuse, drug dependency is not likely, can alleviate patients’ concerns about using these tools. For some patients, the acute care period is the time of greatest stress. For others, the post-transplant period poses the greatest psychological challenge. The support system provided by the hospital and medical personnel has been curtailed, the busy daily schedule of tests, doctor visits and other hospital routines is now replaced by long days of isolation and inactivity, and the challenge of resuming a normal lifestyle can be daunting. When I left the hospital, I thought the tough part was over. I’ve never been so wrong. The inpatient part was the easiest part of the bone marrow transplant process. I was focused and fired up. . . All the worldly things, such as my role as a husband and father, were secondary to winning the inpatient battle. Other routine things such as house payments, medical bill, career and church weren’t even on my mind. Nurses and doctors took care of me. It wasn’t easy, but I felt I was making significant progress toward beating my disease. The clarity of purpose and sense of progress were lost when I came home. Instead of feeling like a successful patient, I felt like a failed person. All those worldly things, such as my roles as husband and father that I had ignored in the hospital, came roaring back. They were once again important and I felt woefully inadequate in those roles. The steroids and cyclosporine made me extremely emotional and irrational. I would cry because I had too much milk on my cereal. I couldn’t sleep (steroids), couldn’t shower (Hickman), couldn’t read (no concentration), couldn’t drink coffee in the morning (nausea), couldn’t exercise (no strength) and couldn’t get close to my children (might get an infection). Even taking my medicine was confusing and overwhelming. I spent time worrying about things I couldn’t control. I was convinced I was going to run out of money, lose my house, my dog, etc. There was no measured progress anymore. I felt I was regressing. I was in an emotional rut. [2]
Emotional difficulties can surface weeks or months after transplant. Unexpected rehospitalization for a complication can cause distress. Some experience symptoms similar to post-traumatic stress syndrome: a sight or smell may trigger an unpleasant memory of the transplant experience, leaving the patient shaken. Returning to work, school or a social world that has moved on during transplantation can make a patient feel isolated. Fear of relapse causes many patients to avoid long-term planning. It can take many months before a patient is able to get through a single day without thinking about the transplant. A recent study has confirmed what many caregivers report: the intensity and duration of transplant-related distress is in some ways greater for caregivers than for patients long term [3]. The caregiver must deal not only with his or her own fears about the patient’s health, but with those of other family members as well. The caregiver may be thrust into new roles, assuming responsibilities once handled by the recovering spouse. These tasks, coupled with his or her own emotional turmoil, can create significant distress. Some who handled the acute phase of transplant well find themselves emotionally drained and unable to fully function many months later:
Hematopoietic Cell Transplantation: The Patient’s Perspective In some ways, it was like we were no longer married. We were never alone. Our whole relationship – everything – was based on illness, medical procedures, hospital and family. Trying to resume a normal relationship was not possible. Family was continually around – always. I understood, but some days I didn’t want to share my husband. I wanted him all to myself. I had to take over all of his responsibilities – paying bills, yard work, etc. Instead of being an equal partner, it was like having another child to worry about. I felt like I’d lost the man I married and just wanted him back.
Psychosocial support networks for patients and their families A number of support networks are available for families facing the emotional challenges associated with transplant. BMT InfoNet has a Caring Connections Program that links HCT patients and family members with survivors who can provide emotional support. Patients and survivors are paired according to diagnosis, type of transplant, and other factors the patient may deem important such as gender, transplant center, religion, etc. Survivors are screened to ensure they provide emotional support, rather than medical information, and the pairs communicate by phone or email. This service is particularly helpful for patients who do not have access to, or prefer not to communicate by email. Patients can access this service at www.bmtinfonet.org/patient.html. Many patients and family members find list-servs on the internet helpful. A list-serv is an electronic mailing list that enables a person to discuss issues with a large number of peers online, and to offer and get support. The list-serv is managed by an individual who typically screens postings before emailing them to the entire list, although the screening is generally limited to weeding out commercial solicitations and hostile emails. Members can opt to receive individual emails from other members after they have been posted, or a daily digest of emails that enables them to determine, by the subject heading, whether any of the issues discussed in the posts are of interest to them. Patients can respond to a posting on the list-serv by emailing the entire list or by privately emailing the individual who posted it. Discussions on HCT-related list-servs are lively, and many patients find the list-serv an invaluable place to vent their feelings and concerns with others who understand and will empathize. A great strength of listservs is that they can validate a person’s feelings, and normalize his or her experience. A downside is the potential for sharing medically inaccurate or inappropriate information, although patients can easily verify the information with their own physician. More often, valuable information about medical interventions that have worked with other patients is shared which the patient can discuss with his or her physician. The Association of Online Cancer Resources (ACOR; www.acor.org) maintains a list-serv for HCT patients (BMT-Talk, 1443 subscribers), one for patients with graft-versus-host disease (GVHD, 348 subscribers), and list-servs organized by diagnosis. Anyone can subscribe via the ACOR website by following the links under Mailing Lists. A real-time online chat room also exists for HCT patients and caregivers. BMT-Support (www.bmtsupport.org) meets several times weekly. There are separate chat rooms for patients and for caregivers. Participants log in at the specified time, and the live online chat is moderated by a survivor with nursing training. Disease-specific organizations, such as the Leukemia & Lymphoma Society or International Myeloma Foundation, offer local or online support programs for patients. While some transplant patients find these helpful, others find discussions with people who have not undergone a transplant unsatisfying. A list of organizations that provide disease-spe-
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cific information and support can be found on BMT InfoNet’s website at www.bmtinfonet.org/resource. General support groups for caregivers are offered by the Well Spouse Foundation (www.wellspouse.org) and by general cancer support organizations such as Gilda’s Club (www.gildasclub.org), the Wellness Community (www.thewellnesscommunity.org), and local cancer support centers. Young adult patients often find resources targeted to their age group helpful. The Ulman Cancer Fund for Young Adults (www.ulmanfund. org) provides a peer support program similar to BMT InfoNet’s Caring Connections Program. Young adults can be linked with others whose life has been affected by transplant and read their stories online. The fund also provides educational scholarships to young adults diagnosed with cancer. Planet Cancer (www.planetcancer.org) also offers online support programs. Some patients or their family members require individual counseling to help deal with their emotional distress. Although transplant centers may provide psychiatric help and counseling while the patient is undergoing transplant, that support system is often unavailable when patients return home. It can be difficult to find a local mental health professional who is experienced in dealing with cancer or transplant related issues. The American Psychosocial Oncology Society (www.apos-society.org) offers a help-line for patients and families seeking a local specialist who is trained in working with cancer patients and can address their needs.
Quality of life post transplant From the patient’s perspective, successful HCT does not simply mean that the patient survives. It also means that he or she resumes a reasonably normal life after transplant. Significant survivorship challenges are often not discussed during the acute phase of treatment, and even at discharge, patients may not be adequately prepared for the psychological stress that ensues. They may also not be equipped with coping strategies for medical difficulties such as depression, infertility, cognitive problems, chronic fatigue, and sexual difficulties that can occur after HCT. In 2006, BMT InfoNet invited HCT survivors and their family members to complete an online survey to determine issues of concern to survivors post transplant. Invitations were issued by email to persons on the Blood and Marrow Transplant Newsletter mailing list, and via the home page of BMT InfoNet’s website. Five hundred and eighty persons completed the survey. Respondents identified themselves as: • an HCT survivor – 68%; • the parent of a survivor – 12%; • the spouse/significant other of survivor – 11%; • the child of a survivor – 1%; • the sibling of a survivor – 1%; • other (caregivers or transplant center staff) – 8%. Forty percent had an autologous transplant, 34% had an allogeneic transplant with a related donor, and 26% had an allogeneic transplant with an unrelated donor. Respondents reported their last date of transplant as follows: • 2001–06 – 55%; • 1996–2000 – 25%; • 1990–95 – 17%; • pre-1990 – 3%. When asked which issues, based on their experience, were most important to transplant survivors, respondents checked the following options (more than one answer being allowed): • Emotional/psychological health of transplant survivor – 72%. • Detecting and preventing long-term complications – 71%. • Fatigue – 63%. • Emotional/psychological health of the survivor’s family – 50%.
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• Chronic GVHD – 49%. • Learning and memory problem – 46%. • Health insurance – 44%. • Educating local physicians about transplant survivors’ needs – 43%. • Secondary cancers – 41%. • Sexuality after transplant – 37%. In response to an open-ended question, survivors expressed frustration over the lack of resources to deal with the psychological burden after transplant, including significant depression. They also expressed concern with problems that interfered with their ability to resume work or school and function effectively, such as cognitive deficits or fatigue. Input from this survey was used to develop a 1-day survivorship symposium for HCT survivors in Oakbrook, Illinois, April 2007. Presentations by experts on fatigue, psychosocial problems post transplant, cognitive issues, chronic GVHD, sexuality, infertility, insurance, and employment issues were featured. Three hundred and fifty survivors and caregivers from throughout the United States attended this educational event, which also featured psychosocial support groups where survivors could network with each other. For most, this was the first opportunity post transplant to hear presentations on issues that were significantly impacting their daily life, such as psychosocial difficulties, cognitive issues, and insurance issues, and to discuss them with other survivors. Not only were their experiences validated, but also they learned tools for managing some of the problems. Evaluations were overwhelmingly positive, and more forums of this type were requested. Many who attended the session on infertility remarked that they had been poorly equipped by their transplant program for this side-effect of treatment. Not only had prophylactic measures, such as sperm banking, been ignored in some cases, but few resources were offered them when it was confirmed they were infertile, either because of prior treatment or because of the transplant. One attendee reported that she had not been advised that infertility was a potential complication of transplant. For some patients, the prospect of infertility post transplant is as upsetting as the diagnosis itself. Disappointment over the prospect of infertility is not limited to patients who have never had children but extends to some individuals who already have children and would like to expand their family. Patients’ emotional distress can be lessened if they are advised that it is sometimes possible to conceive a child post transplant. Men may be able to bank a sufficient quantity of viable sperm before treatment to enable attempts at pregnancy post transplant, provided that prior therapy has not led to aspermia. Some women may be able to conceive a child with assisted reproduction techniques and a donor oocyte. While these steps do not guarantee a successful pregnancy, the technology is improving each year, and it comforts many patients to know that there is at least a chance of conceiving or carrying a child to term. Even if a woman does not become infertile immediately after transplantation, the chemotherapy or radiation administered as part of the HCT can reduce the number of viable oocytes and cause her to become prematurely menopausal later on. Thus, women who wish to conceive a child post transplant should be counseled that their window of opportunity may have shortened, and plans for pregnancy should be pursued as early as practical [4]. Many transplant survivors experience significant, long-term changes in sexuality following treatment. Problems including lack of libido, inability to perform, and pain are reported by persons with both normal and abnormal hormone levels post transplant. As one man stated, “It seems like my brain has been cut off from the rest of my body. I can conjure up all sorts of juicy sexual images in my brain, but can’t get the rest of my body to perform.”
Changes in sexuality can seriously stress a relationship, particularly if the survivor’s partner believes that the problem is purely psychological rather than physical. While fatigue and psychological trauma do, indeed, affect sexuality, it is important that patients and their partners know that physical changes occur following transplant that may affect the survivor’s sexuality as well. Patients should be advised that they may need to develop new approaches to intimacy after transplant to enable both partners to have a satisfying sexual relationship. Patients should be encouraged to report sexual difficulties and be provided with referrals to experts who may be able to help resolve some of the problems. A significant issue that is often overlooked in discussions with patients is changes in cognitive abilities they may experience, both short term and long term. Much has been written about learning difficulties observed in children following HCT, but little note has been taken of the cognitive changes experienced by some adult HCT survivors. Problems reported by survivors include poor concentration, memory problems, stuttering, difficulty in spelling, inability to perform jobs that were previously mastered, and difficulty in learning new tasks. Many survivors have been able to overcome the problem by changing the way that they manage information, such as making lists, writing down all appointments, keeping notes, and so forth. For others, the solution is not so simple, and the problem may interfere with their ability to perform a job or learn a new skill. One survivor who was a sculptor, said, “Before my HCT, conceptualizing the design and drawing it was the most difficult part of my work. When it came to the actual sculpting, I could do it without even thinking. However, after my HCT, I could barely concentrate on the sculpting. Conceptualizing and drawing the design became impossible. No one has been able to explain why this happened, or suggest what I can do to relieve the problem.” One woman who underwent high-dose chemotherapy and autologous HCT was delighted that her company held her computer operator job for her while she underwent treatment. However, when she returned to work 4 months later, she found that the nature of the job had changed. She needed extensive training to perform the new tasks. After 2 weeks of training, she feared she would be fired. As she explained, “When I’m at work and in training, all the explanations make perfect sense. But then I come home, fix dinner, and clean up. When I sit down to review the day’s lesson I’m totally confused. By the following morning, I’ve completely forgotten everything I learned the previous day.” Although most patients do not develop cognitive problems as severe as those experienced by the two persons described above, patients should be advised that changes in cognitive abilities sometimes occur. If cognitive problems do develop, survivors will be less frustrated if they know that their problem is not unique. Advance warning about this potential problem can also relieve tension in the home. Tired and distraught family members will understand that the survivor’s new forgetfulness is not simply laziness, but a consequence of the treatment.
Chronic GVHD Survivors with chronic GVHD face a unique set of challenges post transplant that can severely impact their quality of life. Preliminary results of a 2007 survey conducted by BMT InfoNet of more than 300 transplant survivors who have or had chronic GVHD found that chronic GHVD interfered with the daily activities of nearly two-thirds of patients and nearly 40% of other family members. Forty-one percent reported financial difficulties as a result of chronic GVHD, and nearly a third reported marital difficulties. Fifty-seven percent of survivors with chronic GVHD reported sadness or depression, and 41% said the mental health of other family members had been negatively impacted as well.
Hematopoietic Cell Transplantation: The Patient’s Perspective
Survivors with significant chronic GVHD often feel quite isolated. There are few HCT support groups available for patients after transplant, and disease-specific support groups seldom include a significant number of survivors who have been through transplant, let alone those who have experienced chronic GVHD. Improvements in treatment are rare, and opportunities to network and learn from other survivors are quite limited. For those with internet access, a list-serv on ACOR provides a forum for patients with chronic GVHD. List members discuss their symptoms, therapies they have tried to manage them, and clinical trials they have participated in. Often, a newly diagnosed person or a list member who has become overwhelmed with the difficulties presented by chronic GVHD shares his distress with list-mates, and the outpouring of emotional support is quite impressive. When the patients with chronic GVHD surveyed by BMT InfoNet were asked what resources would be helpful, 98% said a central website with chronic GVHD information, 93% said a print newsletter about chronic GVHD, and more than 8% said a DVD with information about chronic GVHD, a local meeting with transplant doctors to discuss chronic GVHD or a local support group would help. BMT InfoNet is exploring the feasibility of hosting a chronic GVHD website as a centralized resource for patients.
Other resources for patients and survivors As described earlier, a number of resources are currently available for families dealing with the prospect of HCT as well as survivors. BMT InfoNet BMT InfoNet provides online resources for patients during all phases of treatment: • Transplant center directory (www.bmtinfonet.org/centers): an online directory of transplant programs in the United States and Canada providing detailed information on each program, including the medical director and key medical personnel; whether or not the center is accredited by the Foundation for Accreditation of Cellular Therapy and/or is an affiliate of the NMDP; the number of transplants performed, by type, in each of the three previous years; patient age and donor match criteria; and contact information. Patients can search the database by name of center, state, diagnosis, and age of patient (pediatric or adult). Data posted are provided directly to BMT InfoNet from each transplant center and are updated annually. • Blood and Marrow Transplant Newsletter (www.bmtinfonet.org/ newsletters/index.html): an archive of back newsletter issues, written in lay language, featuring advances in research, patient vignettes, and resources of interest to HCT patients and survivors. • BMT InfoNet books (www.bmtinfonet.org/books.html): access to patient handbooks about HCT and being a caregiver, and a calendar featuring transplant survivors, including one patient guide about HCT in Spanish. • Survivorship forum DVD: videotaped presentations and slides from BMT InfoNet’s Celebrating a Second Chance at Life Survivorship Conference. • Caring Connections Program (www.bmtinfonet.org/patient.html): a moderated peer support program linking patients with survivors who can provide emotional support. • Resource directory (www.bmtinfonet.org/resource): a list of organizations that provide disease-specific information and financial support to HCT patients. • E-newsletter: an electronic bulletin with information of interest to patients.
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• Help-line (www.bmtinfonet.org/contact.html): a service enabling patients to pose questions to a panel of medical experts. • Survivorship forums: regional conferences to educate patients and survivors. • Attorney referral service (www.bmtinfonet.org/attorney.html): for patients having difficulty securing insurance approval for their transplant or related treatment. • Directory of drugs used during transplant (www.bmtinfonet.org/ drug): describes the purpose of the drug in the transplant setting, and potential side-effects grouped by likelihood of occurrence. In addition, patients can phone 888-5897-7674 for personalized help with any of the above services. National Marrow Donor Program The NMDP provides resources for patients undergoing a HCT with an unrelated donor, some of which are also appropriate for patients transplanted with a related donor or undergoing an autologous transplant: • Information about the donor search and HCT process. • Transplant center directory: this details the cost of HCT with an unrelated donor at transplant programs in the United States as well as medical personnel at each center; the number of transplants performed for specific diseases; raw and risk-adjusted survival rates; and contact information. • DVDs: for special interest groups including young adults, pediatric patients, and older patients. • List of cord blood banks: including contact information and criteria for donating cord blood. • Financial guide: a workbook to help patients organize their finances. • Survivorship newsletter: discussing some of the issues survivors face 3 months to 2 years after transplant. Some of the online information is available in Spanish and other languages. The NMDP’s Office of Patient Advocacy (OPA) provides phone counseling for patients undergoing HCT with an unrelated donor, including help with tracking donor searches and research on disease- and treatment-related topics. • National BMT-Link (www.nbmtlink.org) offers a guide for HCT caregivers, a DVD entitled The New Normal, and a peer support program for HCT patients. Periodic teleconferences and an in-person support group for Michigan residents are also offered. Center for International Blood & Marrow Transplant Research The Center (http://cibmtr.org/PUBLICATIONS/guidelines.html) provides consensus guidelines on long-term follow-up care for patients after HCT. Recommendations are outlined for 6-month, 1-year, and annual check-ups. A separate version is available for patients and physicians. The information can be downloaded, or hard copies can be ordered. The Children’s Oncology Group The Children’s Oncology Group (www.survivorshipguidelines.org) provides long-term follow-up guidelines for pediatric cancer survivors. National Institutes of Health Sponsored Clinical Trials (http://clinicaltrials.gov) lists federally supported clinical trials with separate descriptions available for health-care professionals and the lay public. Included are enrollment criteria, contact information for principal investigators, and a brief description of the trial’s objectives.
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The Clinical Center (http://clinicalcenter.nih.gov) in Bethesda, MD, provides free clinical care to patients who qualify for one of their clinical trials. Protocols for some HCT patients are available. Other resources • The Children’s Hospital Oakland Research Institute (www.chori.org) offers free cord blood banking for families who have a child with a disease that is treatable by HCT. • Private HLA typing (www.bonemarrowtest.com) to determine whether a person is a potential donor for a family member is sometimes requested by a physician without providing guidance to the potential donor on how to be tested. Some DNA labs provide this service only through a contract with a medical facility. Tepnel Diagnostics will arrange the service directly with the potential donor and provides necessary testing supplies and results via mail. • Disease-specific information and fundraising or financial assistance for HCT patients is provided by a number of organizations. A list of such groups is maintained by BMT InfoNet (www.bmtinfonet.org/ resource). • The ACOR (www.acor.org) provides information about various diagnoses and treatment options, as well as list-servs where patients and survivors can connect. The HCT group (BMT-Talk) and GVHD group are both very active and supportive lists. • BMT-Support (www.bmtsupport.org) offers a real-time online chat room each week for HCT patients and caregivers. The chat is moderated by an HCT survivor with a nursing background.
Although HCT creates enormous challenges for patients and their loved ones, many survivors find personal growth in the experience. When asked what he would advise patients about to go through transplant, Steve Dugan, a 4-year survivor of an allogeneic HCT, offered this advice: Expect to be amazed by how much strength you find in yourself. Expect the shock of what’s happening to you to continue for some time. Stolid acceptance of your situation is just not realistic. Expect to feel very alone and, at times, even detached from your surroundings. Expect to be shown, by the person closest to you, what love really means – maybe for the first time in your life. For me, that was, and always will be, my wife and best friend, Joyce. In Greg Allman’s words: “When life’s game gets so hard, I hold the highest card – the Queen of Hearts.” That’s Joyce! Expect there to be days when you say to yourself, “I just can’t do this anymore.” Expect to make outrageous promises to God in exchange for clemency. Expect to find a whole new group of heroes – nurses. Expect for your real friends to show up. Be prepared for surprises – both good and bad. Expect to be truly shaken – and, at times, depressed, by your physical appearance. Just remember: The mirror reflects what you look like – not who you are. Expect to catch yourself sometimes staring at your children, not wanting to miss a single breath they take.
Conclusion Undergoing HCT is a very difficult experience both physically and emotionally. Working with a compassionate medical team that is attuned not only to the patient’s physical needs, but to his or her emotional needs as well, is a blessing that all patients will deeply appreciate. Being fully armed with information about long-term quality of life issues will help make the HCT patient’s transition from being a successful patient to a successful survivor much easier. Transplant physicians need not be the sole source of information for patients and survivors. Directing patients to resources outside the transplant program’s area of expertise such as experts in fertility, sexuality, cognitive retraining, fatigue management, and psychosocial support will expand a patient’s support network and enhance the likelihood that the survivor and his or her family will live well, even in spite of ongoing medical concerns.
But most importantly: Expect and believe that someday soon you will look at this whole episode of your life – in the rearview mirror.
Kathleen Jones, who underwent two HCTs after being diagnosed with chronic myeloid leukemia, summarizes a perspective shared by many survivors: The bone marrow transplants affected my life in every way. I am thankful to wake up every morning. I have learned compassion and empathy for those unable to be normal. I have learned not to spend time worrying about little unimportant things. I have learned to appreciate sunrises and sunsets. I have learned what it means when others are kind. I have learned the value of my family in my life, and just how precious life is, and how much I want to live it.
References 1. Syrjala K. Relieving pain. BMT Newsletter 1993; 16. 2. BMT Newsletter 1996; 33. 3. Bishop M, Hahn E, Brady M et al. The gift of life comes with a price: the impact of hematopoietic cell
transplant on the long-term quality of life of survivors and their spouses. Clinical Abstract, American Society for Blood and Marrow Transplantation/ Center for International Blood & Marrow Transplant Research Tandem Meeting, 2004.
4. Rinehart J. Director, Division of Reproductive Endocrinology and Infertility at Evanston Northwestern Healthcare, Evanston, IL. Presentation at BMT InfoNet Celebrating a Second Chance at Life Survivorship Symposium, April 2007.
Section 4 Sources of Hematopoietic Cells for Hematopoietic Cell Transplantation
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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Phyllis I. Warkentin & Elizabeth J. Shpall
Hematopoietic Cell Procurement, Processing, and Transplantation: Standards, Accreditation, and Regulation
Introduction Hematopoietic cell transplantation (HCT) is now established therapy for many serious congenital and acquired diseases of hematopoietic origin, as well as many high-risk malignancies. Hematopoietic cells (HCs) obtained from bone marrow, peripheral blood cells, and placental and umbilical cord blood cells are common graft sources derived from autologous, related or unrelated donors. HCs are also being used for functions other than as progenitor cells, such as post-transplant donor lymphocyte infusion [1]. Cellular therapies have also expanded beyond hematopoietic and immunologic reconstitution to new therapies using mesenchymal stem cells, dendritic cells, and gene-modified and other cells, and to efforts at regenerative cellular interventions [2]. Rapid evolution of such therapies has heightened concerns about the safety, purity, potency, and efficacy of these cellular products.
Governmental regulation Federal regulation of human cells, tissues, and cellular and tissue-based products (HCT/Ps) is based upon the authority of the Public Health Services (PHS) Act and the Federal Food, Drug, and Cosmetic Act. Under these Acts, the Food and Drug Administration (FDA) is empowered to enact binding legislation in the form of rules, which are intended to implement, interpret or prescribe law or policy. In 1997, the FDA published its approach to the regulation of HCT/Ps [3]. The goal of the regulatory framework is a unified and tiered approach to the regulation of traditional and new products, including HCs, that would provide more uniformity with only the amount of regulation necessary to protect the public health [3]. Products that are expected to be at a higher risk for disease transmission or at risk for contamination during collection or processing, such as allogeneic cells or cells highly manipulated ex vivo, are subject to more regulation than those products with less risk, such as autologous or minimally manipulated products. The FDA regulations are based on five public health and regulatory concerns: (1) prevention of the transmission of communicable disease; (2) assurance that necessary processing controls exist to prevent contamination of cells and tissues and to preserve their integrity and function; (3) assurance of clinical safety and effectiveness; (4) assurance of necessary product labeling, including permissible promotion for proper
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
product use; and (5) establishment of a mechanism for the FDA to communicate with the cell and tissue industry. A Tissue Reference Group, comprised of representatives of the Center for Biologics Evaluation and Research and the Center for Devices and Radiological Health, was also proposed and established to provide a single reference point within the FDA for all regulatory questions about HCT/Ps. The regulatory structure for the wide range of cellular therapies described above has been defined in Part 1271 of Chapter 21 of the Code of Federal Regulations (21 CFR 1271) [4]. This regulation was created to establish a uniform system for registration of manufacturers of HCT/ Ps, to delineate donor eligibility criteria, and to describe current good tissue practices and other procedures to prevent the introduction, transmission, and spread of communicable diseases. This regulation covers the spectrum of hematopoietic cellular products, from those not manipulated ex vivo, to stem cell-based therapies involving highly processed cells, cultured, differentiated or otherwise manipulated ex vivo, that may be used for other than their normal function, may be used for metabolic purposes, and/or may be combined with nontissue components [5,6]. This regulation includes HCT/Ps regulated solely under the PHS Act, Section 361, those which meet the following criteria: 1 The product is minimally manipulated. 2 The product is intended for homologous use only, as reflected by the labeling, advertising or other indications of the manufacturer’s objective intent. 3 The product does not involve combination of the cells or tissue with another article (excepting water, crystalloid, or sterilizing, preserving or storage agent). 4 The product does not have a systemic effect and is not dependent upon the metabolic activity of living cells for its primary function (unless autologous, first- or second-degree relative donor or for reproductive use) [4]. The regulations in 21 CFR 1271 also apply to those products regulated as “biologic products” under the PHS Act Section 351. These are cells or tissues that are highly processed, used for other than their normal function, combined with nontissue components, and/or are used for metabolic purposes [5]. Other regulations also apply to these “biologic products,” including current Good Manufacturing Practices and other portions of Chapter 21. Part 1271 of 21 CFR includes the Registration Final Rule, the Donor Eligibility Final Rule, and the Current Good Tissue Practices (cGTP) Final Rule. Subpart A of 21 CFR 1271 describes the scope of the regulation and its relevant definitions. Subpart B describes the requirements for establishment registration and listing of products. Any establishment that participates in the manufacture of HCT/Ps must register with the
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FDA initially and annually, and maintain an updated list of HCT/Ps at least every 6 months. Subpart C defines the requirements for donor eligibility, including donor screening and testing, which is considered to be part of manufacturing, and therefore subject to regulation. Donor screening is the review of relevant medical records for indications of past or present infection and for risk factors for a relevant communicable disease. Donor testing is performing laboratory tests on a specimen, generally a blood specimen, collected from the donor to determine if she or he has been exposed to or infected with a relevant communicable disease or its agent. Infectious disease screening and testing requirements are based on risk. Screening and testing are not required for autologous donors. For allogeneic donors of HCs (that include viable leukocyte rich products), relevant communicable diseases and agents include at least the following: human immunodeficiency virus type 1, human immunodeficiency virus type 2, human T-lymphotrophic virus type I, human lymphotrophic virus type II, hepatitis B virus, hepatitis C virus, Treponema pallidum, and human transmissible spongiform encephalopathy, including Creutzfeldt–Jakob disease. Subparts D, E, and F of 21 CFR 1271 constitute the cGTP rule, including provisions for inspection and enforcement. The cGTP rule is a set of regulations intended to prevent the introduction, transmission, and spread of communicable disease by helping to ensure that products do not contain relevant communicable disease agents, that products are not contaminated during manufacturing, and that the function and integrity of products are not impaired through improper processing [4]. GTP regulations include requirements for the establishment and maintenance of a comprehensive Quality Management Program; adequate organizational structure; sufficient personnel; adequate facilities; environmental control and monitoring; adequate equipment, supplies, and reagents for the processes carried out in the facility; proper processing, including process change and process validation; proper labeling, claims, and labeling controls; storage, receipt, distribution, and other records; maintenance of complaint files; reporting adverse reactions and product deviations; and procedures for tracking the product from recipient to donor and from donor to recipient or ultimate disposition. In addition to such rules, the FDA issues guidance documents under the provisions of Good Guidance Practices, delineated in the Code of Federal Regulations [7]. Guidance documents describe the Agency’s interpretation of a regulatory issue related to design, production, labeling, promotion, manufacturing, and testing of regulated products; the processing, content, and evaluation or approval of submissions; or inspection and enforcement policies [8]. Guidance documents may be also related to specific issues within a regulation where there may have been confusion among persons or establishments subject to the regulation. Several of these documents are of particular relevance, including the following: Quality Systems Approach to Pharmaceutical CGMP Regulations [9]; Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) [10]; Regulation of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) Small Entity Compliance Guide [11]; and Certain Human Cells, Tissues, and Cellular and Tissue-Based Product (HCT/Ps) Recovered From Donors Who Were Tested For Communicable Diseases Using Pooled Specimens or Diagnostic Tests [12]. Additional guidances are published as Draft Guidance, but still represent the current Agency thinking related to issues in the manufacture of cellular therapy products [13]. Governmental regulation of HC therapy at the state level is fragmented. Many US states have little specific regulation. Some states have adopted mechanisms of qualifying HCT programs and facilities, such as the certificate of need process. Other states have developed a licensure process, often heavily dependent on the standards established by profes-
sional societies. Some states, such as New York, have identified the public health concerns in cellular transplantation, and have adopted direct and specific regulations for processing of and storage facilities for HCT/Ps [14,15]. A number of states have adopted, or are considering, a mechanism of approval for transplantation that requires accreditation by a professional organization. At the present time, both Massachusetts and Maryland require accreditation by the Foundation for the Accreditation of Cellular Therapy (FACT) to perform HCT within those states.
Voluntary professional accreditation Foundation for the Accreditation of Cellular Therapy Historical background FACT was founded in 1995 to promote quality patient care and laboratory practice in HCT through its program of professional standards and voluntary accreditation for the procurement, processing, and transplantation of HC products [16,17]. FACT was initially founded as the Foundation for the Accreditation of Hematopoietic Cell Therapy (FAHCT). The name was changed in December 2001 to encompass, in addition to HC products and therapies, the new and exciting therapies using mesenchymal stem cells, dendritic cells, targeted lymphocytes, genetically modified cells, pancreatic islets, and others. This change followed the lead of the parent organization, the International Society for Hematotherapy and Graft Engineering, which changed its name in 2001 to the International Society for Cellular Therapy (ISCT). FACT is the accreditation arm of two professional societies dedicated to improvement and progress in cellular therapy. The ISCT was formed in 1992 as a professional society of scientists and physicians working in HC manipulation. Its membership includes most of the major HCT programs worldwide. The Regulatory Affairs Committee of ISCT developed the first draft of Standards for Hematopoietic Cell Collection and Processing in 1994. The other parent society of FACT is the American Society of Blood and Marrow Transplantation (ASBMT), formed in 1993 as a professional society of physicians and investigators involved in the clinical conduct of HCT. The ASBMT Clinical Affairs Committee developed the first Clinical Standards for Hematopoietic Cell Transplantation. Believing that quality care can only be achieved if both clinical and laboratory issues are addressed, the International Society for Hematotherapy and Graft Engineering laboratory standards and the ASBMT clinical standards were merged into a single document in December 1994, forming the foundation for the first edition of FAHCT’s Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation, published in 1996 [18]. A companion accreditation manual was published subsequently to provide guidance to applicant facilities and personnel and to FAHCT inspectors [19]. The accreditation manual included each standard, the checklist items related to that standard to be evaluated during the accreditation process, and additional guidance information, including rationale for the standard, explanations, definitions, and examples of alternative methods, approaches, and organizations that would be considered to be in compliance with the standards. The manual also addressed questions and concerns that had been submitted during the period of public comment to the draft first edition of the Standards. The first edition of FAHCT’s Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation was unique in the breadth and depth of activities covered [18]. Standards applied to hematopoietic progenitor cells (HPCs), defined as: self-renewing and/or multipotent stem cells capable of maturation into any of the hematopoietic lineages; lineage-restricted pluripotent progenitor cells; and committed progenitor cells, regardless of tissue source (bone marrow, umbilical cord blood, peripheral blood or other tissue source). These Standards
Hematopoietic Cell Procurement, Processing, and Transplantation: Standards, Accreditation, and Regulation
also included “therapeutic cells,” defined as nucleated cells from any tissue source (marrow, peripheral blood, and umbilical cord blood) collected for therapeutic use other than as HPCs. FAHCT Standards applied to all phases of the collection, processing, storage, and administration of these cells, including various manipulations such as removal or enrichment of various cell populations, expansion of HC populations, and cryopreservation. These Standards do not address the collection, processing or administration of erythrocytes, mature granulocytes, platelets, plasma or plasma-derived components, or products intended for transfusion support. The FACT Standards define an infrastructure required for the safe and efficacious collection, processing, storage, and use of HCs. They define the minimum education and experience necessary for staff participating in these activities, and require an ongoing assessment of activities. There is a minimum requirement that patient outcome be monitored by at least the tracking of neutrophil and platelet engraftment. FACT Standards do not define a required structure or form for the transplant program, nor do they prescribe the clinical use of HCs. In 1997, it became apparent that the field of placental and umbilical cord blood banking was more complex than addressed in the first edition of the FAHCT HPC Standards, and that additional standards were required to address these complexities. Representatives of FAHCT, ISCT, and ASBMT collaborated with members of NetCord, an international organization of independent cord blood banks, to draft additional standards for cord blood banking and to establish a parallel accreditation program. The first edition of NetCord–FACT International Standards for Cord Blood Collection, Processing, Testing, Banking, Selection and Release was developed by consensus of international experts in the field, initially published in June 2000, and revised in 2002 [20]. These NetCord–FAHCT Standards superseded all relevant sections relating to cord blood in the first edition of FAHCT’s Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation, excepting those clinical standards related to the transplantation of cord blood cells. Now in the third edition, these international standards require all cord blood banks to maintain a comprehensive quality management program, to document the training of all collection and processing staff, to utilize validated methods, supplies, reagents, and equipment, to maintain product tracking, and to maintain details of clinical outcome [21]. These Standards form the basis for the voluntary accreditation of cord blood banks worldwide. Fifteen cord blood banks from the United States, Europe, and the United Kingdom have achieved FACT–NetCord accreditation. FACT representatives have also worked with colleagues from the European Group for Blood and Marrow Transplantation (EBMT) and ISCT-Europe, to establish the Joint Accreditation Committee of ISCTEurope and EBMT (JACIE) [22]. The primary aim of JACIE is to improve the quality of HCT in Europe through its accreditation and education programs, and to work toward international harmonization of standards and regulations. JACIE adopted the first edition of the FAHCT Standards in 1999 [23]. The second edition of the Standards was jointly reviewed by FACT and JACIE [24]. Most recently, the third edition of the Standards, published in 2006, was jointly developed and entitled FACT–JACIE International Standards for Cellular Therapy Product Collection, Processing and Administration [25]. FACT and JACIE collaborated in three training workshops in Barcelona, Spain (January 2000, March 2001, and May 2002) to share accreditation tools and experience, and to initiate the European accreditation program. Following a pilot project in Spain between 2000 and 2003, during which FACT inspectors performed the first on-site survey, the JACIE accreditation program was fully implemented in January 2004 with support from the European Union under the Public Health Programme (2003–08). The JACIE accreditation process is similar but not
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identical to the FACT process described below. Since 2004, 41 centers in 13 countries in Europe have been accredited by JACIE. JACIE has developed various documents based on the FACT model, and made use of on-line publications, registration, and inspection and accreditation processes. During this process, JACIE inspectors and staff found that almost all centers were functioning at a high level of excellence, with the majority having only minor deficiencies noted at the on-site inspection. When formally surveyed, these centers reported that implementation of JACIE accreditation required a significant investment of time and resources, but all believed that the result was a demonstrable improvement in the accredited program [26]. Additional information and documents are available on the JACIE website (www.jacie.org). FACT is now an established nonprofit organization with a central office and staff in Omaha, Nebraska. The core of FACT is its active Board of Directors, comprising an equal number of representatives from ISCT and ASBMT, the Presidents-elect of these two parent organizations, the FACT Medical Director, and the Chairperson of the Standards Committee, who represents ASBMT, ISCT or both. Standing Committees of the Board oversee the activities of the Foundation. The FACT Board of Directors approves all publications and sets the agenda for the Foundation. The infrastructure of the organization includes Chief Executive and Operations Officers, Medical Director, Director of Standards and Training, Quality Assurance Director, Information Technology Director, and Accreditation Coordinators. Standards All FACT Standards are developed by consensus of experts active in the field. Wherever possible, standards are based on established evidence from the literature. Standards are also reviewed by legal counsel and internally for technical accuracy, consistency, and regulatory compliance. Every effort is made to incorporate sound recommendations that foster quality medical and laboratory practice; however, no Standards can guarantee the successful outcome of cellular therapies. Draft standards are published for comment by members of ASBMT, EBMT, ISCT, NetCord, other practitioners in cellular therapy, and the general public. Each comment is discussed and carefully considered by the Standards Committee, and incorporated as appropriate. All Standards require compliance with applicable law, but, as appropriate, requirements of the Standards may exceed the minimum regulatory requirements. In addition, these Standards are not intended to be the only means of complying with the standards of care in a community or industry. Standards are developed by the Standards Subcommittees and Oversight Committees. A Standards Committee Chairperson is appointed by the FACT Board of Directors for a term of 3 years to encompass the development and publication of one edition of each set of standards, cellular therapy and cord blood banking. FACT–JACIE Standards are developed by three subcommittees: Clinical, Collection, and Laboratory Processing. Each subcommittee has a FACT representative and a JACIE representative as a co-chair, as well as additional members representing both organizations. An Oversight Committee, including all co-chairpersons, ensures consistency among the sections of the edition. NetCord– FACT Standards for Cord Blood Banking are developed by separate subcommittees for Collection, Laboratory, and Quality Management and Banking. NetCord and FACT representatives co-chair the subcommittees, which also include additional experts in cord blood banking. FACT–JACIE International Standards for Cellular Therapy Product Collection, Processing and Administration These Cellular Therapy Standards are designed to provide minimum guidelines for facilities and individuals performing HCT and related cellular therapies. FACT Standards require that all clinical, collection,
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and processing facilities develop and maintain a comprehensive Quality Management Plan that includes at least the following components: defined organizational structure; personnel requirements; process development; agreements; outcome analysis; audits; management of errors, accidents, and adverse events; document control; product tracking; and, where appropriate, validation and qualification [27]. The current edition also includes many of the regulatory requirements from the United States FDA and the Directives of the European Union, including donor eligibility and product labeling [4,28–30]. The cellular therapy product proper names as defined in Standards are consistent with the names and definitions proposed for inclusion in the official terminology of ISBT 128 [31,32]. Standards for each of the services or facilities participating in the cellular therapy program describe facility requirements, standard operating procedures, and personnel requirements for the area, including minimum education, training, experience, and competencies for each position. In addition, all services participating in a cellular therapy program are expected to maintain active and clear communications with each other. Histocompatibility testing (human leukocyte antigen-A, B, and DR) must be performed by a laboratory accredited by the American Society for Histocompatibility and Immunogenetics or the European Federation for Immunogenetics. All other laboratory testing must be performed by laboratories appropriately licensed and/or accredited for the specific assays. Clinical standards define a blood and marrow transplant program as an integrated medical team housed in geographically contiguous or proximate space with a single Clinical Program Director and common staff training programs, protocols, and quality management systems. The Clinical Program must use HC collection and processing facilities that meet FACT–JACIE Standards with respect to their interactions with that clinical program. Clinical standards also enumerate support staff; cover donor evaluation, selection, eligibility, and consents; provide minimal guidelines for administration of cellular product therapy, including the preparative regimen of high-dose therapy; describe the appropriate management of clinical research and Institutional Review Board-approved protocols; and require the maintenance of complete and accurate records. Standards for cell collection define elements common to both bone marrow- and apheresis-derived peripheral blood HCs, as well as detail those requirements unique to each cell source, such as administration of mobilizing growth factor, potential need for a central venous catheter, and general anesthesia for marrow harvest. The intent is to provide the framework for donor evaluation and cell collection that will foster safe care for both patient and donor. Comprehensive laboratory standards detail requirements for personnel; process controls; inventory management; validation and qualification of facilities, supplies, reagents and equipment; labels and labeling; storage; transport; and records. The intent is to establish that the laboratory is operated in a responsible and responsive manner, that deviations in processes or products are noted, and that the recipient’s physician is aware of any adverse event that could compromise the cellular therapy product. Laboratory and collection personnel are expected to follow clinical outcome as one measure of product safety and efficacy. There are several notable changes in the third edition of the HPC Standards. The document has been restructured so that the three sections are aligned and consistent throughout. International language, understood uniformly, is utilized whenever possible, and the regulatory requirements of the European Directive have been included with those of the FDA where applicable. Specifically, many of the requirements detailed in the GTP regulations have been incorporated. The Quality Management requirements have been expanded to include specifically detailed elements. Pediatric competencies have been added for the Collection Services as well as the Clinical Program. The minimum number
requirements have been changed to allow smaller programs performing autologous transplant only to be eligible for accreditation; however, programs with more than one clinical site must still treat at least five new patients per year in each site. There are now minimum number or duration requirements for both the collection service and processing laboratory. FACT–JACIE Standards and the accompanying accreditation guidance manual are available in print and online at the FACT website (www.factwebsite.org). NetCord–FACT International Standards for Cord Blood Collection, Processing, Testing, Banking, Selection and Release These Standards are intended for the field of cord blood banking, in which a cord blood bank is defined as an integrated team responsible for the collection, processing, testing, banking, selection, and release of cord blood units [21]. It is important to note that the Standards begin with the processes of maternal donor recruitment, consent, and screening, and the process of collection, rather than only covering those processes occurring in the laboratory. The Standards apply to both the banks responsible for cord blood units collected, stored, and reserved for use by a designated individual or family (“private” banking), as well as those banks responsible for units collected, stored, and donated for use by unrelated recipients. There are some differences between Standards for family units and those for unrelated donor units. Most Standards are similar; however, the methodologies employed to meet these Standards may be somewhat different in the two situations. The nature of the collection sites and the relationships among the bank, the cord blood unit collector, the donor, and the collection facility are among the prominent differences in the Standards for related and unrelated units. Similar to the Cellular Therapy Product Standards, these standards require that each cord blood bank establish and maintain a comprehensive quality management program that covers all aspects of the operation and includes at least the following: organizational structure; personnel requirements, qualifications, training, and competency; systems for document creation, review, control, and maintenance; quality assessments and audits; detection, investigation, reporting, corrective action, and follow-up of errors, accidents, biological product deviations, adverse events, and complaints; validation, qualification, calibration, and maintenance of equipment, supplies, reagents, and materials; inventory control for reagents and products; process controls; systems for product identification, labeling, and tracking; outcome analysis; facilities and safety management; donor suitability determination; vendor qualification; and agreements with third parties. The bank staff is required to follow clinical outcomes from each unit released for transplant in sufficient detail to ensure that the procedures in use continuously provide a safe and effective product. There are standards for unique issues that may face a bank, such as inventory transfer or interruption of operations at established collection or laboratory sites. Comprehensive processing, storage, and labeling standards are consistent with ISBT 128 terminology and labeling requirements [31,32]. To accompany the third edition of the Cord Blood Banking Standards, there is, for the first time, a guidance manual that provides explanations, examples, and clarifications, similar to that accompanying the HPC Standards [33]. Both the third edition Standards and guidance manual are available in print from the FACT office and online at the FACT website (www.factwebsite.org). Accreditation process FACT accreditation is a voluntary process based on documented compliance with the published standards as assessed by an evaluation of submitted written information and an on-site inspection of the applicant program or facility. The evaluation of submitted materials is completed
Hematopoietic Cell Procurement, Processing, and Transplantation: Standards, Accreditation, and Regulation Table 37.1 Foundation for the Accreditation of Cellular Therapy (FACT) inspector qualifications FACT hematopoietic progenitor cell inspector • Meet all educational and experience requirements for the position • Individual member of ISCT, ASBMT, American Society for Apheresis, or NetCord • Affiliated with FACT-accredited or applicant facility or cord blood bank • Has attended a FACT or FACT–NetCord training course, passed a written examination, and completed successfully a relevant inspection as a trainee • Has submitted formal application, confidentiality and other required agreements Clinical program inspector • Is a licensed physician • Has a minimum of 2 years’ experience in hematopoietic progenitor cell transplantation Apheresis inspector • Has a relevant doctoral, nursing or biological science degree • Has completed formal training in apheresis or has at least 1 year’s experience in peripheral blood progenitor cell collection by apheresis as a director, physician, or supervisor or associate supervisor Cell processing facility inspector • Has a relevant doctoral or biological science degree • Has at least 2 years’ experience as director, medical director or supervisor of a cellular therapy processing facility Cord blood bank inspector • Individual member of organizations above, plus ISCT-Europe, EBMT or JACIE Cord blood bank collection inspector • Has a relevant doctoral, nursing or biological science degree • Has at least 1 year’s experience as a collection supervisor in a cord blood bank, or is an active FACT or JACIE clinical or collection inspector Cord blood bank laboratory inspector • Has a relevant doctoral or biological science degree • Has at least 1 year’s experience as director, medical director or supervisor of a cord blood bank laboratory or hematopoietic progenitor cell processing laboratory See text for abbreviations.
by the Accreditation Program Chairman, designee, or appropriate member of the inspection team. One of the strengths of the program is that all inspectors are active in the field of HC therapy, and meet the minimum qualifications for all inspectors for the FACT Accreditation Program. All inspectors are unpaid volunteers who meet the minimum FACT inspector qualifications as listed in Table 37.1. To promote uniformity and consistency in the process and fairness to applicant facilities, it is important that inspectors have a common, up-to-date understanding of the principles of the FACT Standards, and of the approach that the organization takes to the inspection and accreditation. In addition, all inspectors are annually required to submit documentation of potential conflicts of interest, and to verify agreement with the confidentiality and other FACT policies. Although the FACT–JACIE HPC Standards are common, the accreditation processes for FACT and JACIE are separate. Currently, FACT accreditation is open to programs in North America, Australia, and China. JACIE accreditation is applicable in Europe. In addition, the international NetCord–FACT process for cord
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blood bank accreditation, managed by the FACT staff, is a parallel but separate process. The goal of the accreditation process is to raise the bar of performance for all HCT programs and the support services that contribute to their activities, in the expectation that these improvements will lead to better patient outcomes. It is not the goal to be punitive, but rather to be educational and helpful to enable capable and committed personnel to achieve accreditation. Assessments by peers and experts in the field contribute to the ability of FACT to accomplish this goal. The process for initial FACT accreditation of HCT, illustrated in Fig. 37.1, is as follows: 1 Registration. A Program or Facility Director will register interest in accreditation for one or more of the transplant-related services using the demographic registration form available from the FACT office or website. The FACT Accreditation Office staff will review the registration information and determine if the program or facility is eligible for accreditation. 2 Application. An inspection packet is sent to eligible applicants, detailing accreditation requirements and process, including: (a) instructions for completion of the formal application process; (b) a list of documents that must accompany the formal application (licenses, evidence of Board certification, Quality Management documents, etc.); and (c) the Inspection Checklist that lists each of the items to be inspected during the on-site inspection. The applicant personnel will answer each item on the checklist as it pertains to their program, and submit the completed checklist to the FACT office. The inspector will verify each of these items at the on-site inspection, using the checklist as part of the formal inspection report. Use of this checklist ensures that the program is measured against the Standards, and that the inspection will be complete. Completion and submission of the checklist by the applicant is expected within 12 months of its receipt. 3 On-site inspection. The submitted materials are reviewed by a designated FACT Accreditation Coordinator. The results of this review are shared with the applicant, giving the applicant the opportunity to complete missing items or make any corrections to potential problems detected by the Coordinator. The applicant team submits potential dates for the on-site inspection when all key personnel will be available to participate in the process. The inspection team is selected from the available inspectorate based upon the size and complexity of the applicant program, assuring that members of the team have the training and experience necessary to assess all HCT activities. Potential inspectors are asked to recuse themselves if they perceive a potential duplicity of interest that could interfere with the objectivity of the inspection. The proposed inspection team is identified to the applicant facility for approval before the assignment is complete. Inspectors may also be replaced if the applicant perceives a potential conflict of interest. Approximately 3–4 weeks prior to the scheduled on-site inspection, a copy of the application materials is sent to each inspector for review prior to departure for the program. Any missing documents should be requested during this time, and questions clarified. The on-site inspection should be completed in 1 day, starting with introductions and ending with a summation of major observations, but not a final determination of accreditation status. A written report of the observations by the inspection team is submitted to the FACT Accreditation Coordinator as soon as possible after the on-site inspection, including any documents collected on-site, the completed checklist, and a report of all citations observed. 4 Accreditation Committee Review. The FACT Accreditation Coordinator will review the inspectors’ report and all submitted documents, prepare a summary report, and present this report to the Accreditation Committee. The Accreditation Committee is chaired by the FACT
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Application and fees submitted to FACT office
Program or facility eligible?
Inspection scheduled, team assigned
Reapply
No
On-site inspection conducted
Applicant fees returned
Inspection reports and documents returned to FACT office
Yes Inspection instructions and checklist to applicant
FACT staff document review and summary
Inspection materials returned, reviewed by FACT staff
Applicant materials complete?
Inspection results presented to FACT Accreditation Committee No
Applicant contacted for omissions Outcome and next steps determined
Yes
Applicant notified
Deficiencies corrected
Yes
Reinspection required? No Deficiencies corrected
Accreditation awarded
Medical Director, and has a membership of active inspectors representing all three of the major areas (clinical, collection, and laboratory), the chair of the Standards Committee, at least some persons with several years’ experience on the committee, and at least some members of the Board of Directors. Individuals serve 2-year terms on this committee, and are eligible for election to repeat terms. The Accreditation Committee meets at least monthly by conference call, and reviews in detail every inspection report. The possible outcomes are listed in Table 37.2. The Board of Directors retains ultimate responsibility for these outcomes, handles any complaints or appeals, hears and decides contentious issues, and makes any precedent-setting decisions. The Program Director is notified of the outcome, with any instructions necessary to complete the process and achieve accreditation. 5 Accreditation. Accreditation is awarded based on documented compliance with Standards. Any deficiencies identified must be corrected and these corrections documented. The Accreditation Committee may
Fig. 37.1 Foundation for the Accreditation of Cellular Therapy (FACT) Accreditation Program: accreditation process.
review this documentation if needed. Accreditation is valid for 3 years. Accredited Programs are announced in the newsletters of the ISCT and ASBMT, and on the FACT website. 6 Annual report. All accredited programs and facilities report annually on the number of autologous and allogeneic HCT patients treated in the previous 12 months, and on any significant changes in personnel, location or complexity of service. In addition, programs may be asked to document continued compliance with Standards by supplying evidence of implementation of corrections required to achieve accreditation. 7 Accreditation renewal. Programs are expected to have completed the renewal process prior to the expiration of the prior accreditation. At least 9 months prior to expiration of accreditation, each accredited program will receive information and documents from the FACT Accreditation Office to begin the application for renewal. The renewal process is essentially identical to initial accreditation. The applicable standards will be the edition that is current on the day of the on-site inspection.
Hematopoietic Cell Procurement, Processing, and Transplantation: Standards, Accreditation, and Regulation
The first FACT on-site inspection occurred in September 1998; and the first accreditation was awarded in February 1999. Since that time, over 200 programs have submitted an initial application for accreditation. Currently, there are 155 accredited programs in North America, representing an estimated 90% of eligible programs. There are over 160 active inspectors, trained at one or more of 22 North American Training Workshops. Additionally, cord blood bank inspectors have been trained at six International Training Workshops, and 15 cord blood banks have been accredited. In the FACT accreditation process, observations made at the on-site inspection are recorded on the checklist and are determined to be in compliance with the Standards or not in compliance. A deficiency is the failure to comply with a mandatory requirement, stated in the Standards as “shall.” A variance from recommendation is the failure to follow a recommended practice, stated in the Standards as “should.” The inspection summaries of the 145 programs or facilities inspected under the first edition of the FACT Standards (1996) were reviewed by the FACT Accreditation Office to determine the most frequently cited deficiencies and variances from recommendation. This review included only the initial inspection results for these programs, although several Table 37.2 Foundation for the Accreditation of Cellular Therapy Accreditation Program: potential inspection outcomes Accreditation No deficiencies or variances from recommendation observed at the on-site inspection or documented on submitted materials. Full accreditation for 3 years awarded, effective on the date of the Accreditation Committee review Minor deficiencies Minor deficiencies noted at the on-site inspection and/or documented on the submitted materials. Full accreditation can be awarded upon written documentation of satisfactory correction of all deficiencies. The medical director or designee may determine the adequacy of the facility’s response, or the Accreditation Committee may decide to review the responses. Incomplete or unsatisfactory responses may result in a required repeat of the on-site inspection for all or part of the program Significant deficiencies Significant deficiencies, either serious in nature and/or numerous. Full accreditation requires correction of all deficiencies, written documentation of these corrections, and satisfactory completion of a focused reinspection of the areas (clinical, collection, and/or laboratory) where excessive deficiencies have been noted. The results of the focused reinspection will be reviewed by the Accreditation Committee, who will determine the accreditation status of the program Non-accreditation Failure to meet eligibility criteria or to respond to requirement to document corrected deficiencies
541
accreditation renewal inspections were also performed under this first edition of Standards. The results from the first 76 programs have been published [34]. Similar to the results observed by JACIE in its accreditation process, the results of recent on-site FACT inspections demonstrate that most programs are functioning at a high level of quality and have addressed at least most of the Standards. Deficiencies observed generally represent failure to completely address a Standard. The most common deficiencies cited across all areas of the HCT programs were deficiencies related to standard operating procedures, both in format and in content. In many cases, the standard operating procedures for entire processes were absent. In addition, clinical programs frequently had deficiencies in data management, in defining and performing quality management activities, and in documentation of personnel qualifications. Cell collection facilities often had not validated processes in use, and often had elements missing in both the informed consent process and on the product collection label. Cell processing laboratories also frequently omitted required label information. Standard operating procedures most often missing or incomplete were those for assessment of staff training and competency, microbial monitoring of products, resolution of ABO/Rh discrepancies, and processes for the return and reissue of previously released HC products. The most commonly observed deficiencies based on inspections related to the third edition of FACT Standards are listed in Table 37.3. Quality Management and Policy and Procedure deficiencies were the most commonly cited in all three areas: Clinical Program, Collection Facility, and Processing Facility. Specifically, the deficiencies that were observed in all areas included missing policies and procedures for customer-reported product failures, concerns or complaints, lack of procedures and/or approval for planned or unplanned deviations, and absence of documentation of corrective action and/or evaluation of its effectiveness. Management of products with positive microbial cultures was frequently cited; however, most programs addressed some but not all of the specific points required by the Standards. Audits were also commonly cited in Collection and Processing Facilities. Frequently, audits were not used effectively as a means for identifying problems and improving operations, were not reviewed to identify trends or opportunities for improvement, or were conducted by either unqualified personnel or personnel directly responsible for the work being audited. Also across all sections, policies and procedures were commonly cited for lack of specific documents or specific processes, such as annual review or documentation of training prior to implementation. Donor consents were commonly cited for failure to document each of the required elements. Significance of FACT accreditation FACT accreditation helps a cancer program attain its ranking among America’s Best Hospitals, published by US News & World Report [35]. As of April 2007, FACT accreditation for allogeneic HCT was awarded one point toward best hospital status. FACT accreditation for autologous HCT only was awarded one-half point. In addition, US News & World Report improved its usefulness and relevance of ranking of pediatric
Table 37.3 Most commonly cited deficiencies related to the third edition of the Foundation for the Accreditation of Cellular Therapy Standards Clinical program
Collection facility
Processing facility
Quality Management (B4) Policies and Procedures (B5) Donor Selection, Evaluation, and Management (B6) Therapy Administration (B7) Personnel (B3)/Data Management (B8)
Quality Management (C4) Policies and Procedures (C5) Labels (C7) Donor Selection, Evaluation, and Management (C6) Cellular Therapy Product Collection Procedure (C8)
Quality Management (D4) Policies and Procedures (D5) Labels (D7) Storage (D9) Process Controls (D6)
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hospitals by including FACT accreditation as a factor in the selection of the Top Ten America’s Best Children’s Hospitals [35]. FACT–JACIE Standards have achieved international acceptance, as best demonstrated by the joint authorship and committee membership in Standards development. In Australia, the Therapeutics Good Administration has accepted the collection and laboratory standards as the regulation for the field. In the United States, cooperative groups require institutions entering patients on HCT trials to have FACT accreditation. Some states, including Massachusetts, Minnesota, and others require or recommend FACT accreditation to operate an HCT program in the state. Most insurance companies require HCT programs to disclose accreditation status as part of the application for Center of Excellence designation. AABB The AABB (formerly the American Association of Blood Banks) is the professional society of 8500 individuals involved in blood banking and transfusion medicine [36]. It also represents over 2200 institutional members, including community and Red Cross blood collection centers, hospital-based blood banks, transfusion services, and cell processing laboratories that collect, process, distribute, and transfuse blood components and HCs. The AABB has a long history of standard-setting activity, having published its first edition of Standards for a Transfusion Service in 1958, the same year it began its program of on-site inspections and accreditation. AABB also publishes Standards for Immunohematology Reference Laboratories, Perioperative Autologous Blood Collection and Administration, and Relationship Testing Laboratories. Approximately 160 programs involved in HC collection or processing worldwide have been accredited by AABB. Originally published as separate volumes, AABB now has current standards for cellular therapy products and services that encompass HC and cord blood services [37–39]. Standards documents of the AABB are based upon a quality management framework. All standards within the documents are of equal importance. Each standard is stated once; then it applies throughout. The quality management standards, combined with the technical requirements, form the total requirements of AABB. Although very different in structure, the content of the AABB Standards for Hematopoietic Cell Services parallels the FACT Standards as published in the first edition. Accreditation by AABB is based upon submission of demographic information and an on-site inspection, coordinated by a paid, full-time quality assessor. Accreditation is valid for 2 years. National Marrow Donor Program The National Marrow Donor Program (NMDP) was founded in 1987 as a cooperative effort of the AABB, the American Red Cross, the Council of Community Blood Centers, and the United States Navy to facilitate volunteer unrelated donor marrow transplantation. It comprises a network of cooperating facilities, including transplant centers, donor centers, marrow collection centers, apheresis centers, cord blood banks, recruitment groups, cooperative donor registries, contract laboratories, and cell and serum repositories. The Health Resources and Services Administration has regulatory authority over NMDP. Since its inception, NMDP has established standards for membership participation. A Standards Committee composed of experts in various aspects of HCT is in place to provide continuous review and revision of these standards. NMDP Standards cover cells obtained from marrow, peripheral blood, and umbilical cord blood. The Standards include standards for participating centers and groups, personnel qualifications, required support services, policy and procedure requirements, confidentiality, recruitment of the unrelated donor, donor medical and laboratory screening and testing, informed consent, donation and transplant process, a few collection, packaging, labeling, and processing standards, and
records requirements; the informed consent process; progenitor cell packaging, labeling, and transportation; and quality control, patient rights, and records. Standards for laboratory processing are less detailed than those of FACT–JACIE, NetCord–FACT or AABB, since most of the processing laboratories and the procedures performed there are an integral part of the recipient’s transplant program, and participating cord blood banks are required to be otherwise accredited. Oversight of investigational procedures is from the Institutional Review Board, the FDA if applicable, and FACT or AABB if the program participates. NMDP Standards are intended to serve the discrete function of qualifying groups, centers, and banks for participation in the registry. NMDP Standards include standards for quality assessment and improvement, and are generously supplemented by operational policies and procedures of the registry. NMDP regularly audits the data submitted by participating programs, collects annual reports, and may perform on-site inspections as deemed necessary.
Future directions In an era when regulation and voluntary professional standards coexist, it is important that the professional organizations continue to lead the field by setting those quality standards demonstrated to be important in patient outcome, to provide the educational materials necessary to programs to meet and exceed regulations, and to continue communication with regulatory bodies to ensure clear understandings and enable appropriate regulation. Recently, several initiatives have been jointly sponsored by professional organizations to serve the needs of the HCT centers and patients. An interorganizational task force developed a Circular of Information for Cellular Therapy Products that can be used in association with cellular therapy products in any center. Another group developed a donor history questionnaire for allogeneic cellular therapy donors to assist staff in meeting requirements of donor eligibility determination. This questionnaire is periodically updated, and is available on the FACT website. In addition, FDA and professional organizations have several mechanisms to communicate concerns as regulations are promulgated and Standards revised. Some of these mechanisms include the presence of an FDA liaison on various Standards and Advisory Committees, a Uniform Donor History Questionnaire Task Force, and participation in education programs, public workshops, and public advisory meetings. In addition, biannual liaison meetings between representatives of the Center for Biologics Evaluation and Research, the Office of Cellular, Tissue and Gene Therapies, and the professional societies representing active professionals in the cellular therapy community continue as a venue for the communication by industry of issues of concern. In December 2005, the C.W. Bill Young Cell Transplantation Program was enacted as a new structure to support unrelated donor HCT in the United States. This legislation includes provisions to increase the potential use of cord blood units in HCT by increasing the national inventory of such units and managing the distribution of cord blood units. In addition, it creates the Stem Cell Therapeutic Outcomes Database, under which outcomes data must be collected on all persons who are allogeneic donors or recipients of HCT, regardless of the tissue source of the product. This will permit the publication of center-specific survival rates for all allogeneic transplants, and the collection of data on uses of HCs for new therapeutic applications such as regenerative medicine. The impact of this legislation remains to be seen. In concept, the development of meaningful and appropriate outcomes measures is supported by experts in HCT, who believe that scientifically based outcomes reporting will lead to improved center quality and best serve both patients and centers [40]. Participation of members of the HCT community will be essential to optimize the data collection and analysis process.
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References 1. Appelbaum FR. Hematopoietic-cell transplantation at 50. N Engl J Med 2007; 357: 1472–5. 2. Rosenzweig A. Cardiac cell therapy – mixed results from mixed cells. N Engl J Med 2006; 355: 1274– 7. 3. Food and Drug Administration, Department of Health and Human Services. A proposed approach to the regulation of cellular and tissue-based products. Fed Regist 1997; 62: 9721–2. 4. Code of Federal Regulations. Washington DC: Office of the Federal Register National Archives and Records Administration, US Government Printing Office; 2006. Chapter 21, Part 1271. 5. Kessler DA, Siegel JP, Noguchi PD et al. Regulation of somatic-cell therapy and gene therapy by the Food and Drug Administration. N Engl J Med 1993; 329: 1169–73. 6. Halme DG, Kessler DA. FDA Regulation of stem cell-based therapies. N Engl J Med 2006; 355: 1730–5. 7. Code of Federal Regulations. Washington DC: Office of the Federal Register National Archives and Records Administration, US Government Printing Office; 2006. 10.115(a). 8. Witten C. FDA and Tissue Regulation. Presented to the World Health Organization, Geneva, Switzerland, June 7–9, 2006. Available at http://www. fda.gov. 9. Guidance for Industry: Quality Systems Approach to Pharmaceutical CGMP Regulations (2006). Available at http://www.fda.gov/cber/gdlns/qualsystem.htm. 10. Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) (2007). Available at http://www.fda.gov/cber/gdlns/ tissdonor.htm. 11. Guidance for Industry: Regulation of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) Small Entity Compliance Guide. Available at http://www.fda.gov/cber/gdlns/hctpcompl. htm. 12. Guidance for Industry: Certain Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) Recovered From Donors Who Were Tested For Communicable Diseases Using Pooled Specimens or Diagnostic Tests. Available at http:// www.fda.gov/cber/gdlns/hctppool.htm. 13. CBER Guidances/Guidelines/Points to consider. Available at http://www.fda.gov/cber/guidelines. htm.
14. Ciavarella D, Linden JV. The regulation of hematopoietic stem cell collection and storage: the New York State approach. J Hematother 1992; 1: 201–14. 15. Linden JV, Preti RA, Dracker R. New York State Guidelines for cord blood banking. J Hematother 1997; 6: 535–41. 16. Warkentin PI. Voluntary accreditation of cellular therapies: Foundation for the Accreditation of Cellular Therapy. Cytotherapy 2003; 5: 299–305. 17. Warkentin PI. Professional standards for cellular therapies: Foundation for the Accreditation of Cellular Therapy (FACT). In: Gee AP, editor. The Cellular Therapy Book of the Production Assistance in Cellular Therapy Group. New York: Springer (in press). 18. Foundation for the Accreditation of Hematopoietic Cell Therapy. Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation. Omaha, NE: FACT; 1996. 19. Foundation for the Accreditation of Hematopoietic Cell Therapy. Accreditation Manual. Omaha, NE: FACT; 1996. 20. NetCord/Foundation for the Accreditation of Cellular Therapy. NETCORD-FACT International Standards for Cord Blood Collection, Processing, Testing, Banking, Selection and Release, 2nd edn. Omaha, NE: NetCord/FACT; 2002. 21. NetCord/Foundation for the Accreditation of Cellular Therapy. International Standards for Cord Blood Collection, Processing, Testing, Banking, Selection and Release, 3rd edn. Omaha, NE: NetCord/FACT; 2007. 22. Kvalheim G, Urbano-Ispizua A, Gratwohl A. FAHCT–JACIE Workshop on Accreditation for blood and marrow progenitor cell processing, collection and transplantation. Barcelona, Spain. Cytotherapy 2000; 2: 223–4. 23. Joint Accreditation Committee of ISHAGE-Europe and EBMT. Standards for blood and marrow progenitor cell processing, collection and transplantation. Cytotherapy 2000; 2: 225–46. 24. Foundation for the Accreditation of Cellular Therapy. Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation, 2nd edn. Omaha, NE: FACT; 2002. 25. Foundation for the Accreditation of Cellular Therapy and Joint Accreditation Committee of EBMT and Euro-ISCT. FACT-JACIE International Standards for Cellular Therapy Product Collection, Processing and Administration, 3rd edn. Omaha, NE: FACT-JACIE; 2006.
26. Samson D, Slaper-Cortenbach I, Pamphilon D et al. Current status of JACIE accreditation in Europe: a special report from the Joint Accreditation Committee of the ISCT and the EBMT (JACIE). Bone Marrow Transplant 2007; 39: 133–41. 27. LeMaistre CF, Loberiza FR Jr. What is quality in a transplant program? Biol Blood Marrow Transplant 2005; 11: 241–6. 28. Directive 2004/23/EC of the European Parliament and of the Council (7 April 2004). Available at http://eur-lex.europa.eu/en/index.htm. 29. Commission Directive 2006/17/EC (8 February 2006). Available at http://eur-lex.europa.eu/en/ index.htm. 30. Commission Directive 2006/86/EC (24 October 2006). Available at http://eur-lex.europa.eu/en/ index.htm. 31. Ashford P, Distler P, Gee A et al. Standards for the terminology and labeling of cellular therapy products. Transfusion 2007; 47: 1319–27. 32. Ashford P, Distler P, Gee A et al. ISBT 128 implementation plan for cellular therapy products. Transfusion 2007; 47: 1312–18. 33. NetCord/Foundation for the Accreditation of Hematopoietic Cell Therapy. Accreditation Guidance Manual. Omaha, NE: FACT; 2008. 34. Warkentin PI, Nick L, Shpall EJ. FAHCT accreditation: common deficiencies during on-site inspections. Cytotherapy 2000; 2: 213–20. 35. Comarow A. What it takes to be the best. US News & World Report, July 23–30, 2007, pp. 90–4. 36. Warkentin PI. Regulations and standards for hematopoietic progenitor cell facilities. In: Snyder EL, Haley NR, editors. Hematopoietic Progenitor Cells: A Primer for Medical Professionals. Bethesda, MD: AABB Press; 2000. pp. 201– 19. 37. American Association of Blood Banks. Standards for Hematopoietic Progenitor Cell and Cellular Product Services, 3rd edn. Bethesda, MD: AABB; 2002. 38. American Association of Blood Banks. Standards for Cord Blood Services. Bethesda, MD: AABB; 2001. 39. AABB. Standards for Cellular Therapy Products and Services, 2nd edn. Bethesda, MD: AABB, 2007. 40. Committee on Hematopoietic Cell Therapy Quality Outcomes. ASBMT Committee Report White Paper on Measurement of Quality Outcomes. Biol Blood Marrow Transplant 2006; 12: 594–7.
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Dennis L. Confer, John P. Miller & Jeffrey W. Chell
Bone Marrow and Peripheral Blood Cell Donors and Donor Registries
Introduction Hematopoietic cell (HC) donors provide bone marrow or peripheral blood progenitor (hematopoietic) cells (PBPCs) for more than 20,000 allogeneic HC transplant (HCT) recipients annually. Donations from related allogeneic HC donors continue to outnumber those from unrelated donors, even though at least 70% of HCT candidates lack human leukocyte antigen (HLA)-matched sibling donors. This suggests that only a fraction of the potential patient population is currently being served. As unrelated donor registries grow and international collaboration increases, more patients will have the opportunity for transplantation. Even under the most optimistic scenarios, however, alternative approaches will also be necessary. Umbilical cord blood transplantation (see Chapter 39) and haploidentical transplantation (see Chapter 46) are strategies that will play increasingly important roles in meeting the needs of patients. This chapter discusses the evaluation of HC donors, the risks and side effects of donation, and the logistics of HC collections.
Products from HC donors Bone marrow was until recently the most common graft source for allogeneic HCT [1–4], but since 1995, the use of PBPCs has increased dramatically. Data from the Center for International Blood and Marrow Transplant Research (CIBMTR) show that, in the period 2001–04, about 70% of adult allogeneic transplant recipients received PBPC grafts. National Marrow Donor Program (NMDP) statistics confirm the dramatic growth in the use of PBPCs for unrelated donor transplantation (Fig. 38.1). PBPCs are now the most common choice for both autologous and allogeneic transplantation worldwide. Under normal physiologic conditions, a small number of CD34+ HCs circulate in the peripheral blood, but their concentration is too low to enable efficient collection of an adequate HC dose for transplantation. However, in the mid-1980s, it was discovered that HCs could be mobilized from the bone marrow into the blood following chemotherapy and/or the administration of hematopoietic growth factors such as human recombinant granulocyte colony-stimulating factor (rhG-CSF; filgrastim, Neupogen) and human recombinant granulocyte–macrophage colony-stimulating factor (rhGM-
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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CSF; sargramostim, Leukine). Around the same time, advances in apheresis instrumentation and collection techniques occurred such that an adequate number of mobilized HCs (1.0–5.0 × 106 CD34+ cells/kg of patient weight) could be collected from the patient (in the case of autologous transplantation) or an allogeneic donor in one to three daily large-volume apheresis procedures. PBPCs are used almost exclusively for autologous transplantation as durable engraftment occurs more rapidly than with bone marrow. In the allogeneic setting, perhaps because of a larger total dose of T cells and alteration of T-cell function, PBPCs are associated with increased risk of chronic graft-versus-host disease (GVHD). In the matched-sibling setting, both retrospective and prospective studies have demonstrated faster engraftment and higher rates of chronic GVHD with PBPCs compared with marrow, but the impact of PBPC transplantation upon survival and disease-free survival has varied. A recent meta-analysis of nine randomized trials failed to demonstrate a difference in overall survival for PBPC recipients compared with bone marrow recipients [5]. Eapen et al. [6] published CIBMTR data for children receiving HLA-identical sibling grafts showing that PBPCs were associated with higher transplant-related mortality and lower overall survival. The picture in the unrelated donor setting is also unclear. A multicenter prospective randomized trial is currently underway in the Blood and Marrow Transplant Clinical Trials Network to compare survival between recipients of unrelated donor PBPCs and bone marrow. This trial will also assess the impact of PBPCs and bone marrow on both donor and recipient quality of life measures.
Donors of allogeneic bone marrow and PBPC Donors of bone marrow and PBPCs for allogeneic transplantation are either blood relatives of the transplant recipients or unrelated, anonymous volunteers. Related donors Related allogeneic HC donors are usually first-degree relatives of their recipients. Most are full siblings because of the much higher likelihood of complete HLA identity. The family of genes responsible for encoding the proteins of HLA-A, B, C, and DR are colocated in a short region on chromosome 6, and as such are usually inherited en bloc as a single haplotype. Thus, there are only four combinations possible with the two haplotypes of each parent. Between two siblings, the likelihood of two identical HLA haplotypes, a single identical haplotype, and no identical haplotypes is, therefore, 25%, 50%, and 25%, respectively. A parent and
Bone Marrow and Peripheral Blood Cell Donors and Donor Registries 3600
Number of transplants
3300 3000
Marrow
PBHC
Cord blood
2700 2400 2100 1800 1500 1200 900 600 300 2005
2003
2001
1999
1997
1995
1993
1991
1989
1987
0
Year of transplant
Fig. 38.1 Annual transplants from unrelated donors facilitated by the National Marrow Donor Program since its inception. Annual figures include products collected outside the United States and imported, as well as those collected within the United States and exported. Prior to July 1999, only bone marrow was used for initial transplantation, but peripheral blood progenitor cells (PBPCs) were available in the setting of retransplantation. Stippled bars, bone marrow; solid bars, PBPCs; open bars, cord blood units.
their offspring are usually only half-matched (haploidentical), except in the uncommon situation where both parents happen to each possess an identical or nearly identical haplotype. Given that the average size of modern families is just over two offspring, when one child needs an allogeneic transplant, the likelihood of an HLA-identical sibling match is 25–30%. The likelihood of a haploidentical match (full-sibling, halfsibling, parent or child) is much higher, but transplantation using such donors is not yet routine. Unrelated donors and unrelated donor registries Most potential transplant candidates do not have a fully HLA-matched family donor, a limitation that became apparent early in the history of allogeneic transplantation. By the early 1970s, however, case reports began to appear documenting the feasibility of HCT from HLA-matched unrelated donors [7–9]. Such donors were usually gleaned from the files of HLA-typed platelet donors at community blood centers or large hospitals. In 1974 in the UK, the Anthony Nolan Registry was established and dedicated to recruiting and maintaining a file of HLA-typed volunteer bone marrow donors. Within a few years, similar efforts were established in countries throughout Europe, the Americas and Asia. Today, most of the world’s unrelated donor registries collaborate under the auspices of the World Marrow Donor Association (WMDA). The WMDA was organized in 1988 to promote international cooperation and the exchange of HC products [10–12]. The association has established standards and provides guidance on the organization and operation of unrelated donor registries [13,14]. Working groups of the WMDA address issues in quality assurance, registry operations, ethics, finances, and transplant program services [13]. More recently, the WMDA has created an accreditation program for international registries, a Serious Events and Adverse Effects Registry (SEAR) to collect data on serious donor incidents, and a similar registry (SPEAR) to collect productrelated problems and product-related adverse events. The WMDA annually surveys unrelated donor registries around the world [15]. Sixty-seven registries responded to the 2006 survey. Together, these registries listed more than 11,800,000 potential unrelated
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HC donors. Approximately 70% of these donors have been tissue typed for HLA-A, B, and DR, while the remainder has been typed only for HLA-A and B. Most of the registries serve one or more transplant centers in their country; together, the registries reported 1189 transplant centers with potential access to unrelated donors. The Japan Marrow Donor Program reported the largest number of transplant centers, with 192. The WMDA respondents reported collecting 8418 unrelated donor marrow and PBPC products in 2006. In 3269 instances (39%), the collected products were exported to another nation. Germany performed 3029 collections in 2006, which was the most of any nation and represented 36% of the world total. Germany exported 1877 (62%) of the products collected, mostly within the European Union and to North America. The United States was the largest consumer of HC products, with 2293 (27% of the world total), of which 776 (34%) were imported. Worldwide, PBPC and bone marrow collections numbered 5416 (64%) and 3002 (36%), respectively. Registries differ on their policies concerning contact between donors and their transplant recipients. In a recent WMDA survey, 88% of respondents indicated that they allow anonymous contact, for example letter exchanges, between donors and recipients, but only 35% will allow exchange of identifying information. After the first anniversary of transplantation, the NMDP will allow United States donors and recipients to meet provided each consents to release their private information to the other. Much of the world’s available inventory of unrelated adult donors and cord blood units can be accessed through Bone Marrow Donors Worldwide (BMDW). HLA data from 59 registries and 37 cord blood banks, representing more than 11.2 million adult donors and 265,000 cord blood units, were summarized by BMDW in mid-2007. BMDW provides a highly important resource, but is limited by incomplete participation and lack of a comprehensive search function. Given the potential for extreme growth in unrelated donor transplantation, with more than 28,000 transplants annually,1 it is important that a complete worldwide listing of adult donors and cord blood units is made available, together with comprehensive search and matching services. The largest registry in the world is the NMDP, with more than 4.4 million United States registrants, followed by the German National Bone Marrow Donor Registry (ZKRD), with more than 2.8 million. The history of the NMDP registry has been recently reviewed [16]. The NMDP recruits about 350,000 new donors annually. The vast majority of these are recruited at community donor drives, which are held at numerous locations including shopping malls, blood centers, college campuses, community centers, and churches. More than 1000 drives are held each month. New recruits are screened to confirm good health and the absence of high-risk behaviors. Informed consent is administered, after which DNA samples are collected for HLA typing. Blood samples for DNA-based typing have been largely replaced by swabs obtained from the buccal mucosa. HLA typing is performed at an intermediate level of resolution by high-throughput contracted laboratories. Once completed, HLA information is combined with an assigned donor ID and donor demographic information for listing in the NMDP donor database. Private donor information necessary for making direct donor contact is held in separate highly secured computer systems.
1
A total of 12,000 HLA-matched sibling transplants representing 30% of potential allogeneic recipients means that there are 28,000 potential alternative donor recipients (12,000/0.3 − 12,000 = 28,000).
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Donor eligibility and qualification Prior to HC donation, donors must be evaluated to ensure that: (1) they can safely donate, (2) their HCs will be safe for the recipient, and (3) they understand fully what they are being asked to do [17–19].
Regulatory requirements In 1997, the United States Food and Drug Administration (FDA) proposed an approach to the regulation of cells and tissues designed to minimize the risk of contamination and maintain the integrity and function of cell- and tissue-therapy products, including PBPCs and umbilical cord blood HC (bone marrow for transplantation remaining exempt from these regulations because of Congressional legislation) [20]. Subsequently, in 2004, the FDA published three final rules in the US Code of Federal Regulations that describe, among other things, the requirements for registration of establishments that “manufacture” HCs, for determination of donor eligibility, and for compliance with Good Tissue Practices [21]. The FDA determined that minimally manipulated PBPCs and umbilical cord blood HCs, collected from related donors, would be regulated under section 361 of the Public Health Service Act. These regulations are designed to reduce the risk of transmission of relevant communicable diseases, either acquired from the donor or introduced into the products during manufacturing. For HC products from unrelated donors, however, the FDA felt there was a need for additional manufacturing controls, and chose, therefore, to regulate these products under Section 351 of the Public Health Service Act. Section 351 sets additional requirements for Good Manufacturing Practices, which must be followed to ensure the safety, potency, and efficacy of HC products from unrelated donors. The FDA had chosen to phase in its regulatory framework, and this process was incomplete as of mid-2007. Once it is fully implemented, unrelated donor PBPCs must be collected under either an Investigational New Drug application or a Biologics License Application approved or accepted by the FDA. Both related and unrelated donors of PBPCs must be determined to be eligible or ineligible by the screening criteria outlined in the Donor Eligibility Final Rule and the companion FDA guidance document, both of which also provide instructions for correct product labeling [22,23]. The FDA regulatory framework is not unique; similar regulatory frameworks have arisen in the European Union, Canada, Australia and elsewhere. A uniform feature of every regulatory system is establishment of controls to prevent transmission of infectious agents by HC products. There are numerous infectious diseases that, if present in the HC donor, would pose a definite or theoretical risk to the transplant recipient. It is clear that HCs can transmit many of the same infections that are transmissible by blood transfusion including hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV). In addition, some congenital or acquired conditions in the donor, such as genetic defects and immune system abnormalities, may be transmissible to the recipient. Minimizing the risk of disease transmission requires combining the information obtained from a targeted behavioral/medical history, a search for the physical signs of disease, and the results of laboratory testing for specific pathogens [22,23]. HC donors, similar to blood donors, should complete a written questionnaire that has been specifically designed to elicit medical history and identify behaviors or activities that may increase the risk of infectious disease transmission [17–19,22,23]. Questions regarding high-risk behaviors, nonprescription drug use, and skin-breaching procedures, such as tattooing and piercing, are included, as well as questions to assess residence in regions where exposure to malaria or the agent of bovine spongiform encephalopathy may occur. The donor screening
questionnaire is intended to detect information that may place the transplant recipient at increased risk for transplantation-transmissible diseases. Testing of the donor’s blood cannot substitute for direct questioning because the donor might have a “window-period” infection, the blood tests may be falsely negative or, equally important, the testing does not include all potentially transmissible diseases. Indeed, suitable screening blood tests for some disorders, such as Creutzfeldt–Jakob disease and variant Creutzfeldt–Jakob disease, do not currently exist. Positive responses on a proper donor-screening questionnaire may lead to donor disqualification or the donor may be determined to be ineligible by FDA criteria. Donors may also become ineligible as a result of physical examination findings or testing for relevant transmissible infectious diseases, as described below. In the United States, FDA regulations provide a mechanism for collecting HC products from ineligible donors [22,23]. In this case, the transplant physician may determine that there exists a state of “urgent medical need,” which means that a suitable alternative donor or graft source is not readily available, and the risks of alternative therapies are greater than the risk of proceeding with the ineligible donor. If the transplant physician elects to proceed with an ineligible donor, the declaration of urgent medical need must be documented, and appropriate paperwork and labeling must accompany the PBPC product. It should be emphasized that donor eligibility from a regulatory standpoint is distinct from donor suitability. A donor may be eligible, but have medical conditions, as described below, that make him or her unsuitable for donation. The donor physical examination, discussed in greater detail below, should detect behavioral and experiential stigmata, such as recent tattoos, piercings, illicit drug use, and so on, as well as signs of significant conditions, including specifically sepsis and vaccinia [22]. In addition, a sample of the donor’s blood must be tested within 30 days prior to donation for at least the following infectious diseases: HIV 1/2, HBV, HCV, Treponema pallidum, human T-cell lymphotrophic virus I and II, West Nile virus, and cytomegalovirus (CMV). For infusion in the United States, testing must be performed in a certified laboratory, which uses test kits specifically licensed or approved for donor screening and which follows the test kit manufacturer’s instructions. Testing that is performed with an unlicensed test kit or a test kit that is licensed for a different purpose (e.g. diagnosis or monitoring) is a violation of FDA regulations. Testing of HC donors for Chagas’ disease is not currently required, although FDA-licensed kits are available for this purpose. Testing for prior infections with varicella-zoster virus, Epstein–Barr virus, and toxoplasmosis, while not required, may also be desirable. The results of regulated and nonregulated infectious disease testing must be available and reviewed prior to the initiation of preparative conditioning therapy for the recipient [19,22,23]. Donors with a confirmed positive test for HIV must not donate. As already described, donors with prior exposure to HBV and HCV may be used when there are no suitable alternatives and the potential gain outweighs the risk. Strategies for managing hepatitis exposure in donors and recipients have been reviewed [24]. In one instance, interferon-alpha pretreatment of a marrow donor apparently prevented transmission of HCV to the recipient [25]. Donors who have recovered from HBV infection can safely donate marrow [26]. HCT recipients with active HBV infections may, in fact, benefit from transplantation with a donor who shows evidence of HBV immunity [27–30]. Further, it may be that natural immunity, reflecting recovery from HBV infection, is more likely to benefit the transplant recipient than vaccination-related immunity [30]. Transplant recipients whose donors are positive for hepatitis B surface antigen, however, are at high risk for post-transplant hepatic complications and
Bone Marrow and Peripheral Blood Cell Donors and Donor Registries
transplant-related mortality [31,32]. These issues are fully discussed in Chapter 95. CMV-seronegative recipients may benefit from having CMVseronegative donors, but this is a controversial issue. Two analyses of data from the NMDP have shown that, although recipients who are CMV seropositive have poorer outcomes, donor CMV status has no impact on unrelated donor HCT outcome [33,34]. Similarly, it has been suggested that CMV-seropositive patients may benefit from CMV-seropositive donors [35]. Ljungman et al. [36], analyzing data from the European Blood and Marrow Transplant Group, reported that CMV-seropositive recipients of unrelated donor transplantation experienced improved survival and reduced transplant-related mortality when their donors were also seropositive, compared with recipients whose donors were seronegative. This effect was not seen among recipients of HLA-identical sibling transplants. A study of chronic myelogenous leukemia recipients showed those with seropositive donors experienced a more rapid rise of high avidity anti-CMV antibodies following transplant [37]. CMV seropositive recipients with seronegative donors had a slower rise of antiCMV antibodies, which were initially of low avidity. CMV-related issues are further discussed in Chapter 90. Medical eligibility HC donation is not without risk. As described below, adverse events are uniformly encountered, but these are generally minor and short lived. Serious adverse events, including death, may, however, occur. It is essential that, prior to donation, each donor is assessed to ensure the absence of conditions that might increase donor risk to unacceptable levels. To ensure objectivity and preservation of the donor’s best interests, this assessment should be performed by a physician who is not directly involved in the care of the proposed recipient. History and physical The donor undergoes a medical history and physical examination appropriate for the anticipated donation procedure. The history, which addresses risk to the donor, as opposed to the screening history described above, should focus on matters relevant for the anticipated donation, including the psychologic issues discussed below. For all donors, these would include a review of known health problems, a listing of medication and allergies, and a review of the family history. Marrow donors should be questioned about prior surgical procedures and types of anesthesia received. Marrow donors should also have a careful review of systems directed toward neurologic, respiratory, cardiovascular, and musculoskeletal problems. PBPC donors should be questioned about prior whole blood or apheresis donations. The review of systems for PBPC donors should include a careful cardiovascular and neurologic review as well as specific questions about a history of venous access problems, autoimmune diseases, and splenic disorders. The donor’s physical examination should focus upon the neurologic, respiratory, and cardiovascular systems. In addition, marrow donors need an assessment of the oral airways and an evaluation of access to the iliac crests. Donors with a history of musculoskeletal symptoms need a careful examination of the spine and lower extremities. The examination of PBPC donors should additionally include evaluation of venous access, and an abdominal examination for splenomegaly.
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donors should also have determinations of alkaline phosphatase (AP) and lactate dehydrogenase (LDH). Some physicians will also recommend evaluations of immunoglobulin levels and a screen for monoclonal proteins in the blood [38]. Additional evaluations of donors may include urinalysis, chest X-ray, and electrocardiogram. HC donors whose intended recipients suffer from inherited conditions, such as hemoglobinopathies or inborn errors of metabolism, may require specific testing to rule out carrier states that could affect transplant outcome. Donors with sickle cell trait and thalassemia minor can serve as donors in successful bone marrow transplants [39]. As discussed below, however, donors with S-beta thalassemia, SC or other complex sickle hemoglobinopathies should not receive rhG-CSF. Successful transplantation from a donor with hemoglobin H disease has been reported [40]. Barquinero et al. [41] suggested that marrow donors with trisomy 21 and Fanconi syndrome heterozygotes may be less suitable because their recipients experienced a high frequency of poor graft function. A pregnancy assessment must be performed for female donors with childbearing potential [18,19]. Pregnancy is considered a contraindication to marrow donation, but successful marrow collection from pregnant donors has been reported [42,43]. Pregnant women cannot be PBPC donors as hematopoietic growth factor administration is contraindicated. One PBPC donor has been reported, however, who received filgrastim while 2–3 weeks’ pregnant without any adverse impact on the pregnancy or delivery [44].
Ethical and psychosocial issues Psychologic aspects of marrow donation The psychologic condition of the donor should be assessed. In particular, what are the donor’s motivations for considering the HC donation? Is the donor acting out of a genuine desire to help, or are there other motives involved – perhaps an unrealistic expectation of reward or personal gain? Switzer et al. [45] interviewed 343 unrelated marrow donors predonation, and then immediately and 1 year post donation. They determined that donor motives could be classified into six categories, and that an individual donor might express motives in more than one category. Donor motives varied between men and women, and were associated with reported levels of predonation ambivalence and with both psychologic and physical difficulties post donation [45]. Compared with unrelated donors, related donors may have different motivations, and may be subject to increased emotional and physical stress [46,47]. Discussions of the psychologic issues affecting donors post donation are presented in Chapter 33. Occasionally, donors may be subjected to coercion [48,49]. In one instance, a potential donor’s unwillingness to donate for her sister attracted media attention, forcing the donor to reconsider her decision [48]. Switzer et al. [45] have reported that unrelated donors who feel they were pressured, either encouraged or discouraged about donation, are less likely to have a positive donation experience. In an early report of unrelated donors, nine of 20 reported being discouraged from donation by a relative or a friend [50]. Donor consent for HC donation
Laboratory and procedural evaluations of donors The laboratory evaluation of all HC donors should include the following: complete blood count with white blood cell differential, ABO/rhesus grouping, serum electrolytes, glucose, alanine aminotransferase, bilirubin, creatinine or blood urea nitrogen, total serum protein and albumin, and prothrombin time and partial thromboplastin time [19]. PBPC
HC donors must provide written consent prior to donation [18,19]. The model of informed consent for research, as detailed in multiple documents [51,52], is appropriate for the HC donor even in the absence of planned research. Like the research subject, the HC donor is a volunteer who must be provided with full and complete information about the planned procedures. This approach is also consistent with recommenda-
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tions developed by the WMDA. This means that donors must receive a clear description of the proposed donation, its risks, and the alternatives to the procedure. They must also have the opportunity to raise questions and to have these satisfactorily addressed. Materials and programs have been developed to assist with providing donor education and information [53]. HC donors as research subjects It is important to determine circumstances that make the HC donor a bona fide research subject. This can be complicated because much of the research involving HC donors also involves the HCT recipient. Several tests may be applied to help determine whether a particular activity constitutes research involving the HC donor. These include the following: (1) Is there collection of individually identifiable information or product for the purpose of research?; (2) Is there collection of information or product in support of an FDA Investigational New Drug application or an FDA Investigational Device Exemption?; and (3) Is there an interaction with a donor that would not be occurring in the absence of the research? Affirmative responses to any of these questions serve to define research. Examples of research wherein the HC donor becomes a research subject include the following: ex vivo culture or “expansion” of donor cells, insertion of new genes into donor cells, establishment of immortal donor cell lines, and nonstandard genetic analysis of donor DNA. Standard genetic analysis includes, for example, DNA-based HLA typing, hemoglobinopathy testing, and assessment of donor/recipient chimerism. An example of genetic research would be the identification of single nucleotide polymorphisms within particular donor genes for the purpose of assessing impact on transplant outcomes. The complex risks and consent issues created by genetic analysis of DNA have been reviewed [54]. It is worth emphasizing that donors are research subjects when they are asked to provide HCs for patients on experimental transplant protocols where the patient could not otherwise receive a transplant were it not for the protocol (question 3 above). This would include protocols investigating allogeneic transplantation for totally new indications, for example lung cancer or breast cancer, and allogeneic transplantation with a new preparative regimen wherein patients are only eligible for the new regimen if they are ineligible for standard regimens. In both of these situations, the HC donation, even if it is a standard donation, is occurring because of the research protocol.
Donation procedures Bone marrow Marrow is typically collected from the posterior iliac crest of the donor under general or regional anesthesia [55]. The posterior superior iliac spine is the primary landmark for initiating the marrow collection. Marrow is aspirated through large-bore needles into glass or plastic syringes that have been rinsed with a heparin-containing solution. Typically, the iliac crest is entered through a skin puncture or a small skin incision. Once the cortical bone has been penetrated, a small amount of marrow (5–10 mL) is aspirated by applying vigorous suction. The syringe is removed, and its contents are immediately transferred to an anticoagulant solution. Commonly, the aspiration needle is then advanced several millimeters and the process repeated. Several aspirations may be obtained from a single bone puncture as the needle is repeatedly advanced. Through a single skin entry site, the bone may be entered multiple times. A typical collection may involve 200–300 marrow aspirations obtained through a few or several skin punctures or incisions. Occasionally, marrow is also collected from the anterior iliac crests. In
a report from Seattle, prior to 1983, the anterior crests were aspirated in 69% of collections [56]. In current practice, aspiration from the anterior crests is uncommon, but may occur if the posterior marrow space has been compromised by trauma, radiation or some other insult. The aspiration of marrow produces an admixture of marrow cells from the bone cavity and capillary blood. The ratio of marrow to blood appears to depend upon the technique employed. Short, vigorous aspirations are generally thought to produce a higher concentration of marrow cells relative to peripheral blood. Even though the dilution of marrow cells with peripheral blood is variable, the adequacy of marrow collections is determined by the nucleated cell count of the mixture. For many years, the target cell dose for allogeneic marrow collection was over 2.0 × 108 nucleated cells/kg recipient body weight. Sierra et al. [57] evaluated the effect of marrow cell dose in 174 unrelated donor bone marrow transplant patients with high-risk acute leukemia. Their analysis showed a beneficial effect of marrow cell doses above the study group median (3.65 × 108 nucleated cells/kg). As a result of this analysis and similar studies, many centers now routinely seek marrow cell doses of 4 × 108 nucleated cells/kg. Overnight hospitalization of donors was once routine but is now uncommon at most centers. When the marrow collection is completed in the morning, most donors will be ready for discharge by late afternoon. Donors are given instructions about care for their dressings, and precautions about fever or signs of infection. Some centers will routinely provide narcotic-containing analgesics upon discharge, but nonnarcotics will suffice for most donors. Women are often treated with several weeks of oral iron, but this is not necessary for male donors. Decisions regarding transfusions of blood vary between collection physicians. Allogeneic (homologous) blood should not be used without a clear indication and, if indicated, must be irradiated and CMV-safe. When autologous blood units are available, some physicians will routinely transfuse these either intraoperatively or following completion of the collection. Other physicians will not transfuse even autologous blood unless there is an indication upon completion of the collection. The need for autologous blood has been questioned [58,59].
Peripheral blood HCs Requirements for establishment of a PBPC collection program have been reviewed [60]. The PBPC collection site is usually a blood center or the apheresis unit of a hospital. Donors typically remain as outpatients throughout the mobilization and collection process. Filgrastim is currently the most commonly used mobilizing agent, although lenograstim (not available in the United States) yields similar numbers of CD34+ cells [61,62]. Although also not yet licensed in the United States, the chemokine (CXC) receptor-4 (CXCR4) antagonist AMD3100 effectively mobilizes PBPCs in autologous and allogeneic donors [63,64]. The rate of engraftment with cells from related donors appears similar for the filgrastim- and AMD3100-mobilized PBPCs [64]. Combination of AMD3100 with filgrastim may further result in higher numbers of CD34+ cells [63]. Filgrastim is usually administered at a daily dose of 10–16 μg/kg donor weight, which may be given as a single subcutaneous injection or divided into twice-daily injections. PBPCs are collected using a continuous-flow cell separation device. Collections begin after the fourth or fifth dose of filgrastim. Success with shorter courses of rhG-CSF has been described [65]. Usually, cannulae are placed in the antecubital veins. From one line, blood is withdrawn into the apheresis machine at a rate of 30–70 mL/min. The continuous-flow centrifuge is set to separate a fraction composed predominantly of mononuclear cells and platelets from the other blood elements. The mononuclear cell/platelet
Bone Marrow and Peripheral Blood Cell Donors and Donor Registries
fraction, which is rich in HCs, is retained, while the remainder is reinfused to the donor via the return line. During a single 3-hour procedure, it is possible to process between 9 and 12 L of whole blood. The resulting yield of CD34+ cells is correlated with the precollection concentration of CD34+ cells in the donor’s blood. In an effort to reduce donor inconvenience, expedite PBPC collection, and reduce overall cost, there has been increasing interest in largevolume (16–25 L) leukapheresis. Following large-volume leukapheresis, the total yield of CD34+ cells may exceed the calculated maximum based upon the donor’s predonation peripheral blood CD34+ content [66]. This observation suggests that, during the leukapheresis procedure, CD34+ cells are continually released into the blood stream. Several effective apheresis devices are available. Most apheresis instruments have similar CD34+ cell collection efficiencies, but they may vary in the number of platelets collected, which may be of importance in donors with low predonation platelet counts [67,68]. There is wide variability between donors in the number of CD34+ cells that will be mobilized and collected following stimulation with filgrastim [69]. The exact dose of CD34+ cells appropriate for allogeneic transplantation has also not been established, but the usual target is 4–6 × 106 CD34+ cells/kg recipient weight [66,70–74]. For the majority of adult recipients, it appears that one or two 12 L aphereses, or a single large-volume procedure, will provide sufficient HCs for successful engraftment [69,75].
Adverse events associated with HC donation An adverse event is any untoward medical occurrence. Serious adverse events are those adverse events that pose a threat to the individual’s life or functioning. Serious adverse events include at a minimum those adverse events that: (1) are fatal, (2) are immediately life-threatening, (3) require or prolong hospitalization, (4) result in a significant or persistent disability, or (5) represent a congenital anomaly. Both marrow and PBPC donations are associated with adverse events. Fortunately, serious adverse events are not common. In the NMDP experience, hospitalization is the most common reason for an adverse event to be rated Serious. Unexpected adverse events, which may be either Serious or not, are those that are not described in a protocol, consent form, Investigator’s Brochure or the medical literature. Adverse events associated with marrow donation Marrow donors routinely experience minor adverse events [55,56,76,77]. In a review of 1270 related-donor marrow collections in Seattle, it was reported that all donors experienced some component of pain [56]. Stroncek et al. [77] reviewed the experiences of the first 493 unrelated marrow donors in the NMDP. When surveyed 2 days after donation, 75% reported fatigue, 68% had pain at the collection site, and 52% experienced low back pain. Data from 9601 NMDP marrow collections, reported by donor sex, are shown in Table 38.1. Women report more adverse events in general. Only bandage pain is reported more often in men. Older donors report significantly fewer adverse events related to pain, nausea, lightheadedness, and vomiting. Fever is, however, more likely to be reported by older donors. Not surprisingly, in the NMDP experience, some adverse events were dependent upon how the collection procedure is performed. For example, regarding the type of anesthesia, general anesthesia was administered in 78% of cases, epidural anesthesia in 15%, and spinal anesthesia in 7%. Nausea, vomiting, and sore throat are all significantly more common following general anesthesia than following regional anesthesia; fever and fainting are less likely to occur. Similar results were seen in a small controlled study [78].
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Table 38.1 Symptoms reported by National Marrow Donor Program bone marrow donors (n = 9601) Symptom
Women (n = 4106)
Men (n = 5495)
Tiredness* Collection site pain* Back pain Nausea* Sore throat* Pain sitting* Lightheadedness* Headache* Vomiting* Intravenous site pain* Fever Bandage pain* Bleeding at site* Fainting*
85% 78% 67% 63% 62% 62% 53% 40% 39% 37% 22% 19% 10% 7%
76% 75% 68% 40% 57% 57% 42% 32% 17% 23% 22% 26% 8% 5%
* p < 0.05 between men and women.
Observations among unrelated donors may not apply to related marrow donors. In a Seattle study of post-donation pain, male donors tended to report more pain and used more codeine-containing analgesics than did female donors [76]. This study also concluded that pain relief was incomplete with acetaminophen plus codeine, and that donors might benefit from more potent narcotic analgesics. Stroncek et al. [77] examined how side-effects and recovery varied with respect to marrow volume collected, duration of the marrow collection, and duration of anesthesia. The total marrow volume removed, measured as mL/kg of donor weight, was only modestly correlated with most symptoms. The duration of the marrow collection procedure, which reflects not only the volume collected, but also the ease of collection, was the single factor most strongly correlated with increasing frequencies of immediate and delayed symptoms, and with longer time to complete recovery. Longer collections increase the frequency of symptoms and prolong recovery. Sixty-three percent of the 493 NMDP donors reported complete recovery, as defined by the donor, within 14 days. An additional 24% recovered between 2 weeks and 1 month, but 13% of donors took more than a month until they felt fully recovered. Minor complications occur in 6–20% of marrow donations [56,77,79]. These include such events as hypotension, syncope, severe postspinal headache, excess pain, and minor infections. By definition, these complications resolve within a few to several days of onset. Laboratory and radiologic abnormalities also accompany marrow donation. Anemia is the most significant laboratory finding. Marrow harvesting also causes significant, but transient, elevations in serum alkaline phosphatase and ostoecalcin [80]. X-rays, computed tomography scans, and radionuclide scans disclose abnormalities in the pelvis and sacrum that may persist for weeks or months [81]. Abnormalities disclosed by magnetic resonance imaging may correlate with symptoms post donation [82]. Serious adverse events following marrow donation The frequency of serious adverse events following marrow donation is estimated at 0.1–0.3%. A review of data collected between 1969 and 1983 on 2248 allogeneic marrow donations reported to the International Bone Marrow Transplant Registry and 1160 additional donations occur-
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Table 38.2 Major bone marrow donor complications reported to the International Bone Marrow Transplant Registry, 1980–89 (n = 8296) Complication
Number of reports
Myocardial infarction Severe anemia Anaphylaxis during anesthesia Prolonged paralysis after anesthesia Pulmonary embolism Severe back pain Acute renal failure from incompatible blood transfusion Anaphylaxis from incompatible blood transfusion Hepatitis B virus infection Intervertebral disk prolapse Malignant hyperthermia Paroxysmal tachycardia Pulmonary edema Retroperitoneal hematoma Severe hypotension during anesthesia Severe vasovagal reaction
3 3 2 2 2 2 1 1 1 1 1 1 1 1 1 1
Data courtesy of M. M. Horowitz, Center for International Blood and Marrow Transplant Research.
ring in Seattle revealed nine total incidents (0.27%) [83]. These included one case of aspiration pneumonia, two cases of deep vein thrombosis (including one occurrence of pulmonary embolism), three serious bacterial infections (including two with bacteremia), two instances of serious cardiac arrhythmia, and one case of cerebral infarction. The International Bone Marrow Transplant Registry data were updated in 1991 in an examination of 8296 allogeneic donations occurring between 1980 and 1989 (Horowitz, personal communication). Twentyfour instances of life-threatening or incapacitating complications were identified for an incidence of 0.29% (Table 38.2). In the review of the first 493 NMDP marrow donors, only one case, which was potentially a life-threatening complication-apnea and bradycardia, was identified (0.2%) (77). The NMDP has classified major and/or life-threatening complications into five risk categories: anesthesia, infection, mechanical injury, transfusion, and others. Anesthesia risks These are related to the procedures employed and the agents administered. Rarely do significant complications accompany procedures such as intravenous line insertion, endotracheal intubation, and lumbar puncture. Several NMDP donors have experienced profound bradycardia during anesthesia, including regional anesthesia (spinal or epidural), that required emergency treatment. In each case, recovery was prompt and complete. Five serious cases of potential anesthetic complications, including hypotension, hypoxia or cardiac arrest, were identified among 1549 marrow donors at Seattle [79], who were not included in earlier reports [56,83]. A marrow donor who developed life-threatening malignant hyperthermia has also been reported [84]. Infection risks Infections may occur at the sites of marrow collection or line insertions. Infections at distant sites, for example pneumonia, have also been reported [83]. Serious infections have been rare in the NMDP experience. One donor developed bacterial sepsis shortly after marrow dona-
tion. The medical literature also contains additional reports of serious infections following marrow collection [56,83]. Infectious osteomyelitis appears to be a relatively rare complication following marrow collection [79,85]. Mechanical injury risks The procedure of marrow collection may lead to local tissue injuries. These may include bone damage, nerve damage or entry of a collection needle into a blood vessel, an organ or the spinal canal. Hemorrhage, which may be delayed, can create severe pain from compression of soft tissues [86]. Sciatic pain lasting up to 18 months has been reported in a related marrow donor [56]. Prolonged and significant pain may occur following marrow collection, leading to permanent disabilities. Fractures of the iliac crests have also been reported [56,87]. Visceral injuries are apparently very rare, although a case of retroperitoneal hematoma has been reported to the CIBMTR (Table 38.2). Transfusion risks Neither autologous nor allogeneic blood transfusion is free of serious risks. Allogeneic transfusions may cause transfusion reactions, transmit viral infections, and (rarely) transmit bacterial infections. The CIBMTR analysis (Table 38.2) included a case of HBV transmission and two cases of incompatible blood transfusion. Autologous blood has largely replaced allogeneic blood for the transfusion of both related and unrelated marrow donors. Buckner et al. [56], describing the Seattle experience through 1983, reported allogeneic blood transfusions in 21% of 1135 related marrow donors. Allogeneic blood was more often used in women (35%) and in children under the age of 10 years (38%). Among 23 infants under the age of 2 whose marrow was harvested between 1975 and 1986, 22 received allogeneic blood transfusions [88]. In an update of the Seattle experience, only 10% of 1126 adult donors with autologous blood stored required allogeneic blood transfusions [79]. Most NMDP donors receive autologous blood transfusions [77]; NMDP Standards discourage the administration of allogeneic blood [19]. Among 15,400 NMDP marrow collections through 2005, allogeneic blood was administered in only 51 instances (0.3%). In most of these, transfusion of allogeneic blood could have been avoided. Another strategy for avoiding allogeneic transfusion is to recover autologous red cells from the collected bone marrow. This process has been successfully applied in children donors without compromising the quality of the marrow product [89]. Administration of epoetin-alpha (recombinant human erythropoietin, rhEPO) or other erythropoiesis-stimulating agents may also diminish the likelihood of allogeneic blood transfusion following marrow donation [90,91]. Erythropoiesis-stimulating agents marketed in the United States are not approved for administration after bone marrow donation. The FDA issued an alert in November 2006 concerning the safety of erythropoiesis-stimulating agents that included new requirements for label warnings. Other risks Fat embolism has been documented following marrow donation [92]. An unusual case of small bowel mechanical obstruction, which necessitated an exploratory laparotomy, has been reported [93]. The donor had a distant history of prior intestinal obstruction following appendectomy. It was postulated that perhaps the prone positioning of the donor during the marrow collection had allowed an intestinal volvulus to develop. In another report, a donor with unsuspected Addison’s disease experienced grand mal seizures and acute adrenal insufficiency following marrow collection [94]. A flare of systemic lupus erythematosus following marrow donation has also been reported [95].
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Table 38.3 Death occurring in association with hematopoietic cell donation Age
Sex
Hematopoietic stem cell source
Proximity to donation
Cause of death
Reference
40 46 57 35 35 35 47 57 62
M M F M F M F F F
Marrow Marrow Marrow Marrow Marrow Marrow PBPCs PBPCs PBPCs
Prior Prior Immediate Immediate Immediate After Immediate After After
Cardiac arrest Cardiac arrest Ventricular fibrillation Respiratory arrest Myocardial infarction Pulmonary embolism Sickle crisis Stroke Cardiac arrest
[81] * † ‡ [82] § [83] [84], ¶ **
PBPCs, peripheral blood progenitor cells. * Confer, personal communication, 1994. † Scheafer, personal communication, 1997. ‡ Onozawa, personal communication, 1997. § Boogaerts, personal communication, 1997. ¶ Gajewski, personal communication, 1997. ** Aul and Heyll, personal communication, 1997.
Marrow donation and risk of death Death has occurred among normal marrow donors. A review of 7857 marrow collections reported to the CIBMTR revealed two deaths [96]. Table 38.3 lists nine documented deaths occurring within a few days of HC donation [97–100]. The aforementioned cases from the CIBMTR are not included in Table 38.3 because it cannot be determined whether they may represent duplicate reports. As is evident in Table 38.3, two deaths actually occurred prior to scheduled marrow donations. Whether the stress of impending donation somehow contributed to these events is unknown. Similarly, it is difficult to know whether death in the days immediately following HC donation is causally related. Overall, it appears the risk of death in proximity to the procedure is approximately 1 in 10,000.
Table 38.4 Symptoms reported by National Marrow Donor Program peripheral blood progenitor cell donors, excluding reports of bone pain Symptom
All donors (n = 1080)
Adverse events associated with PBPC donation
Myalgia Headache Malaise Insomnia Nausea Sweats Other flu-like symptoms Anorexia Fever Chills Vomiting
54% 52% 49% 28% 15% 14% 12% 11% 6% 6% 2%
Like marrow donors, most PBPC donors will experience adverse events. Most of these are related to the administration of hematopoietic growth factors, most commonly filgrastim (rhG-CSF), lenograstim (a glycosylated rhG-CSF formulation, not marketed in the United States) or sargramostim (rhGM-CSF), that are intended to increase the concentration of HCs in the donor’s blood. Rowley et al. [101] reported the experiences of 69 sibling donors who participated in a randomized comparison of bone marrow and PBPC transplantation. The frequency and intensity of symptoms between the two groups of donors was no different. Bone pain is the most prominent adverse event among PBPC donors, occurring in 25–86% of donors receiving filgrastim [75,102–109]. Bone pain likely results from altered bone metabolism, which is reflected by increased bone-derived alkaline phosphatase and decreased serum osteocalcin [110]. The pain is typically diffuse but most prominent in the spine, hips or pelvis, and ribs [109]. Headache is also common, occurring in 38–70% [109,111]. The discomfort from bone pain improves with mild analgesic therapy, such as acetaminophen or a nonsteroidal antiinflammatory drug. Other symptoms seen with filgrastim include nausea and vomiting (approximately 10%), myalgia (approximately 20%), fatigue (around 15%), insomnia (approximately 10%), and injection site reactions (rare). Symptoms are probably less frequent at filgrastim doses below 10 μg/kg daily. Pain symptoms resolve promptly with discon-
tinuation of filgrastim, and are rarely sufficient to cause premature cessation, or even dose reduction [75,102,107,109,112,113]. The NMDP has evaluated data on more than 1000 adult unrelated PBPC donors, all receiving filgrastim at 10 μg/kg/day for 4 or 5 days. Bone pain is reported by 85% of these donors and is the single most common adverse event. The pain begins after a single filgrastim injection and plateaus after two or three injections. Bone pain resolves promptly after discontinuation of filgrastim. Additional side-effects among NMDP donors are summarized in Table 38.4. HC mobilization with filgrastim also causes numerous alterations in serum chemistries and blood cell counts. LDH, AP, and alanine aminotransferase increase two- to four-fold after five daily doses of filgrastim [75,102,107,109]. Anderlini et al. [75] reported that isoenzymes LDH-4 and LDH-5 comprise most of the LDH increase. Gamma-glutamyl transferase levels are unaffected by filgrastim, suggesting that increased AP is not hepatic in origin. The serum levels of potassium, blood urea nitrogen, and magnesium may show minimal declines during filgrastim treatment [109]. The white blood cell (WBC) count, and in particular the absolute neutrophil count, increase dramatically during filgrastim therapy
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[75,102,107,109]. These increases are dose dependent. At daily filgrastim doses of 10 μg/kg or greater, the total white count may reach 70–80 × 109/L by day 5. Eighty to 90% of the white cells will be neutrophils or band forms. It is generally recommended that the filgrastim dose be reduced if the WBC count exceeds 70–75 × 109/L [60,98]. In the NMDP experience, only five of 1084 donors (0.5%) receiving filgrastim at 10 μg/kg/day demonstrated a WBC count over 75 × 109/L in the preapheresis blood sample on day 5. The median WBC figure among NMDP donors pre apheresis was 36.8 × 109/L, with the 25th and 75th percentiles at 29.9 and 45.2 × 109/L, respectively. A decline in platelet counts accompanies daily filgrastim administration [109,114]. Among NMDP donors, the median decline from baseline to day 5 pre-apheresis is modest, only 16 × 109/L, but the range of platelet count change is large, from a decline of 156 × 109/L to an increase of 103 × 109/L. Frank thrombocytopenia (platelet counts less than 140 × 109/L) was present in 17 of 1084 (1.6%) of NMDP donors by day 5 pre apheresis. The platelet decline appears to be dose independent and occurs even at filgrastim doses of 2 μg/kg daily [109]. The apheresis procedure used in PBPC collection is also a source of adverse events. Securing peripheral venous access at the antecubital veins may produce bruising, hematoma or minor bleeding. Anticoagulation with acid–citrate–dextrose solution may elicit symptoms of hypocalcemia: perioral numbness, paresthesias, and carpopedal spasms [60,66]. These symptoms may be ameliorated with oral calcium supplementation, but are more effectively managed with an intravenous infusion of calcium during apheresis. Reducing the blood flow rate may also improve symptoms of hypocalcemia, but at the expense of prolonging the collection procedure. Another approach that has been used is to supplement the anticoagulation with heparin to diminish the amount of acid–citrate–dextrose required [115]. Peripheral WBC counts will fall after the collection of mobilized PBPCs. Mild neutropenia is usual for a few weeks [111]. Decreases in hemoglobin and hematocrit values are minimal, although in one report hemoglobin levels decreased by an average of 1.2 (range 0.3–2.2) g/dL with each apheresis procedure [74]. Thrombocytopenia is the most consistent and significant finding with PBPC apheresis [66,74,75,106,109,112,114,116–118]. The preapheresis platelet count reproducibly declines between 20% and 30% with each collection, and does not begin to recover until 3–4 days after the last collection. Following two procedures, it is common to encounter total platelet counts below 100 × 109/L [66,74,75,106,112,114,116,117]. In the NMDP experience, the incidence of thrombocytopenia less than 75 × 109/L is 1% (11 of 1084) after a single apheresis procedure (median blood volume processed of 12 L), but rises to 11% (90 of 787) after two procedures. No instances of life-threatening hemorrhage secondary to thrombocytopenia have been reported. Nevertheless, avoidance of aspirin during mobilization and collection, and nonsteroidal antiinflammatory drug therapy during collection is prudent. Some authors have recommended discontinuing PBPC leukapheresis if the platelet count falls below 70–80 × 109/L [60,98]. An alternative to discontinuation of PBPC collection is the recovery and reinfusion of platelet-rich plasma from the leukapheresis product. Each product obtained by standard techniques may contain 3–5 × 1011 total platelets, which is approximately equal to a platelet apheresis product. Mild lymphopenia is routine following PBPC donation and persists for 8–10 weeks [117,119]. The cause of this lymphocytopenia has not been determined, but clinical sequelae have not been reported. The most significant immediate problem of PBPC apheresis is inadequate venous access. Central lines to perform apheresis are required in 0–8% of collections [74,98,108,109,117]. The NMDP has tried to minimize the use of central venous lines. In spite of this, 41 of the first 395 NMDP PBPC donors required a central line. Central lines were 10 times
more common among women than men (37 of 179 women [21%] versus 4 of 216 men [2%]). Central line complications are uncommon, but include pneumothorax, hemorrhage, and infection. To eliminate the risk of pneumothorax, some authors have recommended the femoral approach to central venous access for PBPC collection [60]. A disadvantage of femoral catheterization is the requirement for immobility, which, if more than one apheresis procedure is necessary, will force overnight hospitalization of the donor. Large-volume leukapheresis, which involves the processing of three or more total donor blood volumes, allows the collection of large numbers of HCs in a single procedure and may be appropriate when femoral catheterization is required [66]. Serious adverse events following PBPC donation Filgrastim administration may precipitate severe sickle crisis in persons with sickle cell anemia or complex sickle cell hemoglobinopathies [97,120]. Indeed, a 47-year-old woman with hemoglobin SC disease, who had never had any symptoms from her condition, suffered a fatal sickle crisis during filgrastim mobilization of PBPCs intended for her sister with chronic myelogenous leukemia [97]. It remains to be clarified whether persons with sickle trait (hemoglobin AS) are at any increased risk from filgrastim. Kang et al. [121] safely mobilized and collected PBPCs from nine donors with sickle trait. These donors did experience higher symptom scores during mobilization than did eight simultaneous control donors, but there were no symptoms suggestive of painful sickle crisis. There have been five reports of spontaneous splenic rupture apparently related in some way to extramedullary hematopoiesis [122–126]. In three cases, the spleen was surgically removed, but conservative management was effective in two cases. Platzbecker et al. [127] performed ultrasound evaluations of spleen size before and after rhG-CSF mobilization in 91 healthy PBPC donors. Although no adverse splenic events occurred in this group, significant increases in spleen length and width were routinely documented. No donor factors or clinical findings (change in WBC, increase in AP, etc.) correlated with the degree of splenic enlargement. Similar findings were reported in independent studies by Stroncek et al. [128,129], which also demonstrated that splenic enlargement may persist for up to 10 days in some donors. Based on these studies, ultrasound does not appear to be useful for predicting the risk of hemorrhage into the spleen. Growth factor is probably contraindicated in donors with a history of autoimmune disorders [130–134]. Flares of rheumatoid arthritis and ankylosing spondylitis have been reported in non-PBPC donors following therapy with filgrastim or sargramostim [130,134]. In patients with normal thyroid function, but pre-existing antithyroid antibodies, therapy with sargramostim caused thyroid dysfunction and one case of goiter [131]. A variety of eye inflammatory responses have been reported among allogeneic PBPC donors. These have included marginal keratitis, episcleritis, and iritis occurring during therapy with filgrastim [132,133,135]. Chest pain in association with PBPC donation is occasionally encountered and is noncardiac in origin [109,116]. However, an instance of myocardial infarction following PBPC leukapheresis has been reported [112]. The donor in this instance had a history of coronary artery disease and had previously suffered myocardial infarction. The donor recovered uneventfully, but a planned second leukapheresis procedure had to be canceled. PBPC donation and the risk of death As with bone marrow donation, death at the time of, or in close proximity to, PBPC donation has been reported [97,98]. Table 38.3 lists three
Bone Marrow and Peripheral Blood Cell Donors and Donor Registries
deaths among PBPC donors. Only one of these, the 47-year-old woman with hemoglobin SC disease, was clearly related to the PBPC donation [97]. It is unclear whether the risk of death associated with PBPC donation differs from that of bone marrow donation. Long-term donor follow-up The long-term safety profile of growth factor therapy in normal individuals has not been established. Three cases of leukemia have been reported occurring months to years after PBPC donation [136,137]. In each case, the donor who developed leukemia was the HLA-matched sibling of the transplant recipient, who also had leukemia. It is known that siblings of persons with leukemia have a two- to fivefold increased annual incidence of leukemia [138–140]. Several investigators have performed serial evaluations on the peripheral blood of normal PBPC donors using a variety of techniques to evaluate chromosomal content, DNA stability, replication timing, and gene expression profiling [141–144]. In every instance except one, filgrastim-induced alterations during HC mobilization have been transient; Nagler et al. [143] reported persistent aneuploidy of chromosome 17 in the lymphocytes of PBPC donors 6–9 months after mobilization. There have been several reports providing 1–5 years of follow-up on limited numbers of PBPC donors [70,74,106,145,146]. In no instance have serious adverse events attributable to filgrastim been identified. Cavallaro et al. [44] evaluated 101 PBPC or granulocyte donors who had received filgrastim between 3 and 6 years previously. Ninety-four donors were able to be interviewed; one of the remaining ones had died 15 months after donation from a drug overdose, and the six others could not be interviewed. Among the 94 donors, 11 had experienced adverse medical events during the follow-up period, but none of these related to the hematopoietic system. Two donors had developed cancer during the follow-up period, one breast cancer and the other prostate cancer. Anderlini et al. [147] recently reported on the follow-up of 343 PBPC donors, of whom 281 (82%) were able to be interviewed. With a median follow-up of 39 (range 7–80) months, there were no instances of leukemia or other hematopoietic adverse events. The NMDP conducts annual follow-up of PBPC donors. More than 4000 donors have been followed for between 1 and 9 years, providing nearly 10,000 donor–years of observation. Twenty cases of cancer at various sites have been reported, but there have been no instances of leukemia or lymphoma [148]. PBPCs versus marrow: which is easier for donors? Switzer et al. [149] evaluated 70 unrelated bone marrow donors who subsequently provided a second HC product (25 PBPC and 45 bone marrow). Donors whose second product was PBPCs reported that, in comparison to their prior bone marrow donation, PBPC donation was less difficult physically, required less time, and was more convenient. In randomized trials of PBPC versus bone marrow among sibling donors, the distinction between the two procedures is less clear. Similar levels of anxiety and pain are reported, and time to complete recovery may also be similar [150,151]. When differences are observed, however, in acute symptoms, fatigue, time to recovery or late complications, these tend to favor PBPC donation [151–153].
Special considerations Infants and children as HC donors Safety Children may safely donate marrow [56,88,154–156]. They are probably more likely than are adults to receive allogeneic blood transfusion, but
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serious complications among children donors have been rare [56,88,156]. The use of children as HC donors must consider the ethical and legal issues addressed below. Sanders et al. [88] reported on 23 marrow donors under the age of 2 years. Harvested volumes were between 11.5 and 19.3 mL/kg donor weight. One 14-month-old donor was discovered during predonation evaluation to have a stage 1 neuroblastoma. The tumor was resected at the time of marrow donation. Aside from this case, no serious complications were encountered, but 22 of the 23 infants required allogeneic blood [88]. Successful marrow collection from a 3.95 kg donor has also been reported [156]. The collection volume represented two-thirds of the donor’s total blood volume, essentially necessitating exchange transfusion. Another case reported collecting 335 mL of marrow from a 9.4 kg infant (total blood volume 750 mL) [155]. In this instance, recovery of autologous red cells from the collected marrow enabled avoidance of allogeneic blood transfusion. In a survey of pediatric marrow transplant physicians, only seven of 56 responders were unwilling to collect marrow from infant donors 0–6 months of age [154]. There was little agreement, however, on the management of large-volume collections. Of 52 respondents, six would limit collections to 25% or less of the donor’s blood volume, whereas 24 placed the limit at 50% or higher. Twenty-two respondents preferred to manage large-volume collections in two stages [154]. Children appear similar to adults with respect to their response to filgrastim and PBPC collection [157]. They tolerate filgrastim similarly and provide high yields of CD34+ cells. Central venous access may be required more often for successful collection from children [157]. A report described PBPC collections in five children aged 4–13 years [115]. The children each received filgrastim at a dose of 6 μg/kg every 12 hours for 3 or 4 days. One or two leukapheresis procedures were performed. Three children were donating for siblings, and two were donating for a parent. No child required a central venous line, and no immediate complications were encountered. Pulsipher et al. [158] recently reviewed the clinical, ethical, and research issues surrounding the administration of filgrastim to children. Information, consent, and assent Children who are being evaluated as potential HC donors deserve special attention. They may have very limited understanding of their family member’s illness and of the donation process. Their fears and concerns are often complex and accompanied by significant ambivalence [159– 163]. With hapoloidentical transplantation protocols, children may also be considered as donors for parents, which raises added concerns about understanding, stress, and informed consent [164]. Consent for children presents special concerns. In general, children are only considered for related-donor HC donation. In most instances, parents are expected to consent for their children, which may create conflict of interest situations. When they are later asked, children have reported that they were forced to donate by families or physicians or, short of being forced, that they felt they had no real choice in the matter [162,163]. Consent issues may become very complex as evidenced in one case report concerning a pediatric donor who had been sexually abused by her sibling recipient [165–167]. A survey of pediatric transplant physicians in the United States confirmed that most felt the role of consent appropriately rested with the parents [154]. Outside the United States, “altruism by proxy” has stirred debate in the medical literature [168–170]. In some locales, it remains the standard practice to appoint legal guardians to determine whether HC donation is in the child’s best interest. When governing law allows parents to render consent, it is incumbent on the physician, at a minimum, to recognize the potential for conflict
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of interest, to seek expert ethical guidance when needed, and to ensure to the extent possible that the child donor is a willing and informed participant. Children who are able must provide assent for donation [171]. Whether children can serve as unrelated HC donors is a matter of debate. In 2000, the Washington state legislature passed a law intended to pave the way for minors to become unrelated HC donors [172]. The law states simply, “A person’s status as a minor may not disqualify him or her from bone marrow donation.” This legislation was the direct result of a campaign by a 15-year-old male who wanted to be tested as a potential donor for a young Hawaiian man with leukemia [173]. The NMDP has continued its policy that prospective donors must have reached the legal age of majority (18 years throughout most of the United States) prior to registration. Subsequent donations following an initial HC donation A person can donate HCs repeatedly. Several unrelated donor registries have accumulated experience with donors who donate repeatedly for different recipients. The Japan Marrow Donor Program reported on 137 marrow donors who donated for a second recipient over a 12-year timespan and 5305 total donations [174]. Through 2005, with a total of more than 20,000 donations, the NMDP had had 495 donors provide two donations for separate recipients, and 18 provide products for three different recipients. Such donors may be preferentially selected by transplant physicians because they usually have high-quality HLA-typing data and are known to be highly motivated. Most second donations of marrow or PBPCs are intended for the original transplant recipient and used for treatment of graft failure, poor graft function or disease recurrence. Buckner et al. [56], reporting on 1160 marrow donors, identified 99 who donated marrow twice, usually within a 2-month period. There was a modest decline in the average total number of nucleated cells collected with the second donation. Stroncek et al. [175] reported on 16 second-donation donors at the University of Minnesota. There was a trend toward lower total cells and lower cell concentrations in the second collections. When fewer than 90 days separated the collections, the decrement in cell concentration was statistically significant [175]. Donors whose second donation occurred within 60 days of the first were frequently transfused with allogeneic blood (five of eight within 60 days versus one of eight later than 60 days). Consecutive PBPC collections are also feasible. Stroncek et al. [146] collected PBPC products 1 year apart in 19 healthy volunteers. There were no differences in premobilization blood counts, the response to filgrastim stimulation or the CD34+ cell yield between the first and second collections. Similar results were reported by Anderlini et al. [70], who described 13 allogeneic PBPC donors providing a second donation at a median of 5 (range 1–13) months. No differences were identified in pre-apheresis WBC counts of the donors or in the yield of CD34+ HCs. In more recent studies, de la Rubia et al. [176] noted a decline in the total CD34+ cell yield following a second PBPC mobilization and collection. Donor symptoms were no different between the two donations. Similar results were noted by Platzbecker et al. [177], who also found that female sex and a low peripheral blood CD34+ cell count during the first mobilization predicted for a low CD34+ yield with the second donation. Through 2005, with more than 20,000 transplants facilitated, 684 NMDP donors had provided additional HC support for their original marrow recipients. In 476 instances (70%), the second product was
PBPCs, compared with 208 providing bone marrow. Currently, about one donor in 25 will provide additional HC support for their transplant recipient. Donor experiences during second marrow or PBPC collections have not differed from the first donations. Switzer et al. [149] compared the reactions of second-time NMDP donors to PBPCs and to marrow. All of these donors had initially provided marrow as their HC product. Although the donors asked to provide PBPCs as a second-time product expressed more initial reluctance, they ultimately reported fewer donation-related side-effects than did second marrow donors. The donors who had provided bone marrow followed by PBPCs expressed a strong preference for the latter procedure [149]. In the NMDP experience, the most frequent subsequent donation request today is for peripheral blood lymphocytes collected by apheresis. Indications for donor lymphocytes include accelerated immune reconstitution, treatment of viral infection, immunomodulation (particularly in the setting of reduced-intensity conditioning), and graft-versus-tumor therapy [178]. Approximately 5% of NMDP donors currently provide apheresis lymphocyte products for their recipients. Growth factor-mobilized bone marrow In efforts to accelerate the engraftment rate for bone marrow transplantation, several groups have explored filgrastim administration prior to bone marrow collection [179–182]. These reports, a combination of single-arm trials and comparison studies, suggest indeed that rhG-CSF pretreatment of the marrow donor may accelerate the recovery of neutrophils and platelets. When rhG-CSF was administered in one report at relatively low dose (2 μg/kg/day), accelerated engraftment was not observed [183]. Morton et al. [184] reported a randomized comparison between PBPC- and filgrastim-primed bone marrow. Engraftment of neutrophils and platelets was no different between the two groups, but patients receiving filgrastim-primed bone marrow had significantly less steroid-refractory acute GVHD, less chronic GVHD, and fewer days of immunosuppressive therapy. Taken together, these preliminary observations suggest that it may be possible to achieve the rapid engraftment of PBPCs without the added risk for increased acute and chronic GVHD [182,184,185]. These studies, however, have important implications for HC donors because many of the most significant and nonoverlapping adverse events of PBPC and marrow donation are combined in a single procedure. While reduced volumes of marrow collection may be possible [107,179], this may not fully offset the added risks of filgrastim administration. This would seem to be an area where carefully designed, prospective clinical trials are needed.
Summary Allogeneic HC donation is safe. Serious adverse events are uncommon, and death is exceedingly rare. Nevertheless, all donors must be carefully evaluated and fully informed prior to HC donation. Emerging regulatory requirements must be anticipated and met. The two common procedures for obtaining an HC graft – bone marrow and PBPC collection – differ in their acute and longer-term impacts upon the donors. The long-term safety data for both procedures with respect to preservation of a functional, benign hematopoietic system is incomplete. It is highly likely that annual numbers of allogeneic HCT will continue to grow. Based on estimates of current need, the numbers of related and unrelated allogeneic HCTs will easily double within the next several years.
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39
Hal E. Broxmeyer & Franklin O. Smith
Cord Blood Hematopoietic Cell Transplantation
Introduction Hematopoietic cell transplantation (HCT) from human leukocyte antigen (HLA)-matched related donors has been successfully used for the treatment of children and adults with a high risk of recurrent hematologic malignancies, genetic immunodeficiencies, metabolic disorders, hemoglobinopathies, and marrow failure syndromes. Unfortunately, the majority of patients who could potentially benefit from allogeneic HCT do not have suitably matched related donors. To address this problem, the National Marrow Donor Program (NMDP) was established in 1986 [1,2]. Despite the success of this program, suitable HLA-compatible bone marrow (BM) and peripheral blood (PB) donors cannot be identified for all patients in need of allogeneic transplantation [3]. Therefore, clinical investigators have, over the past two decades, explored the suitability of umbilical cord blood (UCB) hematopoietic cells as an alternative source of hematopoietic stem cells (HSCs). The world’s experience suggests that UCB is an acceptable alternative to BM.
History of UCB transplantation The potential use of umbilical UCB as a source of HSCs was proposed in 1982 in a private discussion held by Edward A. Boyse, Hal E. Broxmeyer, and Judith Bard [4,5]. Dr Boyse felt it was a waste of precious tissue to not use the UCB that was being discarded, and thought that the mature cells in UCB could be used or generated for transfusion purposes. Dr Broxmeyer alerted Dr Boyse to the possibility that UCB likely contains HSCs, and that if there were enough HSCs in UCB, the UCB could be used for transplantation purposes. Subsequent to these discussions, with the stem cell and hematopoiesis expertise of Dr Broxmeyer, and the immunology and genetics background of Dr Boyse, a number of in vitro studies with human UCB [6,7] and in vivo studies with mouse blood [7] were performed to document the feasibility of this proposal. Initial studies used hematopoietic progenitor cell (HPC) assays, which served as surrogate assays for HSCs, to compare numbers of primitive cells in UCB versus BM [6–8]. Early studies assessed whether previously untrained obstetrical healthcare professionals were able to collect UCB that was free of bacterial and fungal contamination, determine the range and average volume of
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
UCB collected, and assay samples for the total number of nucleated cells and HPCs using in vitro culture methods following overnight shipment of UCB units. During the course of these experiments, a number of UCB units were cryopreserved and stored in liquid nitrogen freezers at the Indiana University School of Medicine for potential clinical use as a proof of principle cord blood bank. These studies were followed by experiments in which lethally irradiated mice received transplantations of blood from near-term or term donors, which contained sufficient numbers of HSCs to reconstitute hematopoiesis [7]. The first UCB HCT was performed for a 5-year-old child with Fanconi’s anemia by Dr Gluckman and colleagues in October 1988 [9]. UCB from the patient’s HLA-identical sibling was collected in Durham, NC, by Dr Gordon Douglas, New York University Medical Center; it was shipped to Indiana University where it was cryopreserved by Dr Broxmeyer’s laboratory and hand-delivered to Paris, where the patient underwent his preparative regimen and UCB HCT [4–6,9,10]. The patient had durable engraftment of donor hematopoiesis and survives without hematological manifestations of the treated disease. The next four UCB transplantations, for three children with Fanconi’s anemia and one with juvenile myelomonocytic leukemia, were performed using UCB also collected at a distant obstetrical unit by Dr Douglas and banked at Indiana University [4–7,9–12]. Four of the first five UCB recipients had engraftment of donor cells, with one patient with Fanconi’s anemia having graft failure. In 1991, two children with leukemia received high-dose preparative regimens for UCB HCT from related donors [11,13]. Following these first, largely successful transplant procedures, UCB as an alternative source of HSCs was utilized by others, and these transplants were initially reported by an International Cord Blood Transplant Registry [14]. It is now estimated that at least 350,000–400,000 UCB units have been banked, with at least 6000 UCB transplantations performed worldwide [15]. UCB has now been utilized as a hematopoietic cell source for numerous malignant and nonmalignant diseases in children and adults.
Collection of UCB UCB can be collected for several purposes. Public UCB banks collect UCB for the allogeneic transplantation of any recipient for whom the UCB unit is a suitable HLA match and of sufficient cell dose. The vast majority of the world’s experience with UCB transplantation has been facilitated by publicly banked UCB. UCB can also be collected and stored by expectant parents for potential use by the newborn infant (e.g. autologous transplantation). To date, three autologous transplants
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using previously cryopreserved UCB have been reported for recurrent acute lymphoblastic leukemia [16], neuroblastoma [17], and severe aplastic leukemia [18]. UCB can also be stored for a family member in need of a HCT procedure (e.g. directed allogeneic donor transplantation). While autologous and directed allogeneic collections and storage have typically been performed by private (for-profit) banks, the National Heart, Lung and Blood Institute (NHLBI) of the National Institutes of Health supported a public UCB bank at the Children’s Hospital Oakland in California for this purpose [19]. A similar directed allogeneic UCB bank was established by the National Blood Service of England [20]. These directed allogeneic UCB banks stored UCB from children with hemoglobinopathies and genetic diseases, and from the normal siblings of children with malignant and nonmalignant disease who were in need of a transplant procedure. The number of directed allogeneic transplants is small, with only 17 (3.4% of collected units) allogeneic transplants performed using units from the NHLBI-supported UCB bank at the Children’s Hospital of Oakland [19] and only 13 allogeneic transplants (5.6% of collected units) from the National Blood Service in England [20]. The majority of directed allogeneic transplants facilitated by these UCB banks were for nonmalignant diseases. UCB can be collected from anonymous donors, on local Institutional Review Board-approved studies, for laboratory-based research. Due to the complexities inherent in UCB banking at both public and private programs, the American Academy of Pediatrics issued a policy statement to provide information to physicians so they could provide guidance to parents considering donation of their newborn’s UCB to one of these banks [21]. Recommendations were provided regarding ethical and operational standards, inclusion of policies on informed consent, financial disclosures, and physician, institutional, and organization conflict of interest for those that operate or have a relationship with a UCB banking program. A number of techniques have been proposed for the optimal collection of UCB, considered to be a UCB unit with sufficient volume of blood, total nucleated cells, and CD34+ cells. In addition, the unit would contain a low number of maternal T cells [22], and would be free of transmissible infectious agents. As engraftment is closely correlated with the number of infused cells, numerous variables in the UCB collection process have been examined in attempts to maximize the cell dose. Early studies suggested that factors that may increase these numbers include volume of UCB collected, number of nucleated cells or number of CD34+ cells, larger birth weight, fewer prior live births, and birth order, with a larger number of cells in first-born children [23]. Other factors that appear to increase cell dose include prolonged stress during delivery, placing the infant on the mother’s abdomen after delivery [24], collection prior to delivery of the placenta, cesarean section [25–27], early clamping of the umbilical cord [24,28], and normal saline flush of the umbilical vessels [27,29]. In addition, there is a direct correlation of gestational age with nucleated cell count, but an inverse relation to CD34+ cell count [23,30]. More recent analyses from several large public UCB banks have further identified factors predictive of cell dose [31–33]. In the NHLBIsupported Cord Blood Transplantation (COBLT) study banking program, a total of 11,077 units were cryopreserved [33]. Higher cell doses were obtained from cesarean section deliveries as opposed to vaginal deliveries, and in higher birth weight newborns. Lower cell doses were obtained from African-Americans. There was a significant correlation between CD34+ cell dose and colony-forming unit (CFU)-granulocyte– macrophage (GM), CFU-granulocyte–erythrocyte-macrophagemegakaryocyte (GEMM), burst forming unit-erythroid (BFU-E), and total CFUs [32]. Despite this information, there remains considerable debate about the optimal collection method. The American College of
Obstetricians and Gynecology [34] and the American Academy of Pediatrics [35] has recommended that standard obstetric procedures not be altered to facilitate UCB collections. Cord blood for public allogeneic use is generally collected at a limited number of sites in a single geographic location, with dedicated, trained personnel performing the collections according to standard operating procedures established by the UCB bank. For directed allogeneic or autologous use, UCB is generally collected at the birth location by local obstetrical care providers. The American Academy of Pediatrics has provided guidance regarding recommended procedures for physicians engaged in UCB collection for related and unrelated banking [21]. Recommendations include the collection of UCB in bags containing citrate– phosphate–dextrose anticoagulant, processing and cyropreservation within 48 hours of collection, use of standardized freezing and storage conditions, use of attached segments that can be used for testing and confirmation of identity, storage of extra cells and plasma, infectious disease testing according to United States Food and Drug Administration regulations, use of only UCB banks that are accredited by the Foundation for the Accreditation of Cellular Therapy (FACT) and follow FACT banking standards, and the storage of UCB units under liquid nitrogen or equivalent temperatures. Despite these variable methodologies, the cellular characteristics of UCB units are reasonably constant and distinct from BM and mobilized PB. Single UCB units generally contain a 10fold smaller dose of nucleated cells and CD34+ cells than that typically transplanted with BM or mobilized PB [23]. However, these units are enriched in HPCs [6,8]. Despite efforts to bank as many UCB units as possible, it has been shown that a very large percentage of potential UCB donors are ineligible for donations to public UCB banks [36–38]. In the COBLT study, 34,799 potential donors were screened, with 20,710 giving consent to participate in the study [33]. A total of 17,207 ethnically diverse units were collected between 1998 and 2001, with only 11,077 (64%) of units cryopreserved. Of these quarantined units, only 79% met eligibility criteria with subsequent HLA typing and entry into the search registry. The chief reasons for not using potential UCB donors are the presence of sexually transmitted diseases in the mother, maternal fever during delivery, medications administered to the mother, maternal diseases, complications of delivery, presence of infections, and complications and problems with the placenta or umbilical cord. Overall, experience suggests that UCB donation is a safe procedure for both mother and newborn.
UCB banking The first UCB bank was created at the Indiana University School of Medicine, with the first UCB HCT performed using units cryopreserved and stored in this bank [4–7]. As a result of the interest in UCB transplantation generated from this preliminary transplant experience, other UCB banks were established [39–45]. It is estimated that 350,000– 400,000 UCB units have been collected, tested, and cryopreserved by these worldwide banks [15]. Establishment of a quality UCB bank requires attention to a number of specific issues including donor recruitment, donor consent, donor evaluation, labeling, UCB collection, UCB processing, cryopreservation, histocompatibility testing, infectious disease testing, genetic disease testing, confirmation of recipient histocompatibility testing, tracking and allocation methods, transportation of UCB from the collection site to bank, and from UCB bank to the transplant center, thawing methods, and protection of confidentiality of donors and recipients [46,47]. Therefore, a number of organizations have created standards to ensure the quality of UCB banks and UCB units, including the American Association of Blood Banks (AABB), the American Red Cross, the NHLBI
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COBLT Study, the European Group for Blood and Marrow Transplantation (EBMT), FACT/NETCORD, the Group for the Collection and Expansion of Hematopoietic Cells, the International Society for Cellular Therapy (ISCT), the Joint Accreditation Committee of ISCT-Europe and EBMT, and the NMDP. While minor differences exist in these standards, they are generally consistent. Because of concerns about the loss of nucleated cells, UCB that was initially collected for transplantation was cryopreserved without removal of red blood cells or further separation [6]. With this method of cryopreservation, large volumes of unseparated UCB required a large freezer for storage. Subsequently, numerous investigators explored different methods of UCB separation including the use of Ficoll, Percoll, methylcellulose, gelatin, starch, and lysis to remove red blood cells and recover nucleated cells [28,48–51]. These methods have allowed for more efficient storage of UCB units, although it is not yet clear what the best separation procedure is, or if the collections should undergo any separation at all, in terms of efficient recovery of hematopoietic stem and progenitor cells, and subsequent engraftment of recipients. UCB units that are banked for allogeneic transplantation undergo histocompatibility typing using conventional serological and molecular, DNA-based techniques for class I antigens, and molecular HLA typing for class II alleles. Because the amount of UCB that is available for HLA typing and infectious disease testing is limited, molecular methods, including the polymerase chain reaction and sequence-specific oligonucleotide probe methods, are used to better define class I and class II alleles while using very small amounts of UCB cells [52,53]. While these molecular techniques better define specific alleles, the optimal level of HLA typing for UCB and the clinical impact of higher degrees of resolution are not currently known [54]. Due to the rapidly evolving field of immunogenetics and the need for a standard worldwide nomenclature, the World Marrow Donor Association has provided guidelines for HLA nomenclature, with a recent validation of this system [55]. In addition to ABO, rhesus and HLA typing, UCB banked for transplantation is tested for infectious agents in accordance with the requirements and recommendations of regulatory bodies as listed above. Specifically, UCB units and the mother’s blood are routinely tested for hepatitis B and C, human immunodeficiency virus, human T-cell lymphotropic virus, cytomegalovirus, and syphilis [39,40]. Since some infectious agents can be transferred in liquid nitrogen, newly collected UCB units are typically kept in quarantine until infectious disease testing is complete. If newly collected UCB units are found to be free of potentially transmissible infectious agents, the units are placed into long-term liquid nitrogen storage or equivalent temperatures. In addition to this testing, UCB banks also elicit a history of genetic diseases in the family, travel of donors to places that have a high frequency of transmissible infections, and other high-risk behaviors, including intravenous drug use and high-risk sexual behavior, upon which UCB units may be excluded from the bank. These screening questions are similar to those used for blood donor screening by the AABB. Upon thawing, units of UCB that were cryopreserved for up to 15 years demonstrated 83 ± 12% recovery of nucleated cells, with 95 ± 16%, 84 ± 25% and 85 ± 25% recovery of CFU-GM, BFU-E, and CFUGEMM [56], values essentially equal to those same samples defrosted at 10 years after cryopreservation [57]. Similar results have been noted after 21 years of storage (Broxmeyer, unpublished observations). (Plate 39.1(a) shows colonies derived from UCB multipotential and granulocyte–macrophage progenitor cells of UCB units defrosted after being cryopreserved for 21 years.) The proliferative capacity of these early progenitor cells was intact for colonies formed from the cryopreserved cells, self-renewal potential of CFU-GEMM was high, as assessed by
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replating of colonies into secondary dishes in vitro, and extensive ex vivo expansion was possible. CD34+ cells separated from the 15-year defrosts were able to multilineage-engraft sublethally irradiated nonobese diabetic with severe combined immunodeficiency syndrome (NOD/SCID) mice, suggesting high-quality recovery of HSCs [56]. Laboratory studies demonstrated that UCB cells frozen for several years can be thawed, gene transduced, and ex vivo expanded [56,58,59]. The longest a UCB collection has been stored prior to use for successful cord blood transplantation is in the range of 10–12 years (Rubinstein, personal communication.) UCB banking has raised a number of ethical, regulatory, and legal issues, including questions about recruitment, confidentiality, ownership, informed consent, and fairness in the allocation of this valuable resource. Considerable efforts have been made to define these issues to ensure the appropriate operation of UCB banks [46,47]. It was recognized that there needed to be a national system within the United States to oversee UCB collection, banking, distribution, and usage [60]. Recent federal legislation (discussed below) focused on coordinating these activities and ensuring the safety of this valuable national resource.
Searching for a UCB donor Several potential advantages of unrelated donor UCB transplantation over unrelated donor BM or mobilized PB are the ready availability of banked UCB units, a shorter time to acquisition of UCB with a more rapid time to transplantation, and the ability to tolerate greater degrees of HLA disparity. It was suggested that if four out of six and three out of six antigen matches are clinically acceptable, donors would be identified 99% of the time for patients of all races [61]. Based upon traditional search methods and strategies, several groups have demonstrated that the time required for identification of a suitable hematopoietic cell source and the time to transplantation is more rapid for unrelated donor UCB than for unrelated donor BM [62,63]. A number of questions were raised in a report from the United States Institute of Medicine of the National Academies of Science, including adequacy of cord blood inventories, standardization of the collection of cord bloods, and their processing, storage, documentation, and quality control [64]. In the context of these concerns, the United States Congress provided money under the Stem Cell Therapeutics and Research Act of 2005 to establish a US National Cord Blood Bank Program. This effort fell to the Health Resources and Service Administration (HRSA). Information on the Blood Stem Cells Program of HRSA, kindly provided by Robert L. Baitty, MPP, of HRSA follows. More information on this can be found on the following websites: http://bloodcell.transplant.hrsa.gov and http://marrow.org. This program deals with implementation of the C.W. Bill Young Cell Transplant Program and focuses in part on: the national cord blood inventory; the related cord blood donor demonstration project; program infrastructure including a cord blood coordinating center; and an advisory council. This stem cell therapeutic and research act (public law 109–129) was signed on December 20, 2005 with the aims to increase the number of unrelated donor transplants, to provide a public inventory of high-quality UCB units from diverse populations, and to increase the number of UCB units available for research. HRSA’s implementation was guided by a single point of access for patients and physicians to all sources of blood stem cells, the collection of high-quality diverse UCB units, and complete data on clinical outcomes of transplants. The NMDP was chosen as a Cord Blood Coordinating Center, and six National Cord Blood Inventory Banks were initially chosen. An HRSA Advisory Council on Blood Stem Cell Transplantation has recently been instituted to evaluate amongst other issues, the quality of UCB blood banks and UCB units.
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Clinical results While there is now extensive clinical experience for the transplantation of related and unrelated donor UCB, including a large, prospective national trial (COBLT), to date there are no prospective, randomized clinical trials comparing UCB with BM or mobilized PB. However, there are an increasing number of studies comparing outcomes of UCB transplantation to related and unrelated donor marrow transplantation. Related donor UCB transplantation Two large case series of related donor UCB transplants have been reported by the International Bone Marrow Transplant Registry (IBMTR) [65] and Eurocord [66]. These retrospective series have shown survival rates of approximately 60% at 1 year. However, of greater importance are results of the IBMTR and Eurocord report in a retrospective cohort controlled analysis of children under 15 years of age transplanted for malignant and nonmalignant diseases with HLA-identical UCB or BM [66]. In this study, 2052 unmanipulated BM recipients were compared with 113 UCB recipients. Transplants were performed between 1990 and 1997 at 207 transplant centers worldwide. The median period of followup was 27 (range 3–85) months. Significant differences in these two patient groups were identified, with UCB recipients being younger (5 years versus 8 years; p < 0.001), of lower weight (median weight 17 kg versus 26 kg; p < 0.001), and less likely to receive methotrexate (MTX) for graft-versus-host disease (GVHD) prophylaxis (28% versus 65%; p < 0.001). The median UCB cell dose was 4.7 × 107 nucleated cells/kg (range 0.80). The single institution, case-controlled analysis suggested that, despite significantly greater HLA disparity among UCB recipients, the probabilities of neutrophil and platelet engraftment, acute and chronic GVHD, and survival were similar between recipients of zero to three antigen-mismatched unrelated donor UCB and HLA-matched unrelated donor BM. Similar results were demonstrated in an analysis performed by Eurocord [63]. In this analysis, 541 children with acute leukemia who received zero to four HLA antigen-mismatched UCB (n = 99) were compared with recipients of zero to three HLA-antigen mismatched T-cell-depleted unrelated donor BM (n = 180) or recipients of zero to two HLA antigen-mismatched T-replete BM (n = 262). While comparisons were made after adjustments to these three groups, distinct differences were noted among the three groups. Most notably, UCB recipients received a higher number of HLA-mismatched grafts (92%) versus recipients of T-cell-depleted BM (43%) and versus T-replete BM recipients (18%) ( p < 0.001). The probability of neutrophil engraftment by day 60 was 0.80 (95% CI 0.70–0.90) for UCB, 0.90 (95% CI 0.84–0.96) for T-cell-depleted BM, and 0.96 (95% CI 0.95–0.97) for
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T-replete BM at a median of 32 (range 11–56), 16 (range 9–40), and 18 (range 10–40) days ( p < 0.001), respectively. Platelet recovery occurred at a median of 81 (range 16–159) days for UCB, 29 (range 8–165) days for T-cell-depleted BM, and 29 (range 8–141) days for T-cell-replete BM ( p < 0.001). The probability of grade III–IV acute GVHD was less in recipients of UCB (p = 0.22) and T-cell-depleted BM (p = 0.08) versus T-cell-replete BM ( p = 0.30). Further, the probability of chronic GVHD was lower in UCB and T-cell-depleted marrow recipients compared with T-cell-replete BM ( p = 0.02). While early TRM was highest among UCB recipients ( p < 0.01), nonadjusted estimates of survival (for patient, disease, and transplant differences) at 2 years were similar: 0.35 (95% CI 0.25–0.45) for UCB, 0.41 (95% CI 0.33– 0.49) for T-cell-depleted BM, and 0.49 (95% CI 0.43–0.55) for T-cellreplete BM. Overall mortality was greatest in recipients of T-cell-depleted BM ( p < 0.07). A report from the Center for International Blood and Marrow Transplant Research compared the outcome of matched related sibling donor (n = 101), unrelated donor BM (n = 85), and UCB (n = 81) in children less than 18 months with acute leukemia [76]. Treatment-related mortality was 6%, 15%, and 31% after matched sibling, unrelated donor marrow and UCB, respectively, with no difference in risk of relapse, and overall and leukemia-free survival among the different stem cell sources. A recent meta-analysis compared the outcome of unrelated donor UCB versus unrelated donor BM in children and adults [77]. A total of 161 children and 316 adults received unrelated UCB transplants, with 316 children and 996 adults receiving unrelated donor BM transplants. In children, the incidence of chronic GVHD was lower in unrelated donor UCB grafts (relative risk 0.26), while the incidence of acute GVHD (grade III–IV) did not differ. In adults, TRM and disease-free survival were not different based upon hematopoietic cell source. Taken together, these retrospective and meta-analyses suggest that unrelated donor UCB and T-cell-replete unrelated donor BM have different risks and potential advantages, with similar overall survival. The data support the overall findings suggesting that unrelated donor UCB is a reasonable hematopoietic cell source for children and adults without a matched related donor, giving comparable survival to unrelated donor BM. UCB transplantation in adults As a result of the limited number of cells available in the finite volume of collected UCB, there was initial concern that UCB might not contain a sufficient number of cells to reliably engraft larger children, adolescents, and adults. There is now an emerging experience for UCB transplantation in adults [69,78–86]. The first large series of UCB transplantation in adults reported 68 adults (median age 31.4 years, range 17.6–58.1) with hematologic malignancies, marrow failure syndromes, and a single patient with an inborn error of metabolism [78]. The median weight of patients was 69.2 (range 40.9–115.5) kg. Seventy-one percent of patients received UCB grafts that were mismatched for two or more HLA antigens, with patients receiving a median cell dose of 1.6 × 107 (range 0.6 × 107–4 × 107) nucleated cells/kg and a median of 1.2 × 105 (range 0.2 × 105–16.7 × 10 5) CD34+ cells/kg. The probability of neutrophil engraftment was 0.90 (95% CI 0.85–1.0) at a median of 27 (range 13–59) days. Similar to other reports, this study also demonstrated an association between a higher number of cells in the cryopreserved UCB unit and the rate of neutrophil recovery. Platelet recovery (>50,000/mm3) occurred at a median of 99 (range 42–228) days. The probability of acute GVHD of grade III–IV by day 100 was 0.20 (95% CI 0.11–0.29) without a significant association between the grade
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of acute GVHD and the degree of HLA mismatching ( p = 0.70). The probability of chronic GVHD was 0.38 (95% CI 0.23–0.52). At a median follow-up of 22 (range 11–51) months, 19 of 68 patients were alive (28%) with 18 of them disease free. Thirty-two of 68 (47%) patients died of transplant-related complications. Patients receiving UCB grafts containing more than 1.2 × 105 CD34+ cells/kg had longer survival than patients receiving less than this number of CD34+ cells ( p = 0.05). However, event-free survival was not associated with HLA disparity ( p = 0.07), patient age ( p = 0.42) or the type of malignancy. The results are similar to those reported by Eurocord for 107 adults [75]. The COBLT study prospectively studied the use of UCB in adults [69]. Thirty-four adults with a median age of 34.5 (range 18.2–55) years received four of six (n = 23), five of six (n = 10) and six of six (n = 1) antigen-matched UCB grafts for various malignant hematologic diseases. The cumulative incidence of neutrophil and platelet engraftment by day 42 was 0.66 (median 31 days) and 0.35 by day 180 (median 117 days). The cumulative Kaplan–Meier estimate for survival at day 180 was 0.30. Causes of death included recurrent disease (25%), acute GVHD (25%), chronic GVHD (11%), infection (26%), graft failure (11%), and graft rejection (4%). Recent efforts in adults have focused on the use of reduced-intensity preparative regimens attempting to reduce the high incidence of nonrelapse mortality (NRM). A study of 21 adults with advanced Hodgkin’s lymphoma reported the results of unrelated UCB (n = 9) or matched sibling donation after a reduced-intensity preparative regimen of busulfan, fludarabine, and total body irradiation [82]. All patients had sustained engraftment by day 60, although patients receiving a matched sibling donor graft had a more rapid rate of engraftment. In this small study, the cumulative incidence of acute GVHD, chronic GVHD, and day 100 TRM was no different among hematopoietic cell sources. Progression-free survival at 2 years was also no different, suggesting, with limited patient sampling, comparable outcomes between hematopoietic cell sources in adults with Hodgkin’s lymphoma receiving a reduced-intensity preparative regimen. Figure 39.1 shows the cumulative incidence of neutrophil and platelet recovery and adjusted probability of leukemia-free and overall survival after matched and unmatched BM versus mismatched UCB transplantation in adults with acute leukemia [83]. The issue of low UCB cell doses for large patients has prompted several groups to study the transplantation of 2 UCB units [84–86]. The first of these studies reported 23 adults (median age 24 years) receiving two partially HLA-matched UCB units after high-dose conditioning [84]. The median infused cell dose was 3.5 × 107 (range 1.1–6.3 × 107) nucleated cells/kg. All evaluable patients engrafted at a median of 23 days. Interestingly, by day 100, a single UCB unit predominated. Disease-free survival was 57% at 1 year. Two subsequent trials have studied the transplantation of 2 UCB units after reduced-intensity preparative regimens. In one of these studies [86], 21 adults (median age 49 years) received 2 UCB units after a preparative regimen of fludarabine, melphalan, and antithymocyte globulin (ATG). The median engraftment of neutrophils was at 20 days. NRM was 14%, with a disease-free survival at 1 year of 67%. Patients demonstrated several patterns of donor chimerism, with a single unit predominating in some patients, whereas both units were present in others. The largest study to date for double UCB unit transplantation has recently been reported [84]. One hundred and ten adults (median age 51 years) received 2 UCB units after a preparative regimen of fludarabine, cyclophosphamide, and a single fraction of total body irradiation. Neutrophil engraftment was demonstrated in 92% of patients. The incidence of acute (grade III–IV) GVHD was 22%, with 23% of patient
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(a)
(b)
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Fig. 39.1 Transplantation outcomes with mismatched cord blood, and matched and mismatched bone marrow for treatment of adults with acute leukemia. (a) Recovery of neutrophils, (b) recovery of platelets, (c) adjusted probability of leukemia-free survival, and (d) adjusted probability of overall survival. It is seen that human leukocyte antigen (HLA)-mismatched cord blood should be considered an acceptable source of hematopoietic stem cell grafts for adults in the absence of an HLA-matched adult donor. (Reproduced from [83], with permission.)
developing chronic GVHD. TRM and survival at 3 years were 26% and 45%, respectively. Figure 39.2 shows neutrophil and platelet recovery, and 3 year event-free and overall survival for patients receiving 1 or 2 UCB units after reduced-intensity conditioning in adults with hematologic disease. While mixed donor chimerism was frequently demonstrated soon after transplantation, all patients demonstrated engraftment of a single UCB unit when assessed at later time points. No factors, including CD34+ cell content, total nucleated cell dose, HLA matching, ABO typing, gender match or order of unit infusion were predictive of the predominant unit. The experience in adults suggests that the probability of engraftment and incidence of acute and chronic GVHD are tolerable, but NRM is high with traditional high-dose preparative regimens. Recent preliminary reports of double UCB unit transplants performed after reducedintensity conditioning regimens are encouraging, suggesting that this approach may overcome critical issues of cell dose and high TRM. However, there are unanswered questions and some concerns with transplantation of multiple UCBs. First, only one UCB wins out, and it is not yet predictable which will win. Second, there may be enhanced GVHD with multiple UCBs, which could negate the advantage of UCB, that is, the lowered incidence of GVHD elicited by single UCB transplants.
Immune reconstitution after UCB transplantation As a result of the low number of transplanted cells and the “naïve” pattern of immune cells in UCB, it was initially thought that immune reconstitution following UCB transplantation was delayed in comparison to BM and mobilized PB. It was reported that UCB transplant recipients had a high incidence of infectious complications, although it was not apparent if these infections were the result of delayed immune recovery, delayed neutrophil engraftment or the general use of high-dose steroids for GVHD prophylaxis in UCB recipients. The pattern of immune recovery following UCB transplantation has now been well described for children and adults [68,73,87–93]. The reports demonstrate a remarkably similar pattern, with prompt recovery of natural killer (NK) cells by 2–3 months post transplant, of B cells by 6–9 months post transplant, and of CD8+ T cells by 8–9 months, but delayed recovery of CD4+ T cells with a return to normal numbers at approximately 1 year. In studies involving both children and adults, the recovery of CD4+ T cells was slower in adults, consistent with the pattern of recovery for BM and PB [93]. Mitogen stimulation responses are noted in the normal range by 6–9 months post transplant. Studies of T-cell receptor diversity and T-cell receptor excision circles [89–91] further suggest efficient thymic regeneration mechanisms following
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Fig. 39.2 Cord blood hematopoietic cell transplantation (HCT) after reduced-intensity conditioning in adult patients with hematologic disease. (a) Cumulative neutrophil recovery by day 42, (b) unsupported platelet recovery greater than 50 × 109/L at 6 months, (c) cumulative proportion of 3-year event-free survival, and (d) cumulative proportion of 3-year overall survival. (c and d) Patients receiving either 1 (- -) or 2 (—) cord blood units. The findings support the use of umbilical cord blood after reduced-intensity conditioning as a strategy for extending the availability of HCT, particularly for older patients. (Reproduced from [84], with permission.)
UCB transplantation that are similar, if not superior, to that of BM. The prospective COBLT study included an assessment of immune recovery after unrelated donor UCB transplantation [92]. UCB recipients were studied for the presence of T lymphocytes with herpesvirus antigen-specificity. Overall, 66 of 153 recipients developed antigenspecific T lymphocytes to at least one of the herpesviruses. In a subsequent analysis, it was demonstrated that patients who developed antigen-specific T-lymphocyte responses had a significantly lower probability of relapse (p < 0.003) with improved relapse-free survival (p < 0.0001) [94]. Future directions for clinical UCB transplantation Studies to date demonstrate the importance of cell dose on the pace of engraftment and the ability to engraft larger patients. In addition, cell dose is predictive of NRM. It is also clear that lower cell dose increases the deleterious effects of HLA mismatch [69,95,96]. Several strategies have been explored to address the problems of cell dose and high NRM in adults. The transplantation of 2 UCB units following reducedintensity conditioning regimens are showing encouraging results. These approaches complement the expansion of UCB banks with an increasingly large number of UCB units with a larger cell dose, thereby making larger UCB units more available for transplantation. Hematopoietic growth factors (e.g. granulocyte colony-stimulating factor [G-CSF]) have been routinely used after UCB transplantation. More limited is the use of other growth factors (e.g. interleukin 11 [IL-11]). However, to date, there are no reliable data to suggest the benefit of growth factors following UCB transplantation. Another approach for facilitation of neutrophil and platelet engraftment is the ex vivo expansion of UCB prior to transplantation [97]. Twenty-five adults and 12 children with hematologic malignancies (n =
34) or breast cancer (n = 3) received an infusion of UCB expanded in G-CSF, megakaryocyte growth and development factor, and stem cell factor (SCF; also called steel factor). Patients received a median of 0.99 × 107 nucleated cells/kg (expanded plus unexpanded UCB). The median time to neutrophil and platelet engraftment was 28 (range 15–49) and 106 (range 38–345) days, respectively, with engraftment in all patients who survived 28 days. Grade III–IV acute GVHD occurred in 40% of patients, with extensive chronic GVHD in 63% of patients. This information notes a greatly increased rate of acute and chronic GVHD compared with noncultured cells. With a median follow-up of 30 months, 35% of patients survived. This study and others suggest the ability to expand ex vivo more committed progenitors, with limited ability to expand long-term repopulating cells. The inability of ex vivo-expanded cells to contribute to long-term hematopoiesis may be the result of increased apoptosis, initiation of the cell cycle, and disrupted homing of HSCs. Future studies will explore the safety and efficacy of ex vivo expansion, different expansion conditions, and manipulation of signaling pathways (including Notch, Wnt, bone morphogenetic protein 4, and Tie2/angiopoietin-1), as well as other pathways noted below and intracellular mediators (phosphatase and tensin homolog and glycogen synthetase kinase-3, etc.), to expand HSCs with less effect on differentiation [98].
Characteristics of primitive hematopoietic stem and progenitor cells in UCB The number of nucleated cells available for UCB transplantation is greatly limited compared with that available for BMT. However, the frequency of primitive cells in UCB, as assessed by progenitor cell numbers as determined by assays of colony-forming cells [6,8], is greater than that of BM. Numbers of CFU-GM, BFU-E, and CFUGEMM present in the first 65 UCB collections performed in the
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Broxmeyer laboratory have been published [57]. Most of these values fell within the limits of the corresponding numbers present in BM used for successful BM transplantation [8]. It was noted that the progenitor cell content of UCB units was more rigorously associated with major covariants of post-transplantation survival than was the nucleated cell count, and that progenitor cell content was a better indication of UCB grafts [99]. As important is the quality of these cells in UCB compared with BM. UCB includes cells with extensive proliferative capacity and replating ability in vitro (Fig. 39.3 (a–c)) [6,8,100–108]. UCB progenitors respond very rapidly to stimulation by combinations of cytokines. Moreover, primitive human UCB cells appear to have great capacity to engraft/repopulate the hematopoietic system of mice with severe combined deficiency syndrome (SCID) (Fig. 39.3 (d)). The proliferative characteristics of primitive UCB cells are extensive. However, why engraftment of neutrophils and, especially, platelets is slower with UCB than BM cells still remains a serious unanswered question. This difference may in part reflect the more immature nature of UCB versus BM primitive cells. The more immature status of UCB primitive cells as a total population is suggested by studies evaluating
the loss of telomeric DNA with age [109], and by a higher ratio of more immature to mature colony-forming cells in UCB than in BM [8]. Efforts to accelerate neutrophil recovery in recipients of UCB transplantation have not been very rewarding. Low numbers of megakaryocyte progenitors in UCB grafts have been implicated in the delay in platelet recovery after UCB transplantation [110], but the problem may also be in the maturation capacity of the megakaryocytes from UCB. Cord blood megakaryocytes derived from megakaryocyte progenitors do not complete maturation as well as cells from BM or PB [111]. More intricate knowledge of the cytokine regulation of blood cell production in the context of microenvironmental stromal cell interactions may be necessary for the development of better intervention methodologies to accelerate engraftment of UCB cells. There are a number of cell surface phenotypic markers of primitive cells that help to define these populations. For the most part, these markers, which include CD34, CD38, Thy1, c-kit, fms-related tyrosine kinase-3 (Flt3), and rhodamine-123, are the same for primitive cells found in UCB and BM [112]. These primitive cells express CD34 antigens and low or absent levels of CD38, Thy1, c-kit, and rhodamine-123,
Fig. 39.3 Proliferative Capacity of Human Cord Blood Hematopoietic Progenitor and Stem Cells. A) Colony formation by multipotential (CFU-GEMM) (lower-left)- and granulocyte macrophage (CFU-GM) (upper-right)- progenitor cells from cord blood cells thawed after 21 years in a frozen cryopreserved state, B) Colony formation from a high proliferative potential (Bi and Bii) and a smaller proliferative potential (Biii and Biv) progenitor cell arising from a single high CD34-expressing single cell sorted into a single microtiter plate (i and iii are at original magnification x 20; and ii and iv are at original magnification x 80). Reprinted from Figure 2 of [102] with permission from Blood; C) Secondary CB CFU-GEMM colony derived from replating of a single CFU-GEMM colony into a secondary plate, a measure of self-renewal of CFU-GEMM. See reference104 for more information, D) immunohistochemical staining of CD34+ cord blood cells that engrafted NOD/SCID mouse bone marrow using human Ki67, a marker of cell proliferation. See Orazi et al238 for more information. Conclusion: Cord blood progenitors and stem cells have extensive proliferative capacity.
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and are positive for Flt3. Human CD34+ cells contain HSCs and HPCs, and since CD34 is also found on other cells (e.g. endothelial cells), this marker alone is not a perfect indicator of the actual number of HSCs or the composition of HSCs and HPCs of a graft. CD133 has also been used to identify populations enriched for HSCs and HPCs. In the human system, CD34+ cells can be subtyped into a CD38− population, considered to contain the earliest cells including HSCs, and a CD38+ population in which HPCs are highly enriched and HSCs are few or absent. The complexity and heterogeneity of the CD34+CD38− population is highlighted by the demonstration that, in the human SCID repopulating cell (SRC) assay, fewer than 1 : 600 CD34+CD38− cells appear to be a human SRC [107], and the SCID assay is currently our best assay for evaluating human HSCs. Second generation NOD/SCID (IL-2 common γ chain receptor null) mice are now available. The use of these mice, which manifest enhanced repopulation by human cells and for longer periods of time, may allow adjustments as to the frequency of human SRCs from cord blood. At present, it is possible to more rigorously define murine than human HSCs phenotypically. Mouse HSC functional assays depend on repopulation of lethally irradiated mice in either a competitive or a noncompetitive situation. In the human SRC assay, only sublethal irradiation dosages are given to recipient mice, and human cells have not been proven to rescue lethally irradiated SCID mice. Murine HSCs [113,114] are phenotypically characterized into long-term (Sca-1hiThy1loLin−Mac1−c-kit+) and short-term (Sca-1hiThy1loLin−Mac-1loc-kit+) repopulating cells [115]. The signaling lymphocyte activation molecule (SLAM) family is a group of 10 or 11 cell surface receptors tandemly arranged at a single locus on chromosome 1, and includes CD150, CD244, and CD48. Murine BM HSCs were found to be highly purified in a phenotypically defined population of CD150+CD244−CD48− cells [116]. In contrast, nonself-renewing multipotential HPCs were defined as CD244+CD150−CD48−, and most restricted progenitors were designated as CD48+CD244+CD150−. A similar pattern of SLAM family receptor expression was found on murine fetal liver HSCs [117]. Fetal liver HSCs of mice were CD150+CD48−CD244− , with the vast majority of the fetal liver HPCs found in the CD48+CD244−CD150− or CD48+CD244+CD150− cells. More rigorous phenotyping of murine than human HPCs is also available [118,119]. A recent review documents myeloid lineage commitment from HSCs with the murine system [120]. More definitive phenotypic categorizing of human HSCs and HPCs is needed. In this regard, a clonogenic subpopulation of CD34+CD38− cells was identified that expressed high levels of CD7 and possessed only potential for lymphoid cell development [121]. These cells also expressed CD45RA and HLA-DP, but were low or absent in expression of c-kit and Thy1. Methodologies for assessment of the functional properties of HSCs and HPCs have been published [112]. Efforts are underway to profile HSC and HPC populations at a genomics [122–126] and proteomics [127,128] level, and will no doubt be accelerated for human cells by advances in technology, but may be hindered by lack of more precise phenotypic markers of human HSCs and HPCs that better recapitulate subset functional heterogeneity.
Endothelial progenitor cells and mesenchymal stem/stromal cells in cord blood In addition to HSCs and HPCs, other immature non-HSCs have been identified in UCB. An immature subset of high-proliferative endothelial progenitor cells (EPCs) has been characterized. This cell achieved at least 100 population doublings and had the capacity to form colonies in secondary and tertiary dishes with maintenance of high telomerase activity [129]. EPCs from UCB had greater proliferative potential than those
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from adult tissue sources. EPCs were redefined by clonal analysis and using HSC/HPC principles [130]. Mesenchymal stem/stromal cells (MSCs) produce bone, fat, and cartilage and have great proliferative potential, but whether or not they can be categorized as stem cells is still an open question [131]; hence the cells are sometimes designated mesenchymal stromal, rather than stem, cells (see Chapter 9). MSCs are found in cord blood and have demonstrated immune-modulatory activity [132–141]. Sometimes these cells are found in low frequency in UCB or are absent; thus, great variability is noted in the frequency of these cells in different cord blood collections. Whether or not this variability is technical or has significance for transplant outcome remains to be determined. EPCs and MSCs are still not well defined phenotypically, and enhanced characterization of phenotypes, that recapitulate functions, will increase the potential for use of these cells from cord blood. Efforts towards this for MSCs have been published [142].
Ex vivo expansion of UCB stem cells Since the numbers of HSCs and HPCs that can be obtained from single collections of UCB is limited [6,8,57,100], and these numbers appear to be dose limiting, especially in the context of higher-weight individuals and adults [9,78], a number of different options to deal with this problem have been evaluated. These include attempts at ex vivo expansion of HSCs and HPCs, the use of combined collections of UCB, mixture of UCB with other tissue sources of HSCs, enhancement in the homing capacity of HSCs and HPCs, and intramarrow injections of cells to bypass potential loss of cells through the circulation. Investigators have placed extensive laboratory efforts into expanding UCB HSCs ex vivo. It is clear that primitive UCB cells can be greatly expanded ex vivo. However, these studies demonstrated greater expansion of the more mature cells than the most immature cells in this primitive population. Also, a dissociation of SRC phenotype and function was noted during expansion of UCB CD34+CD38− cells [143], and the fate of functional SRCs has varied greatly among groups studying their expansion ex vivo. These studies leave open the question of whether the long-term marrow repopulating human HSCs are truly being expanded under these conditions and, if so, by how much? Clearly, loss of these most primitive cells is undesirable. While UCB cells that have been subjected to ex vivo expansion culture maneuvers have been transplanted into recipients [97], clinical benefit of these cells has not yet been demonstrated. The capacity to ex vivo expand HSCs is most likely limited by our lack of knowledge of the process of self-renewal. A number of cytokines have been used in attempts to “ex vivo” expand human HSCs/HPCs. The most basic starting combination of cytokines for this effort includes SCF, Flt3 ligand, and thrombopoietin (TPO). Other molecules, such as stromal cell-derived factor (SDF)-1/CXCL12, have also been implicated as useful agents for these efforts. Information on these and other cytokines involved in hematopoiesis, with potential effects on ex vivo expansion, have been described [144]. SDF-1/CXCL12 has recently been found to enhance the replating capacity of multipotential (CFU-GEMM) and macrophage (CFU-M) progenitor cell-derived colonies in vitro [145], a measure of self-renewal capacity of HPCs, and also to greatly enhance the ex vivo expansion of human cord blood HPCs (Broxmeyer, unpublished observations). It remains to be determined if SDF-1/ CXCL12, in combination with SCF, Flt3 ligand, and TPO or other cytokines, will ex vivo expand human HSCs. Information on how the intracellular signals triggered by the cytokines alone and in combination, and in the absence and presence of stromal cell support, are mechanistically mediated for proliferation, self-renewal, and differentiation could greatly enhance the possibilities of modulating HSCs for expansion ex vivo and in vivo. A number of intracellular signaling molecules have
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been identified for such functions; these include the cyclin-dependent kinase inhibitors p21cip1/waf1, p27kip1, members of the STAT transcription factor family and their associated JAK proteins, Notch ligands, Wnt family members, β-catenin, glycogen synthase kinase-3, sonic hedgehog, bone morphogenic proteins, members of the Hox family of transcription factors, and other transcription factors such as PU.1, GATA-1, growth factor independent-1 [144], and Forkhead 0 family members [146]. HSC self-renewal may involve symmetric and asymmetric division of the cells where at least one daughter cell maintains the pluripotentiality of the parent HSC. It may be that the best possibilities for ex vivo expansion will occur in synthetic three-dimensional models with specific types of scaffold [147]. In addition to ex vivo expansion of HSCs/HPCs, fully mature red blood cells have been generated ex vivo from populations of CD34+ UCB cells [148].
Gene therapy of UCB stem cells UCB HSCs are logical cellular vehicles for dissemination of the products of newly introduced genes. There is in vitro evidence that primitive UCB cells may be more efficiently transduced than equivalent cells in BM using retroviral vectors [149,150]. Primitive UCB cells are efficiently transduced by retroviral vectors [58,150,151]. A limited number of attempts at gene therapy were performed utilizing UCB in a clinical setting years ago [152]. Three children with adenosine deaminase (ADA)-deficient SCID received transplants of noncryopreserved autologous UCB CD34+ ADA gene-transduced cells in a reduced-intensity conditioning setting. Engraftment of the retrovirally ADA genetransduced cells was successful, but the level of engraftment was low [153,154]. Enhanced effectiveness of this procedure as a clinical effort awaits elucidation of better vectors and improved methodology to transduce the earliest noncycling HSCs, with maintenance of the self-renewal capacity of the transduced cells and high, but controlled, expression of the introduced genes.
Homing of UCB stem cells Enhanced homing of the small numbers of HSCs found in single collections of UCB is another potential option to increase the effectiveness of UCB transplantation. HSCs are nurtured and produced in the BM, where various cell types provide an appropriate niche for their development [155–162], although what this niche actually encompasses is not completely agreed upon [163]. While some subsets of mouse HSCs appear to home with absolute efficiency to the marrow, as a total population of HSCs they do not. It is likely that only a minority of HSCs that are infused into human recipients actually home to an appropriate microenvironment. Enhanced homing could result in a reduction in the number of cells necessary for engraftment and might shorten the time to engraftment. A clearer understanding of adhesion molecules and the agents that enhance expression and activation of these adhesion molecules will no doubt be of use. The cytokine family of chemokine molecules, along with other cytokines, may be intimately involved in the migration and homing of HSCs. Chemokines are chemoattractant molecules involved in chemotaxis (directed cell movement, as contrasted to chemokinesis, random cell movement) of leukocytes [164]. SDF-1/CXCL12 [165,166], CKβ-11/ CXCL19 [167], and SLC/CCL21 [168] have been implicated in the in vitro chemotactic migration of primitive cells from UCB and BM, and these chemokines act in concert with SCF [166], CKβ-11/CCL19, and SLC/CCL21, and are relatively specific for chemotaxis of macrophage progenitor cells [167,168], while SDF-1/CXCL12 is chemotactic for a wide range of progenitor cells (CFU-E, BFU-E, CFU-GEMM, etc.) [165,166]. SDF-1/CXCL12 and its receptor CXCR4 have been implicated in the homing of human stem cells into SCID mice [169–171],
and retroviral vector-induced overexpression of CXCR4 results in enhanced chemotaxis of cells to SDF-1/CXCL12 [172]. Some intracellular signals have been definitely linked mechanistically to the SDF-1/CXCL12 chemotaxis of primary HPCs. These include the phosphatases SHIP [173] and SHP-1 [174]. HPCs from mice functionally deleted in SHIP and SHP-1 were enhanced in sensitivity to SDF-1/CXCL12 chemotaxis, suggesting that SHIP and SHP-1 act to negatively regulate SDF-1/CXCL12-induced chemotaxis. Also implicated in this migration of HSCs/HPCs are transforming growth factor-β [175] and protein phosphatase 2A [176]. Since SDF-1/CXCL12 also acts as a survival factor for HPCs and HSCs [reviewed in 177–179], and blocks the myelosuppressive effects of inhibitory members of the chemokine family [180], this chemokine may play a dual role in homing to, as well as survival within, the marrow microenvironment. There are a number of different isoforms of SDF-1/CXCL12 [181]. While all demonstrated chemotactic properties, only the α, β, and ε forms enhanced the survival and replating capacity of HPCs. In addition, mice that are null for chemokine receptors, such as CXCR4 (the receptor for SDF-1/CXCL12) [182], CXCR2 (a receptor for IL-8) [183], and CCR1 (a receptor for macrophage inhibitory protein1 alpha) [184], have been used to suggest that these chemokine receptors may also be involved in the trafficking of murine HPCs. CXCR4−/− mice die perinatally with little or no hematopoiesis in the marrow. CXCR2−/− mice demonstrate a hyperplasia of hematopoietic cells, especially in the spleen, and CCR1−/− mice have abnormal tissue distribution of progenitor cells. Treating UCB HSCs or HPCs ex vivo with cytokines, or transducing these cells with genes for receptors or cytokines involved in homing, may permit the use of fewer HSCs for optimal engraftment [185–188]. Decreasing the amount or activity of CD26/DPPIV (dipeptidylpeptidase IV), a membrane-bound extracellular peptidase that cleaves dipeptides from the amino terminus of polypeptide chains, results in enhanced chemotaxis to SDF-1/CXCL12 [189]. CD26/DPPIV can cleave the chemokine CXCL12/SDF-1α at its position 2 proline and inactivate CXCL12/SDF-1. CD26/DPPIV is expressed by a subpopulation of CD34+ cells isolated from UCB and these cells have DPPIV activity. Amino-terminal truncated CXCL12/SDF-1α lacked the ability to induce migration of CD34+ UCB cells, but inhibited normal CXCL12/SDF-1αinduced migration. Inhibition of endogenous CD26/DPPIV activity on CD34+ UCB cells enhanced the migratory response of these cells to CXCL12/SDF-1α. Pretreatment of mouse BM cells with inhibitors of CD26 (e.g. Diprotin A, a tripeptide, or Val-Pyr, a dipeptide), or use of marrow cells from mice functionally deleted in CD26 (= CD26−/−), resulted in enhanced engraftment of self-renewing, long-term repopulating, and competitive HSCs [190]. Enhanced engraftment of human UCB CD34+ cells into NOD/SCID mice was observed after a 15-minute pretreatment of the CD34+ cells with Diprotin A to inhibit CD26 [191,192]. Treating recipient NOD/SCID mice with Diprotin A enhanced engraftment of G-CSF-mobilized human CD34+ cells, which expressed low or absent levels of CD26 [193], and pretreating lethally irradiated congenic mice with Diprotin A enhanced engraftment of untreated donor mouse BM long-term competitive repopulating and self-renewing HSCs [194]. It is possible that pretreating donor cells as well as recipients to inhibit CD26 may be more effective than treating only the donor cells or the recipients. Since CD26 can truncate certain growth factors [187], it is possible that inhibiting CD26 will have additional effects, perhaps enhancing the growth-promoting activities of these cytokines in vivo for enhanced recovery in a stem cell transplantation setting. Efforts are also ongoing to determine if direct injection of HSCs into the bone in a preclinical model will enhance the engrafting capability of these cells [195,196]. The concept here is to bypass loss of cells from
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the circulation that do not home well to the BM microenvironment. There is not enough information available yet in a clinical setting to know how effective this type of maneuver will be.
Immune cells An original concern with UCB transplantation was that UCB collections might contain maternal cell contamination, and thus elicit a severe and life-threatening GVHD reaction [7,9,10]. However, the original studies using relatively insensitive methods did not find any maternal T-cell contamination [7,9–11]. Further studies using much more sensitive technology found that maternal T cells do contaminate UCB collections in many cases [197–201], but the frequency of contaminating T cells was extremely low. This low frequency of contaminating T cells, in the absence of clinical data suggesting an enhanced incidence of GVHD in UCB transplantation [7,9,14,65–67,73], brings into question the clinical meaning of such a low frequency of T-cell contamination. Although the incidence of GVHD is apparently low in UCB transplant recipients, even in an unrelated HLA-disparate setting [65–67,73], the extent of GVHD has thus far not correlated well with the disparity of HLA between donor and recipient [65,73]. The apparently low incidence of GVHD noted in most UCB transplant recipients may reflect the biological activities of UCB T lymphocytes [202,203]. UCB T lymphocytes generate less cytotoxic T-cell activity than do adult T cells, even after repeated allogeneic stimulation in vitro [203–205]. Whereas UCB T lymphocytes respond as well as adult BM or blood T lymphocytes to the proliferation-inducing activity of a primary allogeneic stimulation, UCB T cells become unresponsive to secondary allogeneic stimulation, whereas adult T cells proliferate to an even greater extent in response to secondary allogeneic stimulation compared with their response to the primary allogeneic stimulation [206]. Investigation into the possible mechanisms mediating this UCB T lymphocyte unresponsiveness to secondary allogeneic stimulation has implicated the ras intracellular signal transduction pathway [207]. Stimulated lymphocytes from UCB have reduced the production of cytokines, such as interferon-gamma (IFN-γ) and tumor necrosis factoralpha (TNF-α), which may be involved in the pathophysiology of GVHD. As transcription factors of the nuclear factor of activated T cell (NFAT) family play a role in transcription of IL-4, IFÑ-γ, GM-CSF, IL-13, TNF-α, CD69, FasL, and CD40L genes [208], it is possible that NFAT-1, found to be low or not expressed in UCB T cells [209], might be involved in the lowered amplification of donor UCB T-cell alloresponsiveness against recipient antigens, and in the reduced GVHD noted in UCB transplantation. Cord blood T cells have also been shown to manifest distinct chemokine responsiveness by different chemokine receptor repertoires than T cells from adult blood [210], which may also be involved in differences noted in the extent of GVHD after UCB compared with BMT. Further analysis of UCB T cells focused on CD8+ T cells [211]. CD28 is a co-stimulatory molecule. It is the CD28−CD8+ T-cell subset that has been associated with cytotoxic T-lymphocyte (CTL) effector function. Cord blood is composed of CD8+ T cells with few or no CD28− CTLs. However, a combination of cytokine stimulation and activation of another co-stimulatory molecule in the TNF receptor family (41BB) leads to CTL effector function in the following sequence. IL-15 and IL-12 preferentially induced 41BB on CD28−CD8+ UCB T cells. Costimulation of these cells with anti-41BB restored expression of CD28 on these cells, as well as memory markers such as CD45RO and CCR6. The memory-type CD28+ T cells so generated acquired greatly enhanced CTL activity in association with increased content of granzyme B, a cytolytic mediator. Interestingly, the CTL activity could be almost completely abrogated by a soluble form of 41BB. In a follow-up study, it was noted that transforming growth factorbeta and 41BB modulated UCB CD8+ T-cell effector differentiation
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[212]. Transforming growth factor-beta is an immunosuppressive cytokine that inhibits CTL immune responses, and suppresses differentiation of naïve UCB CD8+ T cells. This effect was abrogated by 41BB costimulation, but not by CD28 or another member of the TNF receptor family, CD30. It was noted that 41BB co-stimulation suppressed TGFβ-induced phosphorylation of Smad2. Critical roles for IL-4 and IL-12 were reported for regulating 41BB effects on TGF-β-mediated suppression. It has been suggested that ex vivo expanded, matured, and activated UCB T cells with IL-2, IL-12, anti-CD3, and IL-7 may be of value for adoptive cellular immunotherapy post umbilical UCB transplantation [213]. It is possible that 41BB activation is also of importance in this type of adaptive immunotherapy. Forkhead Box P3+ (FOXP3+) T cells are unconventional as they suppress other immune cells and play critical roles in immune tolerance [214,215], factors that impinge on GVHD. FOXP3+ regulatory T cells belong to the T-cell receptor-alpha beta group. It was found that FOXP3+ T cells could be divided into CD45RA+ (naïve-type) and CD45RO+ (memory-type) cells. T cells undergo changes in trafficking receptors according to their stages of activation and differentiation [216]. In UCB, CD45RA+FOXP3+ T cells are in the majority, but CD45RO+FOXP3+ T cells become a majority in adult PB. A role for CD45RA+FOXP3+ UCB T cells and the switch to CD45RO+FOXP3+ adult T cells in GVHD is to be evaluated. It was originally thought that a lower GVHD incidence inherent in UCB transplantation might manifest in a lowered graft-versus-leukemia (GVL) effect with UCB cells. While increased relapse rates have not been obvious so far in UCB transplantation for malignant diseases, more experience in this area is needed before any firm conclusions can be drawn. NK cells have been implicated in mediating the GVL effect [217,218]. While UCB generally manifests low NK activity compared with that found in adult BM and blood, the NK cell activity of UCB and adult cells is readily activated in vitro by cytokines, such as IL-2 and IL-12 [217–219], suggesting that UCB NK cells should be as capable as adult BM or blood NK cells to mediate the GVL effect. Interestingly, a subset of NK cells with the surface phenotype CD16+CD56−, which has not yet been identified in the adult blood of normal individuals, has low lytic activity and might represent a precursor of mature NK cells. The lytic activity of CD16+CD56− cells can be enhanced by cytokine stimulation [220]. Functional quantitative activities of other UCB immune cells are also distinct from their counterparts found in the adult [202,217]. Selected cytokine production by T cells and dendritic cell (DC) antigen presentation are reported to be lower in UCB than adult blood cells. DCs are active antigen-presenting cells that initiate and maintain adaptive immune responses. Both lymphoid (HLA-DR+, CD123+, CD11c−, CD33−) and myeloid (HLA-DR+, CD123+, CD11c+, CD33+) DCs have been identified in UCB, although the majority of these cells had a lymphoid morphology [221]. The 41BB ligand was found to mediate maturation of CD11c+ myeloid DCs derived from CD34+ UCB [222]. In addition to enhanced expression of CD11c, other antigens, such as major histocompatibility class II, CD86, and 41BB ligand, were increased after stimulation of UCB CD34+ cells with granulocyte–macrophage colonystimulating factor (GM-CSF), TNF-α, Flt3 ligand, and SCF. Stimulation of the 41BB ligand on these DCs resulted in the capacity of these cells to produce IL-12, results that may be of use in the design of DC-based vaccines with enhanced activity. Freshly isolated UCB lymphoid DCs did not induce a potent allostimulation for naïve UCB T cells. CD34+ UCB cells can generate DCs. A CD7+CD45RA+ subpopulation of CD34+ UCB cells was found to have the dual capacity to generate NK cells and DCs [223]. Immature DCs induce tolerance, while mature DCs induce inflammatory immune responses. The question addressed was whether or not DCs
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could be generated that manifested anti-inflammatory properties in the immature and mature states [224]. Highly purified UCB monocytes were induced to differentiate into IL-10hiIL-12absent DCs in the presence of macrophage colony-stimulating factor (M-CSF) and IL-4, in a GM-CSFindependent manner. These M-CSF/IL-4-generated DCs, in contrast to GM-CSF/IL-4-generated DCs, induced decreased T helper type 1 differentiation and proliferation of naïve CD4+ T cells in both primary and secondary mixed leukocyte reaction, and showed tolerogenic potential, suggesting that these M-CSF/IL-4-generated DCs may be useful for suppressing unwanted immune responses. TGF-β added to the combination of M-CSF and IL-4 induced the generation of immune inhibitory UCB DCs that released cytokines that enhanced ex vivo expansion of human UCB HPCs induced with SCF, Flt3 ligand, and TPO [225]. TGFβ/M-CSF/IL-4-generated DCs secreted lower concentrations of progenitor cell inhibitory cytokines, demonstrated enhanced responses to bacterial lipopolysaccharide (LPS)-induced activation of ERK, JNK, and p38 MAP kinases, and were less potent in activating T cells than were DCs generated with TGF-β/GM-CSF/IL-4. The uniqueness of TGF-β /M-CSF/IL-4-generated DCs to silence immunity while promoting expansion of HPCs may be of potential therapeutic value. Functional activity of DCs in vivo likely relates to the migratory capacity of DCs. GM-CSF/IL-4/LPS DCs generated from UCB monocytes manifested decreased chemotaxis to respective CCR7 and CXCR4 ligands CCL19 and SDF-1/CXCL12 when compared with similarly generated DCs from adult blood monocytes [226]. Decreased chemotaxis of GM-CSF/IL-4/LPS-generated UCB DCs to CCL19 was associated with decreased expression of CCR7 on these cells compared with those generated from adult monocytes. However, UCB DCs expressed higher levels of CXCR4 than the adult DCs, and decreased chemotaxis of UCB DCs to SDF-1/CXCL12 was associated with a stronger and sustained activation of ERK.
Additional information and conclusion Experience with UCB HCT is encouraging. However, worldwide experience with UCB transplantation is still relatively limited. The use of UCB for HCT has advantages and disadvantages [227]. Among the advantages are rapid availability, ability to more rapidly schedule the transplant as the UCB units are stored and ready for use, the apparent reduced need for an exact HLA match, and induction of a less severe GVHD compared with BM. It offers the potential of targeting collections in minority populations, groups that are currently underrepresented in the NMDP registry. Compared with matched or mismatched BM, greater degrees of HLA disparity appear to be tolerated with UCB. As allogeneic UCB banks reach sufficient size, the time required to acquire a donor unit should decrease, allowing transplantation to occur at an earlier time. The establishment of the HRSA-funded UCB Coordinating Center at the NMDP, and enhanced collections of quality UCBs to be banked for future use, should increase the availability of UCB units for transplantation. Disadvantages include the limited numbers of cells collected and stored, a relatively limited inventory enabling at least four-of-six matches with an adequate cell dose for many patients (e.g. differing races and ethnicity), the lack of additional cell collections from the UCB donor, and the noted variability in terms of UCB unit quality after thawing of the stored cryopreserved cells. As with non-UCB sources of hematopoietic cells for transplantation, there are a number of factors associated with transplant outcome [228]. These include the donor, graft, patient, conditioning regimen and prophylactic therapy for GVHD and GVL, treatment-related mortality, relapse, and disease-free survival. While there is increasing information on this with UCB for HCT, there is clearly more information needed,
especially for adult recipients. Thus far, it appears that UCB can be used to treat all malignant and nonmalignant disorders currently treated with BM and mobilized PB cells. As the criteria for selecting the best UCB unit for adults are better defined, and more effective conditioning and prophylactic measures are used, it is likely that the results of UCB HCT will improve. It is known that HLA mismatches play an important role in engraftment, GVHD, and survival, but with UCB, the effect of HLA mismatches is partially abrogated by increased UCB cell dose [228,229]. Results of unrelated UCB HCT for patients with Fanconi’s anemia have demonstrated that factors that can be modified easily, including better UCB unit selection, and a fludarabine-containing regimen has a significantly enhanced therapeutic endpoint for patient survival [230]. In addition to a delay in neutrophil and platelet recovery when UCB is used for HCT, there is also some evidence of delayed immune cell reconstitution, which appears to reflect impaired thymopoiesis and skewing of late memory T cells [231]. There are also reports of an increased risk for opportunistic infections after UCB HCT, mainly of viral origin [232]. Infection-related mortality is a primary or secondary cause of death (in the presence or absence of another major problem such as GVHD) in 50% or more of deaths after UCB HCT. This infection-related mortality within the first 100 days after transplant was higher among recipients of mismatched UCB than HLA-matched or mismatched BM [233], although beyond 100 days there was no significant difference in the three groups. Interestingly, recipients of UCB HCT are reported to manifest similar risks of infection with cytomegalovirus, responses to antiviral therapy, and survival following this viral infection compared with BM or mobilized PB HCT [234]. Not only is UCB HCT associated with lower levels of acute GVHD, but chronic GVHD may be more responsive to therapy after UCB HCT [235]. Post-transplant donor-acquired leukemia is a rare event in HCT, and there is not any evidence that this occurs more frequently in UCB HCT compared with other tissue sources of HCT [236]. However, more information in this area is clearly warranted for all tissue sources of HCT. Since the volume of each UCB available for collection is limited, means to enhance the number or quality of HSCs in UCB is needed. Manipulation of UCB through ex vivo expansion is still a sought-after endpoint, but enhancing the capacity of the UCB HSCs to home to an appropriate microenvironment in the marrow may serve a similar purpose. In the meantime, the problem of limiting numbers of UCB cells is being addressed by use of double UCB for transplantation. In the unrelated UCB setting, it is not possible to reacquire UCB from the donor when the recipient experiences graft failure or relapse. Ex vivo expanded cells stored for future use or the use of another stored UCB unit may circumvent these possible problems. Despite proposals to require long-term tracking of unrelated UCB donors, there is currently little clinical information available on unrelated UCB donors, such that the possibility exists that genetic diseases may be inadvertently transferred to UCB recipients. Enhanced genetic testing in the future will be of use here. Neutrophil, and especially platelet, engraftment in UCB recipients is prolonged. In vivo or ex vivo use of cytokines or expanded mature cells may be of help. Newly identified cytokines and greater knowledge of the intracellular signaling events they trigger for proliferation and self-renewal of HSCs will be helpful. Future anticipated efforts in the field of UCB transplantation include: (1) greater success in efficiently engrafting adults and higher-weight children with a single UCB collection, thus decreasing the potential for enhanced GVHD possible with the use of multiple UCB units; (2) enhanced downmodulation of GVHD and upmodulation of the GVL effect, perhaps by using MSCs, DCs or other immune cell types; mechanistic insight into MSC-mediated immune suppression is increasing [237]; and (3) knowledge of whether UCB is or is not also efficacious for regenerative medicine in a nonhematopoietic situation.
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40
Alan W. Flake & Esmail D. Zanjani
In Utero Transplantation
Introduction For most of human history, the fetus has been shrouded in mystery, obscured by multiple layers of maternal tissue, and protected from the scrutiny and intervention of well-meaning physicians. In the past three decades, this private world of the fetus has been invaded by a vast array of imaging techniques and methods for fetal tissue sampling, allowing the early gestational diagnosis of selected congenital hematologic disorders. Over the past 10 years, progress has accelerated in a number of technologies that are destined to revolutionize the field of prenatal diagnosis. The convergence of knowledge from the human genome project, molecular genetic diagnosis, isolation of fetal cells or DNA from the maternal circulation, and techniques of high through-put gene analysis will, in all likelihood, allow population screening and diagnosis of every genetically based hematologic disorder, early in gestation. In many instances, the early prenatal diagnosis of a disease provides a rationale for prenatal treatment [1]. This rationale is particularly compelling for diseases that cause death or irreversible organ damage before birth. In this circumstance, by the time of birth, the damage has already been done. Examples of successful treatment of lethal fetal disease are now numerous and include fetal transfusion for erythroblastosis fetalis [2] and prenatal correction of selected anatomic malformations [3]. Another compelling rationale for fetal therapy exists when there are biological advantages for prenatal therapy relative to postnatal therapy. In this circumstance, unique aspects of fetal development may allow the treatment of a disease with reduced morbidity, mortality, and cost compared with treatment after birth. This is the rationale for in utero hematopoietic stem cell (HSC) transplantation. It is the purpose of this chapter to review the theoretical, experimental, and, at the present time, limited clinical support for this approach.
An experiment of nature Perhaps the most compelling argument for the potential of in utero hematopoietic cell transplantation (IU-HCT) comes from an experiment of nature. Owen [4], in 1945, observed that dizygotic cattle twins that shared a placental circulation were chimeric for their sibling’s blood cells after birth. Subsequent investigators confirmed that chimeric animals were specifically tolerant for skin grafts [5] and organ grafts [6]
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
from their sibling donor. Natural chimerism arising from shared placental circulation has also been observed in a number of other species, most notably primates [7] and humans [8]. The cotton-top tamarin, a New World primate species, has a high incidence of natural chimerism, with documentation of stable donor cell chimerism of greater than 80% in some animals. The incidence of human chimerism in monochorionic, dizygotic pregnancies is around 8% when sensitive detection methodology is utilized, and natural human chimeras with all levels of donor cell chimerism, including marked donor cell predominance [9], have been observed. These observations are proof in principle for IU-HCT. They confirm that circulating allogeneic stem cells can effectively compete and stably engraft an early gestational recipient, and that, at least in specific circumstances, normal host hematopoiesis is not prohibitive to the engraftment of donor cells. The primary question is, “What are the ideal circumstances to permit experimental and clinical recapitulation of this experiment of nature?”
Fetal immunologic tolerance Owen’s observations of natural chimerism were followed by Burnet’s [10] theory of immunity, which subsequently led to the experimental work of Billingham and Medawar [11] promoting the concept of “acquired” immunologic tolerance. These studies suggested that the early presentation of cellular antigen resulted in specific immunologic tolerance. Their studies documented that tolerance could only be achieved during a period of immunologic immaturity and was best accomplished by the transplantation of living cells. More recent studies have documented the role of the fetal thymus in self-recognition and tolerance induction [12,13]. The human thymus becomes populated by prethymocytes at 8–9 weeks’ gestation [14]. Thymocytes undergo a series of differentiation and selection events in the thymus before becoming mature functional lymphocytes. The details of thymic processing are still being investigated, but the critical events have now been defined [15]. Thymocytes are positively selected for recognition of self-class I or class II major histocompatibility complex (MHC) antigens, which are presented by thymic cortical epithelial cells of thymic stromal origin. Lack of MHC recognition results in programmed cell death. Thymocytes that recognize self-MHC are then negatively selected by high-affinity recognition of “self”-antigen in association with self-MHCs. Thymic deletion occurs in the medullary region of the thymus and is mediated by thymic epithelial cells and thymic dendritic cells that are derived from HSCs. The end result is a repertoire of single positive (CD4+ or
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CD8+) functionally competent lymphocytes that recognize foreign antigen in association with self-MHC. Single positive (post-thymic) lymphocytes are first seen in the peripheral blood circulation at around 12–14 weeks’ gestation [16]. Theoretically, introduction of foreign cells prior to completion of this process would result in thymic processing of foreign cells as “self” with secondary specific tolerance on the basis of clonal deletion. With improved understanding, it is clear that this early assumption in the field of IU-HCT is an oversimplification. In reality, during normal development, self-reactive T-cell clones routinely escape thymic deletion by a number of mechanisms [17–20]. It has been recently appreciated that these cells rarely cause autoimmune disease due to peripheral mechanisms of tolerance primarily involving regulatory T cells [21,22]. In the circumstance of clinical or experimental IU-HCT, donor cells are delivered after the process of thymic selection has begun, and donor antigen presentation in the host thymus may or may not be optimal. While partial deletion of donor reactive lymphocytes after IU-HCT has been clearly demonstrated [23,24], it is likely that a significant number of donor reactive cells escape thymic deletion after IU-HCT, and that the induction of peripheral regulatory mechanisms will be important for success in IU-HCT. We [25], and others [26,27], have documented evidence of an immune barrier after IU-HCT, which will be elaborated on below. In addition, while T cells may be implicated, there are other mechanisms of potential rejection after IU-HCT, including B-cell mediated responses and the innate immune system, that remain relatively poorly understood.
The fetal hematopoietic environment A biologically unique aspect of IU-HCT is the fetal receptive environment. Whereas postnatal bone marrow transplantation (BMT) generally requires myeloablative or at least minimal conditioning to achieve engraftment, such conditioning would be prohibitively toxic to the fetus. Thus, engraftment following in utero HSCs, by necessity, must depend upon competitive population of available receptive sites with a goal of
achieving an adequate level of mixed chimerism to have therapeutic effect for a given target disorder. The receptive sites in the early gestational environment are undoubtedly dynamic and are dependent upon the gestational age of the fetus. Fetal hematopoiesis is characterized by an orderly series of migrational events that proceed from the yolk sac or periaortic splanchnopleura, or both, to the fetal liver, and finally to the bone marrow [28,29]. Thus the migration of fetal hematopoiesis is probably best viewed as a sequential development of organ-specific microenvironmental niches of increasing HSC affinity [30]. At the developmental stage that corresponds to fetal immunologic immaturity in multiple species, the primary receptive site is the fetal liver. Studies in which tracking of transplanted cells have been performed in the sheep [31] and mouse [32,33] models suggest that the pattern of engraftment after IU-HCT recapitulates ontogeny; i.e. prior to bone marrow formation cells home to the fetal liver but once the bone marrow forms, even fetal liver-derived cells will preferentially home to the fetal bone marrow. The early dogma of this field predicted that the rapid expansion of the fetal hematopoietic compartment would favor the availability of open niches for engraftment of donor cells, and that there would be a “window of opportunity” prior to immune competence. While “space” is available prior to population of the bone marrow, this has proven to be only partially true (Fig. 40.1). While it is certainly true that the immunologic opportunity exists, the efficiency of engraftment of bone marrow cells after in utero transplantation is no better than that after postnatal myeloablative BMT, with only approximately 5% of cells homing to and engrafting receptive sites in the fetal liver [32,33]. This suggests that the fetal receptive environment, with a few disease-specific exceptions, is highly competitive and that strategies will need to be developed to improve the competitive capacity of donor cells to achieve significant engraftment.
Animal studies that support in utero transplantation The first experimental studies that supported engraftment of donor cells after in utero transplantation were the classic studies of Billingham and
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Fig. 40.1 A schematic of normal hematopoietic ontogeny depicting the concepts of an early immunologic “window of opportunity” and increasing host hematopoietic competition. The late limit of immunologic tolerance in humans has not been defined and may extend into later gestation in immunodeficiency disorders such as severe combined immunodeficiency disease.
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Fig. 40.2 Maximal donor cell engraftment occurs during the mid- to later stages of the preimmune period. This period is associated with the development of bone marrow in sheep which is populated by donor stem cells in competition with endogenous hematopoietic cells. The failure of donor cells to engraft in very young fetuses may be explained by the absence of suitable receptive sites.
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Fig. 40.3 Levels of engraftment plateau with transplantation of log-fold increases in donor cells. This is most consistent with a model of saturation kinetics with engraftment limited by available receptive sites. An identical curve is seen in the xenogeneic human–sheep model.
40
Medawar [11], who documented donor-specific tolerance for skin grafts after fetal or neonatal transplantation in mice. Although chimerism was not analyzed, it can now be assumed that tolerant animals were chimeric for donor cells. Large animal models In order to determine whether engraftment of allogeneic HSCs could be achieved in a large animal model, we developed an in utero HSC transplant approach in sheep that results in multilineage allogeneic engraftment without the need for myeloablation [34] and, under appropriate conditions, without graft-versus-host-disease (GVHD] [35]. Engraftment after IU-HCT has for unexplained reasons been easier to achieve in the sheep model than in other species and man, raising concern regarding the clinical applicability of findings in the sheep model. However, the sheep model has provided a number of basic observations that have translated well on a developmental stage-for-stage basis with other animal models. The relationship of gestational age at transplantation and engraftment has been examined carefully in the sheep model with confirmation of the concept of a presumed immunologic “window of opportunity.” Late gestational transplantation results in failure of engraftment, with loss of the ability to engraft roughly corresponding to the gestational age at which fetal lambs reject allogeneic skin grafts (75 days gestation) (Fig. 40.2). A similar window exists in the murine model where engraftment can be achieved across full MHC barriers by IU-HCT at embryonic days 14–15, but not in newborn mice. The relationship between dose of donor cells and engraftment has also been examined in the sheep model. Logfold increases in the dose of transplanted cells initially increased engraftment, but this rise rapidly plateaued, suggesting saturation of available receptive sites (Fig. 40.3) [36]. If this theory were true, then transplantation of cells in divided doses should allow the development of new receptive sites between transplants and should improve engraftment. Indeed, in the allogeneic sheep model, transplantation of the same total number of cells in three divided doses increases engraftment by a considerable margin (Fig. 40.4). The importance of route of administration of donor cells has also been examined in sheep. Higher levels of donor cell engraftment have been
Donor cells (%)
35 28.3 ± 6.9
30 25 20
n = 13
15
10.9 ± 3.6
10 5
n=8
0 3
1 Number of injections
Fig. 40.4 The effect of dividing cell dose on engraftment. Transplantation of the same total number of cells as a single injection versus a series of three injections given 1 week apart is shown. The engraftment with divided doses is significantly higher, supporting the concept that engraftment is limited at any particular time by the number of available receptive sites.
obtained when cells are administered by intraperitoneal injection than by intravenous injection. Although we are unable to provide an explanation for this finding, this difference has been observed on many occasions, and for that reason we have utilized the intraperitoneal approach for most studies. In more recent murine studies, we have utilized the vitelline vein for intravascular injection [25,33,37]. This has allowed injection of far greater doses of cells (20 million versus 5 million per fetus), resulting in a higher frequency of engraftment of congenic recipients (100% maintaining long-term chimerism) with similar levels of chimerism. Interestingly, it has not resulted in a significantly increased frequency or level of chimerism in allogeneic recipients. In the context of tracking studies demonstrating equivalent homing, expansion, and migration early after IU-HCT in congenic and allogeneic strains, this suggests that the frequency of chimerism is limited by immunologic barriers and that the level of chimerism is limited by competitive barriers. Other large animal models in which IU-HCT has been attempted include primates [38–43], pigs [44,45], goats [46,47], and dogs [48–51]. Of these, the primate and pig have been most successful. It has been shown in the rhesus model that fetal liver-derived hematopoietic cells
580
Chapter 40
can engraft and maintain long-term low-level multilineage chimerism [38], but that T-cell depleted adult bone marrow resulted in polymerase chain reaction-detectable chimerism at best [43]. In the baboon model, similar low levels of chimerism have been obtained with CD34-enriched adult-derived cell sources [40,41]. In the baboon model, the add-back of T cells to the CD34-enriched donor cells improved the level of chimerism but was limited by GVHD [42], results very similar to those in the sheep model [52]. The use of fetal immune suppression (steroids and antithymocyte globulin) resulted in a transient but not sustained improvement in peripheral blood donor chimerism levels [41]. Recently, interesting results in the swine leukocyte antigen-defined miniature swine model have been reported. Injection of a mixture of T-cell-depleted bone marrow with addition of unmanipulated whole bone marrow to a level of 1.5% T-cell content into the intrahepatic portion of the umbilical vein resulted in long-term, multilineage chimerism at easily measurable levels by flow cytometry in five of seven surviving pigs at 2 months and in two long-term survivors up to 70 weeks of age [45]. Tolerance for donor swine leukocyte antigen was demonstrated by transplantation of donor-matched kidney allografts without the need for immunosuppression [44]. These results need to be reproduced and expanded upon but have major implications for clinical application of IU-HCT as primary therapy or as a means achieving donor-specific tolerance to facilitate postnatal cellular or organ transplantation. The murine model With the exception of the sheep and recently the pig model, engraftment in other species has been difficult to achieve. The first studies in the mouse were the classic studies of the correction of genetic anemia by Fleischman and Mintz [53] in W/Wv mice by transplacental injection of normal HSCs. The W/Wv mouse strain is genetically deficient in the c-kit receptor, resulting in a selective advantage for normal stem cells [54]. In this model, a single normal stem cell can engraft and reconstitute normal hematopoiesis after IU-HCT [55]. Blazar et al. [56] demon-
100 Pep3b n=6 n=4
CD45+ cells (%)
80 70
B6
Congenic
50 40 n=4
20 10
n=6
n=7 n = 28
0 0 (a)
20
90
SJL
Liver Cord blood
B6
80
60
30
100
Liver Cord blood CD45+ cells (%)
90
strated in a murine severe combined immunodeficiency disease (SCID) model that in utero transplantation of congenic or fully allogeneic adult bone marrow could result in full T-cell and partial B-cell reconstitution and a functionally intact immune system. These studies demonstrated the important concept that in experimental circumstances of a selective donor stem cell or lineage advantage, IU-HCT can result in functional reconstitution and disease correction. However, most potential therapeutic applications of IU-HCT do not provide a competitive advantage to donor cells, and cumulative experimental and clinical experience suggests that the prenatal environment presents significant barriers to engraftment. The likely barriers to engraftment have been reviewed elsewhere [57] and can be categorized as a lack of available space, host cell competition, and immunologic barriers. In order to systematically examine the relative importance of these barriers and develop strategies to overcome them, the development of the normal allogeneic murine model was critical. The available inbred and transgenic strains, defined immunology, large litter size, minimal cost, and short gestation of the mouse allow studies to be performed that simply cannot be considered in other species. In addition, the mouse is, stage for stage, very similar to the human with respect to hematopoietic and immunologic ontogeny. In utero HSC transplants can be performed early in gestation (11–15 days) during a time when hematopoiesis is confined to the fetal liver, and prior to completion of thymic processing and the appearance of mature lymphocytes in the peripheral circulation. Finally, the normal mouse initially proved very difficult to engraft, making it a legitimate model for investigation of barriers to engraftment. We, and others, were only able to achieve microchimerism for several years in hematopoietically normal mouse strains [24,27,58–61]. To assess the issue of receptive space available in the fetal environment, we have assessed early events after IU-HCT in different studies with different modes of cell administration (intraperitoneal and intravascular) [32,33]. The results were remarkably consistent, demonstrating that engraftment efficiency was no better in the fetal recipient than in the adult BMT recipient and that only approximately 5% of donor bone marrow cells home to the fetal liver (Fig. 40.5). In response to the initial
40
n = 13
60
100
Allogeneic
40 30 n = 16 n=5
n=8 n=9
n=4
n=4
n = 16
n=8
0
120
Time (hours)
n = 11 n=7
50
10
n = 24 80
60
20
n = 10
n = 28
70
0
20
40
60
80
100
120
Time (hours)
(b)
Donor cell type
FL chimerism at 4 hours (%)
No. of CD45+ cells per recipient FL
No. of homed CD45+ cells
No. of CD45+ cells in donor innoculum
Homing efficiency (%)
Pep 3b BM SJL BM
19 ± 6.3 16.9 ± 7.7
2.6 ± 0.8 × 105 2.6 ± 0.8 × 105
4.8 ± 1.6 × 104 4.3 ± 1.9 × 104
9.9 × 105 9.9 × 105
4.9 ± 1.6 4.4 ± 2.0
(c)
Fig. 40.5 These graphs depict the triphasic kinetic profiles for the frequency of donor cells circulating within the recipient fetal peripheral blood (cord blood) or lodged within the fetal liver at various time points (1.5–96 hours) after in utero transplantation in the murine model. The nearly identical profiles for both the congenic (a) and the fully allogeneic (b) strain combinations are shown. The donor cell frequency is calculated as the percentage of cells expressing the donor CD45.1 isoform among all CD45.1 cells within the host cord blood or fetal liver. (c) Efficiency calculations for the homing of donor hematopoietic cells to fetal liver (FL) in this model. Only approximately 5% of transplanted cells home and engraft. (Adapted from [32], with permission.)
In Utero Transplantation
Donor cells* (%)
study, we improved our cell preparations and performed a dose escalation study to investigate the effect of increasing cell doses. With improved delivery of higher numbers of HSCs, we were able to achieve macrochimerism in multiple allogeneic strain combinations. The level of chimerism was dependent upon cell dose, on cell source, with fetal liver being far superior to adult bone marrow at equivalent doses, and on strain combination (Fig. 40.6). The second barrier of host cell competition may be the most important. It is clear from studies of in utero transplantation utilizing mouse strains that have a genetic deficiency in HSCs [53] or a specific lineage deficiency [56,62,63] that hematopoietic repopulation or lineage replacement can be achieved with very low levels of HSC engraftment and, in the case of c-kit-deficient mice, engraftment of a single HSC [55]. A strategy to improve engraftment would therefore be to selectively improve donor cell competitive capacity by conferring a homing or engraftment advantage at the time of transplantation. We have recently demonstrated that improving donor cell homing to the fetal liver by blockade of the dipeptidyl-peptidase CD26 (preventing inactivation of the chemokine stromal cell-derived factor-1) [64,65] can significantly increase short- and long-term engraftment in the murine model [66]. This is an important proof in principle that manipulation of a single component of the homing/engraftment sequence can have a major effect on engraftment. Similarly, as described below, the conferment of a competitive advantage after birth to donor
1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2
n = 16
cells after establishment of low-level chimerism by in utero transplantation results in conversion to high-level or complete chimerism [23,67,68]. Thus, assuming no immune rejection, after initial engraftment, the size of the donor-derived hematopoietic compartment will depend upon the ability of donor cells to compete with host hematopoiesis. Finally, the immunologic barriers to engraftment remain to be defined. The early gestational fetal environment immunologically differs from the postnatal environment and can be considered in the context of specific and innate immunity. There is little evidence that the fetus, prior to maturation of T cells in the thymus, can mount a specific immune response. Long-term persistence of allogeneic [34] or xenogeneic [69,70] cells and multilineage hematopoiesis in the sheep model suggest that donor-specific tolerance can be achieved by IU-HCT. Our more detailed analysis in the murine system clearly documents that, when engraftment is successful, tolerance is achieved by a predominant mechanism of bidirectional deletion of alloreactive lymphocytes augmented by peripheral mechanisms of tolerance after IU-HCT [23,67,71,72]. Tolerance is consistently present in animals with levels of peripheral blood chimerism of greater than 1% (macrochimerism) [23] but inconsistent in microchimeric animals [24]. We have documented a predominant mechanism of high-level clonal deletion in macrochimeric animals by analysis of relevant Vβ-T-cell receptor consistent with thymic processing of donor antigen (Fig. 40.7).
15
n=6
n = 11
70 60
n = 14
50
10
n = 18
40 30 5
20
n = 16 SJL
Pep
n=9
Donor strain (10 cells)
10
106
Balb/c 6
(a)
581
107 TCD
Donor cell dose
(b)
n = 12 105
(c)
106
5 × 106
Donor cell dose
Fig. 40.6 The effect of strain combination, dose, and cell source on donor cell engraftment after in utero hematopoietic cell transplantation. (a) Peripheral blood chimerism in B6 recipients of adult bone marrow from three different strain donors. (b) Peripheral blood chimerism of Balb/c recipients of B6 bone marrow at low and high doses. (c) Peripheral blood chimerism in B6 recipients of various doses of SJL fetal liver, demonstrating the dramatically higher levels of dose-dependent engraftment achieved with fetal liver-derived donor cells. (All levels measured at 6 months of age.) TCD, T-cell depleted; B6, C57BL6; Pep, C57BL/6; Ly5.1:Pep3B. *Note the difference in scale of the y-axis for the three figures.
B6 control (n = 6) BALB control (n = 6) BALB chimera (n = 12)
6
12
5
10
4 3
*† * *†
2
*
8 6
*
4
1
2
0 (a)
BALB/c control (n = 6) SWR → BALB/c chimera
14
Vβ6+ (%)
Donor CD3+ cells (%)
7
0 Vβ5.1/2
Vβ11
Vβ12
(b)
CD4+
CD8+
Fig. 40.7 Evidence of bidirectional clonal deletion as a mechanism of tolerance in macrochimeric animals created by in utero hematopoietic cell transplantation (IU-HCT). Donor and host strains are chosen based on the presence or absence of class II I–E, which is required for the presentation of mammary tumor virus (mtv) antigens. Mtv antigens are superantigens encoded by Mtv oncogenes in the host genome that are normally deleted in the host strain but not the donor strain. Thus, donor antigen presentation in the thymus can be limited to donor-derived antigen-presenting cells (APCs), i.e. direct antigen presentation, or to host-derived APCs, i.e. indirect antigen presentation. (a) Efficient deletion of donor host-specific Vβ T-cell receptor (VβTCR) in chimeric mice by direct antigen presentation after IU-HCT. *p < 0.001 chimeric mice versus B6 control; †p < 0.05 chimeric mice versus Balb/c control. (b) Partial deletion of host donor-specific VβTCR by indirect antigen presentation in SWR→Balb/c chimeras after IU-HCT *p < 0.001. (Adapted from [23], with permission.)
Chapter 40
These data, along with the observation by ourselves and others that there was no difference in engraftment between congenic and allogeneic cells after IU-HCT [56,58,73–75] formed the basis of our belief for many years that there was not a significant adaptive immune barrier to allogeneic engraftment. We have now re-examined this question in the murine model using much higher doses of cells (enriched HSC and bone marrow mononuclear cells [MNCs]), made possible by the use of the intravascular injection technique [37]. In this circumstance, where there is an excess of donor HSCs, there are major differences in the frequency of engraftment between congenic and allogeneic HSCs or bone marrow. In addition, using new methodology for tracking of injected cells, we have found that all animals are engrafted by allogeneic cells, and that there are no differences in early engraftment, expansion or migration between congenic or allogeneic cells up to 2 weeks after transplantation. However, by 5 weeks, only 30% of the allogeneic animals remain chimeric and go on to become long-term chimeras, whereas 100% of congenic animals maintain their engraftment [25]. This argues against a role for the innate immune system (which should act early after transplantation) and clearly implicates the adaptive immune system. The explanation for the variability in engraftment and inconsistent achievement of tolerance remains to be defined. A possibility, however, is that significant numbers of donor-reactive cells escape thymic deletion after IU-HCT, and that whether a graft is rejected or tolerance occurs depends on the balance of donor-reactive T cells and donor-specific T-regulatory cells. In summary, experimental results from animal models support the rationale and feasibility of IU-HCT. Although engraftment can be achieved clinically in circumstances of a donor cell selective advantage, the primary limitation to clinical application for most target disorders is the relatively low level of donor cell expression achieved. The future of IU-HCT depends upon developing successful strategies to overcome the barriers to engraftment, specifically the immune response, and host cell competition. There are a number of strategies being tested in animal models that show considerable promise and will be discussed below.
Strategies for successful in utero HSC transplantation The potential clinical applications of IU-HCT fall into two general categories. The first is reconstitution or replacement of a stem cell or lineage defect or deficit. The second is prenatal tolerance induction for facilitation of postnatal cellular transplantation. In order for either strategy to be successful, an adequate number of donor HSCs must initially engraft to provide ongoing hematopoiesis at a therapeutic level or to provide adequate thymic presentation of antigen for stable tolerance induction. One of the major problems clinically is the inability to achieve greater than polymerase chain reaction-detectable engraftment using enriched cell populations. The achievement of “microchimerism” is probably inadequate for either purpose as it appears to be associated with the engraftment of very few if any HSCs and does not reliably induce tolerance by a mechanism of clonal deletion. Therefore one of the major clinical challenges is to achieve higher initial levels of HSC engraftment. As conventional myeloablation is not an attractive option in the fetus, an alternative approach has been to attempt to increase available “space” by stromal co-transplantation [76,77]. Although the mechanism remains unclear, it has now been documented in both the allogeneic and xenogeneic sheep models that stromal co-transplantation leads to increased short- and long-term levels of donor cell engraftment in the bone marrow and donor cell expression in the peripheral blood. In the allogeneic model, the co-transplantation of adult stroma (plastic adherent bone marrow cells) resulted in greater enhancement of early donor peripheral
blood expression (60 days after transplantation) than fetally derived stroma, and the enhancement of expression was sustained for at least 30 months (Fig. 40.8). This observation, in combination with previous findings of delayed contribution of bone marrow-engrafted donor cells to peripheral expression in the sheep model [31], suggests that fetal stroma may be deficient or immature in its capacity to support definitive hematopoiesis, adding an additional rationale for co-transplantation of stroma, particularly in circumstances where high levels of donor cells are required early after in utero transplantation. There are a variety of other potential approaches to improve initial engraftment but these await experimental support. At the present time, attempts at in utero reconstitution or replacement have only been successful in circumstances where a competitive advantage exists for donor cells. It would therefore follow that, in order to achieve replacement of host cells in a competitive environment, one would need to either increase the competitive capacity of donor cells or reduce the competitive capacity of host cells, or both. A particularly promising approach is to increase the competitive capacity of donor cells postnatally after establishing low-level chimerism and tolerance by IU-HCT. This approach is based on the observation that engraftment can be achieved without cytoreduction in syngeneic models, arguing that, in the presence of tolerance or effective immunosuppression, cytoreduction may not be required [78,79]. This observation has been validated in allogeneic systems by the success of a variety of nonmyeloablative strategies for postnatal BMT in circumstances of host immune deficiency or tolerance [80,81]. We have applied three nonmyeloablative strategies in the mouse model to alter the competitive balance of the donor and host cell compartments in tolerant mixed chimeric animals after IU-HCT. In the first strategy, donor lymphocyte infusion was utilized to induce a graftversus-host hematopoietic effect. This resulted in a donor lymphocyte dose-dependent conversion of mixed chimerism to complete donor chimerism across a fully MHC-mismatched strain combination (Fig. 40.9). Surprisingly, only one of 56 animals receiving the high dose of donor lymphocytes with conversion to complete donor chimerism developed evidence of GVHD, suggesting that the mixed chimeric recipient created by IU-HCT may be a favorable candidate for donor lymphocyte infusion-induced enhancement of engraftment relative to the postnatal irra-
30 25 Donor hemoglobin (%)
582
20 HSC + stroma 15 10 HSC alone
5 0 5
15
25
35
Months post-transplant
Fig. 40.8 Co-transplantation of adult sheep stromal cells results in a sustained increase in donor-derived hematopoiesis. Sustained long-term engraftment that has persisted for more than 30 months after transplantation is shown, where the donor hemoglobin levels in peripheral blood in animals receiving adult bone marrow stroma (n = 4) are significantly higher (p < 0.01) than those receiving adult T-cell-depleted bone marrow mononuclear cells alone (n = 3). (Reproduced from [76], with permission.)
In Utero Transplantation
diation-induced chimera. Potential reasons for reduced GVHD in our study include the lack of irradiation with its accompanying proinflammatory cytokine milieu, which may lower the threshold for GVHD in nonhematopoietic tissues, or the presence of a highly active, nonirradiated thymus for the production of regulatory T-cell populations. These animals maintain donor-specific tolerance with bidirectional deletion of alloreactive lymphocytes, and normal immune response to third-party or novel T-cell-dependent antigens. This study is indicative of the potential for this approach as it is, to our knowledge, the first time that complete allogeneic chimerism has been achieved across full MHC barriers in the complete absence of cytoreductive or immunosuppressive therapy [23].
n = 17 n = 10
n=6 n = 14
The second minimally ablative approach that we have investigated was suggested by results of marked enhancement of engraftment in the syngeneic nonmyeloablative model when low-dose total body irradiation (TBI) was administered prior to transplantation [82]. When we administered graded doses of minimal TBI to our tolerant mixed chimeric animals following IU-HCT and then retransplanted the animals with 30 million T-cell-depleted donor bone marrow cells, we observed a TBI dose-dependent enhancement of engraftment to levels of greater than 90% donor peripheral blood expression with the highest TBI dose of 276 cGy [67] (Fig. 40.10). Similar results were obtained in a more recent study utilizing single-agent (busulfan) chemotherapy for conditioning the host [68]. These studies confirm the predominant importance of
n=6
n = 17 n=5
80
80
40 20
n=6
60 40 20
0
0 0 1 2 3 4 6 8 12 16 20 24 36 48
(a)
Weeks after DLI n=9 n = 10
0 1 2 3 4 6 8 12 16 20 24 36 48 (b)
n=9 n = 15
Weeks after DLI
n=7
n = 13 n=8
8 week old, low level chimeras ( 10%)
100
Donor cells (%)
Donor cells (%)
n=8 n = 14
4 week old, high level chimeras ( >10%) 100
Donor cells (%)
Donor cells (%)
4 week old, low level chimeras ( 4500; median 14) [24]. In patients with NHL, there was less than 1 log difference in the concentration of tumor cells in BM than in PBHCs. When allowance was made for the greater number of PBHCs used, there was no difference in the total number of tumor cells within the collected products [25]. Taken together, these data demonstrate that it is naïve to assume that PBHC collections are free from contaminating tumor cells. Clinical trials examining the question of tumor contamination and its clinical significance for subsequent outcome after HCT using PBHCs continue.
Strategies for purging of malignant cells At the same time that techniques were being developed to demonstrate the existence of MRD, attempts were being made to develop methodologies to deplete contaminating malignant cells without impairing hematopoietic progenitor cells, including negative purging with pharmacologic agents and mAbs, positive selection with immunoadsorption, immunomagnetic and flow cytometry sorting techniques, ex vivo culture, treatment with cytotoxic T cells, and virally mediated purging (Table 42.2). In the United States, ex vivo manipulation of PBHCs or BM is regulated by the Food and Drug Administration [26]. Most studies performed have utilized pharmacologic or immunologic maneuvers to remove malignant cells from the autologous marrow by a process of negative selection. An alternative strategy is to select positively the hematopoietic stem cells (HSCs) based upon expression of surface antigens such as CD34. These studies are described in detail in Chapter 5. The studies have been hampered largely by the relative inefficiency of CD34 selection techniques [27]. Preclinical studies have examined the potential role of positive selection of CD34 cells followed by negative depletion steps to remove residual contaminating tumor cells [28–31].
Table 42.2 Methodologies for purging tumor cells Physical separation
Pharmacologic Immunologic
T-cell mediated Viral
Size Density Osmotic lysis Lectin agglutination Hyperthermia Pulsed electric field 4-hydroperoxycyclophosphamide Asta-Z Uncoupled monoclonal antibodies Complement-mediated lysis Immunomagnetic beads Directly coupled Chemotherapeutic agent Toxins Magnetic beads Radionuclide
607
Pharmacologic purging A number of antitumor agents have been used for in vitro elimination of tumor cells, but most studies have used active analogs of cyclophosphamide or ifosphamide. The rationale for this approach was the finding that there was relative sparing of HSCs by cyclophosphamide in vivo, and by activated metabolites such as 4-hydroperoxycyclophosphamide (4-HC) [32], due to the relatively high levels of aldehyde dehydrogenase in HSCs that inactivates active metabolites of cyclophosphamide. This was rapidly adapted for clinical use [33–36], with 4-HC used more widely in the United States and mafosfamide used more extensively in Europe. Pharmacologic purging has been hampered by the availability of suitable agents. The Food and Drug Administration did not approve 4-HC for clinical use because of the lack of a prospective, randomized clinical trial, leading to unavailability of this drug for several years. Further development of 4-HC is under review, but studies have continued with mafosfamide. While 4-HC largely spares primitive HSCs, it produces dosedependent toxicity against tumor cells and reduces committed progenitors such as granulocyte–macrophage colony-forming units by up to 99% [37]. The major complication with the use of pharmacologic purging has been the delayed neutrophil engraftment times. Median time to neutrophil engraftment in patients with AML was 29 days using 4HC-purged BM [33] but 22 days using unpurged BM [38]. There is considerable interpatient variability in drug sensitivity, and some centers use assays to assess dose for an individual patient [39]. Amifostine is a prodrug converted by alkaline phosphatase to the active sulfhydryl compound WR-1065, which protects normal cells by scavenging free radicals and binding to active derivatives of antineoplastic agents. Amifostine shortens the engraftment period of 4-HC-purged BM [40]. Other agents that have been investigated include etoposide, which spares hematopoietic progenitor cells and stromal cells compared with leukemia or lymphoma cells [41], 6-hydroxydopamine [42], alkyl lysophospholipids [43], the photosensitizing agent merocyanine with light exposure [44], and guanine arabinoside [45].
Characteristics of ideal mAbs for purging Because of their specificity, mAbs make ideal agents to identify and target such malignant cells. A number of mouse anti-human mAbs have been generated with specificity for human cell surface antigens by immunizing mice with human malignant cells or malignant cell membranes. Despite the hope that unique tumor-specific cell surface proteins would be recognized, all of the cell surface antigens identified to date on neoplastic cells of the hematopoietic or solid tumor malignancies represent normal differentiation antigens, and true leukemia-, lymphoma- or cancer-specific antigens have not been identified. The most important factor to be determined is that the mAb targets the malignant cell as specifically as possible but has no effect on the HSCs necessary for marrow engraftment. The principle for the selective depletion of contaminating residual tumor cells from HSCs is illustrated in Fig. 42.1. Likely mechanisms of failure of immunologic purging include antigenic heterogeneity, whereby not all tumor cells express the targeted antigen, and the relative inefficiency of the purging methodology employed to “purge” the targeted cells. The ideal characteristics of mAbs for purging are shown in Table 42.3. The targeted antigen should be present at high density on the cell surface to increase the efficiency of subsequent cell killing or removal. To limit the effect of antigenic heterogeneity of expression on the target cell, multiple mAb cocktails are employed targeting multiple antigens. Since mAbs are not by themselves toxic, they must be used in combination with other agents to kill the targeted cell. The most widely studied
608
Chapter 42
methods of immunologic purging are complement-mediated lysis, immunomagnetic bead depletion, and immunotoxins. The potential advantages and disadvantages of each of these techniques are shown in Table 42.4. If mAbs are used with complement, complement-fixing isotypes must be used, the most efficient being immunoglobulin M. For immunologic purging using complement-mediated lysis or immunomagnetic bead separation, it is important that the antigen–antibody complex remains on the cell surface and is not internalized. If immuno-
Anti-CD20 mAb Lineage antigens CD19 NHL cells
CD20
Complement
Anti-CD34 mAb Stem cells
CD34
Stem cell collection
Selection/Depletion
Reinfusion of tumor-free stem cells
Fig. 42.1 Principles of immunologic purging. Targeted antigens are present on the surface of malignant cells but are not expressed on hematopoietic progenitors. Malignant cells escaping the purging procedure are likely not to express or express only weakly the targeted antigens. mAb, monoclonal antibody; NHL, non-Hodgkin’s lymphoma.
Table 42.3 Ideal target antigens for tumor cell purging • • • • • •
Not expressed on hematopoietic progenitors Expressed on clonogenic tumor cells High density of expression on malignant cells Limited heterogeneity of expression on tumor cells Lineage restriction Depending on strategy for purging – ability to modulate
toxins are used, the targeted antigen–antibody complex should be internalized to ensure intracellular delivery of the cellular toxin. Complement-mediated lysis The earliest preclinical studies utilized the ability of mAb to fix complement to the mAb-coated cells, which were then eliminated by complement-mediated cytotoxicity. Complement-mediated cytolysis was previously the most commonly employed method for immunologic purging, due in part to its efficiency, specificity, and relatively low cost. In most studies, rabbit complement has been used to circumvent the problem of homologous species restriction, the process whereby cells are generally resistant to lysis by complement from the same species. The ideal complement source must be toxic to cells coated with mAb but not toxic to cells that have not been coated with antibody. There are, however, major disadvantages of using complement. There is considerable variability among different lots of complement so that each new lot must be tested for nonspecific toxicity. There are nonspecific cell losses that occur because of the need for cell-washing steps. In addition, complement-mediated lysis is inefficient when the neoplastic cells only weakly express the targeted antigen. Regulatory issues related to the use of non-human sources of protein for human cell manipulation have markedly decreased the use of complement-mediated lysis for clinical purging. Among the factors that may influence the efficiency of complementmediated lysis are the density of surface antigen expression, antigen modulation, and resistance to complement lysis. In addition, failure of immunologic purging using complement-mediated lysis could be attributed to three possible mechanisms. First, the clonogenic tumor cells might not express, or only express weakly, the cell surface antigens expressed by the majority of tumor cells. Second, modulation of one or more of the surface antigens following attachment of the mAb to its ligand might limit complement-mediated lysis. Third, a subgroup of patients may have malignancies that are intrinsically more resistant to complement-mediated lysis. Tumor cell killing by antibody-mediated complement activation results from osmotic cell lysis following disruption of the semipermeable properties of the cell membrane. An actively metabolizing cell is capable of turning over its cell membrane. This phenomenon may result not only in antigen modulation, but also neutralization of the lytic effects of the complement. Previous studies have shown associations between biochemical events in the cell and sensitivity to complement-mediated lysis [46,47]. An anticomplement factor has been described in normal BM cells that limits complement activation, not only on the cells that
Table 42.4 Comparison of methods of immunological purging Method of tumor depletion
Nature of antibody–antigen interaction
Complement-mediated lysis
Antibody must fix complement High antigen density expression Antigen should not modulate Antibody–antigen complex must internalize High antigen density expression
Immunotoxins
Immunomagnetic bead depletion
All antibodies are candidate targets High antigen density expression
Technical issues
Resistance to tumor cell
Expense
Screen complement lots for toxicity Multiple antibodies additive Multiple cycles are required Nonspecific cell loss Longer incubation time More easy to standardize Difficult to assess efficacy of purge Nonspecific cell loss Easy to standardize Simple and rapid Fewer cycles required
Antigen heterogeneity Resistance to complement lysis
Low
Antigen heterogeneity Few antigen targets internalize Resistance to toxin mechanism
Intermediate
Antigen heterogeneity Antigen shedding
High
Removal of Tumor Cells from the Hematopoietic Graft
produce the factor, but also on antibody-coated cells within the BM [48]. These anticomplementary effects may be overcome by repeated treatments with complement, and previous studies have suggested that the use of repeated treatment cycles is more efficient in removing contaminating tumor cells than single treatment cycles. This approach is timeconsuming, increases the expense of the procedure in terms of both reagents and laboratory staff effort, and may increase the nonspecific loss of HSCs. The continuous infusion of fresh complement while removing media containing the used complement may increase the efficiency and time taken to perform the procedure [49]. Studies have been reported that address whether these mechanisms of complement resistance can be overcome by chemical engineering of mAbs [50] or by neutralization of CD59 activity [51]. Expression of membrane-bound regulators of complement activation including CD46, CD55, and CD59 protect nucleated cells from complement-mediated injury. Increased expression of these molecules may be a mechanism by which tumor cells protect themselves from inflammatory responses and complementmediated injury. Populations of cells that survive following mAb purging appear to be more resistant to subsequent treatments with the same mAb and complement, presumably because of the emergence of subpopulations of cells with a relative decrease in the surface expression of the targeted antigens [52]. Such changes in relative expression of antigen density have also been observed following treatment with chemotherapy. It is important to demonstrate that the tumor to be purged expresses the targeted antigen, not only at the time of diagnosis, but also at the time of hematopoietic stem harvest. This is increasingly the case now that humanized mAbs are in routine use in the treatment of patients with a variety of malignances. Although the emergence of target antigen-negative tumor cells is relatively rare, this has been observed [53–56]. The combination of immunologic and pharmacologic purging appears to be more efficient than either method alone in eliminating clonogenic Burkitt cell lines from human BM [57]. The effectiveness of purging small cell lung cancer cell lines was also significantly increased when mAb and complement-mediated lysis were used in combination with the cyclophosphamide derivative Asta-Z 7557, although there was significant reduction in myeloid colony growth [58]. In T-cell malignancies, 2′-deoxycoformycin has been used in combination with anti-T-cell mAbs to eliminate clonogenic malignant human T cells [59,60]. A combined approach was taken to attempt to eliminate multidrug-resistant leukemic cell lines from BM using a mAb directed against the cell surface product of the multidrug-resistance gene [61]. This treatment did not affect normal committed precursors. Following antibody purging, the addition of etoposide enhanced the purging efficacy and resulted in a 4.6 log reduction of malignant cells. Furthermore, this antibody was effective when used against the patients’ leukemic blasts, suggesting that this approach was effective and selective for removal of multidrug resistance-expressing cells from the graft. Magnetic bead depletion The use of immunomagnetic beads has the advantage that there is no biologic variability between lots as has been observed with complement. A number of particles have been developed that are directly attached to the primary mAbs used for purging. These reagents have the advantage of allowing more rapid and simpler purging procedures. Instruments to remove the immunomagnetic beads are now available commercially, and immunomagnetic bead depletion is used increasingly as a method of eliminating malignant cells for HCT. The use of immunomagnetic beads was originally developed to facilitate depletion of neuroblastoma cells since the available mAb did not fix complement [62]. A number of studies have been performed for a variety of other malignancies,
609
including small cell lung cancer, breast cancer, acute lymphoblastic leukemia (ALL), myeloma, and lymphomas, as described below. The efficacy of mAbs and immunomagnetic beads in removing Burkitt’s lymphoma cells from normal BM has been demonstrated in a number of studies. Clonogenic lymphoma cell assays have demonstrated that different anti-B-cell mAbs differ in their efficiency of depleting lymphoma cells from 1.9 to 2.8 log in one cycle. When three mAbs were used in a cocktail, the efficiency of purging increased to 3.3 log following a single cycle of treatment, and 5 log after two cycles. Treatment of the BM with beads alone or with the mAb did not significantly reduce the number of HSCs as assessed by colony assays [63]. A cocktail of two mAbs was used to assess the relative efficiency of purging of two different immunomagnetic particles [64]. The efficacy of purging the cell line NALM-6 was assessed using immunofluorescence or colony assays. Log tumor cell kill was significantly better using BioMag particles (3.1 log) than Dynabeads (1.8 log) following a single cycle of treatment. These beads differ in size, and the smaller particles appeared to remove cells more efficiently. There was no significant difference in the efficiency of purging after two treatment cycles, and greater than 4.5 log of tumor cell depletion was obtained using either immunomagnetic bead. In preclinical experiments, BM samples from patients with follicular lymphoma that were contaminated with up to 20% lymphoma cells were purged with either a three- or a four-mAb cocktail followed by immunomagnetic bead depletion. This resulted in the loss of all PCRdetectable cells after three cycles of treatment in all 25 patients studied [65]. After two treatment cycles of immunomagnetic bead depletion, four mAbs were more efficient than three mAbs for purging, and immunomagnetic beads were more efficient than complement-mediated lysis. The results also demonstrate that multiple cycles of immunomagnetic bead depletion may still be required to remove PCR-detectable lymphoma cells, although immunomagnetic bead depletion had no significant effect on myeloid colony-forming assays, suggesting that repeated cycles of immunomagnetic bead depletion might be performed safely. Theoretically, attachment of a single magnetic particle may be sufficient to allow removal of the targeted cell in a magnetic field, and some lymphoma cells may have a sufficiently low density of expression of the targeted antigens to allow elimination with immunomagnetic beads but not to allow complement-mediated lysis. Immunotoxins Purging of autologous marrow in vitro using immunotoxins is a particularly promising approach. Several exquisitely effective candidate toxins have been identified that mediate their cytotoxic function by inhibiting cellular protein synthesis. Because the mechanism of killing by toxins is different from that of chemotherapeutic agents, they are capable of killing cells that are resistant to chemotherapy [66]. These toxins are cytotoxic to both normal and malignant cells, and must be targeted to the malignant cell to demonstrate specificity. The combination of these toxins with a mAb to target delivery specifically to the neoplastic cells is a theoretically attractive proposition [67]. If native toxins were to be conjugated to mAb, the resultant immunotoxin would still be capable of binding to nonspecific targets by binding to the toxinbinding site on normal cells. This nonspecific binding is overcome by modification of the toxin moiety to delete the binding site but leave the toxin domains intact. The most widely studied toxins have been ricin, Pseudomonas exotoxin, and diphtheria toxin. Most experience of in vitro marrow purging has been with ricin. Multiple anti-T-cell intact ricin immunotoxins have been evaluated as potential purging agents [68]. The cocktail containing all four immunotoxins in equimolar concentrations eliminated more than 4 log of clonogenic leukemic cells at a dose that spared more than 70% of the pluripotent HSCs.
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Other approaches Flowing hematopoietic cells through pulsed electric fields effectively purges myeloma cells without sacrificing functional stem cells [69]. A novel approach for the separation of malignant cells was reported for 16 patients with B-cell malignancies using floating immunobeads [70], in which low-density polypropylene beads precoated with rat antimouse mAb were added to the harvested autologous BM following incubation with anti-B-cell mAb. This approach resulted in 75% recovery of mononuclear cells with an 83% recovery of myeloid progenitors. Oncolytic viruses are being investigated as a novel purging strategy. DNA viruses such as genetically engineered adenoviral vectors have been used to deliver either a prodrug-activating enzyme or express wild-type p53 selectively in tumor cells in ex vivo purging protocols [71]. Assessment of the efficacy of purging The identical techniques used to assess whether a hematopoietic cell collection contains residual tumor cells can be used to assess whether tumor cells remain present after immunologic purging, including culture systems, clonogenic assays, and PCR analysis. It is more difficult to assess the efficacy of pharmacologic purging using molecular techniques, since the malignant cells are not physically removed. Studies have demonstrated that different anti-B-cell mAbs differ in their efficiency to deplete lymphoma cells [63]. Multiple rounds of treatments were more efficient than single treatments, and the combination of two or more antibodies was more efficient than a single mAb to eliminate tumor cells [72,73]. PCR has been used to assess the efficacy of immunologic purging in both models using cell lines [74] and patient samples [13,65]. The efficacy of mAbs and immunomagnetic beads has also been demonstrated in a number of studies [28,63,65]. Clonogenic lymphoma cell assays have demonstrated that the efficiency of purging increased significantly when three mAbs were used in a cocktail. A cocktail of two mAbs was used to assess the relative efficiency of purging of two different immunomagnetic particles [75]. Log tumor cell kill was significantly better using BioMag particles (3.1 log) versus Dynabeads (1.8 log) following a single cycle of treatment. There was no significant difference in the efficiency of purging after two treatment cycles, and greater than 4.5 log of tumor cell depletion was obtained using either immunomagnetic bead. Addition of more antibodies and the use of immunomagnetic beads compared with complement mediated
lysis results in more efficient depletion of lymphoma cells in all patient samples tested [65]. Using a single cycle of treatment with multiple mAbs and beads, approximately 2.5 log of small cell lung cancer lines could be depleted, although there was variability in the efficiency of purging different cell lines [76]. In parallel studies, there was no significant toxicity noted to myeloid progenitors. Using two small cell lung cancer lines, immunomagnetic bead depletion was shown to result in a 4–5 log reduction of cancer cells and did not adversely affect BM colony growth [77]. AntiCD15 mAb, expressed on a variety of human cancer cell lines, was capable of depleting up to 3 log of breast cancer cells from normal contaminated marrow using immunomagnetic bead depletion but minimally affected normal hematopoietic progenitors [78]. The combination of 4-hydroperoxycyclophosphamide and immunomagnetic bead depletion removed 4–5 log of clonogenic breast cancer cells [79]. Evaluation of the purging efficiency of an immunotoxin prepared by conjugating anti-CD7 with pokeweed antiviral protein revealed that approximately 3 log of clonogenic T cells could be eliminated. The addition of 2′-deoxycoformycin and deoxyadenosine to the immunotoxin resulted in the elimination of up to 6 log of the T-cell line but also resulted in decreased myeloid progenitor colony assay growth [60].
Contribution of infused tumor cells to relapse The finding that the majority of patients who relapse after autologous HCT do so at sites of prior disease has led to the widespread view that purging of autologous marrow could contribute little to subsequent outcome after autologous HCT. Although few direct studies have compared the infusion of purged versus unpurged HSCs, indirect approaches can be made to assess the clinical significance of immunologic purging. Methods to assess the clinical utility of purging are shown in Table 42.5. Clinical studies of purging Pharmacologic purging Pharmacologic purging has been most extensively studied in AML, where it has been shown that ex vivo purging of BM with 4-HC or mafosfamide is feasible and can result in long-term disease-free survival in patients with high-risk AML [33,34,80–82]. Studies have also been
Table 42.5 Impact of immunological purging on engraftment
Disease Complement AML AML ALL NHL Immunomagnetic beads Neuroblastoma ALL Immunotoxins ALL ALL
Number of patients
Antigen/monoclonal antibody
Days to neutrophils >500/μL
Days to platelets >20,000/μL
Reference
138 12 54 100
CD14, CD15 CD33 CD10, CD19, CD7 CD10, CD20, B5
27 (1st CR) 30 24 27
38 45 40 29
[3] [99] [97] [6]
91 8
UJ13A, Thy 1, UJ127.11, UJ181.4 CD10, CD9
28 15
42 27
[104] [102]
13 14
CD7 CD5, CD7
17 27
40 NS
[105] [106]
ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CR, complete remission; NHL, non-Hodgkin’s lymphoma.
Removal of Tumor Cells from the Hematopoietic Graft
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Table 42.6 Approaches to demonstrate clinical efficacy of purging Surrogate endpoints • Depletion of polymerase chain reaction-detectable cell • Depletion of clonogenic tumor cells Gene transfer of marker gene Randomized trial • Purged versus unpurged autologous hematopoietic cells
Fig. 42.2 Cumulative probability of relapse is significantly reduced in children with acute myeloid leukemia in whom in vitro purging has been performed. Reproduced with permission from [89].
performed in NHL [83] and myeloma [84]. An analysis of 1393 patients registered with the European Bone Marrow Transplant Registry (EBMT) demonstrated an advantage of purged BM versus unpurged sources of HSCs [85]. Similar results have been obtained in smaller single-center studies [86–88]. A multicenter analysis of the use of 4-HC for marrow purging in AML patients in first remission also demonstrated an advantage for purging, with a leukemia-free survival of 56% (range 47–64%) for purged versus 31% (range 18–45%) for unpurged marrow [9]. In a retrospective EBMT analysis of 387 children who underwent autologous HCT in first CR, probability of relapse was decreased after in vitro purging as shown in Fig. 42.2 [89]. In contrast to AML, few studies in ALL have addressed the use of pharmacologic purging. A study reporting the outcome in patients registered with the EBMT demonstrated no difference in outcome for patients receiving purged compared with unpurged sources of HSCs [90]. The role of purging in transplants for solid tumor has been reviewed and remains controversial [91]. Immunologic purging Immunologic purging was first performed in NHL and has been most widely studied in this disease [6,8,13,92,93]. Additional studies have been performed in multiple myeloma [27,94,95], ALL [96,97], AML [98], breast cancer [99], small cell lung cancer [58], and neuroblastoma [100,101] among others. These studies have confirmed that immunologic purging can be performed safely and that subsequent hematopoietic engraftment is not significantly delayed. No randomized prospective study has demonstrated whether the removal of occult or overt neoplastic cells resulted in improved disease-free survival [27,95]. Whether the failure to demonstrate an advantage of purging is due to the relative inefficiency of the purging technique or the intrinsic resistance of the tumor cells to the high-dose chemotherapy approach used is not clear. The results obtained in the larger reported trials using immunologic purging are shown in Table 42.6. Complement-mediated lysis In a clinical trial of 138 patients with AML, BM purging was performed using two mAbs and complement-mediated lysis [3]. One hundred and ten patients were in CR at the time of transplantation (23 in first CR, 87 in second or third CR). Engraftment was prompt in most patients, and
only one very heavily pretreated patient in third CR failed to engraft. Engraftment was faster in those patients infused with larger numbers of colony-forming units. This study did not compare results obtained using purged versus unpurged marrow, but the relapse-free survival of the patients in second and third remission appears to be comparable to that obtained following allogeneic transplantation in similar-risk patients. Anti-CD33 mAb and complement-mediated lysis was used to purge the BM of 12 patients with AML [98]. Patients had durable but delayed engraftment, and platelet engraftment was particularly delayed in some patients. Of note, granulocyte–macrophage colony-forming unit colony growth was markedly reduced following purging. In a multicenter study, autologous HCT was used in 54 patients with ALL [96] with purging using anti-CD10, anti-CD19, and anti-CD7 mAbs and rabbit complement. The transplant related mortality was 5% and engraftment was rapid. Of note, this study was not designed to demonstrate efficacy. One hundred patients with B-cell NHL were treated at the DanaFarber Cancer Institute with autologous BM purged when patients were in CR or minimal disease state [6]. Notably, 69 patients had a prior history of histologic marrow involvement, and 37 patients had overt marrow disease at the time of harvest. This study was associated with low treatment-related mortality. Engraftment was rapid in all cases. The results of the use of HCT in patients with follicular lymphoma in first complete or partial remission have also been reported [8,102]. These studies not only address the outcome for those patients receiving autologous HCT, but also enrolled patients on an “intent-to-treat basis” and suggested that virtually all patients with follicular lymphoma could achieve minimal disease state before proceeding to autologous HCT. Long-term follow-up of patients treated at the Dana-Farber Cancer Institute was reported on 153 patients with B-cell NHL with autologous BM purged when patients were in CR or minimal disease state [2]. Notably, 47% of patients had overt marrow disease at the time of collection. This study was associated with low treatment-related mortality, and engraftment was rapid in all cases. Immunomagnetic bead depletion The first clinical studies of purging using immunomagnetic beads were performed for children with neuroblastoma [101]. Immunomagnetic bead depletion was used to purge 123 BMs before autologous stem cell transplantation in 91 cases of neuroblastoma [103]. In this study, 59 patients received a single graft and 32 patients received two sequential procedures. Although the procedure resulted in a significant loss of mononuclear cells, there was little evidence of additional toxic effects on myeloid progenitors. Immunomagnetic beads were used to deplete leukemic cells from the marrows of patients with common ALL [101]. In this study, the BM of 18 patients was purged using a cocktail of three mAbs, although only eight of these patients were subsequently treated with high-dose therapy and autologous HCT. Engraftment was rapid in all cases although slower than that observed in patients with neuroblastoma.
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Immunotoxins Fewer clinical trials have been reported using immunotoxins for purging. Seven patients with high-risk T-cell ALL and six patients with T-cell NHL were treated by autologous BMT following purging with anti-CD7 ricin A immunotoxin [104]. Incubation of the marrow with up to 10−8 mole/L had no significant effect on HSC progenitors as assessed by colony assay growth or by subsequent delay of engraftment. Using a different approach, autologous marrow from 14 consecutive patients with T-cell ALL was purged with a combination of two immunotoxins, anti-CD5 and anti-CD7 linked to intact ricin, plus 4-HC [105]. The efficacy of purging was assessed using multiparameter flow analysis, cell sorting, and leukemic progenitor cell colony assay. Following purging, no blast colonies were observed in the marrows of 11 of 13 evaluable patients. Engraftment occurred in 13 of the 14 patients, and the median time to reach an absolute neutrophil count greater than 500/ μl was 27 days. Despite the apparent efficiency of purging, nine patients relapsed, the majority of them shortly after HCT, suggesting that in this study relapse was most likely due to failure of the high-dose therapy to ablate endogenous disease in these high-risk patients. Outcome after successful immunologic purging
Probability of event-free survival
In studies at the Dana-Farber Cancer Institute, PCR amplification of the t(14;18) was used to detect residual lymphoma cells in the BM before and after purging to assess whether efficient purging had any impact on disease-free survival [13]. In this study, 114 patients with B-cell NHL and the bcl-2 translocation were studied. Residual lymphoma cells were detected in all patients in the harvested autologous BM. Following three cycles of immunologic purging using the anti-B-cell mAb J5 (anti-CD10), B1 (anti-CD20), and B5, and complement-mediated lysis, PCR amplification detected residual lymphoma cells in 57 of these patients. The incidence of relapse was significantly increased in patients who had residual detectable lymphoma cells compared with those in whom no lymphoma cells were detectable. The long-term follow-up results of this patient cohort by the result obtained after purging is shown in Fig. 42.3. This study demonstrates that patients who were infused with a source of hematopoietic cells that was free of detectable lymphoma cells had improved outcome compared with those who had residual detectable
100
PCR neg (57 patients, 14 relapses)
80 60 p or = 60 years old with relapsed or refractory B-cell lymphoma. J Clin Oncol 2007; 25: 1396–402. 123. Vose JM, Bierman PJ, Enke C et al. Phase I trial of iodine-131 tositumomab with high-dose chemotherapy and autologous stem-cell transplantation for relapsed non-Hodgkin’s lymphoma. J Clin Oncol 2005; 23: 461–7. 124. Shimoni A, Zwas ST, Oksman Y et al. Yttrium90-ibritumomab tiuxetan (Zevalin) combined with high-dose BEAM chemotherapy and autologous stem cell transplantation for chemorefractory aggressive non-Hodgkin’s lymphoma. Exp Hematol 2007; 35: 534–40.
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Norbert Schmitz
Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation
Introduction The existence of hematopoietic cells (HCs) in the peripheral blood (PB) was postulated in 1909 by Alexander Maximow [1]. In 1962, Goodman and Hodgson were first to prove that circulating HCs were capable of restoring irradiation-induced marrow aplasia in mice [2]. The first clinical transplantation was published in 1980 by Abrams et al., who transfused large numbers of syngeneic blood leukocytes to a patient with Ewing’s sarcoma [3]. In 1985 and 1986, four different teams reported successful hematopoietic reconstitution by autologous blood-derived HCs in cancer patients [4–7]. Although these latter case reports provided the long-sought proof of principle, the low concentration of HCs in unmanipulated PB remained a major obstacle to the wider use of clinical transplants from PB, especially in the allogeneic setting where the exposure of healthy individuals to mobilizing cytotoxic drugs was not possible. Only with the advent of granulocyte-colony stimulating factor (GCSF) and the discovery that this and other cytokines were able to substantially increase the concentration of CD34+ cells in PB [8,9] did autologous and, 5–6 years later, allogeneic PBHC transplantation (PBHCT) begin to enter the clinic. With regard to PBHCT, in 1989 Kessinger et al. showed that T-cell-depleted blood HCs were able to restore hematopoiesis after myeloablative therapy [10]. Because of fears that the high numbers of T lymphocytes contained in PBHC harvests would induce severe graft-versus-host disease (GVHD) in most recipients, allogeneic PBHCT replete of T cells was first used in emergency situations such as the failure of a previous marrow graft [11] or the inability of the donor to undergo general anesthesia for marrow collection [12]. After pilot studies using unmanipulated PB from human leukocyte antigen (HLA)-identical sibling donors were published in 1995 [13–15], a surge of allogeneic PBHCTs ensued. Currently, about 70% of all allogeneic transplants in Europe and worldwide are performed with PB instead of marrow [16].
The donors When allogeneic PBHCT entered the clinical arena, the experience with healthy individuals donating HCs mobilized into the blood was also
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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limited. The major issues which had to be addressed were the following: 1. How could HCs be harvested most effectively? 2. Did harvest products from the PB contain stem cells in quantities and quality which would allow for timely, complete, and durable engraftment of allogeneic lymphohematopoiesis? 3. What were the acute side-effects and long-term sequelae of the mobilization and collection procedure? For further information regarding donors for HCT see also Chapter 38. Mobilization and collection of HCs from PB The first attempts to “mobilize” HCs into the PB of normal donors followed the experience gained with granulocyte transfusions [17], and with the mobilization of autologous HCs in patients with lymphoma and other diseases where the stem cell pool was not compromised by marrow infiltration or extensive cytotoxic therapy [18,19]. G-CSF at doses between 5 and 16 μg/kg/day was administered to the potential donor for 5–7 days. Such treatment allowed the collection of CD34+ cell numbers ranging from around 2 to more than 20 × 106/kg recipient body weight in most instances. Infusion of the harvest products into recipients with leukemia or lymphoma after myeloablative therapy resulted in reliable and surprisingly fast engraftment [13–15]. Tens of thousands of healthy individuals have now undergone mobilization and collection of PBHCs using a variety of harvest protocols [20–24]. A joint analysis from the International Bone Marrow Transplant Registry (IBMTR) and the European Group for Blood and Marrow Transplantation (EBMT) [25] summarized the experience with 1488 donations of PBHCs from HLA-identical siblings (n = 1322), other relatives (n = 149) or unrelated donors (n = 15). Nearly all donors had received G-CSF (filgrastim, lenograstim) for mobilization. Approximately 40% of the donors had undergone a single apheresis, 45% underwent two, 11% underwent three, and 5% had to undergo four or more leukaphereses to collect the number of HCs deemed necessary for engraftment by the investigator. The Spanish National Donor Registry reported that G-CSF was administered to 466 donors at a median dose of 10 (range 4–20) μg/kg/day for a median of 5 (range 4–8) days. The mean CD34+ cell dose collected was 6.9 (range 1.3–36) × 106/kg, with only 14 donors (2.9%) not achieving the minimum target number of 2 × 106 CD34+ cells/kg [26]. A more recent single-center study reported on 400 related and unrelated donors who had received 10 μg/kg/day subcutaneously of glycosylated or nonglycosylated G-CSF once daily for 4 consecutive days,
Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation
with apheresis commenced on day 5 [27]. The target cell number of 4 × 106 CD34+ cells/kg was reached after one (63%) or two aphereses in 81% of donors. Most other donors collected between 2 and 4 × 106 CD34+ cells/kg, while only 11 donors (2.7 × 108/kg nucleated cells) might result in superior outcome compared with mobilized PB [48]. On the other hand, because PBHC collection products containing high CD34+ cell numbers also contain more T cells, it has been postulated not to use transplants from PB with very high CD34+ cell numbers. Przepiorka et al. evaluated risk factors for acute GVHD after allogeneic PBHCT and found a sharp increase of GVHD if more than 6.3–10.0 × 106 CD34+ cells/kg were transplanted [49]. It will be difficult, however, to confirm this finding with prospective data because such high numbers of marrow or PBHCs are harvested from a small fraction of donors only. A number of investigators have looked at immunophenotypically, genotypically or functionally characterized subtypes of HCs in more detail. Körbling et al. reported that G-CSF at a dose of 12 μg/kg/day increased the concentrations of PB CD34+ cells and more primitive subsets such as CD34+Thy1dim and CD34+Thy1dimCD38− cells by 16.3-, 24.2-, and 23.2-fold, respectively. The mean apheresis yield of CD34+Thy1dim and CD34+Thy1dimCD38− cells per kilogram of recipient body weight and per liter of donor blood processed was 48.9 × 104 and 27.2 × 104, respectively, indicating an additional “peripheralization” effect of G-CSF on primitive CD34+ cell subsets [50,51]. Other reports [52] confirmed these findings but also showed that the immunophenotypic profiles of HCs from blood or marrow differed in several respects (Table 43.1) (reviewed in [53]). The low expression of CD71 and the decreased retention of rhodamine-123 in CD34+ cells from mobilized PB suggest that these cells are not actively proliferating or metabolically active [54]. Kinetic data corroborate the phenotypic findings, further demonstrating that relatively more CD34+ cells from PB exhibit characteristics of quiescent cells [55]. Steidl et al. [56] reported on the molecular phenotype of BM-derived and circulating CD34+ cells from mobilized PB. Gene expression profiling confirmed that CD34+ cells from BM cycle more rapidly, whereas CD34+ cells from PB include more quiescent stem and progenitor cells.
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Table 43.1 Characteristics of CD34+ hematopoietic cells (HCs) from bone marrow (BM) or mobilized peripheral blood (PB) Characteristics
BM
Mobilized PB
Phenotype CD13+, CD33+ CD38− HLA-DR− CD10+ CD19+ CD7+ c-kit VLA-4
Standard Standard Standard Standard Standard Standard Standard
Higher Higher Higher Less Less Less Less
Metabolic activity CD71+ Rhodamine
High High
Low Low retention
Normal Normal Active Standard Standard
Increased Increased Nonactive Less Less Increased Increased Increased
Clonogenicity Colony formation LTC-IC Cycling status Genotype cell cycle progression DNA synthesis GATA 2 N-myc Apoptotic activity LTC-IC, long-term culture-initiating cells.
Prosper et al. [57] investigated the number of long-term cultureinitiating cells (LTC-ICs) in the PB of normal donors treated with GCSF (7.5–10 μg/kg/day) and found that CD34+CD38+ cells and CD34+HLA-DR+ cells sorted from mobilized PB contained 0.5–5% of cells capable of sustaining hematopoiesis in long-term culture for up to 5 weeks. This is at least five times more than the number of LTC-ICs found in steady-state BM or nonmobilized PBHCs. However, 90–95% of the LTC-ICs present in CD34+CD38+ or CD34+HLA-DR+ fractions of mobilized PB were not able to sustain hematopoiesis for 8 weeks, as is the case with close to one-third of CD34+CD38+ and CD34+HLA-DR+ cells from nonmobilized PB. Taken together, phenotypic, genotypic, and functional analyses of HCs from BM or PB demonstrate that stem and progenitor cells are contained in both compartments. HCs from PB may be enriched in LTCICs and other clonogenic cells, they are metabolically less active, cycle less frequently, and show increased apoptotic activity. Stem cell biology including the differences of CD34+ cells from mobilized PB or BM remains an area of active research because a better understanding of basic phenotypes and function is paramount to optimize the peripheralization, harvesting, and clinical use of HCs.
T lymphocytes and other immune cells in mobilized blood There are many differences in the composition of PBHC harvests and marrow grafts if cells other than HCs are considered. Most importantly, PB harvests contain approximately 10 times more T cells than BM; the ratio of CD4+:CD8+ lymphocytes, however, is identical. This had already been noticed with autologous PBHC harvests [58], and was the most
important reason why allogeneic transplants with PB were accepted as an alternative to BM only after studies had demonstrated that the infusion of such high T-cell numbers was not regularly followed by the occurrence of severe GVHD. To some extent, the numbers of T cells contained in PBHC harvests relative to the number of CD34+ cells seem to depend on the mobilization protocol and the day of apheresis. Leukapheresis on day 5 after administration of G-CSF should give the highest stem cell yield with relatively low numbers of T cells contained in the final product [58]. The observation that the high number of T cells in PB harvests did not lead to a dramatic increase in the incidence and severity of GVHD in patients allografted with mobilized PB prompted researchers to investigate the effects of G-CSF administration on the number and function of immune cells in more detail. With regard to T cells, Pan et al. [59] were first to show that pretreatment of healthy individuals with G-CSF polarizes T cells towards the production of type 2 cytokines (IL-4 and IL-10), while type 1 cytokine production (interferon-gamma and IL-2) is reduced. Other effects capable of explaining the surprisingly low incidence of severe GVHD after transplantation of allogeneic PBHCs are downregulation of allogeneic immune responses by posttranscriptional inhibition of tumor necrosis factor-alpha production [60], and the comobilization of CD4+ and CD 8+ T cells. Kusnierz-Glaz et al. [61] demonstrated that CD4+CD8+ αβ T cells are markedly enriched in the leukapheresis products of G-CSF-treated normal donors, and that these cells, further enriched in low-density fractions of mobilized PB, significantly suppress the mixed lymphocyte reaction. Mielcarek et al. [62,63] calculated that the number of CD14+ cells in mobilized PB was 50-fold greater than in marrow. These large numbers of monocytes suppress T-cell proliferative responses to alloantigen by increased IL-10 production and decreased secretion of IL-12 and tumor necrosis factor-alpha. Arpinati et al. demonstrated that the number of T helper type 2 (Th2) cell-inducing dendritic cells (pre-DC2s) was increased in mobilized PB. As pre-DC2s lead to polarization of naïve CD4+ T cells toward a T helper type 2 phenotype, this finding also may explain why transplantation of allogeneic PBHC does not lead to more severe GVHD in a larger fraction of patients [64]. Recently, Franzke et al. [65] showed that T cells do express G-CSF receptors after G-CSF treatment, and G-CSF can thus directly modulate T-cell immune responses. Among other possible mechanisms, G-CSF seems to diminish T-cell receptor-mediated activation and proliferation, downregulate leukocyte function associated antigen-1 alpha (LFA-1α) thus affecting the homing process of the graft and abrogating donor Tcell activation to host antigens), and upregulate GATA-binding protein3 (GATA-3), the master regulatory factor for induction of a T helper immune response. Natural killer cells are five to 10 fold more frequent in mobilized PB than in BM. Joshi and colleagues analyzed peripheral blood mononuclear cells from G-CSF-mobilized blood cell harvests for their immunologic function and compared them with peripheral blood mononuclear cells from steady-state nonmobilized donors [66]. They found a significant decrease in NK- and lymphokine-activated killer cell-mediated cytotoxicity for G-CSF-mobilized effector cells. They also reported a significant decrease in both T-cell and B-cell mitogen responses with G-CSF-mobilized versus nonmobilized cells. Neutrophils not only increase in number after administration of GCSF, but their function also is enhanced immediately after G-CSF administration, as shown by upregulation of Fcγ RIII receptors, cell surface CD11b and CD66 molecules, and elevated levels of plasma elastase [67]. It is not clear which of the mechanisms described contributes to which extent to the striking observation that the infusion of 1 to 2 log more T cells into the recipient does not result in the development of severe GVHD in more patients grafted with PB.
Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation
Side-effects and long-term sequelae of mobilization and collection of HCs The overall short- and long-term effects of G-CSF have repeatedly been reviewed [68,69]. The acute toxicities of G-CSF administration have been reasonably well described, and comprise bone pain, headache, and fatigue as the most frequent side-effects. However, myalgias, chest pain, anxiety, insomnia, night sweats, fever, anorexia, weight gain, nausea and/or vomiting, and local reactions at the injection site have also been described [26,68,70]. Side-effects have been mild to moderate in the vast majority of donors and usually resolve within 48 hours of discontinuation of G-CSF. They can be treated successfully by the administration of minor analgesics (acetaminophen or ibuprofen) in most instances, and these side-effects have not been reported to require cessation of G-CSF administration or cancellation of the PBHC harvest. There is some indication that the frequency and severity of side-effects of G-CSF is dose dependent [71,72]. Therefore, doses as low as possible should be used. A number of side-effects of G-CSF administration and the harvest procedure on blood chemistry, the coagulation system, and baseline hematologic parameters already described in patients with hematologic malignancies also occur in normal individuals. Laboratory abnormalities caused by G-CSF include transient increases of lactate dehydrogenase and alkaline phosphatase, uric acid, alanine aminotransferase, and/or glutamyl transpeptidase, and a decrease in serum potassium and/or magnesium [21,29,73]. Several authors have shown that G-CSF can induce a mild hypercoagulable state, as indicated by in vitro testing. Increases in fibrinogen and factor VIII levels, a reduction in protein C and S levels [74], and increases in prothrombin fragments, thrombin–antithrombin complexes, D-dimers, and platelet aggregation have all been reported [75–77]. Mildto-moderate decrements in platelet, lymphocyte, and granulocyte levels have been described following the collection of PBHCs [17,78]. Thrombocytopenia may partly be due to the administration of G-CSF itself [79] or the leukapheresis procedure. Cytopenias are usually mild and selflimited, although more profound thrombocytopenia following repeated large-volume leukaphereses has been reported [80]. A number of studies have been presented which compare the experience of donors who participated in one of the randomized trials comparing BM transplantation (BMT) with PBHCT [81–84]. It was reassuring that none of these reports described life-threatening complications or long-lasting discomfort in any of the donors. The study reports generally confirm that the side-effects of PBHC harvesting are related mainly to G-CSF administration, while the side-effects of BM harvesting are caused by the harvest procedure itself. The incidence of adverse events occurring in 5% or more of donors of PB or BM who participated in the large European trial are presented in Table 43.2. Some of these studies also measured the emotional status and pain of donors prior to the harvest procedure and at defined time points after the collection of BM or PB. In general, the symptom burden, the levels of maximal and average pain, and anxiety were similar, although symptom peaks occurred at different time points during G-CSF administration (in PB donors) or shortly after BM harvesting. A quicker resolution of symptoms in PBHC donors was also described [82,83]. Only the Scandinavian trial reported a significant difference in the total burden of complaints in the two groups of donors, favoring the PB donors. This study also found that donors would prefer to donate PB rather then BM if asked to donate again [84]. This observation is in line with a report from unrelated donors [85], and a previous report in cancer patients who also preferred to donate autologous PB rather than BM [86]. Serious or lethal complications of G-CSF administration and leukapheresis are rare. At least five cases of splenic rupture associated with the harvest procedure have been reported [87–91]. Therefore, special
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Table 43.2 Subject incidence of adverse events occurring in >5% of donors. Date from the EBMT study comparing bone marrow transplantation (BMT) with peripheral blood progenitor cell transplantation (PBPCT) [44]
Any adverse event
BM donors (n = 166)
PBHC donors (n = 164)
95 (57%)
107 (65%)
Harvesting procedure-related adverse events Any harvest-related 91 (55%) Access pain 39 (23%) Anemia 17 (10%) Back pain 16 (10%) Nausea 10 (6%) Arthralgia 8 (5%) Vomiting 8 (5%) Skeletal pain 3 (2%)
61 2 0 4 1 1 0 10
(37%) (1%) (0%) (2%) (1%) (1%) (0%) (6%)
Filgrastim-related adverse events Any filgrastim-related Musculoskeletal Headache LDH increased Alkaline phosphatase increased
96 71 19 14 8
(59%) (43%) (12%) (9%) (5%)
BM, bone marrow; LDH, lactate dehydrogenase; PBHC, peripheral blood hematopoietic cell.
attention should be paid to donors presenting with an enlarged spleen or a history of splenic disorders. Routine abdominal ultrasound has been recommended to reduce splenic complications. Also, cases of severe cardiovascular events [28,92], acute lung injury [93], acute iritis [94], pyogenic infections [95], gouty arthritis [96], and anaphylactoid reactions [97] have been reported. Such case reports, as well as concerns that G-CSF might perturbate normal hematopoiesis and increase the risk for developing malignant disorders of the blood, including myelodysplasia and leukemia [98], caused investigators to propose an international donor registry more than 10 years ago [28]. The first report on severe donor events in a very large cohort of donors was compiled only recently, emphasizing that a global approach to document and evaluate the acute and long-term effects of stem cell collection in healthy donors is mandatory. The report by Halter et al. [91] summarizes all deaths and severe complications of PB or marrow donations for 51,024 recipients receiving a first HCT. Data were generated retrospectively from questionnaires sent out to the principal investigators of 338 transplant centers who participated in the annual EBMT activity surveys. Any serious event occurring within 30 days after donation and the occurrence of any hematologic malignancy in a donor had to be registered. Overall, five donor deaths occurred (one after BM and four after PB donation; incidence 0.98 per 10,000 donations) and 37 severe complications (12 in BM and 25 in PB donors; p < 0.02) were reported. Nineteen hematologic malignancies (eight after BM and 11 after PB donation; p = 0.4) were reported. Three deaths (pulmonary embolism, subarachnoidal hematoma, and cardiac arrest) were clearly related to the donation, whereas two cardiac arrests on days 15 and 17 after PB donation may or may not have been related to the harvest procedure. The severe, potentially lifethreatening complications mostly were cardiovascular events, hemorrhages, and splenic rupture. The hematologic malignancies reported were acute myeloid leukemia (three), acute lymphoblastic leukemia (three), lymphoma (seven), nasopharyngeal plasmacytoma, chronic
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Chapter 43
lymphoblastic leukemia, and myeloproliferative syndrome (one case each), and other nonspecified disorders. In Chapter 38, Confer et al. summarize data available on nine deaths which occurred worldwide after donation of BM or PB. It is unclear how much overlap might exist between their cases and those reported by Halter et al. Also, it remains uncertain what number of donations formed the basis to Confer’s report. In any case, these data, together with unpublished reports from other registries, suggest that life-threatening and fatal accidents are rare but do occur in donors of PB or marrow stem cells. Therefore, the World Marrow Donor Association recently created a Serious Events and Adverse Effects Registry to collect and evaluate information on serious donor events. The leukapheresis itself is generally well tolerated, but side-effects caused by the anticoagulant and placement of central venous access have been reported. Although any effort should be made to avoid a central venous access, 2–7% of donors ultimately will need it because peripheral veins cannot be used or the necessary blood flow cannot be established [13–15]. Rarely, donors have experienced tetany or severe symptoms of hypocalcemia caused by the anticoagulant used throughout the leukapheresis procedure [81].
The recipients In 1995, pilot studies from the United States and Germany [13–15] indicated that transplantation of allogeneic PBHCs resulted in fast and stable engraftment; GVHD did not seem to occur more frequently than after marrow grafting. Larger series from other transplant centers or cooperative groups [99,100], as well as retrospective comparisons with BMT [101,102], confirmed that transplantation of allogeneic mobilized PB seemed a viable alternative to marrow transplantation. The largest retrospective analysis presented by IBMTR and EBMT compared results of 288 PBHCTs with 536 BMT procedures from HLAidentical sibling donors [103]. The paper confirmed that recipients of PBHCs attained more rapid recovery of neutrophils and platelets; significant differences in relapse rates and the incidence of grade II–IV acute GVHD between marrow and PBHCT were not observed. The incidence of chronic GVHD at 1 year, however, was significantly higher after PBHCT (65%) than after BMT (53%). Treatment-related mortality was lower, and leukemia-free survival rates were higher with PBHCT in patients with advanced leukemias, but were comparable in standardrisk leukemias. The randomized trials Between 1998 and 2002, the results of eight prospective randomized trials comparing the transplantation of allogeneic PBHCs with that of BM were published [41–44,104–107]. These studies were almost exclusively done in patients with leukemia, patients with other hematologic malignancies were included only rarely, and nonmalignant disorders were not represented at all. The donors were HLA-identical siblings, and BM or PBHCs were infused without any manipulation. The trials differed substantially with respect to the number of patients included, the status of the diseases, the conditioning regimes used, the doses of HCs and T cells infused, the GVHD prophylaxis administered, and a number of other variables that may have influenced outcome. Still, the results of these trials allow a number of important conclusions, which are summarized below. Engraftment Transplantation of allogeneic PBHCs results in faster engraftment of HCs of all lineages compared with BMT. Neutrophils exceed a threshold of 0.5 × 109cells/L between 2 and 6 days earlier with PBHC than after
BMT. In the United States trial, the neutrophils surpassed 0.5 × 109/L on day 16 (range 11–29 days) with PB, and on day 21 (range 13–36 days) with BM (p < 0.001) [41]. The Canadian study reported neutrophil recovery on day 19 (range 12–35 days) with PB, and on day 23 (range 13–68 days) with BM [43]. Patients in the EBMT study had the shortest time interval from transplantation to an absolute neutrophil count of greater than 0.5 × 109/L: 12 days for PB and 15 days for BM [44]. However, this trial was the only one of the large randomized studies that used G-CSF (5 μg/kg/day) also after transplantation, and day 11 methotrexate was not included. Two other randomized trials proved that the administration of G-CSF at a dose of 10 μg/kg/day after PBHCT can accelerate neutrophil recovery by 3–4 days [108,109]. Platelet recovery after allogeneic PBHCT is also faster by approximately 6 days: a platelet count of 20 × 109/L was reached on day 13 (range 5–41 days) with PB, and on day 19 (range 7–74 days) with BM in the United States study (p < 0.001). The Canadian study reported platelet recovery after 16 (range 0–100) days for patients grafted with PB, and 22 (range 0–100) days for patients grafted with BM (p < 0.0001). The EBMT study had a median time to platelet recovery greater than 20 × 109/L of 15 days for patients transplanted with PB, and 20 days for patients transplanted with BM (p < 0.0001). This difference translated into a median of 8 (range 1–68) days platelet transfusions in the PBHCT group, and a median of 10 (range 2–71) days in the BMT group (p = 0.0029). It is well recognized that factors other than the source of HCs influence the pace of hematologic recovery. While G-CSF accelerates neutrophil recovery, methotrexate given for prophylaxis of GVHD retards hematopoietic reconstitution [110]. The occurrence of GVHD, infection, the grade of genetic disparity between recipient and donor, and splenomegaly have all been reported to affect the speed of hematopoietic recovery, and may explain the slight variations in times to neutrophil and platelet recovery seen in the randomized trials. Graft failures after PBHCT are not more frequent than after BMT. Comparative studies of hematopoietic chimerism after PBHCT and BMT support the notion that transplantation of PB results in fast, complete, and durable complete chimerism [111]. Immune recovery Recovery of immunity after unmanipulated allogeneic PBHCT is at least as quick as after BMT, but may be faster with respect to some subgroups of cells with immune functions. Circulating naïve (CD4+CD45RA+) and memory (CD4+CD45RO+) helper T cells, B cells, and monocytes are found in higher numbers after PBHCT compared with BMT between 1 and 11 months post transplant [112]. Natural killer cell numbers did not significantly differ between patients grafted with PB or marrow over the same time period. Proliferative responses to T- and B-cell mitogens (phytohemagglutinin, pokeweed mitogen, tetanus toxoid, and Candida) were significantly increased in recipients of PB from HLA-identical donors [112]. Storek et al. [113] studied 115 patients who had been randomly assigned to receive allogeneic marrow or mobilized PB from HLAidentical donors. Between 1 month and one year after transplantation, the counts of CD4+CD45RAhigh and CD4+CD45RAlow/− T cells were significantly higher after PBHCT. T cells appeared equally functional when challenged by phytohemagglutinin or herpesvirus antigen. Median serum immunoglobulin G levels were similar in both groups of patients. Of note, the authors also reported that particularly severe infections were significantly reduced after PBHCT. The reduced risk was most prominent for fungal infections, intermediate for bacterial infections, but low for viral infections. This latter phenomenon may be a consequence of the marked loss of memory T cells which occurs after allogeneic PBHCT [114].
Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation
Another interesting observation published by Lapierre et al. [115] was that, 30 days after PBHCT, anti-A and/or anti-B blood group titers were significantly higher after PBHCT compared with BMT, suggesting that immunohematologic reconstitution after PBHCT is quicker than after BMT. This observation could explain the cases of acute hemolysis described after ABO-incompatible PBHCT [116,117]. On the other hand, transfusion requirements are no different in ABO-, rhesus- or Kell-different transplantation; however, ABO-mismatched patients were reported to show poorer survival than matched patients after PBHCT [118]. Acute GVHD The incidences of acute GVHD grade II–IV were similar for both groups of patients in all larger studies except for the largest one [44], which noted a 13% increase of grade II–IV acute GVHD (p = 0.013) and a 12% increase of grade III–IV acute GVHD with PB (p = 0.0088) (Table 43.3). The EBMT study also was the only study where the maximum grade of acute GVHD was the primary endpoint, and randomization was stratified for some of the known risk factors of GVHD. Other differences in the design of the EBMT study and the other trials were the inclusion of good-risk patients only, the omission of methotrexate on day 11, and the use of G-CSF post transplant. Omission of day 11 methotrexate has been reported to predispose recipients to develop GVHD [110], although this finding was not supported by data from the international metaanalysis [119]. It also remains in question if the high numbers of T cells transferred with a typical PBHC graft are responsible for the higher incidence of acute GVHD. Although such a correlation between GVHD and T-cell numbers was detected in the EBMT study, another study failed to demonstrate such a correlation [120].
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Chronic GVHD The large randomized studies all showed at least a trend to more chronic GVHD with PBHCs, and two studies reported statistically significantly more chronic GVHD after PBHCT [42,44] (Table 43.4). There was also more extensive chronic GVHD in patients having received PBHCs compared with BM cells. This difference was significant in the EBMT trial and in the studies chaired by Blaise et al. [42] and Vigorito et al. [107]. Follow-up reports from the United States trial [121], the French trial [122], and the EBMT trial [123] have been published with a median follow-up of 41, 45, and 36 months, respectively. Flowers et al. still did not observe more chronic GVHD after PBHCT, and the clinical characteristics of chronic GVHD were similar after PBHCT or BMT [121]. However, the updated study showed that chronic GVHD was more difficult to control when it occurred after PBHCT compared with BMT: the number of successive treatments needed to control chronic GVHD was higher after PBHCT, and the duration of glucocorticoid treatment was also longer (p = 0.03 for both endpoints). In contrast, Mohty et al. found a highly significant difference in the 3-year cumulative incidence of chronic GVHD in the PBHC group compared with the BM group (65% versus 36%; p = 0.004) [122]. They also observed significantly more extensive chronic GVHD. Chronic GVHD needed further immunosuppression and led to longer periods of hospitalization. No survival difference was observed thus far. The 3-year follow up of the prospective EBMT trial (Table 46.4) and the 5-year follow up of the retrospective IBMTR/EBMT study also showed more chronic GVHD in patients after PBHCT [124]. Overall, there is evidence that PBHCT causes more chronic GVHD or at least chronic GVHD which is more difficult to treat. The picture is not yet complete, however, and further updates from the large randomized trials are eagerly awaited. Relapse rates
Table 43.3 Acute graft-versus-host disease (GVHD) grades II–IV Reference
PB (%)
BM (%)
p-value
Bensinger et al. [41] Blaise et al. [42] Couban et al. [43] Heldal et al. [104] Powles et al. [106] Schmitz et al. [44] Vigorito et al. [107]
64 44 44 21 50 52 27
57 42 44 10 47 39 19
p = 0.35 Not given p > 0.9 Not given Not given p = 0.013 p = 0.53
All percentages represent cumulative incidences at day +100 post-transplant except for the studies by Blaise et al. [42], Heldal et al. [104] and Powles et al. [106] where the actual percentage of patients with acute GVHD at day +100 is given.
Knowing that the graft-versus-leukemia effect is exerted mainly by donor T cells and that around 10 times more T cells are transferred with a typical PB collection product, it was anticipated that transplantation of PB cells would result in a reduction of relapse rates after PBHCT. Animal studies [125] and results of nonrandomized studies [126] also pointed in that direction. The randomized studies have not consistently confirmed these expectations so far, although the United States study [41] and the small British study [106] indeed reported significantly fewer patients with relapse after PBHCT than after BMT. Transplant-related mortality Retrospective analyses had suggested a trend in favor of lower transplant-related mortality (TRM) after PBHCT. Specifically, the EBMT/ IBMTR study showed a significant advantage in TRM for patients trans-
Table 43.4 Chronic graft-versus host disease (GVHD) First report
Follow-up report at 3 years
References
PB
BM
p-value
Reference
PB
BM
p-value
Bensinger et al. [41] Blaise et al. [42] Couban et al. [43] Schmitz et al. [44]
(46%) (50%) 85% (40%) (67%)
(35%) (28%) 69% (30%) (54%)
0.54
100 g/L) or discontinued (hemoglobin > 120 g/L) for a mean of 3.6 weeks among 11 EPO patients compared with 1.9 weeks among seven controls (p = 0.02) [8]. Ninety-one patients between the ages of 17 and 58 years undergoing allogeneic transplants from sibling donors were entered into a doubleblind randomized trial to evaluate the effect of EPO at a dose of 300 U/ kg/day given three times a week by intravenous injection. Treatment ended when the hemoglobin exceeded 120 g/L and recommenced if hemoglobin fell below 120 g/L, at 150 U/kg/day. If hemoglobin exceeded 120 g/L on a further occasion, the dose of EPO was not given. Patients received 2 U of erythrocytes when hemoglobin dropped below 85 g/L. Univariate analysis revealed a significantly higher reticulocyte count, hemoglobin concentration, and bone marrow erythropoiesis after day 14 in the group receiving EPO, but this was not reflected in decreased RBC transfusions (7 ± 5 in controls versus 6 ± 5 in the EPO group). However, in a multivariate analysis, the administration of EPO was associated with an 18% reduction in RBC transfusion requirement when other variables were taken into account [15]. Allogeneic HCT with major ABO blood group incompatibility may be associated with a markedly prolonged time to RBC engraftment, immune hemolysis or pure red cell aplasia. Several reports suggested a curative effect of EPO for these patients [16,17]. In a patient with normal neutrophil and platelet engraftment and pure red cell aplasia despite documentation of an elevated endogenous EPO level (360 mU/mL; normal value < 19 mU/mL) during the 230-day period of absent erythropoiesis, erythroid engraftment was observed soon after the initiation of EPO at a dose of 50 U/kg daily [16]. A 32-year-old patient with acute myeloid leukemia developed pure red cell aplasia after major ABOincompatible BMT. After receiving rHuEPO and methylprednisolone, she developed reticulocytosis and hemolysis. She promptly recovered from hemolysis and pure red cell aplasia after plasmapheresis [17]. In the era of reduced-intensity conditioning using fludarabine-based conditioning regimens, a higher incidence of pure red cell aplasia may be expected in patients with high antidonor isoagglutinins in comparison
Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation
to myeloablative regimens [18], but this does not seem to be a frequently observed problem [19]. The role of EPO was examined in the context of reduced-intensity conditioning. A phase II trial addressed the role of EPO given either from day 0 (19 patients) or starting on day 28 (27 patients) after reducedintensity conditioning and allogeneic HCT. EPO was administered subcutaneously once weekly at a dose of 500 U/kg/week, and 14 patients receiving no EPO served as the control group. Up to 6 months post transplant, hemoglobin values were significantly higher in patients receiving EPO compared with those receiving no EPO, but transfusion requirements were decreased only in the first month in patients receiving EPO from day 0. Interestingly, a reduced T-cell chimerism below 60% negatively influenced hemoglobin levels post transplant [20]. Summary: clinical use of EPO after HCT In summary, EPO stimulates erythroid engraftment after BMT, resulting in a small but significant reduction of RBC transfusion requirements. Whereas trials after autologous transplantation showed negative results, the beneficial effect after allogeneic HCT was more prominent. Late after allogeneic HCT, individual patients with delayed or inadequate erythropoiesis with inappropriate reticulocyte counts and relatively low serum EPO levels, for example patients with renal impairment, chronic inflammation or chronic GVHD, benefit from the application of EPO.
Granulocyte colony-stimulating factor G-CSF is produced by many cell types and is rapidly induced by inflammatory stimuli. In vivo studies demonstrated that G-CSF is a potent myeloid growth and differentiation factor. The dose-dependent increase of peripheral blood neutrophils is caused by expansion of the myeloid compartment in the marrow, an accelerated production of mature neutrophils, and a rapid shift of mature neutrophils from the marrow sinusoids into the peripheral blood [21]. Side-effects of rHu G-CSF administration are usually mild and include bone pain, occasionally low-grade fever, and weight gain. After highdose therapy and HCT, transient pulmonary infiltrates that cause dyspnea, especially during the early phase of hematopoietic engraftment, can occasionally be observed radiologically and clinically. High-dose corticosteroids and diuretics are the treatment of choice for this complication. The entity of the clinical findings of fever, capillary leak, and pulmonary
647
infiltrates, frequently also including a skin rash, observed during engraftment after HCT has been termed “engraftment syndrome,” and posttransplant G-CSF increases the incidence of this syndrome [22]. Two recombinant preparations of G-CSF are commonly in use: filgrastim produced in Escherichia coli and the glycosylated form, lenograstim, produced in a Chinese hamster ovary cell line. Filgrastim is also available in a pegylated formula, pegfilgrastim, for single-dose use. In the following, the term “G-CSF” is generally used for the various recombinant products.
G-CSF after autologous HCT G-CSF after autologous BMT Early data suggested that neutrophil recovery after autologous BMT was accelerated in G-CSF-treated patients compared with historical controls, exceeding 0.5 × 109/L at a mean of 11 days after marrow infusion compared with 20 days for controls, a significant difference. This reduction led to significantly fewer days of parenteral antibiotic therapy, 11 versus 18 days in controls, and less protective isolation in reverse-barrier nursing, 10 versus 18 days [23]. Similar data were obtained after autologous BMT in lymphoma patients [24]. Several large randomized trials have confirmed the significantly accelerated neutrophil engraftment after autologous BMT compared with placebo (Table 45.2). In the first, G-CSF was given to 315 patients after autologous or allogeneic BMT in a prospective, randomized, placebo-controlled multicenter trial [25]. One day after bone marrow infusion, 163 patients received lenograstim 5 μg/kg/day by 30-minute infusion, and 152 patients received placebo daily for 28 days or until neutrophil recovery. Neutrophil recovery to above 109/L for 3 consecutive days was seen earlier in G-CSF-treated patients (16 versus 27 days; p < 0.001). Time to neutrophil recovery above 0.5 × 109/L was reduced (14 versus 20 days; p < 0.001). The difference was significant both in autograft (20 versus 14 days; p < 0.001) and in allograft (20 versus 14 days; p < 0.01) patients, in children (20 versus 13 days; p < 0.001), and in adults. G-CSF-treated patients had fewer days of infection and of antibiotic administration, and also spent less time in hospital. However, the incidence of clinical and microbiologic sepsis was similar in both groups. There was no significant toxicity ascribed to G-CSF. Survival was similar between groups at both days 100 and 365 after transplantation [25].
Table 45.2 Randomized, placebo-controlled trials of granulocyte colony-stimulating factor (G-CSF) following hematopoietic cell transplantation Absolute neutrophil count >500/μL (day after transplantation)
Number of patients enrolled
Type of transplant
G-CSF given from day
G-CSF
Placebo
p-value
Reference
315 43 41 54 38 62 62 54 42
Auto/allo BMT Auto BMT Auto PBHCT ± BMT Auto BMT Auto PBHCT Auto PBHCT Auto PBHCT Allo PBHCT Allo PBHCT
+1 +1 +1 +1 +1 +1 +5 ±0 +1
14 10 10 12 (14) 10 9 10 11 12
20 18 18 20 14 12.5 12 15 15
0.001 0.0001 0.0001 0.0004 0.0001 0.0001 0.0008 0.0082 0.002
[25] [26] [27] [28] [30] [31] [33] [44] [45]
Allo, allogeneic; Auto, autologous; BMT, bone marrow transplantation; PBHCT, peripheral blood hematopoietic cell transplantation.
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Chapter 45
Patients with lymphoma were treated in a randomized, open-label trial to study the use of G-CSF as an adjunct to high-dose chemotherapy and autologous BMT [26]. Of 43 patients, 19 were randomized to receive filgrastim by continuous subcutaneous infusion at a dose of 10 μg/kg/ day, 10 to filgrastim 20 μg/kg/day, and 14 to a parallel control group that received no filgrastim. The median time to neutrophil recovery of 0.5 × 109/L or more after the day of autologous BMT was significantly accelerated to 10 days in the combined G-CSF groups compared with 18 days in control patients. The median number of platelet transfusions was identical in both groups. Clinical parameters, including the median number of days with fever (1 versus 4) and neutropenic fever (5 versus 13.5), were significantly improved in the G-CSF group compared with the control group. For patients treated with the two different dose levels of G-CSF, the neutrophil recovery and clinical results were similar [26]. Forty-one patients undergoing high-dose therapy followed by infusion of autologous PBHCT with or without bone marrow were randomized to receive G-CSF 5 μg/kg/day beginning on day +1, or no G-CSF [27]. The median time to a neutrophil count of 500/μL or more was significantly shorter – 10.5 days in the G-CSF group compared with 16 days in the control group. G-CSF was associated with statistically significant reductions in the time to neutrophil engraftment among patients who received PBHCs alone (11 versus 17 days) and in patients who received PBHCs in conjunction with bone marrow (10 versus 14 days) [27]. In 54 patients with malignant lymphoma, hematopoietic recovery after high-dose chemotherapy and autologous BMT was compared between patients randomized to receive 10 or 30 μg/kg/day of filgrastim or no growth factor [28]. After high-dose chemotherapy with a cyclophosphamide (CY), VP-16, BCNU (CVB) or a BCNU, etoposide, cytosine arabinoside, melphalan (BEAM) regimen followed by autologous BMT, filgrastim was administered by continuous intravenous infusion from the first day after autologous BMT until neutrophil recovery. When the filgrastim groups were compared with the control group, the major findings were: the median time to reach an absolute neutrophil count (ANC) of 0.5 × 109/L or more was 20 days in the control group and 12 and 14 days, respectively, in the filgrastim groups (p = 0.0004). The duration of neutropenia (ANC < 0.5 × 109/L) was reduced from 27 days in the control group to 11 and 13 days in the G-CSF groups (p = 0.0001). In addition, fewer days of febrile neutropenia were observed in the GCSF groups (5 and 6 days) than in the control group (10 days; p = 0.036). No significant effect on the total number of days with fever was observed [28]. An accelerated neutrophil engraftment was observed in 41 consecutive children undergoing autologous BMT for hematologic malignancies in comparison with a similar historical control group of 38 children who did not receive G-CSF after autologous BMT. Their ages ranged from 2 to 16 (mean 7.2) years. G-CSF was given at a dose of 10 μg/kg/day intravenously in a 2-hour infusion from day +1 until +28 or until the ANC was above 1 × 109/L [29]. G-CSF after autologous PBHCT The results obtained after autologous BMT were essentially confirmed in several phase II and III trials (Table 45.2) addressing the role of GCSF after myeloablative chemotherapy and autologous cytokine mobilized PBHCT [30–33]. Of note, since engraftment after PBHCT is generally accelerated compared with unstimulated BMT, the potential benefit of G-CSF given post PBHCT is likely to be smaller. Thirty-eight patients with lymphoproliferative disorders were randomized to receive low-dose filgrastim (19 patients) or placebo (19 patients) beginning on the first day after HCT [30]. All patients received more than 2.5 × 106 CD34+ cells/kg, which had been mobilized with
chemotherapy and filgrastim 300 μg from day 5. Neutrophil engraftment was significantly more rapid in patients who received G-CSF, with a median number of days until ANC was greater than 0.5 × 109/L of 10 (range 9–13) versus 14 (range 9–19). The time to reach an ANC greater than 1 × 109/L was 12 (range 9–14) versus 16 (range 10–25) days. The total number of patients who required intravenous antibiotic therapy was lower in the G-CSF-treated group (68%) compared with the placebo group (89%); also, the median number of days with fever and the duration of antibiotic therapy were shorter, although these differences did not reach statistical significance. However, although only three of 19 (16%) patients who received G-CSF required amphotericin, 11 of 19 (58%) who received placebo did require it, and amphotericin usage was significantly less in the G-CSF group (p = 0.029). Finally, inpatient stay was significantly shortened in those who received G-CSF, from 16 (range 13–23) to 13 (range 11–18) days [30]. In another prospective study, 34 patients had been randomized to receive lenograstim and 28 to receive no growth factor [31]. The median time to an ANC of over 0.5 × 109/L was 9 days in the G-CSF arm versus 12.5 days in the no-G-CSF arm (p = 0.0001). The median number of days to platelet independence, platelet transfusions, incidence of infection, and RBC transfusion was the same in both arms [31]. Factors influencing hematopoietic engraftment were analyzed within the French multicenter LNH93-3 trial treating patients with aggressive high-grade non-Hodgkin’s lymphoma with intensive chemotherapy including BEAM and autologous PBHCT [32]. In the patient population treated with the same first-line regimen, bone marrow involvement and infusion of fewer CD34+ cells delayed platelet recovery. Administration of G-CSF after PBHCT significantly reduced neutropenia [32]. The effect on accelerated neutrophil engraftment by G-CSF appears to be preserved when the start of application is delayed to day 5 after HCT. Sixty-two adult patients were randomized to receive filgrastim after PBSC infusion (n = 30), and 32 patients, the control group, received no cytokines [33]. G-CSF was administered subcutaneously from day +5 in the treated group at a dose of 5 μg/kg body weight per day. The numbers of CD34+ and mononuclear cells infused were similar in each group. Faster granulocyte engraftment was evident in the treated group (mean of 10 versus 12 days to achieve more than 0.5 × 109/L granulocytes; p = 0.0008), without differences in the incidence and severity of infections, number of days of fever or duration of antibiotic treatment between groups. Considering the economic costs, the median expenditure per inpatient stay was c5961 (range c4386–17,186) in the G-CSF group compared with c5751 (range c3676–15,640) in the control group (p = 0.47) [33]. Several randomized trials showed no delayed neutrophil engraftment when G-CSF was not given before days 3, 5, 6, or 7 after PBHCT [34–36]. Early (day +1) or delayed (day +7) G-CSF after immunoselected CD34+-autologous PBHCT significantly accelerated ANC recovery but did not reduce the amount of supportive treatment or the duration of hospitalization in 21 consecutive patients with hematological malignancies [37]. A randomized trial compared pegfilgrastim with filgrastim after highdose melphalan and autologous PBHCT in 37 patients with multiple myeloma receiving a single 6 mg dose of pegfilgrastim on day 1 post transplant versus daily subcutaneous injections of filgrastim 5 μg/kg starting on day 5 post transplant. The median duration of grade 4 neutropenia in the pegfilgrastim and filgrastim groups was 5 and 6 days, respectively (p = not significant). The results for the two groups were also not significantly different for time to neutrophil and platelet recovery, but the incidence of febrile neutropenia (61.1% versus 100%; p = 0.003) and the duration of febrile neutropenia (1.5 versus 4 days; p = 0.005) were lower in the pegfilgrastim arm, demonstrating the safety and efficacy of this approach [38].
Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation
G-CSF after allogeneic HCT Initial concerns over potential aggravation of GVHD or an increase of relapse in patients with myeloid leukemias tempered the use of growth factors after allogeneic BMT. Essentially, after allogeneic BMT, the same effects can be observed as after autologous transplantation. Schriber et al. reported the experience with 50 patients treated at a single institution using G-CSF after allogeneic sibling (n = 30) and matched unrelated (n = 20) BMT [39]. The time to an ANC or 500/μL of greater was significantly less in patients who received G-CSF and cyclosporine and prednisone for GVHD prophylaxis when compared with historical control patients receiving the same GVHD prophylaxis without G-CSF (10 versus 13 days; p < 0.01). A similar accelerated myeloid engraftment was observed for patients who received additional methotrexate for GVHD prophylaxis when compared with historical control patients receiving the same GVHD prophylaxis regimen (16 versus 19 days; p < 0.05) [39]. Similar results were obtained in children [40]. Effects of G-CSF in patients undergoing allogeneic BMT from volunteer unrelated donors were analyzed retrospectively [41]. Cohorts of patients received G-CSF (n = 22) or no G-CSF (n = 25) in a nonrandomized manner. Median time to ANC of over 500/μl was 14 days with G-CSF versus 16 days without G-CSF (p = 0.048). G-CSF did not influence platelet recovery and incidence of infectious complications [41]. Delayed treatment with G-CSF resulted in a reduction of G-CSF treatment from 19 days to 14 days (p = 0.0017) in 38 patients randomly assigned to receive G-CSF starting on day 1 or day 6 after allogeneic BMT [42]. Reducing the length of treatment by 5 days lowered G-CSF treatment costs by 26.3%, and postponing treatment with G-CSF had no influence on the hematologic recovery after allogeneic BMT [42]. Similar results were obtained in 69 patients after unrelated donor BMT randomly assigned to G-CSF starting on day 0, +5 or +10, resulting in equivalent rapid neutrophil engraftment [43]. A randomized, double-blind, placebo-controlled study was performed by Bishop et al. to determine the effects of G-CSF on hematopoietic recovery after allogeneic PBHCT [44]. Fifty-four patients with hematologic malignancies undergoing related, HLA-matched allogeneic HCT were randomly assigned to receive daily G-CSF at 10 μg/kg or placebo starting on the day of transplantation. The median time to achieve an ANC of greater than 0.5 × 109/L was 11 (range 9–20) days for patients who received filgrastim compared with 15 (range 10–22) days for patients who received placebo (p = 0.0082). The median time to achieve a platelet count exceeding 20 × 109/L was 13 (range 8–35) days for patients who received G-CSF compared with 15.5 (range 8–42) days for patients who received placebo (p = 0.79). There were no significant differences for independence from RBC transfusion, the incidence of acute GVHD or 100-day mortality between the groups. The authors concluded that the administration of G-CSF appeared to be a safe and effective supportive care measure following allogeneic peripheral blood HCT [44]. Forty-two adult recipients of allogeneic PBHCT from HLA-matched related donors were randomized to receive G-CSF 10 μg/kg per day subcutaneously from day 1 through neutrophil recovery or no growth factor support after transplantation [45]. There was no significant difference between the two groups in the number of CD34+ cells infused (median 4.8 versus 4.3 × 106/kg). GVHD prophylaxis consisted of tacrolimus and steroids for nine patients and tacrolimus and mini-methotrexate for 33 patients. The group receiving G-CSF had a shorter time to neutrophil levels above 0.5 × 109/L (day 12 versus day 15; = 0.002) and to neutrophil levels over 1.0 × 109/L (day 12 versus day 16; p = 0.01) [45]. After full haplotype-mismatched HCT in humans, the application of GCSF after transplantation markedly delayed T lymphocyte reconstitution, and omitting G-CSF led to a more rapid increase of T cells and improved anti-infectious defense in this specific situation [46].
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A retrospective analysis of the European Group for Blood and Marrow Transplantation addressed the use of G-CSF after allogeneic HCT in 1789 patients with acute leukemia receiving BMT and 434 patients receiving PBHCT from HLA-identical siblings, from 1992 to 2002 [47]. Among the BMT and PBHCT patients, 501 (28%) and 175 (40%), respectively, were treated with G-CSF during the first 14 days after transplantation. BMT and PBHCT patients treated with G-CSF had a faster engraftment of absolute neutrophils, but platelet engraftment was slower. In the BMT patients, GVHD grade II–IV was 50% in the G-CSF group versus 39% in the controls (relative risk [RR] 1.33; p = 0.007, in the multivariate analysis). The incidence of chronic GVHD was also increased (RR 1.29; p = 0.03). G-CSF was associated with an increase in TRM (RR 1.73; p = 0.00016) and had no effect on relapse, but it reduced survival (RR 0.59; p < 0.0001) and leukemia-free survival (RR 0.64; p = 0.0003) rates. No such effects of G-CSF were seen in patients receiving PBHCs [47]. A meta-analysis based on 34 randomized controlled trials of prophylactic G-CSF and GM-CSF after autologous and allogeneic HCT demonstrated a reduced risk of documented infections and duration of parenteral antibiotics, but not in infection-related mortality [48]. The absolute decrease in the risk of documented infection was 8%, and 13 patients would need to be treated with colony-stimulating factors in order to prevent one infection. Subgroup analyses regarding autologous and allogeneic HCT, the use of G-CSF and GM-CSF, or PBHCT and BMT showed similar results. Specifically, in all allogeneic subgroups, the use of CSFs was associated with less TRM and improved survival, although statistically not significant. Furthermore, colony-stimulating factors did not increase the risk of acute or chronic GVHD and TRM after allogeneic HCT [48]. This analysis demonstrates the importance of randomized trials and highlights the potential pitfalls of premature conclusions from retrospective analyses. G-CSF in murine allogeneic transplant models Apart from these clinical findings, data obtained in murine models of allogeneic HCT point towards possible clinically relevant effects of GCSF on the immune system. G-CSF polarized donor T cells towards a T helper 2 (Th2) cell type, resulting in decreased alloreactivity [49], and furthermore decreased the allostimulatory capacity of antigenpresenting cells [50]. Administration of recombinant G-CSF to C57BL/Ka mice markedly increased the capacity of peripheral blood mononuclear cells (PBMCs) to reconstitute lethally irradiated syngeneic hosts. T- and B-lineage lymphocytes were depleted about 10-fold in the bone marrow of the treated mice, and the T-cell yield in the blood was increased about fourfold. The ability of PBMCs or purified CD4+ and CD8+ T cells to induce acute lethal GVHD in irradiated BALB/c mice was reduced after the administration of G-CSF. This effect was associated with decreased secretion of interferon gamma and interleukin-2 (IL-2), and an increased secretion of IL-4. The donor cell inoculum, which was most successful in the rescue of irradiated allogeneic hosts, was the low-density fraction of PBMCs from G-CSF-treated mice. These low-density cells were enriched for CD4−CD8−NK1.1+ T cells and secreted about 10-fold more IL-4 than the unfractionated cells from the G-CSF-treated donors [49]. Another study investigated whether G-CSF administration to PBHCT recipients, both with and without donor G-CSF pretreatment, further modulated acute GVHD in a murine model of PBHCT [50]. Recipients of G-CSF-mobilized splenocytes showed a significantly improved survival and a reduction in GVHD score and serum lipopolysaccharide levels compared with control recipients. G-CSF treatment of donors, rather than recipients, had the most significant effect on reducing levels of TNF-α 7 days after transplantation. As a potential mechanism of the
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reduction in TNF-α, G-CSF was shown to decrease dendritic cell TNFα and IL-12 production to lipopolysaccharide. G-CSF modulated GVHD predominantly by its effects on donor cells, reducing the production of TNF-α. The authors concluded that G-CSF treatment of BMT recipients, without pretreatment of the donor, did not have an impact on acute GVHD [50]. Summary: clinical use of G-CSF after HCT In summary, G-CSF application after allogeneic or autologous bone marrow or PBHCT significantly accelerates the time to neutrophil engraftment without negatively influencing other parameters such as platelet engraftment or GVHD. This effect is more prominent after BMT and is preserved when the start of G-CSF application is delayed from day +1 to +5 or +7 after HCT. Apart from this, present randomized trials fail to show a clear beneficial effect on other factors such as the outcome of patients after HCT. In contrast to autologous HCT, the clinical situation is more complex after allogeneic HCT, and the present data do not allow definite conclusions regarding specific patient situations. The positive effect of G-CSF on acute GVHD in murine allogenic HCT models is not clearly seen in man. This is likely to be due to a more complex situation in the clinical transplantation of patients with different malignant diseases, to state of remission, and to other influencing factors. Despite the retrospective character of the above-mentioned European Group for Blood and Marrow Transplantation study, its results showing a negative influence of G-CSF after allogeneic HCT remains a caveat. The potential immune-modulating effects of G-CSF on mobilized stem cell grafts, as well as G-CSF application to the recipient and a possible aggravation of an “engraftment syndrome,” should be kept in mind when treating patients with G-CSF after allogeneic HCT. Granulocyte–macrophage colony-stimulating factor GM-CSF can be produced by many different cell types, particularly activated T cells. In vitro, GM-CSF promotes the growth and expansion of granulocytic and monocytic, and, in combination with EPO, also multilineage colony-forming units. Multiple functions of mature neutrophils and macrophages, such as tumoricidal activity, superoxide production, antibody-dependent cell-mediated cytotoxicity, phagocytic activity, microbial killing, and secretion of other cytokines are stimulated [51,52].
Furthermore, GM-CSF, when used in vitro, induces the differentiation of malignant antigen-presenting cells with potent T-cell stimulatory capacity [53]. In humans, GM-CSF causes a dose-dependent increase in mainly blood neutrophils and eosinophils, but also macrophages and lymphocytes. There is no effect on the erythroid or megakaryocytic lineage. After application, patients may experience low-grade fever, fatigue, myalgias, and dyspnea. After higher doses, significant fluid retention, pericarditis, pleuritis, and a capillary leak syndrome may develop [54,55]. Commercially available recombinant preparations of GM-CSF (molgramostin and sargramostim) differ in their state of glycosylation. However, this difference does not appear to be clinically relevant. GM-CSF after autologous HCT In a randomized, double-blind, placebo-controlled trial, 128 patients undergoing autologous BMT for lymphoid malignancies were enrolled [56]. Fifty-six patients received GM-CSF in a 2-hour intravenous infusion daily for 21 days, starting within 4 hours of marrow infusion, and 63 patients received placebo. The patients given GM-CSF had a recovery of the neutrophil count to 500 × 106/L 7 days earlier than the patients who received placebo (19 versus 26 days; p < 0.001), had fewer infections, required 3 fewer days of antibiotic administration (24 versus 27 days; p = 0.009), and required 6 fewer days of initial hospitalization (median, 27 versus 33 days; p = 0.01). There was no difference in the survival rate at day 100 after HCT [56]. The data were essentially confirmed by other large randomized trials in autologous BMT [57] and PBHCT (Table 45.3) [58], and in a trial where GM-CSF and G-CSF were used sequentially after autologous transplantation [59]. In contrast, no statistical difference with regard to hematopoietic engraftment of any lineage or clinical parameters was seen in 50 patients of a randomized trial comparing GM-CSF with placebo after autologous PBHCT with GM-CSF-mobilized grafts [60]. GM-CSF was compared with G-CSF post autologous transplantation in a randomized trial with 42 patients with breast cancer randomized to receive filgrastim versus molgramostim subcutaneously at a dose of 5 μg/kg starting on day 6 after PBHCT. PBHCs were collected in all patients after stimulation with filgrastim and infused following highdose chemotherapy. The median days to reach more than 0.5 × 109/L
Table 45.3 Randomized, placebo-controlled trials of granulocyte–macrophage colony-stimulating factor (GM-CSF) following stem cell transplantation
Number of patients enrolled 128 81 343 44 50 40 57 109
Absolute neutrophil count >500/μL (day after transplantation)
Type of transplant
GM-CSF given from day
GM-CSF
Placebo
p-value
Reference
Auto BMT Auto BMT Auto BMT Auto PBHCT Auto PBHCT Allo BMT (sib) Allo BMT (sib, T-deplet.) Allo BMT (sib; CyA, Pred)
±0 ±0 +1 +1 +1 +1 ±0 ±0
19 15 19 14 12 13 16 13
26 28 27 21 14 16 20 17
0.001 0.001 0.001 0.001 0.22 (n.s.) n.s. n.s. 0.0001
[56] [57] [58] [58] [60] [64] [65] [66]
Allo, allogeneic; Auto, autologous; BMT, bone marrow transplantation; CyA, Pred, prophylaxis for graft-versus-host disease with cyclosporine A and prednisone; n.s., not significant; PBHCT, peripheral blood hematopoietic cell transplantation; sib, matched sibling donor; T-deplet., T-cell-depleted graft.
Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation
granulocytes was similar for patients receiving G-CSF (10.5 ± 0.8 days) and GM-CSF (10.2 ± 0.9 days). No significant differences were observed in platelet engraftment or in the number of platelets transfused. Time of discharge was 2 days earlier in the G-CSF arm (15 ± 4.2 versus 17.4 ± 4.7 days; p = 0.04). Finally, the incidence of adverse side-effects attributable to the cytokines (G-CSF or GM-CSF) was equivalent and only present in 19% of the patients [61]. Another study compared the use of G-CSF (5 μg/kg/day) with sargramostim (GM-CSF) 500 μg/day from day 0 until neutrophil recovery (ANC > 1500/μl) in patients with breast cancer or myeloma who had PBSCs mobilized with the combination of CY, etoposide, and G-CSF [62]. Twenty patients with breast cancer or myeloma received GM-CSF, and 26 similar patients received G-CSF. The patients were comparable for age and stage of disease, and received grafts that were not significantly different. Platelet recovery, transfusion requirements, fever days, and use of antibiotics were similar in both groups. The recovery of neutrophils, however, was faster using G-CSF. Achievement of an ANC of more than 500/μl and more than 1000/μl was reached in the GM-CSF group at 10.5 ± 1.5 and 11.0 ± 1.7 days, respectively, whereas with GCSF only 8.8 ± 1.2 and 8.9 ± 2.2 days were required (p < 0.001). This study suggests that neutrophil recovery occurs more quickly when using G-CSF in comparison to GM-CSF at the specific doses used following autologous PBHCT [62]. A randomized trial addressed the in vivo effect of GM-CSF and GCSF administration on the immune recovery of 24 patients who underwent autologous BMT [63]. G-CSF contributed to a significantly faster recovery of CD8+ cells (p = 0.03). The CD8+ cell regeneration consisted mainly of activated cells (CD38+/HLA-DR+) which lacked the CD11b antigen. In contrast, GM-CSF favored the regeneration of CD4+ cells (through both the CD45RO+ and CD45RA+ subset), leading to a higher CD4+ : CD8+ ratio (p = 0.007). Furthermore, the use of hematopoietic growth factors did not seem to exert a significant influence on the recovery of natural killer cells and B lymphocytes [63].
GM-CSF after allogeneic HCT GM-CSF was also tested in randomized trials after allogeneic transplantation. In a randomized, double-blind trial in patients undergoing HCT for leukemia, 20 received GM-CSF and 20 received placebo for 14 days after allogeneic, matched-sibling BMT [64]. The neutrophil count recovered to 0.5 × 109/L 3 days earlier in the GM-CSF group than in the placebo group (not significant). No difference in GVHD or relapse was reported. In a prospective randomized, placebo-controlled trial involving 57 patients receiving T-cell-depleted marrow transplants, GM-CSF led to higher neutrophil and monocyte counts for 6–10 days during the time period of 2–3 weeks after transplantation, resulting in fewer pneumonias [65]. In a prospective, multicenter, randomized, double-blind, placebocontrolled trial, GM-CSF or placebo was administered by 4-hour intravenous infusion starting on the day of marrow infusion (day 0) to day 20 [66]. All patients received marrow grafts from HLA-identical siblings and cyclosporine and prednisone for GVHD prophylaxis. Fifty-three patients received GM-CSF and 56 received placebo. The time to achieve an ANC of over 0.5 × 109/ L was significantly shortened in GM-CSFtreated patients (day 13 versus 17). The incidence of grade III–IV mucositis and infection were significantly reduced and the duration of hospitalization was modestly shortened by 1 day (p = 0.02) in GM-CSFtreated patients. No differences in platelet recovery, erythrocyte recovery, incidence of veno-occlusive disease, GVHD severity, relapse or survival were observed [66]. Of note, in no trial was methotrexate used as GVHD prophylaxis, and when considering GM-CSF post HCT, the
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concomitant use of a growth factor and an antimetabolite may be counterproductive. A long delay in hematologic recovery after BMT, especially graft failure, can extend and amplify the risks of infection and hemorrhage, compromise patients’ survival, and increase the duration and cost of hospitalization. GM-CSF was evaluated in 37 patients with marrow graft failure after allogeneic (n = 15), autologous (n = 21) or syngeneic (n = 1) BMT [67]. GM-CSF was administered by 2-hour infusion at doses between 60 and 1000 μg/m2/day for 14 or 21 days. At doses of less than 500 μg/m2, GM-CSF was well tolerated and did not exacerbate GVHD in allogeneic transplant recipients. No patient with myelogenous leukemia relapsed while receiving GM-CSF. Twenty-one patients reached an ANC of 0.5 × 109/L or above within 2 weeks of starting therapy, while 16 did not. None of seven patients who received chemically purged autologous marrow grafts responded to GM-CSF. The survival rates of GM-CSF-treated patients were significantly better than those of a historical control group [67]. A prospective, randomized trial compared GM-CSF (250 μg/m2/day for 14 days) versus sequential GM-CSF for 7 days followed by G-CSF (5 μg/m2/day for 7 days) as treatment for primary or secondary graft failure after BMT [68]. Eligibility criteria included failure to achieve a white blood cell count of 100/μL or more by day +21 or 300/μL or more by day +28, no ANC of 200/μL or above by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, eight related allogeneic, and five autologous), and 24 received GM-CSF followed by G-CSF (12 unrelated, seven related allogeneic, and five autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC of 500/μL or more) occurred between 2 and 61 (median 8) days after therapy, while those receiving GM-CSF + G-CSF recovered at a similar rate of 1–36 days (median 6 days; p = 0.39). Recovery to independence from RBC transfusion was slow, occurring 6–250 (median 35) days after enrollment with no significant difference between the two treatment groups (GMCSF median 30 days; GM-CSF + G-CSF median 42 days; p = 0.24). Similarly, independence from platelet transfusion was delayed until 4– 249 (median 32) days after enrollment, with no difference between the two treatment groups (GM-CSF median 28 days; GM-CSF + G-CSF median 42 days; p = 0.38). Recovery times were no different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone, yielding 100-day survival estimates of 96 ± 8% for GM-CSF versus 71 ± 18% for GM-CSF + G-CSF (p = 0.026). Sequential growth factor therapy with GM-CSF followed by G-CSF offered no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT [68]. Summary: clinical use of GM-CSF after HCT In summary, GM-CSF accelerates neutrophil engraftment after autologous and allogeneic transplantation. However, due to a lack of advantage over G-CSF, GM-CSF is not routinely used for this purpose. GM-CSF has been used effectively in bone marrow failure states. The immunostimulating effects of GM-CSF, with its capacity to differentiate antigenpresenting cells, are employed with or without concomitant donor lymphocyte infusions for relapse of leukemia after allogeneic transplantation, and a beneficial role of the combination of alpha-interferon and GM-CSF is suggested [69]. Furthermore, GM-CSF may have a place as an adjunct in post-transplant vaccination trials and in anti-infectious strategies, for example in treating patients with invasive fungal infections by augmenting the phagocytic capacities of monocytes and macrophages.
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Interleukin-3 IL-3, also known as multi-CSF, is a multilineage hematopoietic growth factor produced by T lymphocytes and mast cells that acts on early and committed cell populations of all lineages. In vivo, IL-3 administration is often accompanied by severe side-effects with fever, malaise, chills, headache, arthralgias, and urticaria [70]. Because of these side-effects, a dose-escalating study using IL-3 alone had to be stopped at 2 μg/kg/ day [70]. Used alone, IL-3 did not enhance neutrophil engraftment compared with the use of GM-CSF [71]. IL-3 was used in combination with G-CSF in 54 consecutive patients with refractory or relapsed lymphoma [72]. Four of 31 patients (12.9%) who received IL-3 subcutaneously experienced one severe adverse event, defined as World Health Organization (WHO) grade 3–4 toxicity (fever, n = 2; pulmonary toxicity, n = 2) and were withdrawn from the study. The addition of IL-3 resulted in a significant improvement of multilineage hematopoietic recovery, lower transfusion requirements, fewer documented infections, and a shorter stay in the hospital [72]. In summary, due to the side-effects and only modest positive impact on hematopoiesis, IL-3 or its modifications have not been licensed for clinical use after HCT.
Stem cell factor Stem cell factor (SCF), also known as steel factor, kit-ligand or mast cell growth factor, promotes the proliferation and differentiation of the most primitive hematopoietic progenitor cells into committed progenitor cells. Recombinant human SCF (ancestim) has been developed for clinical use in combination with G-CSF to optimize stem cell mobilization from the marrow into the peripheral blood for apheresis and to provide a sustained increase in the number of hematopoietic stem cells capable of engraftment [73]. When used for hematopoietic cell mobilization, a considerable number of patients reacted with delayed-type hypersensitivity such as skin rash, dyspnea, angioedema, and cardiovascular symptoms. No trials with the use of SCF after HCT have been published. In a single case report, an unexpectedly rapid rise in platelet count was observed with complete hematological recovery after the BEAM regimen in a patient who could not be rescued by autologous transplant but who received G-CSF, epoetin-alpha, and SCF [74]. The authors attributed these findings to this specific growth factor combination, including SCF [74]. Presently, there is no proven indication for SCF after transplantation, and there is no licensed product available for clinical use.
Thrombopoietin/megakaryocyte growth factor Thrombopoietin (TPO) stimulates the growth and differentiation of megakaryocytes and proplatelets via the receptor c-mpl. It is predominantly produced by hepatocytes, and serum levels are inversely correlated with platelet counts. Two ligands for the mpl receptor with broad in vitro megakaryocytic proliferative, maturational, and differentiation capacity have been developed and tested in clinical trials. One is a modified (PEGylated) form of a recombinant truncated mpl ligand, called megakaryocyte growth and development factor (MGDF) [75]. The other is the recombinant glycosylated version of the naturally occurring TPO molecule [76]. In a randomized trial, 47 patients with stage II, III or IV breast cancer undergoing autologous HCT were allocated to placebo (n = 13) or to one of five sequential dose cohorts of PEG-rHuMGDF (n = 34), resulting in a median time to platelet recovery of 11 and 12 days for the placebo and combined PEG-rHuMGDF groups, respectively [77]. In an open-label phase I study, TPO was administered intravenously by bolus
after autologous BMT without the development of neutralizing antibodies [76]. A case of pancytopenia associated with the development of neutralizing antibodies to TPO occurred in a patient who had undergone chemotherapy repeatedly with multiple cycles of subcutaneous administration of PEGylated rHuMGDF [78]. Samples of the patient’s bone marrow showed trilineage hypoplasia with absence of myeloid, erythroid, and megakaryocyte progenitor cells, but with elevated endogenous levels of EPO, G-CSF, and SCF. The subcutaneous route of application of TPO in this trial may have been of importance for the development of anti-TPO antibodies. The role of recombinant megakaryocyte growth factors after HCT has not been thoroughly tested and remains unclear. Furthermore, due to reports on the development of neutralizing antibodies and thrombophilia in vivo, the above-mentioned recombinant products have not been licensed for clinical use. After the experience of these first-generation thrombopoietic growth factors, several second-generation thrombopoietic factors are under development, as summarized in a review by Kuter [79]. AMG 531 is a TPO-mimetic peptide linked to human immunoglobulin G fragments, and weekly subcutaneous applications in healthy volunteers and patients with immune thrombocytopenia resulted in elevated platelet counts without major side-effects apart from mild headache at the day of injection. Phase III trials are presently underway [79]. Further developments include the orally available small molecules eltrombopag and AKR-501 with TPO-agonistic properties [79]. These developments will likely result in clinically available platelet stimulating drugs.
Interleukin-11 IL-11 is a naturally occurring cytokine produced by fibroblasts and bone marrow stromal cells and is a growth factor with pleiotropic effects overlapping those of other growth factors. In vivo, IL-11 administration stimulates megakaryopoiesis and increases peripheral platelet and neutrophil counts [80], an effect that can be enhanced by combination with IL-3. A recombinant preparation of IL-11, oprelvekin, produced in E. coli, is licensed for clinical use for the prevention of severe thrombocytopenia and reduction of the need for platelet transfusions following myelosuppressive chemotherapy. Major side-effects include fluid retention and edema. A placebo-controlled, randomized trial with 80 breast cancer patients tested the effects of recombinant IL-11 on platelet recovery after highdose chemotherapy and autologous transplantation with PBHC support. The results did not demonstrate that treatment with rHuIL-11 significantly decreased platelet transfusion [81]. In a murine allogeneic BMT model of GVHD directed against major histocompatibility antigens and minor antigens, an immunomodulating effect of IL-11 with prevention of lethal GVHD while preserving the graft-versus-leukemia effect was demonstrated [82]. IL-11 decreases cytokine release and increases survival in murine BMT models. In these systems, it reduces gut permeability, partially polarizes T cells to a Th2 phenotype, downregulates IL-12, prevents mucositis, and accelerates recovery of oral and bowel mucosa. In a randomized, double-blind pilot study, IL-11 was administered with cyclosporine/MTX prophylaxis after conditioning with CY/total body irradiation (TBI) and allogeneic stem cell transplantation for hematologic malignancies [83]. Patients received IL-11 50 μg/kg/day subcutaneously or placebo. Only 13 patients (10 IL-11, three placebo) were enrolled because the early stopping rule for mortality was enforced. Of 10 evaluable patients who received IL-11, four died by day 40 and one on day 85. Deaths were attributable to transplant-related toxicity. One of three placebo recipients died of a suicide; the other two are alive. Patients receiving IL-11 had severe fluid retention and early mortality,
Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation
making it impossible to determine whether IL-11 given in this schedule could reduce the rate of GVHD. Grade II–IV acute GVHD occurred in two of eight evaluable patients on IL-11 and one of three patients on placebo. The primary adverse events of the study were severe fluid retention resistant to diuresis (average weight gain 9 ± 4%) and multiorgan failure in five of 10 evaluable patients. The authors concluded that IL-11 as administered in this trial for GVHD prophylaxis in allogeneic transplantation could not be recommended [83].
Interleukin-2 IL-2 is produced by activated T lymphocytes and is an important regulator of the immune system, acting on T, B, and natural killer cells. IL-2 knockout mice develop T cells normally, but they suffer from generalized fatal immunoproliferative disorders and loss of self-tolerance. Recombinant IL-2, aldesleukin, is licensed for the treatment of metastatic renal cell carcinoma. Several phase II trials used IL-2 after autologous HCT for patients with lymphoma or acute myeloid leukemia as an immunomodulator to reduce relapse rates. Fifty-six patients with advanced lymphoma received autologous peripheral blood-derived hematopoietic grafts incubated with IL-2 followed by IL-2 4 mIU/m2 for the first 4 weeks post transplant and IL-2 maintenance. The 3-year progression-free survival of 31% was similar to that of a previous study cohort receiving no IL-2 and was not dependent on the actual IL-2 dose received; IL-2-related toxicity was considerable but reversible [84]. A similar approach was chosen in 59 patients with stage II–IV breast cancer, testing IL-2 in a randomized way [85]. Three-year progressionfree survival was 53% and 48% for patients receiving or not receiving IL-2, without any statistical difference. Of note, some of the patients developed the clinical syndrome of acute GVHD [85]. High-dose IL-2 (9 mIU/m2/day × 4) was given to 39 patients with acute myeloid leukemia in first complete remission, about one-third with good-risk core binding factor leukemias, after engraftment from autologous HCT, resulting in a 2-year progression-free survival of 74% and a TRM rate of 4% [86]. Without large randomized trials, the role of IL-2 in the context of autologous HCT remains unclear. In a pilot trial, the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and nine patients with chronic myeloid leukemia after allogeneic HCT were examined [87]. Overall, IL-2 treatment resulted in a median 1.9-fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a median 9.7-fold increase in FOXP3 expression in CD3+ T cells [87]. Successful treatment with IL-2 0.5 mIU/ m2/day by continuous intravenous infusion of a patient with progressive multifocal leukencephalopathy developing after allogeneic HCT was reported [88], highlighting the role of IL-2 as a potent immunomodulator after allogeneic HCT. Furthermore, IL-2 has an effect by enhancing the antileukemic action of donor lymphocyte infusions for relapsing leukemia after allogeneic HCT [89], a topic covered in a different section of this book.
Keratinocyte growth factor Keratinocyte growth factor (KGF), also called fibroblast growth factor7, is a mediator of epithelial cell proliferation and a growth factor for hepatocytes and pneumocytes. KGF is naturally produced in mesenchymal cells and acts on a wide variety of epithelial cells. It has been shown to be protective in radiation- or chemotherapy-induced organ damage. In murine models of allogeneic transplantation, it protected the gut from lethal GVHD [90]. Interestingly, in murine transplantation models, KGF protected thymic epithelial cells from cytotoxic damage when given prior to conditioning [91]. Furthermore, pharmacologic doses of KGF allow regeneration of GVHD-induced thymic damage, and therefore
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KGF appears to exert a potent effect on thymic epithelial cell function, which in turn allows for normal T lymphopoiesis to occur during acute GVHD [92]. Palifermin, the N-truncated form of rHuKGF-1, is available for the prevention of mucosal damage after intensive chemotherapy or radiation therapy [93]. KGF in autologous HCT A phase I multicenter, dose-escalating trial was performed with rHuKGF for mucositis prevention in lymphoma patients conditioned with the BEAM protocol prior to autologous HCT [94]. A dose of 60 μg/kg/day for 3 days prior to high-dose BEAM chemotherapy was chosen based on safety and preliminary efficacy. With this dose, a reduction in duration of severe ulcerative mucositis from 4.6 (placebo) to 0.8 days was observed [94]. In a case series of patients treated prophylactically with KGF during high-dose chemotherapy and autologous HCT, reduction of severe oral mucositis and related symptoms, but not infections, dietary intake, time to engraftment or cumulative dose and duration of narcotic administration, was observed compared with patients without KGF treatment [95]. KGF improved thymus-dependent T-cell reconstitution with elevated T-cell receptor excision circles after autologous CD34(+) PBHC transplantation in rhesus macaques conditioned with myeloablative TBI [96]. KGF has been proved to significantly reduce the duration and severity of oral mucositis after high-dose TBI-containing therapy in randomized trials [97,98]. In one trial, KGF 60 μg/kg/day was given for 3 days prior to TBI and on days 0, 1, and 2 after transplantation. With this, the incidence of oral mucositis of WHO grade 3 or 4 was 63% in the KGF group and 98% in the placebo group (p < 0.001). The median duration of oral mucositis of WHO grade 3 or 4 was 3 (range 0–22) days in the KGF group and 9 (range 0–27) days in the placebo group (p < 0.001), and patients reported less soreness and use of opioid analgesics in the KGF group [98]. Side-effects were skin rash, pruritus, erythema, and taste alteration of mild-to-moderate severity, and were transient. KGF in allogeneic HCT KGF was tested in a randomized, double-blind, placebo-controlled, dose-escalation study regarding safety, engraftment, and influence on GVHD (n = 69 patients) compared with placebo (n = 31) in patients conditioned with CY/TBI or busulfan/CY and given methotrexate along with a calcineurin inhibitor (cyclosporine or tacrolimus) for GVHD prophylaxis. All patients received three doses before conditioning and either three (cohort 1), six (cohort 2) or nine (cohort 3) doses after HCT. Palifermin doses were 40 μg/kg/day (cohort 1 only) or 60 μg/kg per day (all cohorts). Six patients (two on placebo, four on palifermin) experienced a total of 11 dose-limiting toxicities (most often skin, respiratory or oral mucositis). The most common adverse events included edema, infection, skin pain or rash. Palifermin was associated with a reduced incidence and severity of mucositis in patients conditioned with CY/TBI but not busulfan/CY. KGF was regarded as safe in allogeneic HCT but had no significant effect on engraftment, acute GVHD or survival in this trial [99].
Conclusion In several situations, hematopoietic growth factors have a defined role after HCT. In particular, randomized trials have documented faster erythroid engraftment with EPO after allogeneic BMT, and G-CSF resulted in accelerated neutrophil engraftment after allogeneic and autologous marrow as well as PBHCT. Regarding other endpoints, the results are
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equivocal. The value of these growth factors given after HCT has to be seen in the light of pros and cons of the individual patient and specific transplant situation. Faster engraftment is only one aspect, and other factors, such as number of days in hospital or in isolation, transfusion requirements, possible cytokine-induced engraftment syndromes, or
influence on the immune system, especially after allogeneic HCT, as well as costs, have to be taken into consideration. In the future, growth factors preventing or repairing tissue damage, modulating the immune system post transplantation, and optimizing platelet engraftment will be of special interest.
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93. Blijlevens N, Sonis S. Palifermin (recombinant keratinocyte growth factor-1): a pleiotropic growth factor with multiple biological activities in preventing chemotherapy- and radiotherapy-induced mucositis. Ann Oncol 2007; 18: 817–26. 94. Durrant S, Picot JL, Schmitz N et al. A phase 1 study of recombinant human keratinocyte growth factor (rHuKGF) in lymphoma patients receiving high-dose chemotherapy (HDC) with autologous peripheral blood progenitor cell transplantation (autoPBSCT). Blood 1999; 94(Suppl 1): 708a #3130. 95. Horsley P, Bauer JD, Mazkowiack R et al. Palifermin improves severe mucositis, swallowing problems, nutrition impact symptoms, and length of stay in patients undergoing hematopoietic stem cell transplantation. Support Care Cancer 2007; 15: 105–9. 96. Seggewiss R, Lore K, Guenaga FJ et al. Keratinocyte growth factor augments immune reconstitution after autologous hematopoietic progenitor cell transplantation in rhesus macaques. Blood 2007; 110: 441–9. 97. Stiff PJ, Emmanouilides C, Bensinger WI et al. Palifermin reduces patient-reported mouth and throat soreness and improves patient functioning in the hematopoietic stem-cell transplantation setting. J Clin Oncol 2006; 24: 5186–93. 98. Spielberger R, Stiff P, Bensinger W et al. Palifermin for oral mucositis after intensive therapy for hematologic cancers. N Engl J Med 2004; 351: 2590–8. 99. Blazar BR, Weisdorf DJ, Defor T et al. Phase 1/2 randomized, placebo-control trial of palifermin to prevent graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Blood 2006; 108: 3216–22.
46
Claudio Anasetti, Franco Aversa & Andrea Velardi
Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors
Introduction Hematopoietic cell transplantation (HCT) from human leukocyte antigen (HLA)-matched siblings has become the treatment of choice for many hematologic diseases, but fewer than 40% of patients have an HLAmatched sibling [1]. Registries of HLA-typed volunteers have been established worldwide to provide HLA-matched unrelated donors for HCT (see Chapter 47). The chance of finding an unrelated donor matched for HLA-A, B, C, and DR depends on the HLA diversity of the population and varies with race, ranging from 75% in Caucasians to less than 50% for United States ethnic minorities (http://www.marrow.org). A limitation in the use of unrelated donors derives from the long duration of the search, which may allow disease progression in patients who urgently need transplantation, such as those with acute leukemia. Umbilical cord blood offers the advantages of easy procurement, no risk to the donors, low risk of transmissible infections, and immediate availability of the cryopreserved graft (see Chapter 39). However, engraftment is a major concern when the harvested cord blood contains a low number of mononuclear cells. Older patient age and HLA disparity are poor risk factors for engraftment and survival after transplantation of unrelated cord blood hematopoietic cells [2,3]. An alternative source of hematopoietic cells is from relatives who are partially matched for HLA antigens. Almost all patients have at least one HLA partially matched family member, parent, sibling or child, who is immediately available as donor. Transplants of T-replete marrow or growth factor-mobilized peripheral blood progenitor cells (PBPCs) from relatives mismatched for a single HLA-A, B or DR antigen have met with acceptable success rates using standard protocols commonly employed for transplants from HLA-identical siblings [4,5]. Conversely, transplants of T-replete marrow from donors mismatched for two or three HLA-A, B, and DR antigens have resulted in an extremely high incidence of severe acute graft-versus-host disease (GVHD) [4,6]. Marrow T-cell-depletion has been associated with increased risk of graft rejection, but the use of T-depleted PBPCs has enhanced the probability of engraftment despite donor mismatch for two or three HLA antigens, as well as carrying a low incidence of acute and chronic GVHD [7,8]. In this chapter, we first present principles for the selection of related donors with the least degree of HLA disparity for use in conventional
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
transplants, and later present more recent developments in the field of T-depleted PBPC transplants from donors mismatched for two or three HLA-A, B, and DR antigens.
Donor selection Function and polymorphism of HLA The HLA system (described in detail in Chapter 12) includes at least 12 genetic sites, named HLA loci, located on the short arm of human chromosome 6. Each HLA locus is highly polymorphic because it is occupied by multiple alternative forms of an HLA gene, designated HLA alleles, any of which may be carried by a given individual. HLA alleles encode class I HLA-A, B, and C antigens, and class II HLA-DR, DQ, and DP antigens. Class I HLA antigens are expressed on the surface of all nucleated cells in the body, while class II HLA antigens are expressed on the surface of antigen-presenting cells (APCs) such as dendritic cells, monocytes, B cells, and activated T cells. It is the function of HLA molecules to bind antigenic peptides and activate the immune response in a specific manner. In order to accommodate the need for the binding of ever-changing environmental and microbial antigens, HLA molecules have evolved through gene duplication, gene conversion, recombination, and point mutation to acquire an enormous degree of polymorphism, most evident across different ethnic groups. Class I HLA molecules present antigenic peptides and activate cytotoxic CD8+ T cells, while class II HLA molecules present antigenic peptides and activate helper CD4+ T cells. Both classes of HLA molecule lead to T-cell activation through the binding of specific T-cell antigen receptors. In the case of HLA-incompatible HCT, donor and recipient differ not only for one or more types of HLA molecule, but also for the thousands of antigenic peptides that each mismatched HLA molecule can bind and present to foreign T cells [9]. By this process, HCT leads to the activation of an enormous number of both donor and host T cells that mount a response, its strength depending on the degree of HLA mismatch. T-cell recognition of antigenic peptides presented by mismatched HLA molecules can result both in graft rejection and GVHD. HLA class I molecules also bind and activate specific receptors on natural killer (NK) cells. Recent developments on the role of NK cells in transplantation are presented later in this chapter, in the section on Tdepleted grafts, where the effects of NK cell function have become apparent. HLA class I and II molecules also function as antigens by eliciting antibody responses by B cells. It is thought that anti-HLA antibodies can mediate a hyperacute rejection of allogeneic HCTs [10].
657
658
Chapter 46
HLA haplotypes and segregation in families The HLA antigens inherited together from one chromosome of each parent are referred to as an HLA haplotype (Fig. 46.1). When a family study is performed in search of an HLA-identical sibling donor, parents should also be typed for HLA-A, B, and DR, and each of the four parental HLA haplotypes identified. Parental haplotypes can segregate among the offspring in four different combinations, and the chance that any one sibling is HLA identical with another is 25%. If an HLA-identical sibling is not found, it is possible to find family members who are partially matched for HLA, even though they have not inherited the same two haplotypes, because certain HLA antigens and haplotypes are frequent. The parental haplotypes should be reviewed for homozygosity or sharing of antigens. The parents, siblings and children, and other relatives who share a haplotype with the patient may have a second haplotype that is partially matched with the patient. Figure 46.1(a) illustrates a family in which the parents have matching haplotypes, “b” and “c.” The patient (sib 1, “a/c”) has a unique HLA type, and none of the siblings is HLA identical with the patient. However, the patient and the father (“a/b”) share the paternal “a” haplotype and are matched for the HLA-A, B and DR antigens of their unshared haplotypes (“b” and “c”). Figure 46.1(b) illustrates a family in which the
Parental sharing
Homozygous parent
A B DR
A B DR a b
c
d
A B DR
a b
c d
a c sib#1
a d sib#2
A B DR a c sib#1
a d sib#2
b c sib#3
(a)
b d sib#4 (b)
Fig. 46.1 Segregation of human leukocyte antigen (HLA) haplotypes and partial sharing of HLA antigens within a family. (a) The two parental haplotypes “b” and “c” are identical, so that sibling 1 (haplotypes “a” and “c”) is compatible with one of these parents (haplotypes “a” and “b”). (b) One parent is HLA homozygous (haplotype “c” and “d” being identical), so that sibling 1 (haplotypes “a” and “c”) and sibling 2 (haplotypes “a” and “d”) are compatible.
mother (“c/d”) is HLA homozygous. The patient (sib 1, “a/c”) and sib 2 (“a/d”) share the paternal “a” haplotype and each has inherited one of the two maternal haplotypes (“c” and “d”). Because of the serendipitous similarity of the maternal “c” and “d” haplotypes, sib 1 and sib 2 are HLA matched. Donor and recipient matching Potential donors are those relatives who share by inheritance one HLA haplotype with the patient and are variably mismatched for 0, 1, 2 or 3 HLA-A, B, and DR loci of the unshared haplotype. A more precise classification of matching, useful in predicting the risk of rejection and GVHD after HLA-incompatible T-replete transplants, takes into consideration the vector of incompatibility in situations where donor or recipients are homozygous at one of the mismatched loci (Table 46.1) [6]. If recipient and donor are overall incompatible for one HLA locus but the recipient is homozygous at the mismatched locus, the mismatch does not contribute a risk for GVHD. Conversely, if recipient and donor are overall incompatible for one HLA locus but the donor is homozygous at the mismatched locus, the mismatch does not contribute a risk for graft rejection. Originally, some of the polymorphisms of HLA class I and II antigens were defined serologically by typing with alloantisera obtained from women with a history of pregnancy. The development of genetic typing has demonstrated that few HLA antigens represent unique alleles [11]. For example, HLA-A2 is defined as an antigen by serology, but DNA sequencing has identified as many as 89 distinct alleles, designated from HLA-A*0201 to A*0299, all of which are expressed with a unique amino acid sequence but are recognized by the same anti-HLA-A2 antibody (http://www.anthonynolan.org.uk/HIG). Some other unique HLA-A*02 alleles are not expressed as proteins (for example, A*0215N), they are instead defined as “null” and are immunologically silent. Once a null allele is identified, it should be ignored for donor selection. Distinction between the variants of polymorphic HLA antigens appears to be functionally relevant because, for example, each of the seven B27 antigen variants can be distinguished by specific cytotoxic T-cell clones [12]. The clinical importance of HLA-A, B, C, and DRB1 allelic differences that cannot be distinguished by serologic typing was demonstrated in studies of unrelated donor HCT where mismatching was associated with increased GVHD and worse survival [13–19]. Increasing evidence has related DPB1 mismatching with graft failure, acute GVHD, and protection from leukemia relapse [20–25]. However, the relevance of donor disparity for DQB1 or DPB1 for the post-transplant survival of patients with malignancy remains disputed [21–26]. In the process of
Table 46.1 Mismatching according to vector of genetic disparity for human leukocyte antigen (HLA)-A, B, and DR Vector† Example
Haplotype
Recipient
Donor*
Overall
Rejection
Graft-versus-host disease
Heterozygous recipient and donor
Mismatched Shared* Mismatched Shared Mismatched Shared
A2, B44, DR7 A1, B8, DR3 A1, B35, DR1 A1, B8, DR3 A2, B44, DR4 A1, B8, DR3
A3, B7, DR2 A1, B8, DR3 A3, B35, DR1 A1, B8, DR3 A1, B8, DR4 A1, B8, DR3
3
3
3
1
1
0
2
0
2
Homozygous recipient Homozygous donor
Incompatible antigens are represented in bold. * Recipient and donor are related and share HLA haplotype. † Number of mismatches at HLA-A, B or DR loci.
Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors
donor selection, we must consider that recipients and donor pairs, who have not inherited the same HLA haplotypes but are assumed HLA identical on the basis of serologic typing alone, might be reclassified as mismatched for one or more HLA alleles using high-resolution DNA typing technology. Crossmatching Patients may be alloimmunized by pregnancy or blood transfusions, and sensitization to donor alloantigens increases the risk of graft failure [10,27,28]. Crossmatch testing can determine whether the recipient has been sensitized to HLA antigens of the donor, and is indicated before the selection of an HLA-mismatched donor for HCT. Standard assays for crossmatching the patient’s serum with donor lymphocytes include complement-dependent microcytotoxicity and multicolor flow microfluorimetry [29,30]. HLA-specific antibodies can be detected by enzymelinked immunosorbent assay, flow cytometry or Luminex technologies that are likely to replace the more cumbersome direct crossmatch [31–33]. Probability of identifying an HLA partially compatible family donor One report from Germany found that the probability of identifying an HLA-matched sibling was 40.7%, a matched relative 4.0%, a one HLAA, B or DR locus-mismatched sibling 3.1%, and a one-locus mismatched relative 10.4% [1]. The best chance of finding a donor for patients without an HLA-matched or a one-locus-mismatched sibling is an unrelated donor search, because this is currently successful in up to 75% of cases. If the initial search identifies one or more HLA-A, B, and DR matched unrelated donors, there is no obvious justification to undertake an extended family search beyond siblings, because not only would the probability of finding a suitable donor be lower, but also the donor typing effort and the cost of finding donors would be greater [1]. Computer programs have recently been developed that can calculate the probability of finding a suitable related or unrelated donor based on patient typing [1,34–36], and the results of such programs can be utilized to define the best search strategy for an individual patient. Candidates for allogeneic HCT with no suitably matched donor and a short life expectancy might be better served by immediate transplantation from a relative mismatched for one entire HLA haplotype, or by a partially matched unrelated umbilical cord blood transplant, than by a lengthy search for a completely matched donor.
659
necessary to assess the presence of donor cells. Functional studies in patients with graft failure have demonstrated that residual host T lymphocytes are cytotoxic against donor alloantigens, and the patient’s serum may be active in the antibody-dependent cell-mediated cytotoxicity test against donor cells [37,38]. These findings have indicated that alloimmune-mediated rejection is a mechanism for graft failure after HCT from HLA-incompatible donors. If a high-dose conditioning regimen is administered, either primary or secondary graft failure is usually associated with persistent aplasia. In certain patients, recovery of autologous myeloid cells can occur, especially if the patient is pretreated with reduced-intensity conditioning regimens and administered granulocyte–macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) after graft failure [39]. If there is no recovery of myeloid function despite growth factor therapy, a second transplant from the same or a different donor can be attempted, but the success of second transplants has been limited [10]. Risk factors. HLA mismatch. The relevance of HLA compatibility to sustained engraftment was first analyzed in 269 patients with hematologic neoplasms who underwent T-replete marrow transplantation from a family member who shared one HLA haplotype with the patient, but differed to a variable degree for the HLA-A, B, and DR antigens of the unshared haplotype [10]. These 269 patients were compared with 930 patients who received marrow from siblings with an identical HLA genotype. All patients were treated with cyclophosphamide (CY) and total body irradiation (TBI) followed by infusion of unmodified donor marrow cells. The incidence of graft failure was 12.3% among recipients of marrow from an HLA partially matched donor compared with 2.0% among recipients of marrow from an HLA-matched sibling (p < 0.0001) (Table 46.2). The incidence of graft failure correlated with the degree of HLA incompatibility in the host-versus-graft direction. Graft failure occurred in three of 43 transplants (7%) from donors who were matched with their recipient for HLA-A, B, and DR, in 11 of 121 donors (9%) incompatible for one HLA locus, in 18 of 86 (21%) incompatible for two loci, and in one of 19 (5%) incompatible for three loci (p = 0.03). Therefore, donor HLA incompatibility is a significant risk factor for graft failure. Results of unrelated donor transplants point to HLA-C and DPB1 mismatch as risk factors for graft failure that might account for the graft failure observed after transplantation from family donors matched for HLA-A, B, and DR [13,20]. The reasons for the apparently lower incidence of graft failure with a transplant incompatible for three loci compared with two loci reported in that study are not obvious [10]. Transplants with the highest degree of mismatch were conducted predominantly in children who received a higher marrow cell dose. It is
Transplantation of T-replete marrow grafts Transplant outcome The relevance of HLA incompatibility between donor and recipient has been analyzed in patients with hematologic disorders who received HCTs of T-replete marrow grafts. The patient and related donor shared one HLA haplotype but differed to a variable degree for the HLA-A, B, and DR antigens of the unshared haplotype. The results of these transplants have shown the effect of HLA incompatibility on graft failure, GVHD, and survival, formally demonstrating that HLA antigens constitute the major histocompatibility complex (MHC) in humans. Graft failure Clinical presentation. Primary graft failure is likely to occur if severe granulocytopenia persists for 21 days after transplantation of marrow or PBPCs, but secondary graft failure may also occur after initial engraftment (see Chapter 80) [10]. Studies of chimerism (see Chapter 25) are
Table 46.2 Effect of HLA incompatibility on marrow graft failure
Failure of engraftment Late graft failure All failures
HLA-nonidentical donor (n = 269) number of patients (%)
HLA genotype-identical donor (n = 930) number of patients (%)
p value
23 (8.5%) 10 (4.1%) 33 (12.3%)
15 (1.6%) 4 (0.4%) 19 (2.0%)
20:
BM BM BM PBPC
CY + ATG CY + PCB + ATG CY + Cam CY (120) + FLU
PBPC
PBPC (80%)
FLU (90) + 2 Gy TBI
CY (120) + FLU (180) ± ATG
BM
PBPC (70%)
(180) + ATG CY + ATG
BM (30%)
CY (120) + FLU
(125) + ATG
BM
CY + TAI (n = 98) CY + ATG (n = 31)
PBPC: n = 134
BM: n = 558
various
CY + ATG (54%),
age ≤20: n = 349;
–
CY + ATG
± varied
BM
BM
CY (n = 60) CY + ATG (n = 70) CY
BM
CY + ATG
–
1–67 (18.7)
4–46 (19)
1–51 (24)
2–63 (25)
source
Conditioning program
CSP + MTX
+ MMF
CSP + MTX
CSP + MTX
CSP
CSP + varied
CSP ± Cam
CSP + MTX
CSP + MTX
Varied CSP + MTX
or other
CSP + MTX
CSP + MTX
CSP or −
CSP + MTX
5
3
0
16
0
0
24
15
–
11 11
CSP + MTX
CSP versus CSP + MTX
4
CSP + MTX
GVHD
29
50
11
8
69
14
11
15
42 0
PBPC: 14
BM: 10
–
–
38 30
18 11
24
Acute
GVHD (%)
32
50
4
13
46
4
12
17
82
75
86
84
77
81
89
86
58 90
age >20: 52
age >20: 30 64 42
age >20: 64 age ≤20: 73
age ≤20: 85
76
(1991–96) 74 (1997–02) 80
78 94
74 80
88
Survival (%)
age >20: 31 age ≤20: 27
age ≤20: 12
–
–
44 30
21 32
26
Chronic
(1.8)
0.3–4.3 (2.0)
0.4–3.5 (1.5)
0.2–8.0 (3.9)
0.5–5.4 (1.75)
0.5–12 (4.9)
0.1–6.6 (2.5)
0.2–11.6 (5.8)
(13.6) (4.4)
0.4–11.0 (5)
5-year survival
2.0–13.2 (3.6)
0.6–7.8 (4.0)
0.4–10.2 (6.3)
0.5–16.4 (9.2)
years (median)
follow-up in
Range of
In all series, the majority of patients were previously transfused. ATG, antithymocyte globulin; BM, bone marrow; Cam, alemtuzumab; CSP, cyclosporine; CY, cyclophosphamide (200 mg/kg, unless otherwise indicated); EBMT, European Group for Blood and Marrow Transplantation; FLU, fludarabine (total dose in mg/m2); GITMO, Gruppo Italiano Trapianti di Midoleo Osseo; GVHD, graft-versus-host disease; IBMTR, International Bone Marrow Transplant Registry; MTX, methotrexate; NHLBI, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD; PBPC, granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells; PCB, procarbazine (37.5 mg/kg); TAI, thoracoabdominal irradiation; TBI, total body irradiation; –, data not reported. * Additional interactive Cox survival model analysis for transplant outcome based on EBMTR data of neutrophil count, age, and year of transplant (up to 1990) is available at: http://www.ebmt.org/4Registry/registry5.html.
35
8
13
33
113
21
129
692
352
1275
71
130
81
1988–2004
2005
Seattle, Kahl et al. [53]
years (median)
patients
(%)
rejection
plant
Graft Prevention of
of
Hematopoietic
Age range in
Number
report
Year of trans-
Transplant team, reference
Year of
Table 49.1 Selected recent reports of human leukocyte antigen (HLA)-identical hematopoietic cell transplantation for severe aplastic anemia (SAA)
Hematopoietic Cell Transplantation for Aplastic Anemia
709
710
Chapter 49 100
50
No preceding blood transfusion (n = 53) 75
22/63 Survival (%)
% Graft rejection
40
30
20
Previously transfused (n = 115) 50
25
15/102
p = 0.04
13/110 10
0 0
3/81
3
6
9
12
15
18
Years following marrow transplantation
0 £1975
1976–80
1981–88
>1988
Year of marrow graft
Fig. 49.1 The incidence of graft rejection versus year of transplantation for patients in Seattle receiving marrow from human leukocyte antigen-identical siblings. All patients were conditioned with cyclophosphamide-containing regimens. The numbers above the bars indicate the number of rejections per number of patients transplanted. (Data from [24,53].)
Fig. 49.2 The effect of transfusion status on actuarial survival for patients treated between 1978 and 1991. Tick marks denote censoring times of surviving patients. The p-values were calculated using the log-rank test and are two-sided. (Reproduced from [37], with permission.)
Table 49.2 Transfusion sensitization prior to transplantation with hematopoietic grafts from dog leukocyte antigen-identical littermates after 9.2 Gy total body irradiation
Group
Preceding blood product transfusion on days −24, −17, and −10*
A B
None From marrow donor
C
From two different unrelated dogs on each of the 3 successive days Leukocyte-poor red blood cell transfusions from the donor
D
E
Leukocyte-poor platelet transfusions from the donor
2000 cGy in vitro gamma irradiation
Total number of dogs studied
Number of dogs with sustained engraftment (%)†
Not applicable Yes No Yes No No
62 20 27 16 27 14
61 (95%) 17 (85%) 0 (0%) 15 (94%) 10 (37%) 9 (64%)
No
15
8 (53%)
Statistical significance
} p value < 0.001 } p value = 0.001 p value = 0.001 (compared with group A) p value 90%) present in CP, often diagnosed incidentally during routine examination. Symptoms, when present, usually include fatigue, weight loss, bony aches, and abdominal discomfort from splenomegaly. Patients generally present with leukocytosis, thrombocytosis, and anemia; the marrow is hypercellular. The cytogenetic examination shows the Ph chromosome in over 90% of patients, and in the remaining 10%, cryptic or complex translocations can be detected by fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) assays. Without therapy, CML evolves from CP to AP and eventually to BC. In approximately 25% of patients, there is no intervening AP between CP and BC. Indeed, molecular studies show that there is no clear distinction between AP and BC [19]. Various definitions of AP have been developed. Two of the more commonly employed are those of Sokal et al. [20] and the International Bone Marrow Transplant Registry (IBMTR) [20] (Table 51.1). In general, AP is characterized by symptoms of fever, night sweats, weight loss, and bone pain, difficulty in controlling blood counts using conventional therapy, increased numbers of blasts and early myeloid cells in marrow and peripheral blood, and evidence of karyotypic evolution. The most common cytogenetic changes associated with disease evolution are an additional Ph chromosome, trisomy 8, isochrome i(17q), and trisomy 19 [21]. BC has been defined as having more than 30% blasts and promyelocytes in the bone marrow or peripheral blood, or the development of extramedullary blastic infiltrates [20], although a recent reclassification by the World Health Organization has lowered the blast cut-off to 20%. In approximately two-thirds of patients, the blasts are predominately myeloid, while in the remaining third the blasts have a lymphoid phenotype [22].
Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia
735
Table 51.1 Definition of accelerated phase chronic myeloid leukemia Sokal criteria [20]
IBMTR criteria [82]
Peripheral blood or marrow blasts ≥5% Basophils >20% Platelet count ≥1000 × 109/L despite adequate therapy Karyotype evolution Frequent Pelger–Huet-like neutrophils; nucleated crythrocytes, megakaryocytic nuclear fragments Marrow collagen fibrosis Anemia or thrombocytopenia unrelated to therapy Progressive splenomegaly Leukocyte doubling time 3-log reduction of BCR-ABL transcript level from an aggregate baseline of 30 newly diagnosed patients) at 12 months of imatinib therapy had a 100% probability of remaining progression free at 24 months. In comparison, patients with a CCyR and a less than 3-log reduction of BCR-ABL had 95% progression-free survival (PFS), while patients who did not achieve a CCyR by 12 months had an 86% PFS [57]. Five-year follow-up data from this study revealed that no patient who achieved a MMR by 12 months had progressed to advanced disease [38]. Earlier responses (95%) patients with CP CML, failure to achieve such a response is considered an indicator of a treatment failure and warrants a change in therapy [56]. Cytogenetic response Cytogenetic monitoring of the level of Ph+ metaphases is the strongest prognostic factor for predicting long-term response to imatinib once patients have achieved CHR. Achieving a CCyR is an independent
Mutational analysis of the ABL kinase domain While there is a relatively low incidence of imatinib-resistant BCR-ABL mutations arising in patients with “early” CP CML, mutations are more frequent in patients with late CP or advanced-phase disease [53]. Thus, regular mutation screening is appropriate for patients with advanced disease. Additionally, patients who respond suboptimally to imatinib or show an increase in the level of BCR-ABL transcripts should also be screened [51]. Such regular mutational monitoring could facilitate early identification of potential mutant clones and allow treatment to be switched prior to further disease progression.
Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia
737
Table 51.2 Treatment response to imatinib therapy Response
3 months
6 months
12 months
18 months
Failure Suboptimal Optimal
No HR No CHR CHR and 1–2-log decrease in BCR-ABL
>95% Ph 35–95% Ph 35% Ph 1–35% Ph 0% Ph (CCR) and >3-log decrease in BCR-ABL
>0% Ph 0% Ph and 3-log decrease in BCR-ABL transcripts
CCyR, complete cytogenetic response; CHR, complete hematologic response; HR, hematological response; Ph, Philadelphia chromosome.
Recommendations for response assessment
High-dose imatinib
Time-based landmark responses have been established in order to identify patients who are unlikely to respond to imatinib (Table 51.2) [56,58,67]. Patients can be sorted into groups according to treatment response – that is, failure, suboptimal or optimal. Those in the failure category are considered to be unresponsive to imatinib and should be switched to another treatment (a second-generation TKI or transplantation). Patients who respond suboptimally may still benefit from imatinib, but are less likely to have a favorable long-term outcome, and alternative therapies should be considered. The current recommendations for monitoring response to imatinib treatment in CML are outlined in the recently updated National Comprehensive Cancer Network guidelines [58]. Cytogenetic testing is recommended at 6 months and 12 months after the start of therapy. At 18 months, another cytogenetic evaluation should be conducted if a CCyR has not been achieved by 12 months. Alternative therapies should be considered for patients without: (1) a CHR by 3 months; (2) any sign of a cytogenetic response by 6 months; (3) a partial cytologic response by 12 months; or (4) a CCyR by 18 months of therapy. If the patient appears to be responding to therapy (especially once a CCyR is obtained), quantitative PCR measurement of BCR-ABL in the peripheral blood is recommended every 3 months. If a rise in BCR-ABL is detected and confirmed by two measurements taken approximately 1 month apart, the frequency of measurements can be increased to once a month. Rising levels of BCR-ABL transcripts should also prompt mutation screening. For patients in CP, screening for ABL kinase domain mutations is recommended if there is an inadequate initial response. Additionally, mutation analysis should also be performed on patients with any indication of a loss of response, including hematologic or cytogenetic relapse and a rise in BCR-ABL transcript level. Given the high frequency of BCR-ABL mutations associated with advanced-stage disease, routine mutational screening should be performed in these patients every 3 months regardless of treatment response.
High-dose imatinib (600–800 mg/day) is an option in patients with CP CML who respond suboptimally to treatment with 400 mg/day imatinib. The basis for this therapeutic approach stems from studies demonstrating that some BCR-ABL mutations have only intermediate resistance to imatinib [68], and that BCR-ABL amplification and overexpression sometimes confer resistance. Thus, perhaps increased imatinib may overpower BCR-ABL in certain resistant cases. While studies have shown that high-dose imatinib can produce responses in patients who have relapsed on or are refractory to standard-dose imatinib [69,70], these responses are generally not dramatic or durable. Thus, with the advent of new agents, this strategy is not generally recommended for patients with advanced-phase disease and is not appropriate for patients harboring highly resistant BCR-ABL mutations or BCR-ABLindependent resistance.
Treatment options after first-line imatinib failure There are several treatment options that are now available for patients with imatinib intolerance, who have a poor initial response to therapy or who relapse later. “Second-generation” TKIs are now available and are recommended for the treatment of patients who are resistant to imatinib. For those patients who have a suitable matched related or unrelated donor, hematopoietic stem cell transplantation is an option that has the longest track record of potential cure. Lastly, clinical trials are available for imatinib-intolerant or imatinib-resistant cases, and this option should be considered with high priority.
Secondary TKI therapy Dasatinib is an oral, multitargeted kinase inhibitor, including BCR-ABL, SRC, c-KIT, and platelet-derived growth factor receptor-beta. Dasatinib has greater than 300-fold greater activity against native BCR-ABL in vitro as compared with imatinib, and binds both the active and inactive BCR-ABL conformation. Dasatinib has activity against all imatinibresistant BCR-ABL mutations with the exception of T315I [71,72], and was recently approved by the United States Food and Drug Administration and European Medicines Evaluation Agency for the treatment of imatinib-resistant or imatinib-intolerant CML in any phase and Ph+ ALL [73–76]. Indeed, in CP patients resistant to or intolerant of imatinib, a CCyR was achieved in approximately 40% of cases overall after 8 months of therapy [75]. Moreover, a recent randomized study compared dasatinib with high-dose imatinib in patients with imatinib-resistant CP CML (at doses of imatinib from 400 to 600 mg/day). Patients on dasatinib had CCyR rates of 40%, compared with 16% on high-dose imatinib. The rate of MMR was also greater with dasatinib compared with highdose imatinib, at 16% versus 4%, respectively [54]. Dasatinib may also play a role in the treatment of advanced-phase CML. Several studies have demonstrated efficacy in advanced-phase disease. For example, 27% and 43% of myeloid and lymphoid BC, respectively, achieved a CCyR on dasatinib. Unfortunately, the response was rather short lived, as the median PFS was 5 and 3 months for myeloid and lymphoid BC, respectively [73,74]. Nilotinib has also shown activity in patients with imatinib-resistant or imatinib-intolerant CP and AP CML [77,78]. Nilotinib has yielded CCyR in approximately 40% of CP and 20% of AP cases, respectively. The estimated overall survival for CP patients was 95%, and 79% for AP patients. It should be noted that it is, however, impossible to directly
Chapter 51
compare the results for dasatinib and nilotinib in these trials, since the definitions of imatinib resistance and intolerance differ between them.
Management of the newly diagnosed CML patient in the imatinib era Given the success of imatinib, and the promise of newer TKIs, one must ponder if a chapter devoted to transplantation of CML is destined to the medical history shelf. Is transplantation for CML dead? Here is what we know: 1 Imatinib is remarkably effective for patients treated in CP, as greater than 75% of patients obtain a CCyR. In the IRIS trial, approximately 70% of cases remain in a CCyR at 5 years of follow-up. Treatment of advanced-phase (accelerated or blast disease) is associated with much poorer outcomes. 2 Allogeneic transplantation is generally associated with 10-year survivals of 75% or better for patients in CP, but survival likewise falls in accelerated or blast-phase disease. 3 Relapse occurs for patients in CP treated with imatinib, but outcome can be effectively monitored by sensitive RT-PCR assays. Current recommendations from advisory panels, such as the European Leukemia Net and the National Cancer Care Alliance, state that all CP patients start on imatinib therapy, but allow for consideration of transplantation based on the patient’s age, preference, and response to initial imatinib [58,67]. The tacit assumption is that, given the excellent results of both imatinib and transplantation, a contemporary randomized trial comparing the methods would be very unlikely. There are also questions that make the decision on how early to transplant problematic. For example, given that TKI do not appear to kill the CML stem cell, is relapse inevitable? Given that many relapses stemming from ABL point mutations act aggressively, will these patients be especially hard to cure with transplantation? And, while short-term TKI therapy does not seem to impact negatively on transplant results, what will happen with long-term exposure to these agents? There have been a few attempts to compare up-front transplantation with nontransplant therapy [79]. A recent trial enrolled 621 patients with CP CML; of these, 354 were considered eligible for transplantation and “biologically randomized” based on the availability of a related donor. Of the 123 patients who received a transplant, the 10-year estimate of survival was 53%. Those 219 patients without a related donor were treated with IFN until imatinib became available later in the trial. Imatinib was then offered to patients whose disease did not respond to IFN. The 10-year estimate of survival in this group was 52%. The survival curves of these two groups show that the transplant group suffered a higher early mortality, with a relative flattening of the survival curves, whereas the nontransplant group had a better early outcome, with the survival curves continuing to drop. The crossover of the curves came at 8 years. It should be noted that the “nontransplant” group contained patients who later underwent an unrelated transplant (their survival being 69%). It is unfortunately hard to translate this study to contemporary practice. Given the remarkable success of imatinib as first-line therapy, it would be expected that the survival curves for patients treated with imatinib up front would be superior to that of the IFN–imatinib group in this study. In addition, most transplant centers would be disappointed with the survival statistics in the transplant arm of this study of long-term outcomes, which were approximately 50%. All things considered, imatinib is a reasonable initial treatment of choice for patients with CML, and in most treatment guidelines it is recommended as the initial therapy for CP patients. For patients with CP disease, imatinib can be initiated, with a concurrent work-up of family donors, and if needed, unrelated donors. Criteria for imatinib failure or suboptimal response are in evolution (Table 51.3). Certainly
failure to achieve a CHR at 3 months of treatment is an indication to switch therapy. At 6 months of therapy, patients should have some cytogenetic response, and by 12 months, patients should achieve an MCyR, with the elimination of two-thirds of their Ph chromosomes on cytogenetic examination. By a conservative approach, patients should achieve a CCyR by 18 months of therapy. Patients who relapse after a CCyR, especially those with ABL point mutations, should consider alternative therapy, including transplantation. For patients diagnosed in AP or BC, initial treatment with imatinib or dasatinib is recommended, but since responses are generally short, all such patients should be evaluated for transplantation and, if found to be appropriate candidates with suitable donors, should proceed to transplantation as soon as possible.
Allogeneic hematopoietic cell transplantation The outcome of allogeneic hematopoietic cell transplantation (HCT) in CML is influenced by many factors, most importantly the phase of disease, but also the type of donor used, the nature of the stem cell product, and the age of the patient. Phase of disease As with all treatments of CML, outcomes are far superior for HCT for patients in CP compared with advanced-phase disease. The Seattle team began studying HLA-matched sibling transplants for CP CML in 1979, and in 1982 reported initial results from 10 patients [80]. Shortly thereafter they published the first large study on 167 patients transplanted through 1983 from matched siblings [81]. These results have been updated through November 2002, as shown in Figs 51.1 and 51.2, and show that 40% of patients transplanted in CP survive; results deteriorate with transplantation done in advanced phases. Data from the International Bone Marrow Transplant Registry (IBMTR) between 1994 and 1999 show a probability of survival of 69% (standard deviation 2%) for 2876 patients transplanted within the first year from diagnosis, and 57% (standard deviation 3%) for 1391 patients transplanted more than 1 year from diagnosis (Fig. 51.3) [82]. Contemporary results from selected single institutions continue to demonstrate the excellent outcomes with HCT. For example, the Seattle group has recently reported on their most recent trial using a preparative regimen of targeted BU plus cyclophosphamide (CY) in 131 consecutive CP CML patients. Survival 3 years
Annals paper (Tx through 1983) Updates as of November 2002
1
0.8
Survival
738
0.6
Blast phase in remission (n = 12)
0.4
Chronic phase (n = 67)
0.2
Accelerated phase (n = 46) Blast phase (n = 41)
0 0
5
10
15
20
25
Years
Fig. 51.1 Kaplan–Meier probabilities of survival by phase at the time of transplantation for 166 patients with chronic myeloid leukemia who received transplants through 1983 from human leukocyte antigen-identical siblings, first published in 1986 [81]. The results are updated as of November 2002. Tx, treatment.
Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia Annals paper (Tx through 1983) Updates as of November 2002
1
1.0 Overall survival
0.8
0.8 Disease-free survival
0.6
0.4
Probability
Relapse
739
Blast phase (n = 41) Chronic phase (n = 67)
0.2
Accelerated phase (n = 46)
0.6
0.4
0.2
0 5
0
10
15
20
Relapse
25 0.0
Years
0
Fig. 51.2 Cumulative evidence of cytogenetic relapse by phase at the time of transplantation for 166 patients with chronic myeloid leukemia who received transplants through 1983 from human leukocyte antigen-identical siblings, first published in 1986 [81]. The results are updated as of November 2002. Tx, treatment.
1
2
3
4
5
6
Years after transplant
Fig. 51.4 Kaplan–Meier probabilities of survival and disease-free survival and the cumulative incidence of relapse among 131 patients with chronic myeloid leukemia in chronic phase transplanted from matched siblings following a preparative regimen of targeted busulfan and cyclophosphamide [83].
100
Probability (%)
80
HLA-identical sibling, 50 chromosomes 6% 25%
TCR translocations 7q35/TCRb 14q11/TCRad
2%
41%
6% Hypodiploid 100,000/μL in 70%) CD13/CD33 coexpression (>50%) Incidence increasing with age (75% if >55 years) Partly CD20+ (45%) Large tumor mass (lactate dehydrogenase increased in >90%) Organ involvement (32%) CNS involvement (13%) CD20+ (>80%) Mediastinal tumor (60%) CNS involvement (8%) High white cell count (>50,000/μL) (46%) Subtypes: Early T (6%) Thymic T (12%) Mature T (6%)
70% t(4;11)/ALL1-AF4 (20% Flt3 in ALL1-AF4+) 4% t(1;19)/PBX-E2A (pre-B only)
High risk
Common-ALL (49%) Pre-B ALL (12%) Mature B ALL (4%) (L3-ALL, Burkitt’s leukemia)
T ALL (25%)
Adapted with permission from Gökbuget and Hoelzer [19].
White cell count >30,000–50,000/μL t(9;22)/BCR-ABL t(1;19)/PBX-E2A
t(8;14)/c-myc-IgH
20% t(10;14)/HOX11-TCR 100,000/μL HOX11L2
794
Chapter 55
induction, the mortality is related to age, with the major cause of death being infection. Patients who fail to achieve a remission, analogous to those patients who are slow to achieve a remission, have a very poor prognosis and are candidates for early HCT [19]. Several studies have demonstrated that allogeneic HCT performed after failure to achieve a CR can be curative in approximately 20% of patients [21,22]. Based on the intensity of the induction treatments now being utilized in adult treatment regimens, failure to achieve a remission is an indication for early HCT, rather than utilizing repetitive cycles of therapy that is not likely to be effective. Thus, for adult patients, human leukocyte antigen (HLA) typing of the patient and family members is an important test to perform early after diagnosis. CNS prophylaxis CNS leukemia occurs in about 6% of patients, with a higher incidence in T-cell ALL (8%) and mature B-cell ALL (13%). Treatment and prophylaxis of CNS leukemia usually consists of intrathecal methotrexate (MTX) alone or in combination with ARA-C or prednisone. Adult ALL patients who do not receive specific CNS treatment have a CNS relapse rate of 30%, similar to that observed in children. Thus, all patients should receive some form of CNS therapy. In most adult ALL trials, prophylaxis has included CNS radiation to 24 Gy, which reduces the CNS relapse rate to about 9%. It is not clear whether systemic high-dose treatment alone prevents CNS prophylaxis, but it may be possible to avoid radiation by combining early high-dose chemotherapy and intrathecal therapy. The risk of CNS relapse is associated with other prognostic factors, including T-cell ALL, B-cell ALL, extreme leukocytosis, elevated lactate dehydrogenase, and extramedullary organ involvement. Management of CNS relapse prior to HCT In general, the development of a CNS relapse in patients with ALL portends a poor prognosis, both for the effect it has on the CNS and because it is a harbinger of systemic relapse. Most patients with ALL have had some form of CNS prophylaxis with radiation or chemotherapy prior to their first systemic relapse. Although children with an isolated CNS relapse can still do well with the treatment of CNS and systemic reinduction therapy, adults with an isolated CNS relapse fare poorly, and such an event is an indication for HCT [23]. Patients who relapse in the CNS require additional therapy to control the disease prior to HCT. Previous prophylactic intrathecal therapy and CNS radiation does not necessarily preclude the use of total body irradiation (TBI) in the preparation for HCT. However, if a patient has had CNS irradiation and relapses, a second course of irradiation prior to a TBI-containing preparative regimen is not recommended as this may lead to more neurotoxicity following transplantation. In general, these patients can be managed with either intrathecal MTX or the combination of MTX, ARA-C, and hydrocortisone until there is clearing of leukemic cells from the spinal fluid. Following HCT, such patients should receive five intrathecal MTX injections during the first 100 days, followed by one monthly injection for 12–18 months or until toxicity precludes additional treatments. This approach, designed in Seattle, can result in control of the leukemia without substantially increasing the risk of leukoencephalopathy [24]. The risk of CNS damage caused by cranial radiation, intrathecal chemotherapy, and high-dose chemotherapy has been documented, and is related to the amount of intrathecal chemotherapy following the HCT procedure [24]. Currently, a common approach for patients with ALL who will be undergoing HCT, without any evidence of CNS disease, is to administer intrathecal MTX as prophylaxis for a total of five times prior to the HCT procedure. Cranial radiation is not necessary if the
patient then receives a preparatory regimen containing fractionated TBI. Maintenance therapy The optimal duration and form of maintenance therapy in adult ALL is unknown. The goal of maintenance treatment is to eliminate minimal residual disease (MRD) that may have persisted after induction and consolidation treatment. Standard maintenance is usually based on a combination of 6-mercaptopurine and MTX. Attempts in previous trials to omit maintenance after induction and consolidation therapy resulted in inferior results. In general, the cure rate with chemotherapy for adults with ALL is 35–40%. These results may be better in adolescents and younger adults utilizing more pediatric chemotherapy-based regimens, and are poorer in older adults, especially over the age of 60 [25,26]. Treatment of Ph+ ALL As noted above, translocation 9;22 is the most frequent genetic alteration in adult ALL, found in 20–30% of patients, and the incidence increases with age. The phenotype of this leukemia is almost exclusively CD10+ precursor B ALL, and there are only rare reports of its presence in Tlineage ALL. In this circumstance, the disease is likely related to the blast crisis of CML. Clinically, patients present with a variable white blood cell count, surface expression of CD19, CD10, and CD34, as well as the coexpression of myeloid markers, especially CD13 and CD33. The principles of initial diagnosis and treatment are similar to those of non-Ph+ ALL. Cytogenetic and molecular genetic analyses are required to establish the diagnosis of Ph+ ALL and can be obtained within a week of diagnosis. These studies are important because of the evolving treatment of the disease with targeted therapy. Historically, the prognosis of adult patients with Ph+ ALL treated only with chemotherapy has been poor, with less than a 10% probability of long-term disease-free survival (DFS). CR rates after induction therapy range from 60% to 90%; however, the median remission duration was inferior, between 9 and 16 months, with almost no long-term survivors. As discussed below, allogeneic HCT has been the treatment of choice for such patients, with many series showing better than 50% long-term survival in patients who undergo HCT in first remission. The disease has been even more difficult to treat in older patients with ALL where this cytogenetic abnormality actually is more common. The understanding of the leukemogenic role of the BCR–ABL oncogene and its dependency on the constitutive activation of the ABL tyrosine kinase led to the development of selective ABL inhibitors, with imatinib being the first to gain clinical approval. Its initial activity was demonstrated in phase I and phase II trials in CML, but also in ALL [27]. Although many patients with Ph+ ALL achieved a remission, there were very few long-term survivors. Only the subset of patients who then went to allogeneic HCT while in remission had a favorable outcome, with 50% of the patients disease free at 1 year [27]. Poor results of single-agent imatinib led to various investigations to explore the efficacy of imatinib as front-line therapy in combination with chemotherapy. Phase II trials combining imatinib (days 1–14 of each cycle), with hyperfractionated CVAD (CY, vincristine, adriamycin, and dexamethasone) chemotherapy had a very high remission rate after 21 days of treatment, suggesting synergy between imatinib and concurrent chemotherapy, with some patients achieving a molecular remission [28]. Similarly, encouraging results have been achieved in other centers in which imatinib was started after 1 week of induction therapy and then co-administered with chemotherapy during the remaining induction. In these trials, the 1-year event-free survival and overall survival was
Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults
78–88%, with tolerability comparable to that seen with chemotherapy alone. Although the optimal schedule for combining imatinib with chemotherapy has not yet been established, both alternating and concurrent imatinib chemotherapy combinations have been studied, with a clear advantage of the simultaneous over the alternating schedule in terms of the achievement of a negative PCR for the BCR–ABL gene [29]. Those patients who undergo allogeneic HCT for ALL do not seem to have any detrimental effect of prior imatinib treatment. Given the increased incidence of Ph+ ALL in older patients with ALL and the difficulty of achieving a remission in these patients, imatinib has also been studied as front-line treatment in older patients with ALL, with an improved outcome compared with chemotherapy, and with better tolerability [30]. Several studies now suggest that imatinib-combined chemotherapy in newly diagnosed Ph+ ALL with subsequent treatment with allogeneic HCT has improved the results of treatment. With regard to the CNS, patients with Ph+ ALL are at significant risk for developing CNS leukemia [31]. Imatinib concentrations in the cerebrospinal fluid reach about 1–2% of serum levels and are not therapeutic. Thus, all patients with ALL, independent of their chromosome abnormality, require CNS prophylaxis [31]. Additional novel tyrosine kinase inhibitors with significantly more potent activity against the BCR–ABL gene have been developed and are in clinical trials. Currently, trials are being developed to compare combination chemotherapy and imatinib to allogeneic HCT in the era of tyrosine kinase inhibitors. As discussed below, the use of these drugs may have an impact on the pretransplant disease burden of the patient and reduce the risk of relapse following HCT. Prognostic factors in ALL The major prognostic factors for achieving a CR are advanced age and Ph+ ALL. These prognostic factors are of even greater importance in predicting the durability of remission and survival, and are utilized to assess the need for allogeneic HCT. Table 55.2 identifies some of the adverse prognostic factors for remission duration in adult ALL that have been identified in previous clinical trials and are utilized in determining a patient’s risk of relapse. Role of MRD In addition to age and cytogenetic analysis at the time of diagnosis, the most important prognostic factor, and a direct reflection of sensitivity to chemotherapy, is the rapid achievement of a CR. Thus, a slower time to achieving a remission is an indicator of relative chemoresistance, similar to what has been observed in pediatric patients. Those patients who take more than one cycle of induction chemotherapy have a poor long-term prognosis and a shorter remission duration [32–34]. A more quantitative approach to assess the response of an individual patient to chemotherapy is the measurement of MRD at various time
Table 55.2 Adverse prognostic factors for remission duration in adult acute lymphoblastic leukemia (ALL)
795
points after therapy. Quantitative assessment of tumor cell kill is emerging as an independent prognostic factor that reflects the resistance of the cells to chemotherapy and allows potential individualization of treatment [35,36]. The assessment allows the identification of potential patients at high risk for relapse despite achieving a morphologic remission and who may benefit from early HCT. Studies are being performed to determine the most predictive time point for measurement. It appears that, after consolidation, a high level of MRD at 10−4 is associated with a high risk of disease relapse, with a rising level of MRD on treatment also portending relapse [37,38]. In some studies, a high level of MRD after induction and consolidation has been identified as a high-risk feature despite the achievement of a morphologic remission and the absence of high-risk cytogenetics [37,38]. Conversely, the identification of patients who are sensitive to chemotherapy and achieve a low level of MRD (nondetectable) may identify a group of patients who do not need transplantation or can wait until there is clear evidence of relapse [37,38]. It also remains to be determined what the benefit of HCT may be in patients who are in first remission but have evidence of a new factor defining high-risk disease, that is, high MRD. At the present time, MRD measurement has certain limitations related to the technical procedure, which is time-consuming, expensive, and requires a specialized laboratory to conduct the studies. The testing also involves multiple evaluations with either immunophenotypic flow cytometry analysis or molecular analysis with patient-specific probes for gene rearrangements. Thus, the future of treatment of adult patients with ALL in first remission may be refined to determine those patients who are unlikely to benefit from further chemotherapy and should be considered for transplantation during first remission. Allogeneic HCT in first CR of ALL Allogeneic HCT in first CR (CR1) has generally been reserved for those patients who present with poor-risk features, such as those described earlier. In several phase II studies, patients with high-risk disease treated with allogeneic HCT had a DFS longer than would have been predicted, especially those with Ph+ ALL. Depending on the risk factors present at diagnosis in an individual patient, standard chemotherapy leads to continued remissions ranging from less than 10% to more than 50% [39,40]. Studies have been conducted indicating that HCT offers some groups of high-risk patients long-term disease survival rates of between 40% and 60% [41–47]. At the City of Hope and Stanford University, two series of patients with high-risk features who underwent allogeneic HCT in CR1 have been recently updated. Selection criteria included a white blood cell count of over 25,000/μL, chromosomal translocations t(9;22), t(4;11) or t(8;14), age older than 30 years, extramedullary disease at the time of diagnosis, and/or the need for more than 4 weeks to achieve a CR. Two-thirds of the patients had at least one risk factor, and the
Clinical characteristics
Immunophenotype
Cytogenetics/molecular genetics Treatment response
Higher age >35 years High white blood cell count >30,000/μL in B-lineage, 100,000/μL in T-cell ALL Pro B (B-lineage, CD10−) Early T (T-lineage, CD1a−, sCD3−) Mature T (T-lineage, CD1a−, sCD3+) t(9;22)/BCR–ABL or t(4;11)/ALL1-AF4, 1;19 Late achievement of complete remission >3–4 weeks Minimal residual disease positivity in remission
796
Chapter 55
1.0 0.9 0.8
Probability
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
1
2
3
4
5
6
7 8 9 Time in years
10
1.0 Median DFS
DFS Probability
0.8
Sibling donor 25.6 months No sibling donor 13.8 months
3-year DFS 5-year DFS 47% 34%
45% 23%
11
12
13
14
15
Fig. 55.2 Probability of event-free survival (upper curve), overall survival (middle curve), and relapse (bottom curve) for 55 adult patients with high-risk acute lymphoblastic leukemia transplanted in first remission. (Reproduced and updated from [48], with permission.)
Table 55.3 European Cooperative Oncology Group–Medical Research Council UKALL XII/EC2993: outcome after allogeneic hematopoietic stem cell transplantation in Philadelphia chromosome-negative patients who had donors versus those who did not have donors
No.
5-year overall survival (%)
5-y relapse rate (%)
2-year nonrelapse mortality (%)
High risk* Donor No donor
401 171 230
40 36
39 62
39 12
Standard risk Donor No donor
512 218 294
63 51
27 50
20 7
0.6 45% 0.4 P = .007 0.2
0
18%
2
4 6 8 Time from CR achievement (years)
10
Fig. 55.3 Disease-free survival (DFS) according to genetic randomization. The group with a sibling donor comprised 100 patients, whereas that with no sibling donor included 159 patients. CR, complete remission. (Reproduced from [39], with permission.)
* High risk is defined as age 35 years or over, white cell count over 30,000/μL for patients with B-cell disease or over 100,000/μL for patients with T-cell disease, or time to attain complete remission of longer than 4 weeks.
undergoing allogeneic transplant in first remission. Table 55.4 shows a comparison of a number of other trials comparing chemotherapy to HCT. remaining patients had two or more high-risk features at presentation. The majority of these patients underwent allogeneic HCT within the first 4 months after achieving a CR. Allogeneic HCT during first remission led to prolonged DFS in this patient population, who would otherwise have been expected to fare poorly. At a median follow-up of greater than 5 years, the probability of event-free survival was 64%, with a relapse rate of 15% (Fig. 55.2) [48]. The French Group on Therapy for Adult ALL conducted a study comparing chemotherapy to autologous and allogeneic HCT [39]. Although the overall results of treatment did not show a treatment advantage for the group treated with allogeneic HCT, subgroup analysis revealed that those patients with high-risk disease, including patients with a t(9;22) and t(1;19) translocation, had a higher 5-year survival of 44% as opposed to 20% in the other two groups (Fig. 55.3). The recently completed Eastern Cooperative Oncology Group–Medical Research Council (ECOG–MRC) trial showed that allogeneic HCT resulted in improved disease control, with long-term benefit seen mostly in younger patients [49]. Table 55.3 shows the DFS and response rate for patients on this trial with the risk of relapse reduced following allogeneic HCT when compared with either chemotherapy or autologous HCT, showing the improved outcomes and better leukemia control for those patients
HCT for Ph+ ALL Historically, the dismal outcome with chemotherapy has led to trials focusing on the use of allogeneic transplantation for the treatment of adult Ph+ ALL. Most have been single-institution studies utilizing a variety of regimens, and the cure rate varies from 30% to 65%, dependent upon age and remission status [50,51]. Investigators from City of Hope and Stanford University have analyzed their experience in 79 patients with Ph+ ALL transplanted from HLA-identical siblings while in CR1 between 1984 and 1997 to determine long-term survival and disease control. All patients but one were conditioned with fractionated TBI (1320 cGy) and high-dose etoposide (60 mg/kg). The 3-year probability of DFS and relapse was 55% and 18%, respectively, with the latest relapse at 27 months. Beyond first remission, HCT is curative in a much smaller minority of patients but remains the treatment of choice (Fig. 55.4). The development of imatinib and other tyrosine kinase inhibitors for the treatment of BCR–ABL+ hematopoietic malignancy has changed the up-front treatment strategy, and also may affect the outcome after HCT. Recently, the feasibility of performing allogeneic HCT after first-line
797
Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults Table 55.4 Comparison of several large trials for patient outcome in first complete remission in acute lymphoblastic leukemia (ALL) CR1
Group study, citation
Number of patients considered for HCT
Outcome measure
Chemo autologous therapy (%) HCT (%)
Allogeneic HCT (%)
p
ECOG–MRC JALSG-ALL93 LALA-87 LALA-87, high risk LALA-94 LALA-94, high risk GOELAMS 02, high risk PETHEMA 93, high risk
913 142 257 156 259 211 156 183
OS at 5 years OS at 6 years OS at 10 years OS at 10 years DFS at 3 years OS at 5 years OS at 6 years OS at 5 years
45 40– 31 11 34 21, 32 40 at 6 years 49
53 46 46 44 47 51 but median OS not reached 75 at 6 years 40
0.02 NS 0.04 0.009 0.007 Not stated 0.0027 0.56
HCT, hematopoietic cell transplantation; NS, not significant; OS, overall survival. (a) 1.0
0.9 p value = 0.0114
Survival probability
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21
Time (year) from date of transplant Disease status
1CR
>1CR
(b) 1.0 0.9
p value = 0.2780
Probability of relapse
0.8
Fig. 55.4 Probability of long-term disease-free survival and risk of relapse in 79 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia following allogeneic hematopoietic cell transplantation in first complete remission (CR1) and beyond first remission (>CR1). (a) Overall survival; (b) relapse rate. (Reproduced from [103], with permission.)
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
1
2
treatment with imatinib plus chemotherapy has been reported [52]. In this series, 29 adult patients who completed induction therapy were treated with allogeneic HCT, and the authors compared their results with those from 31 patients who had received transplantation in their unit
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21
Time (year) from date of transplant Disease status
1CR
>1CR
prior to the availability of imatinib treatment. The data suggest that the risk of relapse was significantly less in the imatinib group (3.5% versus 47.3%; p = 0.002), potentially reflecting a lower burden of residual disease at the time of HCT, and allowing a higher percentage of patients
Chapter 55 100
P < .001
Probability of DFS (%)
80 Imatinib group (78.1 ± 11.6%) 60 Historical group (38.7 ± 8.8%) 40
20
0 0
12
24 36 48 Months after SCT
60
72
P < .001
80 Imatinib group (78.1 ± 11.6%) 60 Historical group (38.7 ± 8.8%)
40
Fig. 55.5 Probabilities of disease-free survival (DFS) and overall survival in the imatinib group versus the historical group of patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. SCT, stem cell transplantation. (Reproduced from [52], with permission.)
20
0 0
12
24 36 48 Months after SCT
to come to transplantation in a “good” first remission. The results also indicated a superiority in DFS (76% versus 38%; p < 0.001) without much difference in transplant-related toxicities (Fig. 55.5). Thus, in the same way that imatinib may improve the up-front success of induction therapy and potential long-term outcome of patients with Ph+ ALL, entering transplantation with a lower burden of disease may improve the cure rate for such patients. Of additional interest is the follow-up of patients with ALL and the impact of the detection of MRD after allogeneic HCT. Radich et al. reviewed the results of 36 patients with Ph+ ALL who underwent allogeneic HCT and were monitored with sensitive assays of MRD [53]. Seventeen patients were transplanted in relapse, and 19 were transplanted in remission. Twenty-three patients had at least one positive BCR–ABL PCR assay after transplantation either before a relapse or without subsequent relapse. Ten of these 23 patients relapsed after a positive assay at a median time from first positive PCR assay of 94 (range 28–416) days. By comparison, only two relapses occurred in the 13 patients with no prior positive PCR assays. The unadjusted relative risk of relapse associated with a positive PCR assay compared with a negative assay was 5.7. Recent studies have also demonstrated the feasibility of administering imatinib following allogeneic HCT for BCR–ABL+ hematologic malignancy and can be used either preemptively or to treat any MRD detected after transplant prior to relapse instead of using donor lymphocyte infusions (DLIs) [54]. This strategy may improve the outcome of high-risk patients and facilitate improved long-term control of disease after HCT. The observation concerning the impact of pretransplant MRD suggests that it may be desirable to utilize second- and third-generation BCR–ABL inhibitors to reduce disease burden before transplant. Relapsed or primary refractory ALL ALL is refractory to primary chemotherapy in approximately 10–15% of patients, and allogeneic HCT can be successfully used to achieve both a remission and long-term control in approximately 20% of such patients. Of all those patients who do achieve a CR1 to primary therapy, approximately 50–70% will relapse. Relapsed ALL in an adult is not curable with standard chemotherapy, but remissions are sometimes achieved with reinduction using either a standard vincristine, prednisone, and anthracycline or an ARA-C-based regimen, particularly high-dose ARAC combined with an anthracycline or clofarabine [55–57], especially in patients with a long first remission. Recent studies confirm that, in the absence of HCT, adult patients with relapsed ALL have an extremely poor prognosis regardless of initial
60
72
1 P < 0.001 0.8 Probability of DFS
100
Probability of overall survival (%)
798
0.6 1st Remission (n = 41) 0.4 ≥2nd Remission (n = 46) 0.2
Relapse (n = 95)
0 0
2
4
6
8
10
12
Years after transplantation
Fig. 55.6 Long-term survival in patients with acute lymphoblastic leukemia demonstrating the impact of remission status on the outcome of transplant. DFS, disease-free survival. (Reproduced from [59], with permission.)
remission duration, and that transplantation, when feasible, is the only possible curative therapy [58]. Available data from the Center for International Blood and Marrow Transplant Research (CIBMTR) show that patients transplanted with an HLA-identical sibling donor for ALL in second CR (CR2) have an approximately 35–40% chance of longterm DFS, while those transplanted with disease not in remission have a DFS of only 10–20%. Figure 55.6 shows the overall DFS for patients with ALL, depending on their remission status, who underwent allogeneic HCT [59]. As in all series, these data do not account for those patients who suffer a relapse and do not undergo transplantation. Nevertheless, for an adult patient with ALL after a first relapse, allogeneic HCT is the best curative option. Unrelated HCT for ALL Historically, the outcome after transplantation from unrelated donors has been inferior to that observed after matched-sibling transplantation because of increased rates of graft rejection and graft-versus-host disease (GVHD) resulting from increased alloreactivity in this setting [60]. The National Marrow Donor Program (NMDP) reports a 5-year DFS of 35% in CR1 in adults and 46% in children, decreasing to 25% and 40%, respectively, in CR2. Over the past few years, improved results have been reported from several single-center studies, reflecting improvements in donor–recipient allele-level molecular matching in both class I and II histocompatibility genes, GVHD prophylaxis, and supportive
Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults
799
care [61]. An NMDP study showed that younger donor and recipient age were associated with significantly improved outcomes. Recent reports suggest equivalent results for high-risk patients from either related or unrelated donors [62–64], making this a reasonable option for a patient who needs an allogeneic HCT for treatment of their disease.
relapsed ALL. It is for these reasons that HCT after reduced-intensity conditioning is of limited effectiveness in patients with ALL who are not in remission. Studies focused on developing antigen-specific T-cell immunotherapy for ALL may help augment the GVL activity of donor T cells (see Chapter 18) [71–76].
Role of graft-vs.-leukemia effect in patients with ALL
HCT after reduced-intensity conditioning for treatment of ALL
The low response rate in patients with ALL following DLI has led to questions about the significance of the graft-versus-leukemia (GVL) effect in preventing relapse in this disease. The GVL effect is derived from observations of a higher relapse rate after autologous or syngeneic HCT compared with allogeneic HCT, a lower incidence of relapse in patients who had GVHD, as well as increased relapse rates in recipients of T-cell-depleted marrow grafts. The most compelling argument for a strong GVL effect in ALL comes from both single-institution and registry data [65,66]. These studies show a consistent decrease in relapse rates in patients who develop GVHD compared with those patients who do not. Table 55.5 shows the rate of relapse after HCT for ALL in CR1 and the correlation with GVHD. The occurrence of acute, chronic or both forms of GVHD correlated with the best DFS. A study of 192 patients with ALL, most of whom were transplanted in second remission [67], evaluated the probability of relapse among patients without or with GVHD. Relapse was significantly higher in the group that had less than grade II GVHD. In patients without significant GVHD, the actuarial risk of relapse approached 80%, compared with 40% in those who developed grade II or higher GVHD. A subsequent study [59] confirmed this observation for both relapse and overall DFS. An evaluation of 1132 patients with T- or B-lineage ALL supports the observation that both acute and chronic GVHD are associated with a decreased risk of relapse in both of the major immunophenotypes of adult ALL [68]. Recent studies have also demonstrated the beneficial impact of chronic GVHD on reducing relapse in patients undergoing allogeneic HCT for ALL, including Ph+ ALL [69,70]. Although the data support the importance of a GVL effect in mediating a clinically useful antileukemic response in patients with ALL, the reasons for the limited beneficial effect for patients with relapsed ALL treated with DLI are not clear. The different outcomes may reflect differences in the ability of ALL cells to present antigen targets, the low frequency of T-cell precursors reactive with minor antigens presented by ALL cells, the susceptibility of ALL targets to lysis or kinetic differences in the way leukemic cells grow after HCT. Thus, cytoreduction with chemotherapy prior to DLI is a better strategy for patients with
Table 55.5 Relapse after transplantation for acute lymphoblastic leukemia in first complete remission Group
Relapse probability at 3 years (%)
Allogeneic, non-T depleted No GVHD Acute only Chronic only Both
44 ± 17 ± 20 ± 15 ±
Syngeneic
41 ± 32
Allogeneic, T-depleted
34 ± 13
GVHD, graft-versus-host disease. Reproduced with permission from Appelbaum [66].
17 9 19 10
Although there has been a large number of studies performed in evaluating the role of allogeneic reduced-intensity transplantation in patients with myeloid malignancies, multiple myeloma, and low-grade nonHodgkin’s lymphoma, there have been fewer studies conducted in patients with ALL. In general, the consensus has been that, for patients with ALL, high-dose chemoradiotherapy is required for an improved cure rate, but this approach is of limited use in patients over the age of 50. In addition, an evaluation of outcomes suggests that the graftversus-tumor effect is more effective against myeloid malignancies such as AML and CML, and B-cell malignancies of mature B cells such as low-grade non-Hodgkin’s lymphoma and multiple myeloma, but less so with a more undifferentiated B-cell disease such as pre-B ALL, especially if not in remission (see Chapter 71) [77–79]. Nevertheless, a few small studies have been conducted that suggest that there may be a role for reduced-intensity allogeneic transplantation even in this disease, particularly in older patients, with 34% achieving long-term remission in a report from the European Group for Blood and Marrow Transplantation (EBMT) [77]. A recent report of patients undergoing HCT utilizing either related, unrelated or cord blood donor and a fludarabine/ melphalan regimen showed an optimistic outcome in a group of patients who were either at high risk during first remission or transplanted after achieving a second or subsequent remission [80]. The results of the recent UKALL XII/ECOG E2993 study of adult ALL show high toxicity and limited improvement in DFS despite better disease control in older adult patients. Thus, there is increased interest in the development of clinical trials exploring these reduced-intensity approaches in older patients with ALL in remission who would otherwise be candidates for transplantation based on age, cytogenetics, MRD, and response to initial treatment. The poor outcome of older patients with ALL using standard chemotherapy makes this an important consideration for treatment in those patients. Regimen development for allogeneic HCT for ALL Historically, the most commonly used regimen for transplantation of patients with ALL is CY plus TBI. Several other different preparative regimens have been developed, each based on substituting a different chemotherapeutic agent for CY in combination with TBI for patients with ALL. High-dose fractionated TBI in combination with high-dose ARA-C has been employed by several centers, and, with the exception of a small series of pediatric patients at Case Western Reserve, there has been no significant improvement in DFS with this regimen in recipients of allogeneic HCT from sibling donors [81,82]. Investigators at Johns Hopkins University approached the problem by conducting trials substituting busulfan (BU) for TBI in order to decrease the long-term side-effects of TBI and to determine the efficacy of highdose combined alkylating therapy in eliminating leukemic cells [83,84]. These nonradiation-dependent regimens have shown activity in the treatment of advanced ALL, suggesting that TBI is not an absolute requirement for successful treatment of ALL by HCT. A retrospective analysis from the CIBMTR found that a conventional CY/TBI regimen was superior to a non-TBI-containing regimen of BU plus CY in children, with a 3-year survival of 55% versus 40% for BU/CY [85]. However,
Chapter 55
Radioimmunotherapy-based transplant regimens for ALL Studies in AML have shown lower relapse rates with higher doses of TBI, suggesting that methods that can selectively deliver radiation to sites of leukemia without increasing systemic toxicity might be of benefit to the patient. The use of tumor-reactive monoclonal antibodies conjugated with local acting radionucleotides such as iodine-131 (131I) or yttrium-90 (90Y) are being explored to accomplish the goal of decreased relapse (see Chapter 24). Initial studies conducted in Seattle in an animal model showed the feasibility of this novel approach, and subsequent phase I and II studies in patients have been initiated. These studies have demonstrated that initial targeting of marrow and other sites of leukemia could be accomplished utilizing 131I-conjugated monoclonal antibodies. The most recent studies have focused on a monoclonal antibody reactive with CD45, an antigen that is found on leukemic cells as well as normal hematopoietic tissue and, unlike CD33, does not internalize after antibody binding. A phase I trial of 131I anti-CD45 monoclonal antibody plus CY and TBI for advanced leukemia was completed [90]. This study focused on the biodistribution and toxicity of escalating doses of targeted radiation combined with 120 mg/kg CY and 12 Gy TBI followed by matched related or autologous HCT. Among 44 patients, five had ALL in relapse
(a)
Adjusted probability of LFS, %
despite these differences in survival, the risk of relapse was similar. A recent study of BU, fludarabine, and 400 cGy of TBI, which would be considered a high-dose regimen, showed a low transplant-related mortality (TRM) of 3% and a projected DFS of 65% [86]. The group at the City of Hope studied the substitution of etoposide (VP-16) for CY in combination with fractionated TBI (13.2 Gy) followed by allogeneic HCT [87]. A phase I–II trial indicated that a dose of etoposide of 60 mg/kg is the maximum tolerated dose when combined with a TBI dose of 1320 rads. In that study, 36 patients with ALL were treated, 20 of whom were in relapse. The actual DFS was 57%, with a 32% relapse rate, suggesting that the regimen had significant activity in patients with advanced ALL, a result confirmed in a subsequent trial from the Southwest Oncology Group [88]. A subsequent study from City of Hope/Stanford showed a 64% DFS for adult patients undergoing transplantation with this regimen in CR1 (see section on allogeneic transplant for ALL in first remission). The recently completed UKALL XII/ECOG E2993 trial, a comparative study of chemotherapy and autologous and allogeneic HCT, utilized this regimen for patients in CR1. A comparative analysis of TBI combined with either CY or etoposide chemotherapy was conducted to determine the relative efficacy of the chemotherapy in the transplant regimen [89]. The outcomes of 298 patients with ALL in CR1 or CR2 receiving HLA-matched sibling allografts after CY/TBI conditioning were compared with 204 patients receiving etoposide and TBI. In this analysis, four groups were compared based on the radiation dose: CY/TBI 13 Gy (n = 81), etoposide/TBI 13 Gy (n = 151). Analyses of relapse, leukemia-free survival (LFS), and overall survival were performed separately for CR1 and CR2 patients. TRM did not differ by conditioning regimen. In CR1, there were also no significant differences in relapse, LFS or survival by conditioning regimen. In CR2, the outcomes differed among conditioning groups. In comparison with CY/TBI 75% reduction in a bulky mass or no nodes >2 cm, and 2 mm in diameter), micrometastases (>0.2 and ≤0.2 mm), and isolated tumor cells (≤0.2 mm), the latter group considered N0; 2 classification of lymph nodal status by number of involved axillary nodes: one to three, N1; four to nine, N2; 10 or more, N3; 3 new classifications for metastases to the: internal mammary (N1 if not apparent clinically, or N2 if clinically apparent), infraclavicular (N3), and supraclavicular (N3) lymph nodes. Local treatment with breast-conserving surgery followed by radiation offers the same local control as mastectomy [15,16], and is, therefore, the preferred option in most cases. Preoperative chemotherapy does not improve outcome compared with the same regimen administered postoperatively, but allows a higher rate of breast-conserving surgery by downsizing the tumor [17]. The likelihood of achieving a pathologic complete remission (pCR) to preoperative chemotherapy is inverse to the tumor size. Overall, a pCR is obtained in 15–25% of cases, which is associated with improved outcome. Thus, pCR is currently used as an important intermediate endpoint, a surrogate of final outcome, in trials of preoperative chemotherapy.
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Nontransplant approaches Nonmetastatic breast cancer Relapse rates, particularly at distant sites, increase proportionally to the number of positive axillary lymph nodes, and can be estimated to be approximately 20% at 5 years for patients with no involved nodes, 40% for patients with one to three positive nodes, 45–65% for patients with four to nine involved nodes, and over 70% for those with 10 or more positive nodes [18]. There have been recent important advances in postoperative standarddose chemotherapy (SDC) for patients with node-positive disease, affecting mostly the group with one to three positive nodes, compared with the group with high-risk primary breast cancer (HRPBC), defined by four or more nodes involved. Large studies have shown the benefit of a minimum of six to eight cycles (compared with the old standard of four cycles of adriamycin and cyclophosphamide) of anthracyclines combined with paclitaxel or docetaxel, either in sequential [19–21] or concurrent [22,23] fashion. The use of a dose-dense schedule of anthracyclines administered every 2 weeks with granulocyte colonystimulating factor (G-CSF) support also seems to improve the outcome of patients with node-positive disease [24]. Several trials enrolling patients with HER2+ tumors have demonstrated a large benefit at shortterm follow-up by adding trastuzumab to postoperative chemotherapy, either concurrently with taxanes [25,26] or upon completion of all chemotherapy [27]. Likewise, the addition of trastuzumab to preoperative chemotherapy improves pCR rate (greater than 50%) and outcome in HER2-overexpressing tumors [28]. The superiority of the new aromatase inhibitors over tamoxifen as adjuvant hormonal therapy for postmenopausal women with hormone receptor-positive tumors has been demonstrated. Several trials have shown improved outcomes using anastrazole [29], letrozole [30,31] or exemestane [32]. Although these three agents vary in their chemical properties and potency, it is unclear whether one is superior. It is also unclear which of the different schedules tested in these trials is best: use up front for 5 years, treatment for 3 years after 2 years of tamoxifen (“early switch”), or a “late switch” after 5 years of tamoxifen for an additional 5 years.
Metastatic breast cancer Since most patients receive an anthracycline regimen in the adjuvant setting, nonanthracycline regimens, with docetaxel, paclitaxel or capecitabine, have been developed for use as first-line treatment for metastatic disease. Several such combinations have been shown to improve survival, such as docetaxel–capecitabine [33], gemcitabine– paclitaxel [34], and gemcitabine–docetaxel [34]. The triweekly schedule of paclitaxel, previously considered standard, has been shown to be inferior to newer paclitaxel schedules or formulations, such as its weekly administration [35], or to nanoparticle albuminbound paclitaxel [36]. As in the adjuvant setting, several trials have demonstrated a benefit from combining trastuzumab with anthracyclines (although frequent clinical cardiac toxicity prevents the broad use of this combination with doxorubicin) [37], paclitaxel [37] or docetaxel [38] in patients with HER2+ tumors. Recently, the new dual EGFR–HER2 tyrosine kinase inhibitor lapatinib has been shown to improve significantly event-free survival (EFS) and response rate, but not overall survival (OS), in combination with capecitabine when compared with capecitabine alone, in patients with advanced tumors co-overexpressing both receptors, even after failure of prior trastuzumab [39]. Similarly, the addition of the anti-vascular endothelial growth factor antibody bevacizumab to paclitaxel has been shown
to extend EFS and increase response rate, but not OS, over paclitaxel alone, as initial treatment for metastatic breast cancer (MBC) [40]. The new aromatase inhibitors have shown superiority over tamoxifen for postmenopausal women with metastatic ER+ tumors [41]. The new bisphosphonates pamidronate [42] and zoledronic acid [43] have proven efficacy in preventing bone fractures and other skeletally related events in patients with bone metastases.
Hematopoietic cell transplantation for breast cancer Autologous hematopoietic progenitor cell support (AHPCS), derived from bone marrow or from peripheral blood progenitor cells (PBPCs), allows for the dose escalation of chemotherapy up to 10-fold, exploiting the dose–response effect of many drugs. While AHPCS circumvents myelosuppression, extramedullary organ toxicities become dose limiting. These substantial dose increments clearly increase the antitumor activity of high-dose chemotherapy (HDC) compared with SDC, with the hope that it will translate into improvements in outcome. Widespread substitution of PBPCs for bone marrow support and other improvements in patient care have increased the safety of HDC to current treatmentrelated mortality (TRM) rates below 5%. Despite the recent advances in standard therapy, more than 50% of patients with HRPBC and virtually all patients with MBC receiving SDC experience treatment failure and ultimately die from their disease. The initial prospective phase II trials of HDC in breast cancer, dating back to the late 1980s, were followed by randomized studies in both settings. Some argued that the impressive results of the phase II trials could be explained by patient selection, staging biases or short follow-up. Thus, controversy has surrounded HDC for breast cancer for more than a decade [44]. HDC trials in MBC Prospective HDC trials in MBC targeted in a sequential fashion patients with refractory [45,46], untreated [47], and responding disease [48–50]. It soon became clear that HDC produced not only the highest response rate and complete response (CR) rates ever reported in MBC, but also a consistent long-term EFS rate of 10–25% in patients transplanted after response to first-line chemotherapy, which appeared unprecedented (Fig. 63.1(a)). These results triggered phase III evaluation of HDC, employing a variety of regimens, for MBC. In the “Philadelphia” study, 184 patients responding to first-line therapy were randomized to receive HDC with cyclophosphamide, thiotepa, and carboplatin (STAMP-V) or maintenance cyclophosphamide, methotrexate, and fluorouracil (CMF) for 18 months or until progression [51]. In its latest update at a median follow-up of 67 months, there were no differences in EFS (4% versus 3%) or OS (14% versus 13%) [52]. A significant interaction was detected between age and treatment, with young and older patients apparently benefiting from HDC and CMF, respectively. Only 45 patients in CR were treated in study, which conferred only a 20% power to detect a 20% absolute OS difference in this subset. Strikingly, the partial remission to CR conversion rate was higher after CMF than after STAMP-V (9% and 6%, respectively). The low CR conversion rate in the transplant arm seems much lower than in most HDC trials, where CR conversion rates of 20–50% are typically reported. In the National Cancer Institute of Canada (NCIC) trial, 224 responsive MBC patients were randomized to additional SDC or HDC with cyclophosphamide, mitoxantrone, and etoposide [53]. In this trial, 20% of patients randomized to the HDC arm were never transplanted. Transplant-related mortality was 6%. At median follow-up of 48 months, significant differences in favor of transplantation were observed in EFS
1.0
1.0
0.9
0.9
0.8
0.8 Cumulative proportion
Cumulative proportion
Hematopoietic Cell Transplantation for Breast Cancer
0.7 0.6 0.5 0.4
OS
0.3 0.2
p = 0.001
0.7 0.6 0.5
Soft tissue (n = 46)
0.4 Visceral, not liver (n = 38)
0.3 0.2
Bone / BM (n = 94)
DFS
0.1
0.1 Liver (n = 14)
0.0
0.0 0
(a)
933
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2
3
4
5
6
7
8
9
10
11
12
Years post transplant
0 (b)
1
2
3
4
5
6
7
8
9
10
11
12
Years post transplant
Fig. 63.1 High-dose chemotherapy for metastatic breast cancer (MBC). (a) Long-term disease-free survival (DFS) and overall survival (OS) of prospective phase II trials with cyclophosphamide, cisplatin, and BCNU (STAMP-I) as first-line therapy for MBC patients at the University of Colorado. (b) Event-free survival of STAMP-I trials according to specific metastatic sites. BM, bone marrow.
(median 11 versus 8 months; p = 0.006), but not OS (median 24 versus 28 months; p = 0.4). In the French PÉGASE 03 study, 180 patients with responsive tumors were randomized to HDC with cyclophosphamide and thiotepa, or observation [54]. The CR rate increased from 11% to 24% after HDC (p = 0.0002). At a median follow-up of 48 months, a significant EFS advantage was observed in favor of HDC (27% versus 10%; p = 0.0005), without significant OS differences in this first analysis (38% versus 30%; p = 0.7). Investigators from the International Breast Cancer Dose Intensity Study group treated 110 patients with four cycles of doxorubicin and docetaxel, followed by either six cycles of CMF or sequential high-dose cycles of ifosfamide, carboplatin, and etoposide and cyclophosphamide/ thiotepa with AHPCS [55]. The TRM rates in both arms were 4% and 9%, respectively. The HDC arm produced higher response rate (71% versus 44%) and CR (29% versus 6%) rates. At a median follow-up of 42 months, the study was positive for its primary endpoint, EFS (HDC 16% versus SDC 9%; p = 0.01). There was a trend for an OS advantage in the HDC arm in an intent-to-treat analysis (45% versus 37%; p = 0.1), which reached significance in an analysis based on actual treatment received (49% versus 35%; p = 0.02). In the PÉGASE 04 trial, 61 responding MBC patients were randomized to additional SDC or HDC with cyclophosphamide, mitoxantrone, and melphalan [56]. In its preliminary analyses, nonsignificant differences favoring HDC in EFS and OS were noted [56]. However, in its final analysis at a median follow-up of 7.5 years, significant benefits from HDC in progression-free survival (PFS) (18.7% versus 0%; p < 0.005) and OS (36.8% versus 13.8%; p = 0.02) were demonstrated, with median PFS times of 12 versus 6 months, and median OS times of 44 versus 19 months [57]. In the German Breast Cancer Dose Intensity Study group trial, 92 untreated MBC patients were randomized to receive six to nine cycles of adriamycin and paclitaxel or tandem cycles of high-dose cyclophosphamide, mitoxantrone, and etoposide, 6 weeks apart with AHPCS [58]. At a median follow-up of 52 months, there were no significant differences between the high-dose and control arms in PFS (27% versus 20%; p = 0.5) or OS (18% versus 21%; p = 0.75). Investigators at Duke University conducted two randomized trials with a crossover design, comparing early versus late use of HDC, using
cyclophosphamide, cisplatin, and BCNU (carmustine) (STAMP-I). In the first trial, 100 hormone-refractory MBC patients in first CR were randomized to immediate HDC or observation with HDC at subsequent relapse [59]. At a median follow-up of 10.6 years, EFS was superior in the immediate transplant arm (26% versus 10%; p = 0.03), with a median EFS of 10 versus 4 months (p < 0.006). Six-year OS rates were 26.5% versus 35% (p = 0.69), with median OS times of 2.1 versus 3.3 years (p = 0.2). The combined TRM from transplant in both arms was high (8.6%). In the second Duke trial, 69 patients with hormone-refractory boneonly MBC were randomized after first-line SDC to immediate HDC and radiotherapy of all bony metastases, or to radiotherapy and observation, again with HDC upon progression [60]. At a median follow-up of 8.1 years, EFS was superior in the immediate transplant arm (17% versus 0%; p < 0.0001) with median EFS times of 12 versus 4 months (p < 0.0001). The OS rates were not significantly different between the immediate and late transplant arms (17% versus 9%; p = 0.1) with median OS times of 3 versus 1.8 years. Interestingly, all three survivors in the observation arm received a transplant after progression, two of them subsequently remaining event-free. As in the other Duke trial with STAMP-I, overall transplant TRM was high (9.7%). In summary, a significant benefit in EFS in favor of HDC has been noted in six of the eight trials, but only one study (PÉGASE 04) shows an advantage in OS (Table 63.1). Longer follow-up is clearly needed in the NCIC and PÉGASE 03 trials to determine whether the early HDC advantage in EFS ultimately translates into an OS benefit. The lack of a direct comparison between HDC and non-HDC arms in the two Duke crossover trials obviates OS analyses and ultimately complicates their interpretation. The 2004 Cochrane Collaboration review of this issue included six trials (excluding the two Duke crossover studies) and a total of 850 patients [61]. A significant overall EFS benefit from HDC was detected at 1 year (risk ratio 1.81, 95% [confidence interval] CI 1.39–2.34; p < 10−5), and at 5 years (risk ratio 2.84, 95% CI 1.07–7.5; p = 0.04). While there was no OS difference at 1 year (risk ratio 0.99, 95% CI 0.91–1.08; p = 0.9), it approached statistical significance at 5 years (risk ratio 1.5, 95% CI 0.96–2.34; p = 0.08). Of note, at the 5-year time point analysis of this review, the final analysis of PÉGASE 04 reported in 2005, which was significant for OS, was not included. The overall TRM rates in the
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1.0
1.0
0.9
0.9 OS
0.7 0.6
RFS
0.5 0.4 0.3 0.2
0.8 Cumulative proportion
Cumulative proportion
0.8
4–9+
0.7 0.6 IBC
0.5
≥10+
0.4 0.3 0.2
0.1
p = 0.39
0.1
0.0
0
1
2
(a)
3
4
5
6
7
8
9
0.0
10 11 12 13
Years
0 (b)
1
2
3
4
5
6
7
8
9
10
11
12
13
Years
Fig. 63.2 High-dose chemotherapy for high-risk primary breast cancer. (a) Long-term relapse-free survival (RFS) (67%) and overall survival (OS) (70%) of 264 patients enrolled in prospective phase II trials of cyclophosphamide + cisplatin + carmustine at the University of Colorado, at a median follow-up of 7 years. (b) RFS in the different populations enrolled in those trials: four to nine positive nodes (4–9+), 10 or more positive nodes (≥10+), and inflammatory breast cancer (IBC).
HDC and SDC groups were 3.4% and 0.4%, respectively (risk ratio 4.07, 95% CI 1.39–11.88; p = 0.01). HDC in HRPBC The groups at Duke University [62] and the National Tumor Institute in Milan [63] pioneered the evaluation of HDC in patients with HRPBC. Long-term results of the Duke phase II trial in patients with 10 or more involved (+) axillary nodes showed a 10-year EFS rate of 72% [64]. In the Milan study, which explored a sequential high-dose single-agent approach, a 57% EFS rate was reported at median follow-up of 4 years [63]. The encouraging prospective phase II data of HDC in HRPBC (Fig. 63.2) prompted randomized trials across the world. Two very small randomized phase II trials were first reported in 1998. In a study conducted at the MD Anderson Cancer Center, 78 patients with 10 or more positive nodes after up-front surgery, or four or more positive nodes after preoperative chemotherapy, received eight cycles of fluorouracil, adriamycin, and cyclophosphamide, followed by either two cycles of dose-intense cyclophosphamide, etoposide, and cisplatin (a regimen that is not currently considered HDC) or no further therapy [65]. At latest follow-up of 11.8 years, there were no significant differences between the control and experimental arms in EFS (40% versus 26%; p = 0.1) or OS (47% versus 42%; p = 0.1) [66]. The other early study was a small Dutch pilot trial enrolling 81 patients with axillary level III involvement, who were treated with neoadjuvant 5-fluorouracil, epirubicin, and cyclophosphamide (FEC), surgery, and one postoperative cycle of FEC, and were then randomized to high-dose cyclophosphamide, thiotepa, and carboplatin (CTC; administered at higher doses and an optimized schedule, compared with STAMP-V) or observation [67]. The high-dose arm had a 15% drop-out rate. The final analysis of this trial, at median follow-up of 7 years, did not show differences between the HDC and SDC arms in EFS (49% versus 47.5%; p = 0.3) or OS (61% versus 62.5%; p = 0.8) [68]. Of note,
this trial employed a nonstandard infraclavicular lymph node biopsy, and not a standard axillary node dissection, to determine eligibility. Both of these trials were clearly underpowered to detect realistic differences. It is worth noting that the minimum detectable benefit in either trial would have been required to be of greater magnitude than the overall impact of adjuvant chemotherapy, versus no postoperative treatment at all, for breast cancer. Subsequent to their pilot trial, Rodenhuis and colleagues conducted a larger population-based phase III study that enrolled 885 patients with four or more positive lymph nodes, randomized to receive four cycles of FEC followed by one more cycle of FEC or high-dose CTC [69]. At its latest analysis at a median follow-up of 84 months, a trend for an EFS advantage was noticed in favor of HDC (64.3% versus 59%, hazard ratio 0.84; p = 0.07), with no detectable OS differences (73% versus 70%; p = 0.2) [70]. Prospectively planned subset analyses showed a substantial improvement in EFS among patients with 10 or more positive nodes (68% versus 49%; p = 0.01). In the group with four to nine positive nodes, EFS rates were 72% (HDC) versus 65% (SDC) (p = 0.5). Other subgroup analyses, which were unplanned, suggested that young patients and those with HER2− or with lower-grade tumors might benefit from HDC. Specifically, patients with HER2− disease (n = 621) experienced a marked benefit from HDC in EFS (72% versus 59%; p = 0.002) and OS (78% versus 71%; p = 0.02), with a hazard ratio for relapse decreased by about one-third after HDC [70]. In Cancer and Leukemia Group B (CALGB) 9082 trial, Peters and colleagues enrolled 785 patients with 10 or more positive nodes, who received four cycles of cyclophosphamide, doxorubicin, and 5fluorouracil (CAF) and were randomized to HDC with STAMP-I or to one additional cycle of those drugs at intermediate dose (ID) with GCSF support [71]. At a median follow-up of 7.3 years, there were no significant differences between the HDC and ID arms in EFS (61% versus 58%; p = 0.2) or OS (71% for both groups; p = 0.7). There was a 31% reduction in the number of relapses in the HDC arm, particularly among women younger than 50, which is certainly consistent with a
Hematopoietic Cell Transplantation for Breast Cancer
dose–response effect. However, the high TRM rate of 8.1% in the HDC arm (versus 0% in the ID arm) offsets this benefit. In the Anglo-Celtic trial, 605 patients with four or more positive nodes were randomized to four cycles of doxorubicin followed by either highdose cyclophosphamide and thiotepa or eight cycles of CMF [72]. At median follow-up of 6 years, there were no differences in EFS (57% and 54%, respectively; p = 0.7) or OS (62% and 64%, respectively; p = 0.4). Investigators from the Eastern Cooperative Oncology Group (ECOG) randomized 540 patients with 10 or more positive nodes to receive six cycles of CAF, with or without high-dose cyclophosphamide and thiotepa, administered with AHPCS from bone marrow or, towards the end of the study, PBPCs [73]. At a median follow-up of 6.1 years, there were no significant differences between HDC and SDC in EFS (55% versus 48%; p = 0.1) or OS (58% versus 62%; p = 0.3). Among patients meeting strict protocol eligibility, EFS appeared higher in the HDC arm (55% versus 45%; p = 0.04). In the transplant arm, there were nine toxic deaths (eight of the patients having received bone marrow support) and nine cases of secondary leukemia/myelodysplastic syndrome. The Southwestern Oncology Group (SWOG) trial randomized 539 patients with four or more positive nodes (92% of them with four to nine positive nodes) to receive conventional treatment with sequential dosedense adriamycin, paclitaxel, and cyclophosphamide, or four cycles of doxorubicin and cyclophosphamide (AC) followed by HDC (using STAMP-V in 86% and STAMP-I in 14% of patients) [74]. At a median follow-up of 70 months, there were no differences in EFS (78% versus 73%; p = 0.35) or OS (87% versus 83%; p = 0.4). In the Scandinavian trial, 525 patients with eight or more positive nodes (or five or more positive nodes plus an ER− and high S-phase fraction tumor) received nine cycles of individually tailored doseintensive FEC, or three cycles of conventional FEC followed by HDC with STAMP-V [75]. The tailored FEC regimen resulted in fewer breast cancer relapses than STAMP-V, but, probably due to its high cumulative epirubicin dose, it induced a relatively high 4% incidence of secondary leukemia/myelodysplastic syndrome. In the latest update of this trial at a median follow-up of 5 years, there was a trend in favor of tailored FEC in EFS (51% versus 45%; p = 0.07), with no significant differences in OS (60% versus 56%; p = 0.3) [76]. A major problem in interpreting this study is the fact that the control arm received higher cumulative doses of chemotherapy than the transplant arm. Nitz and colleagues from the West German Study Group (WSG) randomized 403 patients with 10 or more positive nodes to a dose-dense arm with four cycles of epirubicin and cyclophosphamide (EC) followed by six cycles of CMF, all every 2 weeks with G-CSF, or a transplant arm with two initial cycles of EC followed by tandem cycles of highdose epirubicin, cyclophosphamide, and thiotepa administered 4 weeks apart with AHPCS [77]. Neither arm had any TRM. At a median followup of 48 months, there was significant superiority of transplant in EFS (60% versus 44%; p < 0.001) and in OS (75% versus 70%; p = 0.02). An unplanned retrospective analysis showed a large benefit from HDC for tumors with a basal-like phenotype (ER/PR negativity, HER2 negativity, and basal cytokeratin positivity), with a hazard ratio for relapse of 3.06 (95% CI 1.41–6.06) if receiving dose-dense chemotherapy compared with tandem HDC [78]. The Michelangelo study compared the Milan high-dose sequential single-agent approach to SDC in 382 patients with four or more positive nodes [79]. At its most recent analysis at a median follow-up of 11 years, nonsignificant differences in favor of the high-dose arm in EFS (52% versus 44%) and OS (60% versus 51%) were noted. In the International Breast Cancer Study Group (IBCSG) study, 344 patients with 10 or more positive nodes (or five to nine positive nodes and an ER− or a T3 tumor) received standard four cycles of AC/EC fol-
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lowed by three cycles of CMF, or a more dose-intense arm of three cycles of EC above conventional doses, every 3 weeks with AHPCS [80]. At a current follow-up of 5.8 years, the EFS rates were 52% and 43% for the dose-intense and standard arms, respectively (p = 0.07), with OS rates of 70% versus 61% (p = 0.1). In the PÉGASE 01 trial, 314 patients with seven or more positive nodes were randomized to receive SDC followed by high-dose cyclophosphamide, mitoxantrone, and melphalan or observation [81]. The TRM in the HDC arm was 0.6%. At a short median follow-up of 33 months, there were significant differences in EFS (71% versus 55%; p = 0.002), without an OS advantage (84% versus 85%; p = 0.3). Zander and colleagues of the German Autologous Bone Marrow Transplant Group (GABG) randomized 307 patients with 10 or more positive nodes to receive four cycles of EC followed by HDC with cyclophosphamide, thiotepa, and mitoxantrone, or three cycles of CMF [82]. Its most recent update at a median follow-up of 6.1 years showed nonsignificant trends in favor of HDC in EFS (51% versus 41%; p = 0.1) and OS (62.5% versus 57%, hazard ratio 0.84; p = 0.3) [83]. Bliss et al. randomized 281 patients with four or more positive nodes to receive six cycles of FEC, or three cycles of FEC followed by HDC with STAMP-V [84]. At a median follow-up of 68 months, there were no differences in EFS (59% versus 57%; p = 0.76) or OS (67% versus 66%; p = 0.4). In summary, at least 15 randomized trials, 11 of them enrolling over 300 patients, have compared diverse forms of HDC with lower doses of chemotherapy for HRPBC. Their results, in most cases still preliminary, appear contradictory at this point (Table 63.2). There are clearly negative trials, such as the ECOG, Scandinavian, CALGB, SWOG, and Anglo-Celtic studies. In contrast, other studies, such as WSG or PÉGASE 03, show significant superiority of HDC. Other trials, such as the National Dutch, GABG, IBCSG, and Michelangelo studies, show nonsignificant trends in favor of transplant at this point. The 2006 Cochrane review on HDC for HRPBC analyzed 13 of those trials, totaling 5111 patients [85]. Mature results, defined as all study participants followed for a minimum of 3 years, were available for only the small MD Anderson and Dutch pilot trials, and the CALGB study. A significant EFS benefit in favor of HDC was detected at 3 years (risk ratio 1.19, 95% CI 1.06–1.19), 4 years (risk ratio 1.24, 95% CI 1.16– 1.45), and 5 years (risk ratio 1.06, 95% CI 1–1.13), despite not including the positive PÉGASE 01 and WSG trials in this last time point analysis. There were no significant OS differences at any time point. Overall TRM rates were 2.5% and 0.1% in the transplant and conventional treatment groups, respectively (risk ratio 8.58, 95% CI 4.13–17.8). Quality of life scores were significantly worse immediately after HDC, but with no differences after 1 year. The recently reported MD Anderson Cancer Center/European Group for Blood and Marrow Transplantation meta-analysis of individual patient data from 15 randomized trials, including 6102 patients, has detected an overall absolute EFS benefit of 13% in favor of HDC (p = 0.0001) at an overall median follow-up of 6 years, with separation of the curves only appreciable after the second year of follow-up (Fig. 63.3(a)) [86]. The difference in OS (which, not surprisingly, was not noticeable until later than 5 years of follow-up) did not reach statistical significance (p = 0.16) (Fig. 63.3 (b)). Critical commentary on the available results in MBC and HRPBC Adequate length of follow-up is critical. Many of the results reported to date are still preliminary. An overall EFS benefit for HDC is apparent in both HRPBC and MBC. As has been pointed out [87], EFS is clinically relevant in the adjuvant setting, and has become a valid endpoint
Responsive
Responsive Untreated Untreated
CR
Untreated
HR, Bone only Responsive
NCIC [53]
Philadelphia [51,52] PÉGASE 03 [54] IBDIS-1 [55]
Duke crossover1(Patients in CR) [59] GEBDIS [58]
Duke crossover-2 (Bone only) [60] PÉGASE 04 [57] CPA/Mito/Mel
61
69
Tandem CPA/Mito/Eto ×2 STAMP-I
CPA/TT/Cb(STAMP-V) CPA/TT Sequential Ifos/Cb/Eto → CPA/TT CPA/CDDP/BCNU (STAMP-I)
CPA/Mito/Eto
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92
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52
127
67 48 42
48
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NR Median 11 months 4% Median 10 months 27% 25% Median 14 months 25% Median 10 months
HDC
Event-free survival rates
0% Med: 6 months
0% Median 4 months
20% Median 11 months
10% Median 4 months
3% Median 9 months 10% 20% Median 9 months
NR Median 9 months
Control
18% Median 27 months 17% Median 3 years* 37% Median 44 months
5th year
0 1st year
2nd year
3rd year
4th year
Fig. 63.4 Time distribution of relapses after high-dose chemotherapy for high-risk primary breast cancer at the University of Colorado (n = 264). The number above each bar corresponds to the number of relapses during each year of follow-up.
for new drug approval in the adjuvant setting for breast and colorectal cancer. It can be argued that EFS differences in MBC are of little benefit unless they translate into an improved quality of life or prolonged OS. However, it is obvious that trials showing significant EFS differences in their first analyses, such as the NCIC or PÉGASE 03 studies, need longer follow-up. It is important to consider the different natural history of breast cancer after SDC and HDC. Median time to relapse of HRPBC patients after SDC is generally between 2 and 3 years. In contrast, the majority of relapses after HDC for HRPBC occur within the first 2 years following treatment, and very few patients experience recurrence after the fifth year (Fig. 63.4) [88,89]. Observations from other settings, such as lymphoma [90] or myeloma [91], where survival benefits were only noticed after mature analyses, may be also applicable to breast cancer, and longterm follow-up and second publication of major trials will be important for definitive conclusions. While we tend to discuss HDC as a single approach, there are substantial differences in the HDC regimens used in the randomized trials.
We can now predict which patients with MBC are most likely to achieve long-term remissions after HDC, such as those with low tumor burden, in CR after SDC, or without liver involvement (Fig. 63.1(b)) [92,93]. Prospective studies of conventional multimodal treatment for patients with isolated relapses had shown long-term EFS rates of 32–39% [94–96]. The hypothesis that these MBC patients with low tumor burden might attain major benefit from HDC early in the course of their disease was prospectively tested at the University of Colorado [97]. A phase II study of four cycles of adriamycin-based induction therapy followed by HDC with STAMP-I, as first-line therapy for oligometastatic disease, was conducted in 60 consecutive patients. Oligometastases were defined as one or more sites of macroscopic tumor that could be either resected en bloc and/or encompassed within a single radiotherapy field, and/or less than 5% of bone marrow involvement. Most patients had received previous adjuvant chemotherapy. At a median post-transplant follow-up of 5 years, the EFS and OS rates were 52% and 62%, respectively, with median EFS and OS times of 4.3 years and 6.7 years, respectively (Fig. 63.5(a)). HER2− status and a single metastatic site were independent favorable predictors of outcome (Fig. 63.5 (b)) [98]. Likewise, retrospective analyses have identified prognostic factors for HDC for HRPBC. Somlo et al. identified PR positivity as an independent favorable predictor of EFS in 114 patients enrolled in their studies using two different HDC regimens at City of Hope National Medical Center [99]. In an analysis of 176 patients treated at the University of Colorado with STAMP-I, tumor size, presence of either or both of ER and PR (ER/PR), and nodal ratio (number of positive nodes to number of sampled nodes) were independently associated with relapse [100]. Those three variables formed the basis for the following scoring system, which can assign a relapse risk to each patient before HDC: Score = (Nodal ratio × 3.05) + (Tumor size × 0.15) – (ER/PR × 1.15) In this formula, tumor size is entered in centimeters, and ER/PR is assigned “1” if positive (i.e. ER and/or PR are positive) and “0” if negative (if both are negative). Scores of 2.41 or more, and less than 2.41, allocate patients to a high- or low-risk category, with risks of relapse of 65% and 11%, respectively. The differences in EFS (p < 0.000001) and OS (p < 0.00001) were highly significant (Fig. 63.6(a)). This predictive model was subsequently validated both externally and prospectively [89]. The prognostic value of the nodal ratio, probably superior to that of the absolute number of positive nodes, has been subsequently confirmed by other authors in HDC-treated patients [101,102], and is being actively investigated in breast cancer populations treated with SDC [103]. HER2 overexpression has been shown to have major prognostic implications after HDC in HPRBC (Fig. 63.6 (b)) [104,105,106] and MBC (Fig. 63.5(b)) [97,107]. Additionally, HER2− status may predict benefit from HDC compared with SDC [70]. Other molecular independent adverse prognosticators include EGFR overexpression (Fig.
Hematopoietic Cell Transplantation for Breast Cancer
Cumulative proportion surviving relapse free
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Fig. 63.5 High-dose chemotherapy for oligometastatic disease. (a) Relapse-free survival (RFS) and overall survival (OS) results of a prospective trial of STAMP-I. (b) RFS curves in this trial according to HER2 status and number of metastatic sites.
63.6(c)), particularly when co-overexpressed with HER2 (Fig. 63.6(d)) [108], and increased tumor angiogenesis (Fig. 63.6(e)) [109]. Future comparative trials could focus on the populations most likely to benefit from HDC. Purging of progenitor cell grafts Although tumor contamination may be lower in PBPC than in bone marrow fractions [110], breast cancer cells in PBPCs can still be detected in substantial fractions of MBC and HRPBC patients. Most post-HDC relapses in patients with MBC occur in sites of prior disease, suggesting an insufficient cytoreductive capacity of HDC, rather than a direct effect from tumor cells contaminating the graft. Recent analyses have shown a significant, but not independent, adverse effect from tumor contamination of PBPC products in MBC patients receiving HDC (Fig. 63.7(b)) [111,112], which suggests that tumor cells may constitute a marker of systemic micrometastases, rather than being directly responsible for failure of transplant. In contrast, the presence of occult tumor cells in apheresis products constitutes an independent adverse prognostic factor for recurrence in recent analyses of HRPBC patients (Fig. 63.7(a)), providing indirect proof of a role of contaminating tumor cells in relapse in this population [111,113]. While direct evidence for a contribution of contaminating tumor cells to relapse after autografting, as demonstrated for other tumors [114,115], is lacking, purging the graft of tumor cells holds promise, particularly for HRPBC patients. Negative purging, which eliminates contaminating cells by pharmacologic or immunologic methods, achieves 2–4-log tumor cell depletion, albeit with marked engraftment delays [116–118]. Positive selection targets the CD34 antigen, expressed on 0.5–3% of normal cells in the bone marrow and PBPC fractions, but not on breast cancer cells [119]. Shpall and collaborators tested CD34 selection in 155 patients, showing a mean 2-log tumor cell depletion, with normal post-transplant neutrophil and platelet recovery and immune reconstitution [120]. In a separate trial, 92 HRPBC patients were randomized to receive HDC with CD34selected or unselected PBPCs [121]. No short-term differences in EFS or OS were noticed in this small trial. Since most patients with contaminated products still have detectable cancer cells present in their stem
cell grafts following CD34 selection, effective purging may require a combination of different procedures [122,123]. Other potential ways of future progress There is a pressing need to improve HDC for breast cancer. It appears unlikely that first-generation high-dose regimens developed two decades ago would end up being the optimal stem cell-supported, high-dose combinations. It is necessary to develop new synergistic HDC combinations of more active drugs showing a dose–response effect, such as docetaxel [124] or gemcitabine [125]. In parallel, the traditional use of a late single HDC cycle after long SDC induction does not appear to constitute optimal treatment, and earlier use of sequential HDC cycles after shorter induction SDC courses [126], or used up front as in the randomized WSG trial in HRPBC [77], needs to be further explored. Independent of whether the superior tumor debulking capacity of HDC translates into improved outcomes, post-transplant minimal residual disease provides an optimal scenario for application of novel therapies. HDC and novel therapeutics targeting signal transduction pathways are not mutually exclusive. In solid tumors, available data suggest that these promising new agents may help improve outcome when combined with chemotherapy, but they are unlikely to have a substantial impact alone. The anti-HER2 monoclonal antibody trastuzumab or other biologic agents can be easily administered after transplant, or perhaps even integrated into the high-dose regimen. A pilot study has shown the safety of concurrent administration of trastuzumab with HDC, exploiting their in vitro synergy [127]. While immune therapies for breast cancer are still in development, their testing post HDC has a robust rationale. Retrospective analyses of the post-transplant recovery of the absolute lymphocyte count suggest a major role of the immune system in controlling post-HDC residual tumor, particularly in MBC (Fig. 63.8) [93,128]. A post-transplant immune role appears to be more important for the subset of memory T cells [129]. Combination of HDC with immune therapies holds substantial promise. In summary, achievement of a minimal residual disease status with HDC may allow institution of innovative therapies post transplant to prevent recurrence by such immunologic approaches, or novel targeted
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1.0 1.0
0.9
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0.0 0 (e)
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Fig. 63.6 Effect of prognostic factors on event-free survival (EFS) of patients with high-risk primary breast cancer after high-dose chemotherapy. (a) Effect of the clinical score (tumor size, hormone receptor status, and nodal ratio). (b) Effect of HER2 overexpression. (c) Effect of endothelial growth factor (EGFR) expression. (d) Combined effect of HER2 and EGFR expression. (e) Effect of intratumor microvessel density (MVD).
Hematopoietic Cell Transplantation for Breast Cancer
1.0
1.0
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–
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941
0.7 0.6 0.5 0.4 OTC+
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Fig. 63.7 Prognostic effect of detection of occult tumor cells (OTC) by immunocytochemistry in the apheresis products on event-free survival. (a) High-risk primary breast cancer. (b) Metastatic breast cancer. HDC, high-dose chemotherapy.
1.0
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0.8
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10
11
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Fig. 63.8 Effect of immune recovery, determined by the absolute lymphocyte count (ALC) on day 15 after transplantation (cut-off 500/mm3), on freedom from relapse (FFR) after high-dose chemotherapy. (a) No effect in high-risk primary breast cancer. (b) Significant effect in metastatic breast cancer.
agents. All new therapies will require the scrutiny of controlled clinical trials.
Allogeneic transplantation for MBC Allogeneic hematopoietic cell transplant has been shown to confer an immune graft-versus-tumor (GVT) effect against hematologic malignancies. Following anecdotal reports suggesting the existence of a potential GVT effect in breast cancer [130,131], Ueno and colleagues at MD Anderson Cancer Center treated 10 MBC patients with high-dose cyclophosphamide, thiotepa, and BCNU, followed by allogeneic PBPC transplantation from a matched sibling [132]. Four patients who experienced tumor progression after transplant had their immunosuppression reduced, and one received a donor lymphocyte infusion. Two of those patients experienced regression of liver metastases in association with exacerbation of acute graft-versus-host disease (GVHD).
Allogeneic transplantation using HDC is associated with significant morbidity and mortality. The introduction of reduced-intensity conditioning (RIC) regimens, generally based on fludarabine or lower doses of busulfan, which can provide enough immunosuppression to allow allogeneic stem cell engraftment, produced a substantial reduction in TRM. This strategy relies most of its antitumor efficacy on a GVT effect. The demonstration of a therapeutic effect of RIC allogeneic transplantation in metastatic renal cell carcinoma [133] led to trials of this approach at different institutions in MBC (Table 63.3). Bishop and colleagues employed an elegant design to differentiate the antitumor effects of the conditioning regimen from a GVT effect in 16 patients with MBC involving in most cases the liver or lungs, pretreated with a median of four prior regimens [134]. Patients first received fludarabine and cyclophosphamide to attain tumor control and sufficient immunosuppression (estimated as a CD4+ count 7) breast cancer patients: the PEGASE 01 trial. Proc Am Soc Clin Oncol 2001; 20: abstract 27. Zander AR, Kröger N, Schmoor C et al. Highdose chemotherapy with autologous hematopoietic stem-cell support compared with standard-dose chemotherapy in breast cancer patients with 10 or more positive lymph nodes: first results of a randomized trial. J Clin Oncol 2004; 22: 2273–83. Zander AR, Kroeger N, Schmoor C et al. Randomized trial of high-dose chemotherapy with autologous haematopoietic stem cell support vs. standard-dose chemotherapy in breast cancer patients with 10 or more positive lymph nodes: overall survival after 6 years of follow up. J Clin Oncol 2006; 24(Suppl): abstract 672. Coombes RC, Howell A, Emson M et al. High dose chemotherapy and autologous stem cell transplantation as adjuvant therapy for primary breast cancer patients with four or more lymph nodes involved: long-term results of an international randomised trial. Ann Oncol 2005; 16: 726–34. Farquhar C, Marjoribanks J, Lethaby A, Basser R. High dose chemotherapy for poor prognosis breast cancers: systematic review and metaanalysis. Cancer Treat Rev 2007; 33: 325–37. Berry D, Ueno NT, Johnson MM et al. High-dose chemotherapy with autologous stem-cell support versus standard-dose chemotherapy: metaanalysis of individual patient data from 15 randomized adjuvant breast cancer trials. Breast Cancer Res Treat 2007; 106(Suppl 1): S5. Efelbein GJ. Stem-cell transplantation for highrisk breast cancer. N Engl J Med 2003; 349: 80– 2. Damon LE, Wolf JL, Rugo HS et al. Long followup of patients with primary breast cancer and ≥10 involved axillary lymph nodes after high-dose chemotherapy and autologous stem cell transplant. Biol Blood Marrow Transplant 2002; 8: abstract 71. Nieto Y, Nawaz S, Shpall EJ et al. Long-term analysis and prospective validation of a prognostic model for patients with high-risk primary breast cancer receiving high-dose chemotherapy. Clin Cancer Res 2004; 10: 2609–17. Philip T, Guglielmi C, Hagenbeek A et al. Autologous bone marrow transplantation as compared
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Christie J. Moore, Brandon Hayes-Lattin & Craig R. Nichols
Hematopoietic Cell Transplantation in Germ Cell Tumors
Introduction
Initiation of salvage therapies in germ cell tumors
Malignant testicular tumors are the most common solid tumors among young men, with a rising annual incidence of approximately 9000 new cases in the United States. Ninety-five percent of malignant testicular tumors are germ cell tumors [1]. Germ cell tumors can also arise in extragonadal sites such as the retroperitoneum or mediastinum, often portending a poorer prognosis. Staging is based on pattern of spread, and initial therapy is guided by the presenting stage (Tables 64.1 and 64.2) [2]. The importance of germ cell tumors in the field of oncology is highlighted by the fact that standard therapies for early stage disease routinely cure more than 95% of patients. Current research goals focus on limiting therapy while preserving high cure rates for these patients. Before the advent of modern cisplatin-based chemotherapies, metastatic germ cell tumors were almost always fatal, but today the cure rate for metastatic disease reaches 70–80% (Tables 64.3 and 64.4) [3,4]. Unfortunately, 20–30% of patients with advanced disseminated disease require additional therapy after primary cisplatin-based treatments, including salvage surgery or chemotherapy. In an effort to better care for and study patients with metastatic disease, many prognostic models based on the pattern of disease spread and serum tumor markers at disease presentation have been employed [5–14]. In 1995, a uniform system was developed based on 5202 patients with nonseminomatous germ cell tumors and 660 patients with seminomas treated with cisplatin-containing regimens, to guide decisionmaking and standardize patient enrolment in clinical trials (Table 64.5) [15]. Additional poor prognostic factors may include a high proliferative index or the presence and number of cytogenetic abnormalities such as an isochromosome of the short arm of chromosome 12 [16,17]. Conventional-dose salvage approaches to patients with poor risk, recurrent or refractory germ cell tumors lead to remission in 30–60% of patients, but only 20% of patients gain long-term survival. Such a relatively low rate of cure with salvage therapy spurred investigation of alternate therapies including high-dose chemotherapy with autologous hematopoietic cell transplantation (HCT) (Table 64.6). Herein, we will review the management of the small fraction of patients for whom initial treatments are not fully effective, with emphasis on the role of high-dose chemotherapy.
The decision to pursue salvage therapy for germ cell tumors is important, and consultation with experts in the intricacies of managing this disease is strongly recommended. Several clinical situations may mimic progressive or recurrent disease and lead to inappropriate initiation of salvage therapy.
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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Tumor markers Management of testicular cancer has come to depend on the accurate determination and clinical interpretation of serum tumor markers. Lactate dehydrogenase is a nonspecific marker that probably correlates with disease bulk. The most sensitive and specific markers are alphafetoprotein (AFP) and β-subunit human chorionic gonadotropin (HCG). Among patients with disseminated testicular or primary retroperitoneal nonseminomatous germ cell tumors, HCG will be elevated in 75% and AFP in 40–60% of patients. HCG is elevated in 15–20% of patients with metastatic seminoma [1]. AFP is a glycoprotein normally produced by the fetal yolk sac or embryonal carcinoma elements of germ cell cancers, and is not detectable in normal adults. The half-life of AFP in the serum is about 5 days. False-positive elevation of AFP is quite rare, with differential considerations including laboratory error, liver inflammation, and other tumor types such as hepatoma, pancreatic, biliary, and gastric cancers. Additionally, a hereditary persistence of AFP has been documented in rare cases [18]. Persistent elevation of AFP in a patient with a seminoma is viewed as evidence of nonseminomatous elements, and management should proceed as such. β-Subunit HCG is a smaller glycoprotein that is normally produced by trophoblastic tissues, such as the syncytiotrophoblastic components of germ cell tumors. The HCG protein comprises antigenically distinct α and β subunits, and the serum half-life of the entire protein is 18–24 hours. False elevations may occur in patients who use marijuana, and there is some cross-reactivity in the radioimmunoassay with luteinizing hormone. In cases of persistently elevated HCG, patients should be asked about marijuana use, and testosterone should be given to ensure that a hypogonadal state with resultant high levels of luteinizing hormone is not interfering with the measurement. If the level remains elevated, restaging procedures and investigation of sanctuary sites are in order. Pure seminoma is often associated with normal AFP and HCG levels, but some patients, particularly with advanced disease, may have lowlevel elevation of HCG (usually 2 cm but not >5 cm in greatest dimension, or multiple lymph nodes, none >5 cm in greatest dimension Metastases in a lymph node, >5 cm in greatest dimension
Distant metastasis (M) MX Cannot be assessed M0 No distant metastasis M1 Distant metastasis: M1a: nonregional nodal or pulmonary metastasis M1b: distant metastasis other than nonregional nodes and lungs Serum Markers (S) SX Not available or not performed S0 Marker study levels within normal limits S1 LDH 10.000 AFP, α-fetoprotein; HCG, human chorionic gonadotropin; LDH, lactate dehydrogenase.
Table 64.2 American Joint Commission on Cancer (AJCC) staging system [2] Stage groupings Stage 0 Stage I Stage IA Stage IB Stage IS Stage II Stage IIA Stage IIB Stage IIC Stage III Stage IIIA Stage IIIB Stage IIIC
pTis, N0, M0, S0 pTI–4. N0, M0, SX pT1, N0, M0, S0 pT2–3, N0, M0, S0 Any pT, N0, M0, S1–3 Any pT, Nl–3, M0, SX Any pT, NI, M0, S0–1 Any pT, N2, M0, S0–1 Any pT, N3, M0, S0–1 Any pT, any N, M1, SX Any pT, any N, M1a, S0–1 Any pT, N1–3, M0, S2 Any pT, any N, M1a, S2 Any pT, N1–3, M0, S3 Any pT, any N, M1a, S3 Any pT, any N, M1b, any S
level over a 3-week period is consistent with disease eradication. Less steep declines of HCG levels usually correlate with residual disease post surgery or the emergence of drug resistance to chemotherapy. The reappearance of a marker elevation often precedes radiographic appearance and is an invaluable method of detecting early relapse. However, particularly among patients with levels of HCG over 50,000 mIU/mL, the disappearance of HCG from the serum may be unpredictable and occasionally quite prolonged. In a retrospective review of 41 patients with presenting HCG levels of over 50,000 mIU/mL, less than 10% had a normal HCG at the institution of the fourth course of chemotherapy [19]. However, 22 of 41 (53.7%) were continuously without evidence of disease despite no further therapy. We feel that the optimal strategy for such patients is close observation with initiation of salvage therapy if and when there is serologic progression. Importantly, salvage therapy should not be initiated on the basis of a persistent HCG elevation alone, but rather reserved for the clinical setting of a well-documented rise in the markers. In cases of rising markers after standard therapy, investigation of sanctuary sites such as the central nervous system or the contralateral testis should be performed before initiating salvage therapy.
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Table 64.3 Conventional primary therapy results Stage
Treatment
Disease-free survival
I II, III
Radical orchiectomy followed by active surveillance and appropriate treatment at time of relapse Radical orchiectomy plus bleomycin–etoposide–cisplatin (BEP) × 3–4 cycles, plus retroperitoneal lymph node dissection for partial response, plus BEP × 2 cycles for residual disease
~100% 80% (good risk) 60% (poor risk)
Table 64.4 Conventional-dose chemotherapy regimens
Indication
Regimen
Primary treatment of disseminated disease (stage II–III)
BEP [52]: Bleomycin 30 units IV on days 2, 9, and 16 Etoposide 100 mg/m2/day IV on days 1–5 Cisplatin 20 mg/m2/day IV on days 1–5 Repeat cycle at 21-day intervals
Primary salvage treatment of relapsed disease
VeIP [18]: Vinblastine 0.11 mg/kg/day IV on days 1–2 Ifosfamide 1.2 g/m2/day IV on days 1–5 Cisplatin 20 mg/m2/day IV on days 1–5 Repeat cycle at 21-day intervals VIP [18]: Etoposide 75 mg/m2/day IV on days 1–5 Ifosfamide 1.2 g/m2/day IV on days 1–5 Cisplatin 20 mg/m2/day IV on days 1–5 Repeat cycle at 21-day intervals TIP [57]: Paclitaxel 250 mg/m2 IV continuous infusion on day 1 Ifosfamide 1500 mg/m2/day IV on days 2–5 Cisplatin 25 mg/m2/day IV on days 2–5
IV, intravenously.
Table 64.5 International Germ Cell Consensus Classification prognostic staging system Non-seminoma Good prognosis
Intermediate prognosis
Poor prognosis
Seminoma
• Testis or retroperitoncal primary and • No non-pulmonary visceral metastases and • Good markers (AFP 10 × upper limit of normal)
16% of patients PFS 41% @ 5 years OS 48% @ 5 years
OS 92% @ 5 years
OS 80% @ 5 years
• Any primary site and • No non-pulmonary visceral metastases and • Normal AFP, any HCG, any LDH
90% of patients PFS 82% @ 5 years
• Any primary site and • Non-pulmonary visceral metastases and • Normal AFP, any HCG, any LDH
10% of patients PFS 67% @ 5 years
AFP, α-fetoprotein; HCG, human chorionic gonadotropin; LDH, lactate dehydrongenase; OS, overall survival rate; PFS, progression-free survival rate.
OS 86% @ 5 years
OS 72% @ 5 years
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Table 64.6 Possible indications for high-dose chemotherapy in the management of germ cell tumor (GCT) Salvage therapy for multiply relapsed or refractory GCT Initial salvage for relapsed GCT Primary treatment for poor-risk GCT
A small proportion of (otherwise incurable) patients will be cured Large single-center studies suggest there may be a role. However, recent European prospective randomized trial failed to show any effect on treatment outcomes Routine inclusion of high-dose chemotherapy in first-line treatment did not improve treatment outcome in a prospective randomized North American trial. Not indicated outside of a clinical trial
Radiographic abnormalities Depending on the stage at the time of diagnosis, 20–50% of patients who undergo induction chemotherapy for disseminated germ cell tumor will have significant residual radiographic abnormalities. Postchemotherapy surgical resection should be considered only in the setting of normalization or continuing decline of serum tumor markers. Resection of residual abnormalities is rarely urgent, and repeat imaging of the areas of abnormality should be performed prior to surgery, as often tumors will continue to involute. Patients with residual abnormalities and persistent elevations of AFP or HCG often have unresectable, viable tumor, and should therefore be considered for salvage chemotherapy regimens rather than surgical debulking [20]. Other clinical situations may mimic progressive or recurrent disease. For example, in patients with an appropriate fall in serologic markers but radiographic progression during induction chemotherapy, enlarging benign teratomatous portions of the tumor may be mistaken for progressive disease [21]. Appropriate management of such a patient includes completion of induction chemotherapy with subsequent surgical resection of residual radiographic abnormalities and not the administration of salvage therapies. The development of nodular lesions on chest imaging at the end or soon after completion of chemotherapy may represent bleomycininduced pulmonary injury rather than recurrent disease. These nodules are characteristically located in a subpleural region, and the diagnosis should be considered in a patient who is otherwise responding to therapy [22].
Conventional salvage therapies for germ cell tumors A unique feature in the management of germ cell tumors is the ability to achieve long-term disease-free survival with secondary chemotherapies after initial treatment failures. The standard for comparison for initial salvage therapies in germ cell tumors is the combination of vinblastine, ifosfamide, and cisplatin (VeIP) [23]. In one series, 135 patients with progressive disease after cisplatin- and etoposide-based chemotherapy were treated with VeIP regardless of metastatic site or performance status [24]. A 50% complete response (CR) rate after chemotherapy with or without surgical resection of residual disease was seen, with a long-term overall survival rate of 32% and a disease-free survival rate of 24%. Importantly, none of the 32 patients with extragonadal nonseminomatous disease was continuously disease free. Subsequently, paclitaxel was found to have significant single-agent activity in patients with refractory germ cell tumors, leading to its incorporation into salvage combination chemotherapy regimens [25–28]. Kondagunta et al. published promising results with the combination of paclitaxel, ifosfamide, and cisplatin (TIP) as second-line salvage therapy in patients with relapsed testicular germ cell tumors [29]. Forty-six patients with progressive germ cell tumor were given up to four cycles of TIP. Of note, all treated patients had prior treatment limited to six or fewer cycles of cisplatin, a gonadal primary tumor site, normal postchemotherapy markers, and either a CR or a partial response (PR) for
more than 6 months after completion of initial chemotherapy. CR was achieved with chemotherapy alone in 63%, and an additional 7% achieved CR with surgical resection of residual mass containing viable malignancy after chemotherapy. A durable CR rate of 63% was observed at a median follow-up of 69 months. In this highly selected group of patients with favorable prognostic factors for response to standard-dose salvage chemotherapy, TIP is an active salvage regimen and can be considered as an alternative to VeIP. However, these salvage regimens have never been compared in a phase III trial of unselected relapsed or refractory patients. Salvage surgery may play a significant role in the treatment of patients with chemorefractory germ cell tumors. Murphy et al. retrospectively reviewed all patients felt to have progressive chemorefractory yet resectable disease who underwent salvage surgery at Indiana University from 1977 to 1990 [30]. The majority underwent isolated retroperitoneal lymphadenectomy (69%). Thirty-eight of 48 patients (79%) were grossly disease free after surgery, and 29 (60%) obtained a serologic remission. Ten patients (21%) achieved event-free survival for 31–89 months. Six additional patients who relapsed after surgery achieved disease-free survival after additional surgery (four patients) or high-dose chemotherapy and autologous HCT (two patients). Notably, no patients with more than one site of metastatic disease, even when resectable, achieved long-term disease control. This series confirmed many of the conclusions drawn by Wood et al. regarding the experience with surgical salvage at the Memorial SloanKettering Cancer Center [31]. Surgical salvage was performed on 15 patients with chemorefractory disease and persistently elevated serum markers. The entire residual mass or solitary metastasis was entirely resected in each case. Twelve of 15 patients achieved normalization of serum markers after resection, and 7 of these patients remained without evidence of disease on follow-up 3–53 months after resection. Five of the 12 patients achieving CR with salvage surgery relapsed within 2 months, and 2 of them were rendered disease free with further chemotherapy. Of the three patients who did not achieve CR with salvage surgery, two achieved disease-free status with postoperative salvage chemotherapy. Overall, 11 of 15 patients (73%) were rendered disease free after salvage surgery with or without additional therapy. Isolated AFP elevation and retroperitoneal site of disease correlated with favorable outcome. These series and others point to a definite potential for cure with salvage surgery in selected patients with recurrent chemotherapyrefractory germ cell tumor.
High-dose chemotherapy for recurrent germ cell tumors An active strategy to improve outcomes for patients with relapsed or refractory germ cell tumors is the use of high-dose chemotherapy with autologous hematopoietic cell rescue. Initial attempts at cisplatin dose escalation (without hematopoietic stem cell support) increased toxicity without a survival advantage [32]. However, several advances led to
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substantial progress towards defining the role of high-dose therapy for germ cell tumors. Drugs such as carboplatin, etoposide, and alkylating agents such as thiotepa or the oxazaphosphorines (ifosfamide and cyclophosphamide) demonstrated antitumor activity, dose responsiveness, and a wide dose range between dose-limiting myelotoxicity and doselimiting extramedullary toxicities. Additional improvements in transplant procedures (hematopoietic cells from peripheral blood rather than bone marrow), supportive care (hematopoietic growth factors and selective antibiotics), and patient selection have advanced the field. The first investigations of high-dose chemotherapy with autologous HCT for germ cell tumors involved single-agent etoposide or etoposide and cyclophosphamide [33–35]. Although some patients were refractory to these agents in standard doses, 20–40% response rates and rare longterm cures were observed.
I–II study and none of 11 patients in the phase II study achieving a complete remission. In an update of this series, there were no failure-free survivors at 2 years in 13 patients with primary mediastinal nonseminomatous germ cell tumor treated with tandem high-dose chemotherapy [39]. However, in an international multicenter analysis of second-line chemotherapy in patients with relapsed extragonadal nonseminomatous germ cell tumor, 48 patients received high-dose chemotherapy followed by autologous bone marrow transplant at the time of relapse [40]. Ten of those patients (21%) were continuously disease free; seven of the patients had retroperitoneal germ cell tumor, and the other three had primary mediastinal disease. This is a population for whom conventional salvage therapies also have poor outcomes, and such patients should be the focus of investigational approaches [41,42]. Dose escalation and additional agents
Early trials Most modern phase I–II trials of high-dose chemotherapy have included carboplatin and etoposide, sometimes with ifosfamide, cyclophosphamide, or thiotepa. Several centers intended high-dose therapy to be repeated once after recovery (tandem transplants) to maximize potential benefit. Initial investigations focused on patients with either relapse after best standard salvage chemotherapies or cisplatin-refractory germ cell tumors that were felt unlikely to be cured by any available treatment. Nichols and colleagues at Indiana University pioneered this field with the first phase I–II study of two courses of high-dose carboplatin and etoposide in patients with germ cell tumors refractory to cisplatin (defined as progression on or within 4 weeks of the last cisplatin dose) or recurrent after primary cisplatin-based therapy and a salvage regimen containing ifosfamide and cisplatin [36]. The results were updated after the first 40 patients [37]. Over half of the patients had received three or more prior regimens, and 70% were considered cisplatin refractory. Therapy consisted of carboplatin 900–2000 mg/m2 and etoposide 1200 mg/m2. Ifosfamide was added to the regimen in three patients. Twenty-six of 40 (65%) received both courses of high-dose therapy. Overall, 7 of 40 (18%) died as a consequence of therapy, primarily due to infection. There were 12 patients (30%) who attained a CR, and 14 (35%) who achieved a PR, for an overall response rate of 65%. Six patients (15%) were long-term disease-free survivors, and a seventh patient died at 22 months free of germ cell tumor from a therapy-related acute myeloid leukemia. These results were confirmed in a phase II multi-institutional trial of 38 similar patients receiving tandem high-dose carboplatin and etoposide, where 58% completed both transplants, a 13% treatment-related mortality was observed, 9 patients (24%) achieved a CR (2 after posttransplant surgical resections), and 5 patients (13%) were alive and free of disease with a minimum of 18 months follow-up [38]. These initial phase I and II trials of high-dose chemotherapy and HCT provided multiple insights. First, these trials clearly demonstrated that a portion of patients (perhaps 13–24%) with multiply recurrent germ cell tumors could achieve long-term disease-free survival. Second, nearly all relapses occurred in the first 18 months post transplant, and disease status after high-dose therapy predicted long-term outcome. Third, the tandem transplant approach was feasible. In the phase I–II study, all of the long-term disease-free survivors received both transplants, and 8 of 12 patients in partial remission after the first transplant achieved complete remission after the second. Fourth, surgical salvage remained an important treatment modality after transplant, leading to complete remissions and likely contributing to cures in several patients. Lastly, patients with primary mediastinal germ cell tumors failed to respond, with none of 8 patients in the phase
Subsequent trials of high-dose therapy began to enroll less heavily pretreated patients, and incorporated the use of peripheral blood hematopoietic cells and growth factors, which all served to shorten the duration of cytopenias. These improvements led investigators to attempt further dose escalations or add additional agents such as ifosfamide or cyclophosphamide. One trial of dose escalation involved treating 33 patients with relapsed or refractory germ cell tumors with carboplatin 1650– 2100 mg/m2 and etoposide 1200–2250 mg/m2 for tandem cycles [43]. The dose-limiting toxicities for this regimen were mucositis and peripheral neurotoxicity, and reversible transaminase abnormalities were common. Results were similar to earlier studies, with 20 of 33 patients (61%) completing both transplant procedures. Treatment-related mortality was 18%, including four patients who died prior to response evaluation. Four of the remaining 29 evaluable patients (14%) achieved complete remission, and 8 of 33 (24%) became long-term survivors. This trial served to establish the maximum tolerated doses of carboplatin (2100 mg/m2) and etoposide (2250 mg/m2), which continue to represent the current standard of care conditioning regimen. The German Testicular Cancer Study Group added ifosfamide 0– 10 g/m2 to carboplatin 1500–2000 mg/m2 and etoposide 1200–2400 mg/ m2 as a transplant regimen in 74 patients with relapsed or refractory germ cell tumors [44,45]. Therapy was delivered regardless of response to two preceding cycles of reinduction standard-dose cisplatin, etoposide, and ifosfamide (VIP). Treatment-related mortality was only 3%, but late toxicities included renal toxicity (21%), paresthesias (29%), and ototoxicity (18%). Approximately 50% of patients achieved a complete remission with this therapy, and 28 of 74 (38%) were alive from 3.2 to 5.6 years post treatment. In an attempt to decrease the toxicities observed with ifosfamide, investigators at Memorial Sloan-Kettering Cancer Center studied the addition of cyclophosphamide to carboplatin and etoposide in patients with cisplatin-refractory germ cell tumors [46]. In this series, 58 patients with an incomplete response to initial conventional-dose cisplatin-containing chemotherapy, or incomplete response or relapse from CR with a cisplatin-based salvage regimen, were enrolled. Patients received carboplatin 1500 mg/m2, etoposide 1200 mg/m2, and a range of 60–150 mg/ kg cyclophosphamide followed by infusion of bone marrow-derived hematopoietic stem cells. Thirty-one patients (53%) received one cycle of therapy, and 27 (47%) received two. CR was achieved by 23 patients (40%); 20 of these were treated with two cycles of therapy. With a median follow-up of 28 months, 12 patients (21%) were alive and free of disease. There were seven (12%) treatment-related deaths, the majority of which were related to bleeding or sepsis with multiorgan failure. Hepatic and renal toxicities were observed in 26% of patients. Two independently predictive variables for survival were identified: pretreatment HCG level and presence of retroperitoneal metastases.
Hematopoietic Cell Transplantation in Germ Cell Tumors
Several investigators have revisited the incorporation of newer and older drugs in an attempt to improve the results of high-dose chemotherapy. A phase II multicenter French study (TAXIF) treated 45 patients with relapsed poor-prognosis germ cell tumors with two cycles of epirubicin and paclitaxel, followed by three consecutive high-dose chemotherapy cycles (cyclophosphamide 3g/m2 plus thiotepa 400 mg/m2 followed by two cycles of ifosfamide 10 g/m2, carboplatin area under the curve 20, and etoposide 1500 mg/m2) supported by stem cell transplantation [47]. Twenty-five patients died from disease progression, and five patients from toxicity. Of the 22 patients who received the complete course, the overall response rate was 37.7%, with 8.9% achieving a CR. Median overall survival was 11.8 months, with a 3-year survival and progression-free survival rate of 25.3%. Notably, patients with adverse prognostic factors (absolute cisplatin refractoriness, or HCG >1000 mIU/ mL before transplant, or a combination of progressive disease before transplant, cisplatin-refractoriness prior to transplant, or primary mediastinal tumor) did not benefit from the approach. At Memorial Sloan-Kettering Cancer Center, 47 patients germ cell tumor patients with cisplatin resistance and one or more unfavorable prognostic features for achieving CR with conventional-dose salvage therapy (including extragonadal primary site, progressive disease after an incomplete response to first-line therapy, and poor or lack of response to prior treatment with cisplatin plus ifosfamide conventional-dose therapy) were treated with two cycles of paclitaxel plus ifosfamide, followed by three cycles of carboplatin and etoposide with peripheral blood stem cell support [48]. Etoposide was administered at a total dose of 1200 mg/m2 and carboplatin was dose escalated according to target area under the curve. Three of the 47 patients were treated with paclitaxel– ifosfamide but did not receive carboplatin–etoposide. Of the 44 patients, 77% received all three planned cycles of high-dose chemotherapy. All 47 patients were evaluated for response and after completion of highdose chemotherapy; 23 patients (49%) achieved a CR. Three additional patients (6%) achieved a CR to chemotherapy and surgery, for an overall CR rate of 55%. With a median follow-up time of 40 months, 24 patients (51%) were alive and free of disease. When the results of this study were combined with the results of a prior phase I–II trial of similar design [49], an overall CR rate of 56% was reported, with 50% of patients alive with no evidence of disease. Finally, the value of sequential high-dose chemotherapy in patients with relapsed or refractory germ cell tumors was addressed in a prospective randomized multicenter trial of the German Testicular Study Group [50]. Two hundred and sixteen patients were randomized to either one cycle of conventional-dose VIP followed by three sequential cycles of high-dose carboplatin and etoposide followed by autologous stem cell transfusion, or three conventional-dose cycles of VIP followed by a single cycle of high-dose carboplatin, etoposide, and cyclophosphamide followed by autologous stem cell reinfusion. There was no statistically significant difference in event-free, progression-free or overall survival between the two arms. However, treatment-related deaths were less frequent in patients receiving sequential high-dose chemotherapy
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(p 1000 IU/L before transplant
1 1 1 2 2
Good (score 0) Intermediate (score 1–2) Poor (score >2)
51% 27% 5%
61% 34% 8%
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Summary 100
Overall survival rate (%)
80 60 40 20 0 0
10 20 30 40 50 60 70 80 90 100 110 120 Months since first day of high-dose chemotherapy
No. at risk
184 161 128 103 80 61 49 27 23 17
7
4
In summary, the use of high-dose chemotherapy with autologous HCT for the treatment of relapsed or refractory germ cell tumors can produce long-term survivors in 15–50% of patients. The principal toxicities include nephrotoxicity, peripheral neurotoxicity, reversible transaminase elevations, and gonadal toxicity. Treatment-related mortality rates ranged from 0% to 18%, with most recent series reporting 3–5%. Patients with primary mediastinal tumors only rarely benefit from salvage highdose therapy. Treatment earlier in the course of relapsed or refractory disease appears to be associated with better outcomes. Current standard of care at most high-volume centers consists of a tandem transplant strategy utilizing the conditioning regimen of carboplatin 2100 mg/m2 and etoposide 2250 mg/m2. The addition of a third agent requires dose reductions of these two most active drugs, and it is unlikely that an adequately powered randomized trial to determine the benefit of adding a third agent will ever be performed given the rarity of this clinical circumstance.
Fig. 64.1 Kaplan–Meier estimates of overall survival. The top and bottom lines show the 95% confidence interval.
Initial salvage with high-dose chemotherapy for germ cell tumors
Overall survival rate (%)
100 Low risk (0 points)
80 60
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10 20 30 40 50 60 70 80 90 100 110 120 Months since first day of high-dose chemotherapy
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73 63 59 44 34 26 21 10 9 64 45 37 35 28 22 19 12 10
7 7
47 23 17 13 10
1
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5
2
1
4 3
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Fig. 64.2 Disease-free survival according to the Indiana Prognostic Scoring System. The prognostic scoring algorithm, based on the three-variable model, assigned a score of 3 points for third-line chemotherapy, 2 points for platinum refractoriness, and 2 points for advanced International Germ Cell Cancer Collaborative Group stage. High scores indicated a low probability of disease-free survival.
line or later therapy. Notably, 18 (45%) of 40 patients with cisplatinrefractory disease achieved disease-free status. In patients with cisplatin-sensitive disease, 98 (68%) of 144 were disease free. Three drug-related deaths (2%) were reported, and three additional patients developed acute leukemia after therapy. Based on this retrospective review of the Indiana University experience, a three-variable prognostic model was elucidated that included timing of high-dose chemotherapy, platinum sensitivity or refractoriness, and International Germ Cell Cancer Collaborative Group stage (Fig. 64.2). However, this scoring system was not validated by outside groups, and there currently is no universally accepted prognostic score for patients at initial relapse [55,56]. An international effort to elucidate prognostic factors for first-salvage treatment is currently under way.
The modest results of standard-dose salvage chemotherapies (the 24% long-term disease-free survival with VeIP discussed above), combined with the improving rates of durable responses and decreasing morbidity and mortality associated with high-dose therapy, have led to the investigation of high-dose therapy for initial salvage of relapsed germ cell tumors. Indiana University published one of the first series to incorporate high-dose chemotherapy as initial salvage [57]. Sixty-five patients with relapsed or persistent testicular cancer were given initial salvage treatment with tandem courses of carboplatin 2100 mg/m2 and etoposide 2250 mg/m2 followed by autologous hematopoietic cell rescue. All patients had primary testicular tumors. Fifty-six of the 65 patients (86%) received at least one cycle of standard-dose salvage chemotherapy (usually VeIP) prior to transplant. Ten of 65 (15%) received ex vivo cytokine-stimulated, mdr-1-transduced autologous CD34+ cells, and 26 of 65 (40%) received post-transplant maintenance oral etoposide. Using the Beyer Prognostic Score for outcomes with high-dose therapy (Table 64.7), 61% were good risk, 31% intermediate risk, and 8% poor risk. There were no treatment-related deaths. Complete remission was achieved in 28 patients (43%) with high-dose chemotherapy, and in another patient with additional VeIP after only one transplant, and 13 patients (20%) with post-transplant surgical resections. With follow-up ranging from 16 to 91 months, 37 patients (57%) were continuously disease free, and 40 (60%) remain disease free after definitive therapy. Only one phase III randomized prospective trial of high-dose chemotherapy in the initial salvage setting has been reported [58]. In this study, 280 patients with relapsing germ cell tumors who had achieved complete or partial remission with first-line platinum combination chemotherapy were randomized to either four cycles of VIP or VeIP (arm A) or three cycles of VIP or VeIP followed by high-dose carboplatin, etoposide, and cyclophosphamide with hematopoietic stem cell support (arm B). Overall CR was observed in 51 (42%) patients in arm A and 53 (43%) patients in arm B. Additionally, 21 (17%) versus 23 (18%) patients achieved a tumor marker-negative partial remission. There was no clinical benefit to the replacement of a fourth cycle of VIP or VeIP by high-dose chemotherapy in terms of either 3-year event-free survival (35% versus 42%; p = 0.16) or overall survival (53%; 95% confidence interval 46–59%). In a subgroup analysis of the 104 patients achieving a CR, the 3-year disease-free survival rates were 55% and 75% (p = 0.04) in favor of the high-dose chemotherapy arm.
Hematopoietic Cell Transplantation in Germ Cell Tumors
This study was criticized for being underpowered (only 80% of patients in arm A and 73% of patients in arm B receiving the fourth cycle of chemotherapy) to detect a significant difference in 3-year eventfree survival. Additional sources of criticism included the high degree of variability in initial treatment regimens, and the single-transplant approach. In light of the previously discussed superiority of sequential high-dose versus single-dose chemotherapy, it is difficult to conclude from this trial alone that there is no role for high-dose chemotherapy in the initial salvage setting.
Primary treatment with high-dose chemotherapy for poor-prognosis germ cell tumors Incorporating high-dose chemotherapy as a first-line strategy for the treatment of patients with poor-risk germ cell tumors at presentation has also been investigated. Two randomized trials of high-dose chemotherapy with autologous transplant support for the treatment of patients with poor-risk germ cell tumors have now failed to show a benefit [59–61]. In a French trial, Droz reported 115 patients deemed poor risk by Institut Gustave Roussy criteria (an older prognostic model) who were randomized to receive either four cycles of cisplatin 200 mg/m2, vinblastine, etoposide, and bleomycin (PVeVB), or two cycles of PVeVB plus one cycle of high-dose therapy including cisplatin 200 mg/m2 followed by autologous transplant. With a median follow-up of 9.7 years, 31 and 27 patients have continuously shown no evidence of disease in the respective arms. There was no significant difference between the overall survival curves (p = 0.167). This study was criticized because the four-drug regimen is not considered a standard therapy, the dose intensity and total cisplatin dose were lower in the “high-dose” arm, and a substantial number of patients randomized to the high-dose arm did not receive the assigned therapy. Additional criticisms included the extensive accrual period, and significant variability in initial treatment regimen [59,60]. The question was more definitively answered by a United States trial reported in early 2007 [61]. In this randomized Intergroup (Cancer and Leukemia Group B–South-Western Oncology Group–Eastern Cooperative Oncology Group) phase III study, 219 previously untreated patients with intermediate- or poor-risk germ cell tumors were randomized to either four cycles of standard bleomycin, etoposide, and platinum (BEP) or two cycles of BEP followed by two cycles of carboplatin 600 mg/m2, etoposide 600 mg/m2, and cyclophosphamide 50 mg/kg administered on days 1–3 before transplantation followed by autologous stem cell infusion. The trial was designed to detect a 20% improvement in durable CR at 1 year. CR was achieved in 61 of 111 patients (55%) on the BEPalone arm, and in 61 of 108 patients (56%) on the BEP + high-dose chemotherapy arm. With a median follow-up of 51 months, the 1-year durable CR rate was 48% after BEP alone and 52% after BEP + highdose chemotherapy (p = 0.53). The median time to treatment failure was 11.3 months in the BEP-alone arm, and 23.2 months in the BEP + highdose chemotherapy arm (p = 0.40). No difference in survival was seen between the two arms (p = 0.94). Rate of tumor marker decline was correlated with treatment outcome as a secondary endpoint. In a subset analysis, 67 patients with unsatisfactory marker decline were identified. In this subset, the proportion achieving 1-year durable CR was 61% for patients receiving high-dose chemotherapy versus 34% for those on the BEP-alone arm (p = 0.03). The authors concluded that routine inclusion of high-dose chemotherapy in the first-line treatment of germ cell tumor patients with metastases and poor predicted outcome to chemotherapy did not improve treatment outcome. The subset analysis of patients with unsatisfactory marker decline is provocative and will likely be incorporated into future trials analyzing
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the role of high-dose chemotherapy. With collaborative international clinical trial efforts, it may be possible to adequately power studies to detect smaller yet clinically relevant differences in outcome in this rare group of patients.
Surgical management after high-dose chemotherapy The role of surgical resection after high-dose chemotherapy was assessed in a retrospective review of the German experience [62]. This retrospective review identified 216 patients treated on three consecutive high-dose chemotherapy trials between 1989 and 1999. Partial remissions with negative or positive tumor markers after high-dose chemotherapy were achieved in 128 patients (59%). Of these patients, 57 (45%) proceeded to residual tumor resection; the remaining 71 (55%) could not be operated on because of progressive disease, inoperability due to tumor mass or poor performance status, or patient refusal. With a median follow-up of 87 months, 37 (65%) of 57 patients were alive, and 34 (59%) of 57 patients remained continuously disease free. Viable cancer was documented in the resected specimen of 26 (46%) of the 57 patients. The projected overall and event-free survival rates after 5 years in patients with viable cancer were 42% and 38% (p < 0.01). The projected overall and event-free survival rates after 5 years in patients without viable cancer were 84% and 77% (p < 0.01). Of note, all patients in this series had been treated with a single cycle of high-dose chemotherapy, which likely influenced the incidence of viable cancer in resected specimens. However, this series confirmed that postchemotherapy resection contributed to overall treatment outcome, and thus residual tumor resection continues to be offered to patients with partial remission after high-dose chemotherapy.
Management of relapse post high-dose chemotherapy Outcomes have been unfavorable in patients who experience relapse after high-dose chemotherapy with stem cell support. A retrospective review of the Indiana University experience identified 186 patients with relapsed germ cell tumors who were treated with high-dose chemotherapy and peripheral blood stem cell transplantation between 1986 and 1997 [63]. Of these, 101 patients relapsed after high-dose chemotherapy after a median interval of 10 months. Forty-seven of these patients received further chemotherapy, and seven patients underwent surgery alone. A total of 66 chemotherapy regimens were administered to 47 patients, with an overall response rate of 18.2% and a CR rate of 5%. Of the patients who received surgery alone, only one patient was a longterm survivor. Finally, two recent trials have examined the use of additional salvage chemotherapy for patients relapsing after high-dose chemotherapy. In a phase II trial at Indiana University, 32 patients with progressive disease after two cycles of high-dose carboplatin and etoposide were treated with paclitaxel 100 mg/m2 and gemcitabine 1000 mg/m2 intravenously on days 1, 8, and 15 every 4 weeks for a maximum of six cycles [64]. Patients who had received either agent prior to high-dose chemotherapy were ineligible. An objective response was achieved by 10 patients (31%); this included six CRs and four PRs (of 2–6 months’ duration). Of the six CRs, four patients (12.5%) were continuously disease free with paclitaxel and gemcitabine alone at more than 20, 40, 44, and 57 months from treatment. One additional CR patient was rendered disease free after two subsequent resections of carcinoma. There was no treatment-related mortality. The German Testicular Study Group treated 32 patients with relapse after high-dose chemotherapy and nine patients with cisplatin-refractory disease with gemcitabine 800 mg/m2 and oxaliplatin 130 mg/m2 on day
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1, and paclitaxel 80 mg/m2 on day 8, of a 3-week cycle for a minimum of two cycles [65]. Of the 41 patients, 2 patients (5%) achieved a CR, 34% achieved a marker-negative PR, and 12% achieved marker-positive PR, for an overall response rate of 51%. With a median follow-up of 5 months, 15% of patients remained in complete remission after gemcitabine, oxaliplatin, and paclitaxel chemotherapy with or without residual tumor resection. There were no toxic deaths with this regimen. These trials point to some possibility of chemotherapeutic salvage even in heavily pretreated and cisplatin-refractory patients.
receiving high-dose carboplatin and etoposide, three patients (2.6%) developed secondary leukemias [71]. This was not significantly different from the expected rate of secondary leukemia in patients receiving additional cycles of standard-dose etoposide as salvage chemotherapy. Advances in adjuvant or maintenance therapies with oral etoposide or immunmodulators such as interleukins have been considered [72]. Development of an international prognostic scoring system for firstsalvage treatment is currently under way. Ultimately, advances in the molecular understanding of germ cell malignancies may lead to targeted therapies.
Ongoing studies and future directions Newer compounds with single-agent response rates are being incorporated into trials including bexarotene, paclitaxel, gemcitabine, bendamustine, and oxaliplatin [66–68]. Unique dosing strategies such as multiple cycles or alternating agents may be incorporated in future highdose algorithms. Adjunctive agents may reduce the toxicity of high-dose therapy such as amifostine or keratinocyte growth factor [69]. Concern has been raised over the generation of secondary hematologic malignancies, particularly with the use of high-dose etoposide. However, in one series, the incidence of secondary leukemia among 302 recipients of autologous HCT with cumulative doses exceeding 2 g/m2 of etoposide was only 1.3% [70]. In another series of 113 patients
Conclusion High-dose carboplatin and etoposide-based chemotherapy with autologous HCT is accepted as a standard therapy for patients with germ cell tumors who have failed two prior standard-dose regimens. Prospective randomized trials have thus far failed to support the use of high-dose chemotherapy as primary treatment or initial salvage for poor-prognosis patients. Due to the complexity of clinical decision-making, it is of paramount importance that patients in this rare clinical situation be treated at high-volume referral centers. Clinical trial participation should be encouraged for all patients requiring high-dose chemotherapy.
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Cancer Cooperative Study Group. J Clin Oncol 1994; 12: 1223–31. Beyer J, Kingreen D, Krause M et al. Long-term survival of patients with recurrent or refractory germ cell tumors after high dose chemotherapy. Cancer 1997; 79: 161–8. Motzer RJ, Mazumdar M, Bosl GJ et al. High-dose carboplatin, etoposide, and cyclophosphamide for patients with refractory germ cell tumors: treatment results and prognostic factors for survival and toxicity. J Clin Oncol 1996; 14: 1098. Lotz JP, Bui B, Gomez F et al. Sequential high dose chemotherapy protocol for relapsed poor prognosis germ cell tumors combining two mobilization and cytoreductive treatments followed by three high dose chemotherapy regimens supported by autologous stem cell transplantation. Results of the phase II multicentric TAXIF trial. Ann Oncol 2005; 16: 411–18. Kondagunta GV, Bacik J, Sheinfeld J et al. Paclitaxel plus ifosfamide followed by high dose carboplatin plus ifosfamide in previously treated germ cell tumors. J Clin Oncol 2007; 25: 85–90. Motzer RJ, Mazumdar M, Sheinfeld J et al. Sequential dose intensive paclitaxel, ifosfamide, carboplatin, and etoposide salvage therapy for germ cell tumor patients. J Clin Oncol 2000; 18: 1173–80. Lorch A, Kollmannsberger C, Hartmann JT et al. Single versus sequential high-dose chemotherapy in patients with relapsed or refractory germ cell tumors: a prospective randomized multicenter trial of the German Testicular Cancer Study Group. J Clin Oncol 2007; 25: 2778–84. Margolin BK, Doroshow JH, Ahn C et al. Treatment of germ cell cancer with two cycles of highdose ifosfamide, carboplatin, and etoposide with autologous stem-cell support. J Clin Oncol 1996; 14: 2631–7. Ayash LJ, Clarke M, Silver SM et al. Double doseintensive chemotherapy with autologous stem cell support for relapsed and refractory testicular cancer: the University of Michigan experience and literature review. Bone Marrow Transplant 2001; 27: 939–47. Beyer J, Kramar A, Mandanas R et al. High-dose chemotherapy as salvage treatment in germ cell tumors: a multivariate analysis of prognostic variables. J Clin Oncol 1996; 14: 2638–45. Einhorn LH, Williams SD, Chamness A et al. High-dose chemotherapy and stem-cell rescue for metastatic germ-cell tumors. N Engl J Med 2007; 357: 340–8. Shamash J, Stebbing J, Powles T. In reply. N Engl J Med 2007; 357: 1771. Lorch A, Beyer J, Bokemeyer C. In reply. N Engl J Med 2007; 357: 1772. Bhatia S, Abonour R, Porcu P et al. High-dose chemotherapy as initial salvage chemotherapy in patients with relapsed testicular cancer. J Clin Oncol 2000; 18: 3346–51. Pico JL, Rosti G, Kramar A et al. A randomized trial of high-dose chemotherapy in the salvage treatment of patients failing first-line platinum chemotherapy for advanced germ cell tumors. Ann Oncol 2005; 16: 1152–9. Chevreau C, Droz JP, Pico JL et al. Early intensified chemotherapy with autologous bone marrow transplantation in first line treatment of poor risk
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non-seminomatous germ cell tumours. Preliminary results of a French randomized trial. Eur Urol 1993; 23: 213–17. Droz JP, Kramar A, Biron P et al. Failure of highdose cyclophosphamide and etoposide combined with double-dose cisplatin and bone marrow support in patients with high-volume metastatic nonseminomatous germ-cell tumors: mature results of a randomized trial. Eur Urol 2007; 51: 739– 48. Motzer RJ, Nichols CR, Margolin KA et al. Phase III randomized trial of conventional-dose chemotherapy with or without high-dose chemotherapy and autologous hematopoietic stem-cell rescue as first-line treatment for patients with poor-prognosis metastatic germ cell tumors. J Clin Oncol 2007; 25: 247–56. Rick O, Bokemeyer C, Weinknecht S et al. Residual tumor resection after high dose chemotherapy in patients with relapsed or refractory germ cell cancer. J Clin Oncol 2004; 22: 3713–19. Porcu P, Bhatia S, Sharma M, Einhorn LH. Results of treatment after relapse from high dose chemotherapy in germ cell tumors. J Clin Oncol 2000; 18: 1181–6. Einhorn LH, Brames MJ, Juliar B, Williams SD. Phase II study of paclitaxel plus gemcitabine salvage chemotherapy for germ cell tumors after progression following high dose chemotherapy with tandem transplant. J Clin Oncol 2007; 25: 513–16. Bokemeyer C, Oechsle K, Honecker F et al. Combination chemotherapy with gemcitabine, oxaliplatin, and paclitaxel in patients with cisplatin-refractory or multiply relapsed germ cell tumors: a study of the German Testicular Cancer Study Group. Ann Oncol 2008; 19: 448–53. Bokemeyer C, Gerl A, Schoffski P et al. Gemcitabine in patients with relapsed or cisplatinrefractory testicular cancer. J Clin Oncol 1999; 17: 512–16. Bokemeyer C, Kollmannsberger C, Harstrick A et al. Treatment of patients with cisplatin-refractory testicular germ-cell cancer. German Testicular Cancer Study Group (GTCSG). Int J Cancer 1999; 83: 848–51. Kollmannsberger C, Gerl A, Schleucher N et al. Phase II study of bendamustine in patients with relapsed or cisplatin- refractory germ cell cancer. Anticancer Drugs 2000; 11: 535–9. Cronin S, Uberti JP, Ayash LJ, Raith C, Ratanatharathorn V. Use of amifostine as a chemoprotectant during high-dose chemotherapy in autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 2000; 26: 1247–9. Kollmannsberger C, Beyer J, Droz JP et al. Secondary leukemia following high cumulative doses of etoposide in patients treated for advanced germ cell tumors. J Clin Oncol 1998; 16: 3386–91. Houck W, Abonour R, Vance G, Einhorn LH. Secondary leukemias in refractory germ cell tumor patients undergoing autologous stem cell transplantation using high dose etoposide. J Clin Oncol 2004; 22: 2155–8. Cooper MA, Einhorn LH. Maintenance chemotherapy with daily oral etoposide following salvage therapy in patients with germ cell tumors. J Clin Oncol 1995; 13: 1167–9.
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Richard W. Childs & Ramaprasad Srinivasan
Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors
Introduction Our perception of the mechanisms through which malignant cells are eradicated following allogeneic hematopoietic cell transplantation (HCT) has evolved substantially over the past four decades. No longer merely thought of as a means of rescuing hematopoietic function following high-dose conditioning, allogeneic transplantation is now known to be a powerful type of immunotherapy capable of curing patients with otherwise fatal malignant diseases. This conceptual evolution has translated into a diversification of the indications for allogeneic HCT and led to the development of reduced-intensity transplant approaches whose beneficial antineoplastic effects can be attributed largely to the transplanted donor immune system. Recently, investigators have begun to test whether nonhematologic malignancies might likewise be susceptible to allogeneic immune attack. In this chapter, we highlight recent work that has improved our awareness and understanding of the graftversus-tumor (GVT) effect, and discuss the preliminary results of its application as an investigational therapeutic modality in the treatment of metastatic solid tumors.
Allogeneic transplantation as immunotherapy: the GVT effect Acute leukemias have traditionally been viewed as “chemosensitive” malignancies. The inability to “cure” the majority of adults with leukemia following conventional chemotherapeutic regimens was attributed at least in part to a failure to deliver the sufficiently high doses of antineoplastic agents required to eradicate all malignant cells. Dose-limiting toxicities, including prolonged delays in lymphohematopoietic recovery, significantly hindered efforts to intensify such regimens. Pioneering work by E.D. Thomas and others led to the development of allogeneic HCT as a method to allow for accelerated recovery of lymphohematopoietic function following the administration of high-dose chemotherapy [1,2]. For the next two decades, allogeneic HCT became increasingly used as a therapeutic option to treat patients with a variety of different treatment-resistant hematologic malignancies. Initially, the curative
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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potential of the procedure was ascribed solely to the cytotoxic effects of the conditioning regimen, be it chemotherapy alone or in conjunction with total body radiation. It was not until years following its inception that investigators first documented the very important and powerful immune mechanisms that contributed to the curative potential of the approach in humans. Throughout the 1980s, several landmark observations provided insight into this phenomenon known as the “graft-versus-tumor” effect. First, in an analysis of over 200 patients with hematologic malignancies undergoing human leukocyte antigen (HLA)-matched HCT from a sibling donor, investigators noted that patients who experienced moderateto-severe acute graft-versus-host disease (GVHD) or chronic GVHD had a significantly lower incidence of leukemic relapse compared with patients with little or no GVHD [3]. Similar benefits in disease-free survival were observed in recipients of HLA-identical (nontwin) sibling donor allografts compared with those receiving syngeneic allografts [4], as well as in recipients of T-cell-replete grafts versus those receiving T-cell-depleted transplants [4–7]. These observations suggested a very important role for donor T cells in mediating this effect, and also implicated the targets of GVT as falling outside the realm of antigens encoded by the major histocompatibility complex (MHC). Definitive evidence that GVT effects were clinically meaningful came from studies in patients with chronic myelogenous leukemia who relapsed posttransplant and were subsequently induced back into remission following a donor lymphocyte infusion (DLI) [8]. Over the last decade, numerous investigators have successfully exploited GVT effects against a variety of malignancies of hematopoietic origin, including both acute and chronic leukemias, lymphomas, multiple myeloma, and Epstein–Barr virus (EBV)-related lymphoproliferative disorder [9–17]. The ability of GVT to induce remission varies among these malignancies, with the critical determining factors dictating neoplastic susceptibility to GVT being not entirely understood.
The immune system and solid tumors Tumors such as metastatic melanoma and renal cell carcinoma (RCC), with their ability to undergo occasional spontaneous regressions or maintain prolonged periods of stable disease, have long fascinated investigators and led to the concept that the immune system might play a role in the regulation or control of malignant cells. Indeed, the first spontaneous regression of metastatic RCC after nephrectomy was described in 1928 and was attributed to an antibody-mediated immune response [18].
Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors
The propensity of nude (athymic) mice to develop lymphoreticular tumors, and the increased incidence of malignancies in patients with congenital or acquired immunodeficiency states, lent further credence to the notion that the immune system might play a role in the control of cancer [19–21]. The development of immune-based therapeutic strategies against metastatic solid tumors was a logical consequence of these observations. At the National Cancer Institute (NCI), pilot immunotherapy trials in patients with advanced solid tumors – particularly metastatic RCC and metastatic melanoma – were conducted in the early 1980s by Rosenberg and colleagues [22]. The earliest trials were intended to nonspecifically stimulate innate immune responses against tumors using cytokines such as interleukin-2 (IL-2). In their preliminary experience using IL-2 in patients with either metastatic RCC or melanoma, overall response rates of 20% (RCC) and 17% (melanoma) were observed. Importantly, some patients achieved complete and durable remission of disease, providing valuable proof of concept of the therapeutic potential of immune-based therapeutic strategies. Subsequent studies conducted at the NCI as well as by the Cytokine Working Group employed adoptive transfer of lymphokine-activated killer cells or ex vivo-expanded tumor-infiltrating lymphocytes in conjunction with IL-2. Although response rates in some trials approached 35%, it remains unclear if tumor-infiltrating lymphocyte therapy provided any additional therapeutic benefit over cytokine treatment alone [23–29]. The low response rate and significant morbidity that was frequently associated with such therapy (particularly highdose IL-2) provided the impetus for the exploration and development of immune strategies that would selectively target the tumor. The foundation for tumor-specific cellular immunotherapy was laid following the identification of a number of different tumor-associated antigens (TAAs) in melanoma and other solid tumors [30–32]. Several clinical trials have evaluated the safety and efficacy of cancer vaccines designed to enhance immune responses to TAAs. Although immune correlative studies have shown that some vaccines significantly expand TAA-reactive T-cell populations in vivo, early clinical results from such trials have been modest. Several factors likely contribute to the poor clinical efficacy of conventional vaccine strategies designed to enhance “self” immunity against TAAs. To date, the vast majority of trials have used an MHC class Irestricted peptide-based approach. As a consequence, resultant immune responses are limited to a single antigenic epitope devoid of a critical CD4+ helper T-cell component [33]. Furthermore, such vaccine strategies may favor the selection of tumor cells lacking the targeted antigen, a phenomenon described as “antigen escape.” Modifying this approach by simultaneously immunizing with multiple tumor antigens that are both MHC class I and II restricted, or by using the tumor itself as a vaccine (tumor lysates, tumor apoptotic bodies, etc.), may partly overcome this limitation [34]. It has also been suggested that tumors may evade cytotoxic T lymphocytes (CTL) by downregulating expression of accessory molecules required by CTLs to mediate lysis. Perhaps one of the greatest limitations of conventional immunotherapy is that the host immune system it attempts to enhance has intrinsic functional compromise. A number of lines of evidence support this concept. First, the host immune system is often rendered incompetent by prior chemotherapy and/or by tumor-related factors. Second, longstanding immune tolerance to tumor antigens, including TAAs targeted by conventional cancer vaccines, may exist. Theoretically, these limitations could be circumvented by allogeneic HCT, a procedure that culminates in complete host immune replacement. Perhaps more importantly, unlike innate host immunity, an allogeneic immune system would have the capacity to mount immune responses to polymorphic variants of tumor-specific or broadly expressed minor histocompatibility antigens (mHAs) [35–37].
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Solid tumors as a GVT target Animal models exploring graft-versus-solid tumor effects The notion that immune responses following allogeneic HCT can be directed against solid tumors was first tested in animal models in the early 1980s. Using a mouse model, Moscovitch and Slavin showed that the high incidence of spontaneous lymphosarcomas in NZ B/W hybrids could be significantly diminished by the transplantation of allogeneic hematopoietic stem cells from BALB/c mice [38]. Subsequently, this group demonstrated the ability of minor and major histocompatibility antigen-mismatched transplants to protect mice from developing metastatic disease following mouse mammary adenocarcinoma (4T1) tumor challenge [39,40]. These experiments showed that alloimmune responses against mHAs that were expressed on tumors could be generated following hematopoietic stem cell transplantation. Early clinical data suggesting GVT effects against solid tumors in humans Compelling clinical evidence supporting the therapeutic utility of the GVT effect in hematologic malignancies prompted investigators to explore allogeneic HCT as a therapeutic tool against metastatic solid tumors in the late 1990s. The first report suggesting a possible GVT effect against a tumor of epithelial origin noted the incidental regression of a metastatic breast adenocarcinoma lesion following allogeneic HCT for relapsed acute myeloid leukemia [41]. Subsequently, Eibl et al. [42] reported tumor regression coincident with acute GVHD in a patient with chemotherapy-resistant metastatic breast cancer following a high-dose transplant from an HLA-identical sibling. The ability to expand mHAspecific CTLs (obtained from the patient at the time of clinical response) that lysed breast cancer cell lines in vitro lent credence to the argument that the tumor regression was at least in part alloimmune mediated. Investigators at the M.D. Anderson Cancer Center reported their experience in 10 patients with metastatic breast cancer who received an allogeneic HCT from an HLA-matched sibling following high-dose conditioning [43]. One complete response (CR) and five partial responses (PR) were noted. GVT effects were implicated in at least two patients whose tumor regression was temporally associated with the onset of acute GVHD and withdrawal of immunosuppression. Delayed regression of metastatic lesions following high-dose allogeneic HCT in a woman with ovarian cancer offered further evidence that some tumors of epithelial origin were indeed susceptible to GVT effects [44].
Use of reduced-intensity conditioning in allogeneic transplantation for solid tumors By the late 1990s, there was sufficient interest in investigating for GVT effects against solid tumors based on available preclinical and clinical data. The major factor limiting the initiation of pilot trials was the significant morbidity and mortality associated with conventional allogeneic HCT. Dose-intensive conditioning used to provide both tumor cytoreduction and a means to allow donor engraftment contributed in part to the morbidity and mortality associated with HCT. Subsequently, it was recognized that reducing the intensity of the preparative regimen might translate to a reduction in the risk of early, conditioning-related morbidity and mortality. In the late 1990s, transplant regimens using reduced-intensity conditioning (RIC) were designed by a number of investigators, and were evaluated for their engraftment potential and toxicity profile [45–49]. Two major factors impacted on the design and development of these dose-reduced conditioning regimens. First was the recognition that GVT
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effects alone might be sufficient to eradicate some hematologic malignancies in the absence of dose-intensive therapy. Second was the realization that the primary role of the conditioning regimen might be limited to preparing the recipient for engraftment by inducing adequate host immunosuppression. Thus, RIC regimens were designed using agents that would induce adequate immunosuppression to facilitate donor immune engraftment while maintaining a low toxicity profile. Such “low-intensity” regimens were first evaluated in hematologic malignancies known to be responsive to allogeneic HCT. Pilot trials utilizing RIC HCT demonstrated that such regimens were generally well tolerated, having a decreased incidence of transplant-related morbidity and mortality, while achieving sufficient donor immune engraftment to induce sustained remissions of some hematologic malignancies [45–49]. One of the major contributions of RIC was that it allowed for the extension of allogeneic HCT to debilitated and older patients who, because of the high risk of treatment-related mortality (TRM) with a high-dose transplant, would ordinarily have been denied the benefit of a procedure with curative potential. Importantly, investigators anticipated that the overall risk of TRM would be lower with a reducedintensity approach, a notion which emboldened them to explore for GVT effects against solid tumors.
Clinical results of reduced-intensity allogeneic HCT transplantation in solid tumors RCC: the National Institutes of Health experience The current worldwide clinical experience of RIC HCT for solid tumors is limited, and the approach continues to be experimental. Because there was no convincing evidence to support the existence of a GVT effect in solid tumors, and given the potential for considerable morbidity with HCT, early pilot trials were restricted primarily to terminally ill patients with advanced treatment refractory metastatic disease. At present, RCC remains the solid tumor in which allogeneic antitumor responses have been best characterized. The following factors provided an incentive to study the susceptibility of this malignancy to a GVT effect. First, metastatic RCC is a uniformly fatal cancer in which the majority of patients succumb to their disease within a year of diagnosis. Second, despite the recent introduction of targeted agents against the vascular endothelial growth factor (VEGF), therapeutic options are extremely limited, with conventional chemotherapy and radiotherapy being largely ineffective [50]. Third,
RCC is considered an “immunoresponsive” tumor based on its susceptibility to cytokine therapy [22,51,52], reports of occasional spontaneous regression [18,53], and the existence of tumor-infiltrating T lymphocytes in regressing metastatic lesions [23,25]. These considerations led to the development of a clinical protocol at the National Institutes of Health (NIH) that sought to test the safety and efficacy of RIC HCT in patients with cytokine-refractory metastatic RCC [48,54]. The primary considerations in the design of the allogeneic HCT trial for patients with metastatic kidney cancer were the following: 1 devising a conditioning regimen that would ensure maximal host immunosuppression to favor rapid and complete donor immune engraftment with relative sparing of recipient myeloid progenitors; 2 devising a post-transplant immunosuppressive approach that would provide optimal prophylaxis for GVHD without precluding the ability to initiate a donor immune mediated GVT effect; 3 judicious use of DLIs to break donor tolerance to host antigens for the promotion of a GVT effect; 4 appropriate patient selection: choosing patients with cytokinerefractory metastatic disease who would be anticipated to survive at least as long as would be required for a delayed GVT effect to occur (i.e. 4–6 months). Therefore, the presence of evaluable metastatic disease, a good performance status (Eastern Cooperative Oncology Group grade 0–1), adequate organ function, and a life expectancy of at least 3 months were prerequisites for patient enrolment. Cyclophosphamide and fludarabine have profound immunosuppressive effects and are well tolerated when given in combination to treat patients with low-grade lymphoproliferative disorders [55,56]. Accordingly, we devised an RIC HCT approach in which patients received a conditioning regimen consisting of cyclophosphamide (60 mg/kg × 2 days) and fludarabine (25 mg/m2 × 5 days), followed by infusion of an unmanipulated, granulocyte colony-stimulating factor-mobilized peripheral blood hematopoietic cell graft from a six of six or five of six HLA antigen-matched sibling donor (Fig. 65.1). Lineage-specific engraftment of donor myeloid (CD14+/CD15+) and T cells (CD3+) was quantitated from post-transplant peripheral blood lymphocyte samples using a polymerase chain reaction-based analysis of either variable number tandem repeats or short tandem repeats polymorphic between patient and donor (Fig. 65.2). Based primarily on animal data showing a decreased risk of GVHD following RIC HCT, we chose to use single-agent cyclosporine (CSP)
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Fig. 65.1 Cyclophosphamide- and fludarabine-based reduced-intensity conditioning hematopoietic cell transplantation protocol for renal cell carcinoma. CSP, cyclosporine; DLI, donor lymphocyte infusion; G-CSF, granulocyte colony-stimulating factor.
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as GVHD prophylaxis in our initial patient cohort. Much to our surprise, a high probability of grade II–IV acute GVHD (actuarial probability 56%) was observed in the first 25 patients with RCC treated (which was lethal in three cases). Consequently, mycophenolate mofetil (MMF) was added to CSP as GVHD prophylaxis in the second cohort of RCC patients. Gradual withdrawal of CSP/MMF was initiated on day 30 or 60 after transplantation, with the timing and rapidity of immunosuppression tapering being dictated by the rate of tumor progression, degree of donor T-lymphoid chimerism, and presence or absence of GVHD. One or more dose-escalated DLIs were administered to all patients with mixed T-cell chimerism, progressive disease or partial remissions in the absence of acute or chronic GVHD. Since interferon (IFN) has been shown to upregulate MHC expression on RCC tumor cells in vitro (Fig. 65.3), we hypothesized that treating with this cytokine post
Fig. 65.2 Example of polymerase chain reaction-based analysis of two different minisatellites polymorphic between patient and donor. Patient (lane 1) and donor (lane 5) lanes show two patient-specific and two donor-specific bands. Also shown are: control mixtures of donor-into-patient DNA representing 30% (lane 2), 50% (lane 3), and 90% donor chimerism (lane 4); and post-transplant blood sample sorted into lineages using magnetic beads showing 100%, 60%, and 99% donor chimerism in CD2+/CD3− natural killer cell (lane 6), CD14+/15+ myeloid cell (lane 7), and CD3+ T-cell fractions (lane 8), respectively.
transplant might make the tumor a better target for a GVT effect. Therefore, patients without GVHD who failed to respond to DLI were eligible to receive post-transplant cytokine therapy with IFN-α, either alone or in combination with IL-2. The initial experience of RIC HCT in metastatic RCC was published in 2000 [48]. Ten of the first 19 patients treated with this transplant approach had tumor shrinkage, including three who had a CR and seven patients who had a PR. At present, 74 patients have undergone RIC HCT for RCC at the NIH. Of these, 73 patients demonstrated durable engraftment, achieving 100% donor T-cell chimerism by day 100 post transplant. Twenty-nine of 74 (39%) patients have had a disease response, including seven CRs and 22 PRs. Five patients who were deemed to be “nonresponders” had radiographic evidence for a mixed response. Disease regression was temporally associated with acute and chronic GVHD in many patients, was typically delayed in onset and did not occur until CSP was tapered, consistent with an alloimmune-mediated GVT effect. Several patients have proven to have a durable disease response, including the first complete responder, who remains without evidence of metastatic disease now over 9 years post transplant (Fig. 65.4). Regression of disease in multiple metastatic foci has been observed, although pulmonary responses appear to occur most frequently. On occasion, disease responses have been dramatic and have included complete resolution of large pulmonary metastases with bulky adenopathy (Fig. 65.5). Preliminary data would suggest that disease response following RIC HCT is a clinically meaningful phenomenon since regression of metastatic RCC appears to be associated with a trend towards improved survival. Survival in nonresponders has been less than 6 months in contrast to those achieving a PR, who survived a median 2.5 years post transplant. In general, patients with metastatic kidney cancer have tolerated this conditioning well. Although virtually all patients have developed febrile neutropenia, we observed no sinusoidal obstructive syndrome of the liver or chemotherapy-associated mucositis. Twentyeight percent (21 of 74) of patients developed cytomegalovirus antigenemia, with only one case of cytomegalovirus disease (esophagitis) that was responsive to gancyclovir therapy. Mortality associated with transplant-related complications occurred in 8 of 74 (11%) patients, with either infection or acute or chronic GVHD being the major cause of TRM. Since kidney cancer frequently metastasizes to the lungs, some patients with RCC may be at high risk for the development of postobstructive pneumonia following transplantation. Such a complication occurring in the profoundly immunocompromised can lead to disastrous infectious sequelae, as was observed in two of our RCC patients who
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Fig. 65.4 Renal cell carcinoma metastatic to the lungs (pretransplant images A1 and A2). There was no change in metastatic disease 30 days following conditioning (B1 and B2). Following cyclosporine withdrawal, metastasis regressed completely by 110 days post transplant (C1 and C2). The patient remains without evidence of disease over 9 years after transplantation.
Fig. 65.5 Delayed regression of renal cell carcinoma. Pretransplant images showing bulky anterior mediastinal (A1) and hilar (A2) adenopathy. Stable disease seen 6 months post transplant (B1 and B2). Regression of bulky adenopathy was observed 9 months post transplant (C1 and C2).
died from bacterial sepsis related to obstructive bronchial pneumonia. Most deaths related to acute GVHD occurred during the first 2–3 years following inception of the trial; more recently, advances in our ability to manage severe or steroid-refractory GVHD have translated into a greatly reduced risk of mortality from this complication [57]. Acute GVHD has been the greatest contributor to morbidity and mortality in our RCC patients undergoing RIC HCT. Because MMF is a potent inhibitor of inosine monophosphate and has been shown in vitro to block proliferative responses of cytotoxic T cells, we added MMF to CSP as prophylaxis for GVHD in our second cohort of patients with RCC. Unfortunately, an interim analysis of the first 30 patients treated with both drugs revealed no difference in the incidence of grade II–IV or grade III–IV acute GVHD between the CSP alone and the CSP + MMF cohorts (actuarial probability of acute grade II–IV GVHD 56% and 61%, respectively). Based on these observations, we have withdrawn MMF from the transplant regimen and are currently investigating the effect of adding low-dose methotrexate (MTX) to CSP as GVHD prophylaxis. A recent analysis of 230 patients with a variety of malignancies and nonmalignant hematologic disorders undergoing RIC HCT with cyclophosphamide and fludarabine conditioning at our institution revealed that the incidence of acute GVHD grade II–IV and III–IV was
significantly less in those patients who received CSP and MTX as GVHD prophylaxis compared with those who received either CSP alone or CSP plus MMF. Importantly, the addition of MTX to CSP did not appear to abrogate the ability to induce GVT effects against RCC, with four of 18 patients who received this regimen demonstrating an objective response (including one patient with a CR). Assessment of donor chimerism following RIC HCT is important not only to document the establishment of donor engraftment, but also to allow for post-transplant manipulation of the donor immune system to optimize the chances of inducing a GVT effect. In our experience, GVT effects following RIC HCT typically do not occur until the immune system has converted from mixed to predominantly donor T-cell chimerism. As a consequence, detailed serial measurements of donor engraftment in both lymphoid and myeloid lineages are performed on all patients undergoing this approach. Representative lineage-specific engraftment profiles and their relationship to clinical outcome are shown in Fig. 65.6. Although donor T-cell engraftment typically precedes myeloid engraftment in patients receiving cyclophosphamide- and fludarabinebased conditioning, engraftment patterns may vary considerably among individual patients [58]. Several factors influence the degree and rapidity of donor engraftment, including the agents used in the conditioning
Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors Progression GVHD
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Importantly, however, tumor shrinkage has also been observed to occur in the absence of or temporally distant from GVHD, an observation which might imply that tumor cells are particularly sensitive to allogeneic immune attack or that tumor-specific immune effectors might be involved in mediating RCC regression in some patients. Finally, regression of RCC following a DLI or after treatment with low-dose subcutaneous IFN-α has also been observed. Disease regression after DLI suggests that the mediators of the GVT effect may be analogous to those mediating leukemia regression. Interestingly, some patients who had failed to respond to IFN-α before transplantation had disease regression when the drug was given post transplant. This observation suggests that the mechanism by which IFN-α promotes tumor regression in the post-transplant setting relates to the ability of the drug to make the tumor a better target or enhance the allogeneic immune system rather than to a direct antineoplastic effect.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Weeks post transplant
Fig. 65.6 Representative lineage-specific engraftment profile following cyclophosphamide- and fludarabine-based conditioning. The percentage donor chimerism in T cells (䊉) and myeloid cells (䉱) is shown in a patient who had a disease response 17 weeks post transplant. CSP, cyclosporine; GVHD, graft-versus-host disease.
Table 65.1 Tumor response patterns consistent with a graft-versus-tumor effect after reduced-intensity conditioning hematopoietic cell transplantation • • • • •
Delayed onset of response (>100 days post transplant) Response following withdrawal of immunosuppression Response following or concomitant with graft-versus-host disease Response following donor lymphocyte infusions Prolonged duration of response in tumors previously treatment refractory
regimen, the prevailing state of host immunity, and allograft cell doses. In a multivariate analysis of 36 patients with metastatic solid tumors who underwent RIC HCT at the NIH, pretransplant exposure to chemotherapy and increased allograft CD34+ cell dose were found to significantly facilitate the engraftment of donor myeloid and T cells [59]. Therefore, tailoring the intensity of RIC in solid tumor patients based on prior chemotherapy exposure would seem worthy of exploration. While the exact mechanisms underlying the regression of metastatic RCC following allogeneic HCT are yet to be unraveled, several observations (both laboratory and clinical) would suggest an alloimmune effect mediated at least in part by donor T cells is at work (Table 65.1) [48]. The majority of patients who ultimately achieved a disease response showed early tumor growth in the first few months after transplantation, a time period when the newly engrafted donor immune system was kept in check by immunosuppressive therapy or when mixed T-cell chimerism prevailed, leading to “tolerance” of host tissues (including the tumor). Tumor regression was typically delayed (4–8 months) and followed conversion to predominantly donor T-cell chimerism after immunosuppression had been withdrawn or was being tapered. These observations highlight the importance of utilizing a transplant approach that favors rapid donor T-cell engraftment and incorporates judicious and timely withdrawal of GVHD prophylaxis. As had previously been described in patients with hematologic malignancies, a prior history of acute GVHD was associated with an increased probability of having a tumor response. One might speculate that disease regression in this setting could be the consequence of alloreactive T cells targeting mHAs that are broadly expressed on both normal tissues and tumor cells.
Emerging role of RIC HCT for RCC Although allogeneic HCT is clearly an investigational approach for the treatment of metastatic RCC, several other investigators have begun to report promising results in this disease (Table 65.2). Investigators at the University of Chicago initially reported on 15 patients undergoing HCT following a fludarabine- and cyclophosphamide-based conditioning regimen [60]. The first four patients treated were conditioned with an extremely low-intensity regimen consisting of fludarabine (90 mg/m2) and cyclophosphamide (2 g/m2) given at doses that failed to achieve adequate levels of host immunosuppression. As a consequence, three of the four patients (75%) failed to achieve stable donor T-cell engraftment and ultimately rejected their transplant. Subsequent patients received higher doses of the same conditioning agents (fludarabine 150 mg/m2 and cyclophosphamide 4 g/m2), and achieved successful and sustained donor engraftment. Of their 12 patients who engrafted and were evaluable for a disease response, four (33%) had radiographically documented disease regression consistent with a PR, including one patient who had tumor regression in his primary kidney tumor. These data have since been updated to include the first 19 patients transplanted, four of whom achieved a PR. Although all patients have relapsed at a median of 609 days following HCT, all responders were alive with a median follow-up of 41 months (compared with a median overall survival of 14 months for the entire cohort) [61]. As with other trials of RIC HCT for solid tumors, a dominant donor immune system (determined by T-cell chimerism studies) was a prerequisite for the generation of a meaningful GVT effect. Only those patients who had stable and predominantly donor immune engraftment demonstrated a disease response. In fact, one patient with graft rejection (a nonresponder) underwent a second transplant with the intensified conditioning regimen, achieved complete donor engraftment, and subsequently had a PR. Only two of 12 (17%) patients experienced grade II or greater acute GVHD, perhaps the consequence of a more gradual termination of GVHD prophylaxis. Whether delaying the withdrawal of post-transplant immunosuppression will decrease the incidence of acute GVHD without negating beneficial GVT effects will need to be determined from larger studies. A Cancer and Leukemia Group B (CALGB) intergroup trial evaluated the feasibility of performing HCT for metastatic RCC in a multiinstitutional setting in the United States [62]. Twenty-two patients underwent HCT from an HLA-matched sibling donor following cyclophosphamide plus fludarabine-based conditioning. Seventeen of nineteen evaluable patients achieved greater than 90% donor chimerism by day 120 after transplantation. Acute (grade II–IV) GVHD was seen in 50% of patients, while 23% of patients experienced chronic GVHD. There were no objective responses seen, and median overall survival
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Table 65.2 Summary of clinical trials of reduced-intensity conditioning hematopoietic cell transplantation in metastatic renal cell carcinoma
Investigators
Conditioning agents
GVHD prophylaxis
Childs et al. [48] and unpublished data
CY + FLU
Rini et al., Artz et al. [60,61] Bregni et al. [67] Pedrazzoli et al. [87] Blaise et al. [88] Nakagawa et al. [89] Ueno et al. [90] Hentschke et al. [91] Massenkeil et al. [92] Tykodi et al. [93] Barkholt et al. [63] Rini et al. [62] Peres et al. [94]
CY + FLU CY + FLU + thiotepa CY + FLU FLU + BU + ATG FLU /CLA + BU + ATG FLU + MEL FLU + TBI ± ATG FLU + CY + ATG FLU + TBI Multiple FLU-based regimens FLU + Cy FLU + CY or FLU + TBI
CSP (first 25 patients) CSP + MMF (subsequent patients) Tacro + MMF CSP + MTX CSP + MTX CSP CSP Tacro + MTX CSP + MMF CSP ± MMF CSP + MMF CSP ± MMF or MTX Tacro + MTX CSP + MMF
Acute GVHD (II–IV)
Chronic GVHD
Treatment-related mortality
Response (partial or complete)
53%
21%
11%
53%
22% 86% 0% 42% 44% 47% 50% 29% 50% 40% 32% 44%
39% 71% N/A 60% 44% 27% 30% 57% 50% 33% 23% 38%
14% 0% 29% 9% 0% 33% 40% 14% 13% 16% 9% 12%
22% 57% 0% 8% 11% 27% 0% 29% 13% 32% 0% 32%
ATG, antithymocyte globulin; BU, busulfan; CLA, cladribine; CSP, cyclosporine; CY, cyclophosphamide; FLU, fludarabine; GVHD, graft-versus-host disease; MEL, melphalan; MMF, mycophenolate mofetil; MTX, methotrexate; N/A, not available; Tacro, tacrolimus; TBI, total body irradiation.
was only 5.5 months, with most patients dying from disease progression (with a median time to progression of 3 months). Inclusion of a number of patients with multiple adverse prognostic factors, sparing use of DLI (only two of 22 patients receiving DLIs despite disease progression in the majority), and inclusion of patients with nonclear cell histology are some of the factors that may account for the poor outcome observed in this trial. This trial clearly highlights the importance of appropriate patient selection and the need for identifying prognostic factors likely to predict for a favorable outcome. The European Group for Blood and Marrow Transplantation (EBMT) reported on their experience in 124 patients with metastatic RCC undergoing HCT at multiple centers from an HLA-identical (n = 106) or partially matched (n = 5) family donor, or from an HLA-matched unrelated donor (n = 13) (Table 65.2) [63]. A variety of fludarabine-based RIC regimens were utilized. CSP alone or in combination with MMF or MTX was used for GVHD prophylaxis in the majority of patients. Durable engraftment was achieved in 121 patients. The incidences of acute grade II–IV and chronic GVHD were 40% and 33%, respectively. Twenty-eight of ninety-eight evaluable patients had an objective tumor response including four patients with a CR. Responses were typically delayed, with PRs and CRs occurring a median of 135 days and 265 days, respectively. A short time interval from diagnosis of RCC to HCT, the presence of acute GVHD, and the use of an HLA-mismatched donor were each associated with a higher probability of response. Transplantrelated mortality was 16% at 1 year, while overall survival at 2 years was 30%. In a multivariate analysis, chronic GVHD, good performance status (Karnofsky score ≥80), DLI administration, and fewer than three sites of metastatic disease were identified as factors favorably impacting survival.
Toxicities and limitations of RIC HCT in metastatic RCC Although there is ample evidence now to support the susceptibility of metastatic RCC to an allogeneic GVT effect, there are a number of potentially life-threatening toxicities of RIC HCT that currently limit the
Table 65.3 Limitations of reduced-intensity conditioning hematopoietic cell transplantation in solid tumors Incidence (%) HLA-matched sibling donor available Acute GVHD Chronic GVHD Cytomegalovirus reactivation Graft rejection Treatment-related mortality
25–30% 30–60% 40–70% 20–40% 5–10% 10–20%
GVHD, graft-versus-host disease; HLA, human leukocyte antigen.
broader application of this approach (Table 65.3). While the incidence of early TRM appears to be less with RIC, overall 5–20% of patients will ultimately die from a transplant-related cause. Infection and complications related to acute GVHD are the greatest risks associated with the procedure. In fact, three of the first 25 patients with RCC transplanted at the NIH died from GVHD-related causes [48]. Unfortunately, the addition of MMF to CSP as GVHD prophylaxis did not reduce the incidence of grade II–IV or grade III–IV acute GVHD. However, new second-line therapies for severe steroid refractory GVHD such as daclizumab (monoclonal antibody directed against the alpha chain of the IL-2 receptor) and infliximab (monoclonal antibody directed against the tumor necrosis factor-alpha receptor) have shown early promise [64,65]. Our preliminary experience suggests that these monoclonal antibodies are effective in reducing mortality from steroidrefractory GVHD, particularly when given with lipid-complexed amphotericin B or voriconazole as Aspergillus prophylaxis, concomitant with a rapid reduction in corticosteroid dose once steroid-resistance is apparent [57]. Larger studies will be required to determine whether these agents will ultimately result in a reduction in the risk of GVHD-related mortality without compromising the ability to generate a GVT effect.
Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors Table 65.4 Patient characteristics likely to predict a favorable outcome after reduced-intensity conditioning hematopoietic cell transplantation for solid tumors • • • • • •
Good performance status (Eastern Cooperative Oncology Group grade 0–1) Younger patient age (i.e. 8 cm in maximal diameter or >100 mL in volume historically carried a poor prognosis for survival. Intensification of therapy by the addition of ifosfamide and etoposide improved event-free survival (EFS) from 54% to 69% for patients with localized disease, and eliminated pelvic primary location and large tumor size as prognostic factors [26]. Unfortunately, their use did not improve the EFS of patients with metastatic disease, which was 22% regardless of treatment regimen. The recently completed Children’s Oncology Group study tested increasing dose intensity by randomizing patients to receive this same treatment regimen with cycles given at 2-week intervals versus the standard 3-week intervals. Four-year EFS was 65% for patients with localized disease treated at 3-week intervals, but 76% for patients treated with the time-compressed regimen (a statistically significant difference; Womer, personal communication), lending further support to the importance of dose and time intensity for the cure of pediatric solid tumors. In addition to systemic chemotherapy, adequate local control is essential to eradicate the disease. Local control measures include radiation therapy and surgery. The relative efficacy of radiotherapy compared with surgery remains controversial. The most recent studies, utilizing dose-intensive chemotherapy, show little difference in survival between patients treated with surgery and patients treated with radiation [27], with respect to both local recurrence and overall survival (OS). In a single-institution study of 76 patients with localized disease, the local failure rate was identical in the patients treated with radiation, surgery or both [28]. Interestingly, in a multivariate analysis, only the use of chemotherapy was a prognostic factor for local control (the 10-year local control rate for patients treated with chemotherapy being 83.7%, compared with 51.1% for patients who did not receive chemotherapy), supporting the concept that systemic treatment contributes to local control.
Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors
Hematopoietic cell transplantation It is clear that patients with high-risk ESFT, including patients with metastatic or recurrent disease, have a very poor prognosis. Doseintensive chemotherapy has not improved the outcome for patients with metastatic disease, and patients with recurrent disease are not cured with standard chemotherapy. For these patient populations, HCT has been investigated as a way to improve outcomes. One of the earliest and largest reports of high-dose therapy investigated the use of total body irradiation (TBI) with autologous peripheral blood progenitor cell (PBPC) support as consolidation therapy for high-risk ESFT patients. Over a 5-year period, 91 patients were treated on three successive chemotherapy protocols at the National Institutes of Health, and remissions were consolidated in 65 of the patients with 8 Gy of TBI [29]. Nineteen patients were not given TBI because they failed to achieve remission or relapsed prior to treatment, and seven refused. Twenty of the 65 treated patients (31%) became long-term survivors, a higher rate than expected with chemotherapy alone in this group of patients, and superior to a group of contemporary patients treated without high-dose therapy. However, only patients who did not progress after chemotherapy were eligible for HCT, so OS for the total patient population was only 22%, no better than results with chemotherapy alone. These results do suggest, however, that a select group of patients might benefit from HCT. At the same time, the groups in Vienna and Dusseldorf reported very impressive results using a combination of TBI, melphalan, and etoposide for poor-risk patients with ESFT. These patients had either multifocal disease (seven of 17 patients) or early or multiple relapses (10 of 17 patients). The dose of TBI was 12 Gy – higher than utilized by the National Institutes of Health. Relapse-free survival for these patients was 45% at 6 years [30]. Unfortunately, these results did not hold up with additional enrollment and follow-up. Updated results, analyzing 36 patients with up to 139 months of follow-up, showed an EFS of only 24% [31]. Similar results were reported from the UK, where a group of 18 patients with poor-risk ESFT (defined as metastatic at diagnosis, very large primary tumors or a patient in second complete remission) were treated with high-dose busulfan and melphalan and HCT. OS in this group was good (13 of 18 patients, EFS of 72%, with a range of followup from 2 months to 7 years), but OS of the patients with metastatic disease was only 25% [32]. More promising results have been reported from a review of European Bone Marrow Transplant Registry (EBMTR) data. While the DFS rate for patients with ESFT metastatic to bone or bone marrow who underwent HCT between 1982 and 1992 was only 21% [33], results were superior for the subgroup of patients treated with the combination of busulfan and melphalan, and for the patients who did not receive TBI. An updated review of these data reported 5-year OS of 44% for the patients treated with a busulfan-containing regimen, compared with 23% for patients treated without this drug [34]. These results have been confirmed in two smaller studies. The group at the University of Washington reported their results utilizing triple alkylator therapy (busulfan, melphalan, and thiotepa) followed by total marrow irradiation with HCT after each of these treatments for 16 patients with poor-risk ESFT. Six of their patients (36%) survived for 27–66 months [35]. Davies et al. have reported 62% DFS at 3 years for a similar group of 11 patients also treated with the same triple alkylator therapy (but no irradiation) [36]. Further support for a possible benefit from alkylator-intensive therapy comes from two other European groups. The group in London recently reported results of HCT for 33 patients with recurrent or progressive ESFT between 1992 and 2004 [37]. The 22 patients treated with busulfan and melphalan had OS of approximately 50%, with follow-up as long as 12 years. Similarly, the Société Française des Cancers de l’Enfant
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treated 97 newly diagnosed patients with metastatic ESFT using doseintensive chemotherapy and consolidation with busulfan and melphalan for patients with a complete or very good partial remission. Five-year EFS for all 97 patients was 37%, and EFS after HCT was 47% (52% for patients with metastases limited to the lungs) [38]. Although none of these reports presents results of a randomized trial, the data suggest a survival advantage for patients treated with regimens incorporating high doses of alkylating agents, especially busulfan. A recent publication from the University of Washington looked specifically at patients with relapsed ESFT to determine the benefit of high-dose therapy and HCT for this particular subset of patients. Patients were treated with a variety of salvage induction chemotherapy regimens, and patients who responded to this therapy were offered high-dose therapy with busulfan, melphalan, and thiotepa with HCT support, some with total marrow irradiation as discussed above. While the 5-year OS for this group of patients was poor (23%), the subgroup of patients with metastases limited to the lungs who had chemosensitive disease and were treated with HCT had 62% OS (eight of 13 patients). In fact, among patients who responded to their retrieval therapy, progression-free survival and OS were superior with HCT compared with chemotherapy alone (PFS 61% versus 21%, and OS 77% versus 21%) (Fig. 67.1). Thus, for a subset of patients with chemosensitive recurrent disease, HCT appears to offer a survival advantage compared with chemotherapy alone [39]. All of the studies summarized above share the same weakness – none is prospective and randomized. This limits the confidence with which HCT can be recommended as “standard of care” for ESFT patients. An ongoing study, Euro-Ewing 99, was designed to address this deficiency. In this study, patients are assigned to one of three risk groups, based on the presence or absence of metastases, the size of the primary tumor, and the response to neoadjuvant therapy. Patients in the lowest-risk group are treated with 14 cycles of chemotherapy. Patients judged to have intermediate risk disease (PR rather than CR to neoadjuvant therapy, a tumor >200 mL or pulmonary metastases) are randomized to consolidation with chemotherapy versus high-dose therapy with busulfan and melphalan, and patients with the highest risk (extrapulmonary
Fig. 67.1 Survival from time of first recurrence for patients with a recurrent Ewing’s sarcoma family tumor who responded to salvage induction chemotherapy, by use of high-dose therapy (HDT). (Reproduced from [39], with permission.)
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metastases) are randomized between two different preparative regimens for their HCT. This study is expected to resolve some of the controversies surrounding the utility of HCT for patients with ESFT. Rhabdomyosarcoma Epidemiology and etiology Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood, accounting for 5–8% of childhood cancer [10]. About 250 new cases of rhabdomyosarcoma are diagnosed in the United States annually, slightly more frequently in boys than in girls. Little is known about the etiology of rhabdomyosarcoma. These tumors sometimes arise as second malignant neoplasms in patients previously treated with ionizing radiation, but radiation exposure accounts for a very small minority of cases. Molecular and cellular biology There are two major histologic variants of rhabdomyosarcoma: embryonal and alveolar. Embryonal rhabdomyosarcoma, named for its resemblance to immature skeletal muscle, has been subdivided into solid and botryoid variants. Embryonal histology accounts for 57% of cases of rhabdomyosarcoma and usually affects children between 3 and 12 years of age. Alveolar histology, named for its resemblance to normal lung parenchyma, accounts for 19% of cases of rhabdomyosarcoma, and usually strikes patients between 6 and 21 years old [10]. Embryonal rhabdomyosarcoma carries a more favorable prognosis than alveolar. As in ESFT, recurrent translocations have been identified in rhabdomyosarcoma, although only in the alveolar subtype. In 55% of cases, a translocation between chromosomes 2 and 13, t(2;13)(q35;q14), is seen, and in 22% of cases a similar translocation involving chromosome 1, t(1;13)(p36;q14), is identified [40]. These translocations involve related transcription factor genes, paired box 3 (PAX3) and 7 (PAX7), respectively. In each case, the DNA-binding domain of the PAX gene is fused to the transactivation domain of the forkhead box (FKHR) gene. Disruption of PAX genes leads to abnormal muscle development [41], suggesting a direct etiologic relationship between the translocation and the development of malignancy. Moreover, ectopic expression of these chimeric genes can transform cells. The PAX3-FKHR translocation appears to carry a poorer prognosis than PAX7-FKHR [42]. Clinical description Signs and symptoms present at diagnosis depend on the site of the primary tumor, the involvement of surrounding normal organs, and the presence of distant metastases. Typically, patients present with an asymptomatic mass, although involvement of cortical bone may cause pain, and genitourinary disease may cause hematuria or urinary retention. Different subtypes of rhabdomyosarcoma arise in different sites. Embryonal rhabdomyosarcoma typically arises in the head and neck region or the genitourinary tract, while alveolar rhabdomyosarcoma tends to arise in the extremities and the trunk [10]. Head and neck sites, including orbit and parameningeal primaries, account for 40% of cases; trunk and extremity sites account for 25–30% of cases; and genitourinary sites account for 20% [40]. The most common sites of metastasis are the lungs, lymph nodes, cortical bone, and bone marrow. Nontransplant approaches Rhabdomyosarcoma is treated with multimodality therapy, including chemotherapy, radiation therapy, and surgery. Surgery alone is insufficient to cure even localized disease. In the era before the development of effective systemic chemotherapy, the OS for patients with localized disease was 25% or less [43]. With current multimodality therapy,
Table 67.1 Clinical group definitions for rhabdomyosarcoma patients Group
Definition
Group I Group IIa Group IIb
Localized disease completely resected Gross total resection with microscopic residual disease Regionally involved lymph nodes, completely resected with the primary Regional disease with involved nodes, totally resected with either microscopic residual disease or histologic evidence of involvement of the most distant lymph node in the dissection Incomplete resection Distant metastases
Group IIc
Group III Group IV
failure-free survival rates for patients with localized disease are as high as 90% [44]. The most important predictor of treatment failure is the clinical group identified in early Intergroup Rhabdomyosarcoma Study Group studies (Table 67.1). In addition to classification by clinical group, rhabdomyosarcoma is also staged using a specific tumor–node–metastasis staging system [44] that not only takes into account tumor size, lymph node status, and distant metastases, but also incorporates information regarding the site of the primary tumor, reflecting prognostic differences between favorable and unfavorable anatomic locations. Patients are then classified as high, intermediate or low risk based on clinical group and stage, and treatment is based on this assessment of risk. The principles of nontransplant therapy for rhabdomyosarcoma, as established by the Intergroup Rhabdomyosarcoma Study Group clinical trials, include local control (consisting of surgery, radiation therapy or both) and systemic therapy to address either gross or microscopic metastatic disease. Primary surgical resection is indicated, unless the tumor is deemed unresectable. In this case, radiation therapy is employed and can provide definitive local control for patients with unresectable rhabdomyosarcoma [45]. The specifics of adjuvant systemic chemotherapy vary depending on risk stratification. In general, the chemotherapy used for low-risk patients is significantly less intensive than the therapy for intermediate- or high-risk patients. Unlike osteosarcoma or ESFT, patients with rhabdomyosarcoma undergo surgical resection prior to the institution of systemic therapy, as reflected in the staging system, which takes into account the extent of resection. Upon recovery from surgery, patients are treated with chemotherapy with or without radiation, which is usually delivered relatively early in the treatment regimen. Hematopoietic cell transplantation The role of high-dose therapy and HCT for high-risk rhabdomyosarcoma is not established. Several retrospective analyses have been reported. Between 1982 and 1994, the EBMTR received reports of 98 transplants performed in children and young adults with relapse or progression of initially localized rhabdomyosarcoma. The proportion surviving diseasefree is approximately 20%, and median survival remains short, at only 8.3 months from HCT [46]. The German–Austrian Pediatric BMT Group in 1997 published the results of 36 transplants performed for metastatic or recurrent rhabdomyosarcoma between 1986 and 1994. Thirty-four of the 36 patients received high-dose melphalan-based regimens, usually augmented with etoposide and carboplatin. The estimated EFS at 2 years after HCT was 36% (standard error [SE] 7%) [47]. This rate is better than expected, but the small number of patients and possible effects of selection bias preclude conclusions about the efficacy of HCT for rhabdomyosarcoma.
Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors
The European Collaborative MMT4–91 trial was the first prospective trial to evaluate the efficacy of HCT as consolidation therapy in first remission for patients who presented with metastatic rhabdomyosarcoma. This study resulted from the amendment of MMT4–89 for patients with newly diagnosed metastatic rhabdomyosarcoma, so that institutions could choose to substitute HCT with high-dose melphalan for the fourth and final 9-week cycle of chemotherapy in patients who had achieved CR before the third cycle of therapy. Thus, there was a nearly contemporaneous control population of patients either enrolled on MMT4–89 or enrolled on MMT4–91 at centers not participating in HCT. Fifty-two patients in CR after six courses of chemotherapy were treated with highdose melphalan, either alone or in combination with other agents such as carboplatin, etoposide, thiotepa, and/or busulfan. Outcomes were compared with those of 44 patients who were also in CR after six identical cycles of chemotherapy but went on to receive further conventional dose chemotherapy rather than HCT. Although patients were not randomized to HCT or conventional therapy, decisions to administer HCT consolidation were made at the center level, reducing the potential for selection bias. Unfortunately, the patient population treated with HCT (n = 52) in fact had poorer prognostic factors: lymph node involvement was more common (56% versus 34%), alveolar histology was more common (44% versus 30%), fewer patients were less than 10 years old (60% versus 68%), and more patients had large tumors over 5 cm in diameter (73% versus 61%) than in the control population (n = 44). Despite these differences and the small sample size, the median time from the end of therapy to relapse was significantly longer for the HCT group than the conventional chemotherapy group (168 versus 104 days; p = 0.05), but there was no difference in EFS or OS [48]. Single-institution protocols for high-risk rhabdomyosarcoma that incorporate HCT consolidation show similar results, with DFS for stage IV patients ranging from 14% to 28% at 3 years or more [49–52].
Wilms’ tumor Epidemiology and etiology Wilms’ tumor is the most common renal malignancy of childhood, with an annual incidence in the United States of 8.1 cases per million children under age 15 years, with a slight female predominance [53]. Children with bilateral disease have an earlier age of onset, 23.5 months for boys and 30.5 months for girls, than those with unilateral disease, 36.5 months for boys and 42.5 months for girls [54]. This age difference was a key observation in the development of Knudson’s classic two-hit model of tumorigenesis [55]. The additional variation in age of onset by gender and ethnicity suggests heterogeneity in the pathogenesis of Wilms’ tumor [56]. Tumors associated with intralobular nephrogenic rests have an earlier onset [57], while those with perilobular nephrogenic rests are associated with a higher birth weight and a later age of onset [58]. Molecular and cellular biology Molecular correlates of these epidemiologic observations have recently been identified. In most normal tissue, imprinting of the insulin-like growth factor-2 (IGF2) gene silences the maternal allele, so only the paternal allele is expressed. In a study of 36 informative Wilms’ tumor specimens, loss of imprinting that led to expression of the normally silent maternal allele was strongly associated with perilobular nephogenic rest histology and a 2.2-fold increased expression of IGF2 [59]. Conversely, mutation of the Wilms’ tumor 1 (WT1) suppressor gene is associated with earlier age at onset, stroma-predominant histology, intralobular nephrogenic rests, and poorer response to therapy [60].
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Clinical description Children with Wilms’ tumor typically present with a painless abdominal mass or abdominal distention. Other findings include gross hematuria and fever. Hemorrhage into the tumor can result in a rapidly expanding abdominal mass and anemia. Compression of renal vasculature can produce hypertension. Radiographic findings can usually distinguish Wilms’ tumor as an intrinsic renal mass from neuroblastoma arising in the adrenal gland and displacing the kidney. Patterns of spread include local extension beyond the tumor pseudocapsule into the renal sinus, the renal vasculature, and lymphatics. The tumor can be locally aggressive, violating the renal capsule, and can disseminate through lymphatics. Hematogenous dissemination is primarily to the lungs, and less commonly to the liver. Nontransplant approaches Wilms’ tumor is the exceptional childhood solid tumor that is often curable by conventional chemotherapy when metastatic at diagnosis, and even after relapse. However, high-risk groups can be defined. The National Wilms Tumor Studies, NWTS-2 and NWTS-3, accrued 2757 untreated patients age 15 years or younger, with stage I–IV disease. The prognosis of the 367 patients (14%) who relapsed after achieving an initial CR was analyzed in 1989. Histology was an important predictor of outcome, with unfavorable histology defined by the presence of diffuse anaplasia (Fig. 67.2) [61]. In NWTS-3, 3-year survival after relapse was 42% with favorable histology versus 16% with unfavorable histology. Similar numbers of patients relapsed at less than 6 months, 6–11 months, and over 12 months from diagnosis. Their respective 3year survival was 18%, 30%, and 41%. Among patients with favorable histology, the 3-year survival by initial stage was 57% for stage I, 36% for stage II/III, and 17% for stage IV; for patients with unfavorable histology, 3-year survival by stage was 17% for stage I, 14% for stage II/III, and 7% for stage IV. Among stage II/III patients with favorable histology, those randomized to receive three-drug initial therapy had 16% 3-year survival from relapse, compared with 42% among those initially treated with two-drug regimens. Intra-abdominal relapse was unfavorable when this field had been previously irradiated, precluding further radiation therapy to the site of relapse [62]. An analysis of the long-term survival outcome of patients relapsing on the United Kingdom Children’s Cancer Study Group Wilms Tumor 1 trial, which accrued 381 patients from 1980 to 1986, confirmed the negative prognostic importance of unfavorable histology, high initial stage, and early relapse, given salvage with ifosfamide or cisplatin and etoposide [63,64]. Taken together, these results have led to the accepted definition of a high-risk subgroup of relapsed Wilms’ tumor patients whose expected 3-year survival is under 20%, including those with any of the following features: unfavorable histology, relapse within 6 months of diagnosis, failure of a three-drug regimen, and involvement of sites other than lung and abdomen, or involving the abdomen after irradiation. However, NWTS-5 treated patients who relapsed after vincristine and dactinomycin therapy without prior irradiation on a uniform regimen consisting of vincristine, doxorubicin, CY, and etoposide, combined with radiation to involved organs. Of 72 patients enrolled, 68 were evaluable, and the 58 patients age 18 and younger with unilateral disease at initial diagnosis were analyzed. Only 10 of the patients had no adverse prognostic features, 32 had one, and 16 had two. Nevertheless, 4-year EFS was 71% and 4-year OS 82% [65], showing that aggressive conventional therapy can overcome adverse prognostic factors in minimally pretreated patients. In contrast, results of the NWTS-5 study for patients who relapsed after initial therapy with vincristine, dactinomycin, and doxorubicin were less favorable. One hundred three patients were enrolled in this stratum, and 91 were evaluable. The 60 patients with initially unilateral
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Fig. 67.2 Top panel: Patterns of anaplasia within Wilms’ tumor that constitute diffuse anaplasia. Bottom panel: Anaplastic focus on the right, sharply demarcated from adjacent nonanaplastic Wilms’ tumor on the left. (Reproduced from [61], with permission.)
disease who relapsed after achieving a CR were analyzed. For these 60 patients, 4-year EFS was 42% and 4-year OS was 48%. Twenty-five patients had only one adverse prognostic factor, 22 had two or three adverse prognostic factors, and 13 had four or more adverse prognostic factors. More adverse prognostic factors, male gender, and sites of relapse other than the lungs conferred significantly poorer EFS [66]. In summary, there is room for improvement over the results of nontransplant chemotherapy in patients with high-risk relapses following threedrug initial therapy. HCT for high-risk relapse of Wilms’ tumor Limited published information is available regarding high-dose therapy for patients with high-risk relapse of Wilms’ tumor. The experience collected by the European Group for Blood and Marrow Transplant (EBMT) has been reviewed [67]. Twenty-five patients with Wilms’ tumor received high-dose therapy over a 7-year period from 1984 to 1991. Twenty-one of these children had had one to four relapses, and four patients had
stage IV disease refractory to first-line therapy. Of 11 patients transplanted in second CR (CR2), 10 had one or more high risk features as defined above. Twenty of the 25 patients received high-dose melphalanbased regimens, although seven different regimens were used. Eight of 17 children transplanted in CR became long-term disease-free survivors. Of patients with measurable disease, five of eight achieved CR and one of eight achieved PR to high-dose therapy, demonstrating the activity of these regimens in refractory Wilms’ tumor. However, only one of eight children with measurable disease at the time of HCT became a long-term survivor [67]. Small case series have suggested that patients with multifocal, generally multiply recurrent Wilms’ tumor may benefit from high-dose thiotepa-based preparatory regimens. In addition, a single institution treated 13 patients with recurrent Wilms’ tumor with HCT from 1991 to 2001. Twelve of 13 had at least one adverse prognostic factor. Four different preparative regimens were used, and four of the patients received tandem HCT. There was no transplant-related mortality, and 4-year EFS was 60% [68]. These results are sufficiently encouraging to warrant prospective evaluation, first in phase II trials to identify active regimens, and then in a randomized comparison with chemotherapy for patients with highrisk disease. The French Society of Pediatric Oncology (Société Française de l’Oncologie Pédiatrique [SFOP]) has completed the first prospective trial of HCT for high-risk relapsed Wilms’ tumor [69]. From 1988 to 1994, 31 patients underwent HCT, including 29 relapsed (16 in CR2, four in PR2, three in CR3, five in PR3, and one in CR5) and two patients with stage IV anaplastic Wilms’ tumor transplanted in CR1. All patients had at least one high-risk feature, and were heavily pretreated with five or six chemotherapy drugs before HCT. The preparatory regimen consisted of melphalan 180 mg/m2, etoposide 1000 mg/m2, and carboplatin, dosed to achieve an area under the curve of 20 mg–min/mL over 5 days. Radiation therapy was delivered to sites of bulky metastatic disease after recovery from HCT. Seven patients sustained renal tubular damage, one developed sinusoidal obstruction syndrome of the liver but recovered fully, and three developed interstitial pneumonitis. Only one of nine evaluable patients failed to achieve CR after HCT. Sixteen patients relapsed at a median of 8.5 (range 3–53) months after HCT, and 12 patients remained in continuous CR a median of 48.5 (range 36–96) months after HCT. DFS was 50% (SE 17%) and OS 60% (SE 18%) at 3 years [69]. The only statistically significant prognostic factor was the number of disease progressions before HCT: in second response, DFS was 63.1 +/− 20%, compared with 22.2 +/− 24% in third response or beyond. The results are better than historical results for high-risk patients with such advanced disease. However, over the same period, 15 patients in the participating centers with at least one adverse prognostic factor failed to undergo consolidation because of uncontrolled progressive disease, and they would need to be included in an intent-to-treat analysis to estimate survival from relapse in an unselected population. The German Cooperative Wilms Tumor Studies confirmed these results, treating 23 patients with HCT from 1992 to 1998 because of defined high-risk criteria including relapse, progression or incomplete response of initial stage IV disease (12), second or subsequent relapse (nine), relapse within a radiation field (four), bone or brain metastases (three), relapse at less than 6 months after nephrectomy (three), or relapse of a tumor with unfavorable histology (one). The preparative regimen was identical to that of the SFOP in 19 patients. One patient developed renal failure requiring dialysis for 1 month, and six developed tubular dysfunction. For the entire cohort, EFS was 48% and OS 61%, but disease status at the time of HCT strongly predicted outcome: 11 of 13 patients in CR but only three of 10 patients in PR survived at a median follow-up of 58 months [70].
Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors
The recently completed NWTS V Pediatric salvage protocol was initially designed to incorporate HCT for patients who failed to achieve a CR after two courses of salvage induction chemotherapy. However, the study was amended to eliminate HCT. Although the results of this salvage protocol are not yet mature, the St. Jude institutional experience and the results of the Children’s Cancer Group pilot salvage protocols suggest that new chemotherapy approaches have improved the outcome of patients with high-risk relapse even without HCT [71,72], and it would therefore be appropriate to launch an international trial to assess the value of HCT compared with conventional-dose chemotherapy in high-risk recurrent Wilms’ tumor, with the greatest benefit expected in patients in second response (Fig. 67.3). HCT as consolidation for Wilms’ tumor Because of the excellent curability of Wilms’ tumor with conventional chemotherapy, few patients have been treated with HCT for Wilms’ tumor in first response, all those who have having stage IV disease. All
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five patients reported by EBMT, including two with unfavorable histology, survive disease free at a median of 62 (range 14–67) months after HCT [67]; both patients with stage IV anaplastic Wilms’ tumor transplanted by the SFOP died of progressive disease [69], while the one similar patient treated by the German Cooperative Wilms Tumor Studies survived [70].
Osteosarcoma Epidemiology and etiology Osteosarcoma is the most common primary bone tumor of childhood and adolescence, and usually involves the long bones [10]. The incidence of osteosarcoma is three new cases per million per year, or 5.6 cases per million Caucasian children younger than 15 years in the United States [73]. The peak age of onset is during the second decade of life, during the adolescent growth spurt [74], somewhat earlier in girls than in boys. Boys are affected more frequently than girls, a finding speculated to reflect the larger bone volume in boys compared with girls [75]. A second incidence peak after the age of 50 years [76] may reflect tumors induced by environmental exposures. Because of its association with the adolescent growth spurt, the relationship between height and the risk of developing osteosarcoma has been investigated. Several contradictory reports on this subject have been published, most having significant methodological flaws. A recent population-based study from the United Kingdom analyzed 364 patients with osteosarcoma and confirmed that patients with osteosarcoma were significantly taller than the general population [77]. Interestingly, although these findings were later confirmed in an even larger study from Italy, the latter group found that this difference was limited to patients diagnosed while still growing [78]. They found that in patients diagnosed while still growing, height at the time of diagnosis exceeded age-matched controls, but that patients diagnosed after completing their growth were the same height as their peers. These findings support the hypothesis that growth of long bones is etiologically involved in the development of osteosarcoma, but the precise relationships remain unclear. The only proven exogenous risk factor for the development of osteosarcoma is exposure to ionizing radiation [79]. Unlike ESFT, however, osteosarcoma has been associated with several inherited syndromes, including bilateral retinoblastoma, Li–Fraumeni syndrome, Bloom’s syndrome, Rothmund–Thomson syndrome, Werner’s syndrome, and multiple exostoses. Osteosarcoma is also associated with Paget’s disease. These findings have led to the identification of numerous genetic changes involved in the development of this tumor. Thus, a good deal is known about the molecular biology of osteosarcoma. Molecular and cellular biology
Fig. 67.3 Metastatic recurrent Wilms’ tumor. Computed tomography scans showing first chemoresponsive relapse subsequently consolidated with autologous HCT.
The recognition that patients with bilateral retinoblastoma are at increased risk of developing osteosarcoma implicates the RB1 gene in the pathogenesis of osteosarcoma. As expected from this observation, loss of RB1 at chromosomal locus 13q14 is a frequent finding in sporadic osteosarcoma [80]. Osteosarcoma is one of the defining tumors of Li– Fraumeni syndrome, and this has led to investigation of mutations in p53 in spontaneous osteosarcoma. Mutations in p53 occur in 40–60% of cases [81]. Both of these genes, TP53 and RB1, are implicated in cell-cycle regulation and proliferation. Numerous other cell-cycle regulatory genes have also been implicated in this disease, including MYC, CDKNA2, CDKN2A, CDKN2B, and MDM2 [81–83]. Chromosomal instability appears to play a key role in the molecular pathogenesis of osteosarcoma. No recurrent chromosomal translocation has been identified, but numerous loci with amplification, point mutation or loss of heterozygosity have been reported (reviewed in [84]). This is
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consistent with the observation that osteosarcomas are diagnosed with increased frequency in patients with Bloom’s syndrome, Rothmund– Thomson syndrome, and Werner’s syndrome, disorders caused by germline mutations in RecQ helicases, which are involved in maintaining genomic stability [85]. In addition to these general regulatory genes, a loss of function of genes implicated in bone differentiation has also been seen in osteosarcoma. Deregulated bone differentiation is commonly seen in osteosarcoma [86]. Runt-related transcription factor-2 is a primary regulator of bone development [87] and is upstream of other regulatory genes, including OSX [88] and TWIST [89]. The TWIST locus, 7p21, was found to be rearranged in 31 cases of osteosarcoma, deleted in 22, and amplified in nine out of a total of 68 cases examined [90]. Additionally, a high-expression ezrin has been correlated with a poor outcome in osteosarcoma patients [91], ezrin being a protein implicated predominantly in metastatic disease [92]. Further investigation into the molecular biology of osteogenesis will undoubtedly uncover further genes that contribute to the development of osteosarcoma. Clinical description The most common presenting complaint of osteosarcoma is pain [10]. Swelling and pathologic fracture are often also seen. Radiographic findings typically include a mixed osteolytic/sclerotic lesion with disorganized soft tissue calcification. At diagnosis, the disease is localized in 80% of cases, with metastases, most commonly to lung, seen in the other 20%. Bone is the other common site of metastatic disease [93], with metastases rarely seen in lymph nodes. Osteosarcoma arises most frequently in the limbs (80% of cases), the femur being the most common bone [94]. It occurs predominantly in the metaphysis of long bones, arising from the medullary cavity and invading into the epiphysis, even in the presence of a growth plate. Nontransplant approaches The mainstays of therapy for osteosarcoma remain surgery and chemotherapy. Until the 1970s, surgery was the only available therapy, but complete resection cured only 20% of patients. Three out of four patients died within 2 years of diagnosis, almost exclusively due to pulmonary metastases. The introduction of adjuvant chemotherapy has increased OS to almost 70% for patients with localized disease [95], although the cure rate for patients with metastatic disease remains unacceptably low. The first combination chemotherapy regimens consisted of methotrexate, doxorubicin, and cisplatin, and this combination remains the standard 30 years later. Neoadjuvant chemotherapy was introduced in the late 1970s to facilitate limb salvage surgery [96], and led to the recognition that histologic response to therapy was a strong prognostic feature. Patients with a good histologic response (>90% tumor necrosis) had 68% OS at 5 years, compared with 52% survival in poor responders [97]. Based on this finding, there have been several attempts to improve survival by augmenting chemotherapy for the poor responders. No such attempt has shown a statistically significant benefit [98]. Thus, standard therapy for osteosarcoma remains the same today as it was in 1980 – surgical resection and a three-drug regimen of methotrexate, doxorubicin, and cisplatin – and is the same for patients with localized or metastatic disease, despite the vast difference in prognosis between these groups. Hematopoietic cell transplantation HCT is not commonly used to treat osteosarcoma, despite the lack of effective therapy for relapsed patients and the poor prognosis of patients who present with metastases. In fact, the EBMT received reports of only seven patients transplanted for osteosarcoma as of 1992. Of the five patients with measurable disease, one died of toxicity, three patients had no response, and one transplanted in refractory relapse achieved a PR
that was improved to CR by surgical excision of lung metastases, and remains in CR2 18 months post HCT. The two patients transplanted in CR3 relapsed 8 and 11 months after HCT [99]. The Cooperative German–Austrian–Swiss Osteosarcoma Study Group retrospectively analyzed 15 patients who received HCT for recurrent osteosarcoma. These patients had all achieved a CR when initially treated. Their sites of relapse were lung in nine patients, lung plus local in two, mediastinal in two, and local only in two. All underwent resection of their recurrent disease before HCT; only two had macroscopic residual disease, and neither responded to the high-dose preparative regimen. The preparative regimens all incorporated a combination of etoposide with melphalan or carboplatin, or both. Six patients received two courses of HCT, first with thiotepa and CY and then with melphalan, but two of these patients died of toxicity. The 3-year OS was 29%, and DFS was 20%. Both disease-free survivors received melphalan-based preparative regimens [100]. The Italian Sarcoma Group reported the results of a trial of surgery and tandem autologous HCT for patients with relapsed osteosarcoma. Thirty-two patients were treated. PBPCs were mobilized with CY and etoposide. Fourteen patients had surgery before chemotherapy, and 11 had surgery at the completion of all therapy. Eleven of 32 patients were in CR before their first course of high-dose therapy, while 21 were treated with gross disease. At the end of treatment, 25 patients (78%) were in CR, six were in progressive disease, and one had died of toxicity. Despite this excellent response rate, the 3-year OS was only 20%, and 3-year DFS was only 12% [101]. These few studies suggest that there is little if any benefit to high-dose chemotherapy with HCT for relapsed osteosarcoma. In light of the lack of benefit to treatment intensification for patients with a poor response to neoadjuvant therapy, this suggests that future improvements in the treatment of patients with relapsed or refractory osteosarcoma will not come from treatment intensification, but that new approaches are required. Retinoblastoma Epidemiology and etiology Retinoblastoma affects one child of every 18,000 live births in the United States in the first 5 years of life [102]. Approximately 25–30% of cases are bilateral and follow an autosomal dominant inheritance pattern; in addition, approximately 10–15% of unilateral cases are hereditary. The observation that hereditary cases occur earlier in life and are bilateral or multifocal was key in the development of Knudson’s two-hit model of carcinogenesis [103]. Knudson proposed that patients who inherit one mutation need only a single somatic mutation in a given cell for malignant transformation, while sporadic cases required the accumulation of two independent somatic mutations to transform a single cell. Molecular and cellular biology The postulated inactivation of a tumor suppressor gene was confirmed by the demonstration of loss of heterozygosity at 13q14 [104]. The cloning of the RB1 tumor suppressor gene [105,106] was a pivotal step in understanding the pathogenesis of retinoblastoma as well as its role as a tumor suppressor gene involved in many other malignancies. The RB1 gene product is commonly absent in both sporadic and heritable retinoblastoma, as predicted by the two-hit model. The messenger RNA is ubiquitously expressed and encodes a nuclear protein with DNAbinding sequences [107]. The protein is unphosphorylated in quiescent cells, but is phosphorylated in proliferating cells [108,109], and regulates the cell cycle in its unphosphorylated form by binding the E2F1 transcription factor [110]. In addition to loss of function of both alleles of the RB1 gene, amplification of MDMX (65%) or MDM2 (10%) occurs
Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors
in the majority of human retinoblastomas, inactivating p53 and promoting the development of retinoblastoma [111]. Clinical description Retinoblastoma typically presents as leukocoria when the tumor becomes visible through the pupil or causes retinal detachment or vitreous hemorrhage. Patients may lose central vision, leading to strabismus. Less commonly, heterochromia can result from neovascularization of the iris. In developing countries, the diagnosis may not be made until the eye becomes enlarged or the orbit is invaded, but extensive disease is uncommon in the United States. In patients with germline RB1 mutations, new primary tumors may appear in the retinas for the first 3–5 years of life. In addition to bilateral retinoblastoma, these patients are at risk for primitive neuroectodermal tumors in the pineal and suprasellar regions, a presentation termed trilateral retinoblastoma. Nontransplant approaches Early detection of retinoblastoma is the norm, and the treatment outcome of patients with localized disease is excellent. Enucleation is curative for early solitary unilateral lesions, while adjuvant chemotherapy is indicated for patients with pathologic risk factors such as extension beyond the cribriform plate. Orbital extension is curable in 60–85% of patients with multimodal therapy, including external beam irradiation and systemic chemotherapy. In patients with bilateral disease, additional methods are used in an effort to preserve useful vision, including brachytherapy, cryotherapy, laser photocoagulation, and systemic as well as subconjunctival chemotherapy. In contrast, advanced central nervous system (CNS) involvement and “trilateral retinoblastoma” [112] are incurable: multimodal therapy including systemic and intrathecal chemotherapy and cranial irradiation has produced remissions which, however, have not been durable [113]. Hematogenous dissemination to bone, marrow, and viscera, and involvement of soft tissue and lymph nodes carries an extremely poor prognosis. Only ifosfamide and CY have efficacy as single agents, and have produced CRs when used in two- and three-drug combinations [113]. There is a single case report of long-term survival of hematogenously disseminated retinoblastoma in a patient treated aggressively with MAD-DOC (mechlorethamine, doxorubicin, cisplatin, dacarbazine, vincristine, and CY); however, 2 years later, the patient developed secondary myelodysplasia requiring allogeneic bone marrow transplantation [114]. The patient became a long-term survivor without recurrent retinoblastoma or myelodysplasia. HCT as consolidation for disseminated disease There are several individual case reports and small case series [115–119] of HCT for patients in CR2 after metastatic recurrence in bone or marrow. These reports describe a total of 25 patients, of whom only five experienced further progression of their disease. The remaining patients had no evidence of disease 18–107 months after relapse. The SFOP formally evaluated HCT for patients with high-risk retinoblastoma, using a single-arm protocol active from 1989 to 1994 [120]. The target population was patients with extraocular disease or histologic evidence of tumor at the cut end of the optic nerve or its subarachnoid space. Measurable extraocular disease had to be chemosensitive in order for patients to be eligible to proceed to HCT. During the study period, 34 high-risk patients were identified: eight with microscopic residual optic nerve involvement, 10 with extraocular disease confined to the orbit, 11 with extraocular involvement including distant bones or marrow, and five with CNS involvement. Nine patients did not proceed to HCT because of CNS progression (six), parental refusal (two), or toxicity (one). Therefore, 25 patients (six with optic nerve disease, seven with orbital disease, eight with distant bone or marrow disease,
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and four with CNS involvement) proceeded to HCT, with a preparatory regimen of carboplatin 1250–1750 mg/m2, etoposide 1750 mg/m2, and CY 3200 mg/m2. Five of six patients who had measurable disease at the time of HCT achieved CR. On an intent-to-treat basis, five of eight patients with optic nerve involvement, six of 10 patients with orbital involvement, five of 11 patients with bone or marrow involvement, and one of five patients with CNS involvement survived with no evidence of disease. Compared with historical results using much longer courses of conventional chemotherapy, this approach produced comparable survival for patients with optic nerve or orbital involvement, and was superior for patients with distant bone or marrow involvement [120].
Desmoplastic small round cell tumor Epidemiology and etiology Desmoplastic small round cell tumor (DSRCT) is a highly malignant abdominal small round blue cell tumor with epithelial, mesenchymal, and neural characteristics, initially described in 1991 [121]. It occurs in children and young adults and is probably not rare; over 101 cases were reported between 1989 and 1996. There is a strong male predominance, and a median age at diagnosis of 25 years [122]. There are no known associations with exposure to carcinogens [123]. Molecular and cellular biology A balanced translocation, t(11;22)(p13;q12) results in EWS-WT1 fusion transcripts considered diagnostic of DSRCT [124]. As in EWS-FLI1 fusion transcripts in Ewing’s sarcoma, these fusion transcripts retain the amino-terminal effector domain of EWS and replace its DNA-binding domain with that of its fusion partner. The fusion transcripts are heterogeneous both within and between tumors with respect to the use of specific EWS and WT1 exons, the presence of internal deletions, and the insertion of heterologous DNA. However, the splice variants are in-frame and the chimeric proteins bind WT1 response elements [125]. The transcriptional regulatory activity of EWS-WT1 differs from that of WT1. The platelet-derived growth factor A chain is directly induced by EWS-WT1, and may contribute the stromal content of DSRCT [126]. In addition, expression of IGF1 receptor, which has mitotic and antiapoptotic functions, is normally repressed by WT1, but is activated by EWS-WT1 chimeras [127]. The interleukin-2/15 (IL-2/15) receptor β chain promotes cell growth by downstream activation of signal transducer and activator of transcription-3 (STAT3) and STAT5 [128]. The brain-specific angiogenesis inhibitor-1-associated protein-3, when expressed ectopically in tumor cells, enhances anchorage-independent and low-serum growth, and may assist the secretion of fibroblast growth factor-1 (FGF-1) and FGF-2 to produce the desmoplastic reaction characteristic of the tumor [129]. The leucine-rich repeat containing 15 gene normally expressed only in placenta, in the trophoblast cell layer responsible for implantation of the embryo, is hypothesized to contribute to the invasiveness of DSRCT [130]. Clinical description Patients typically present with abdominal distention and pain, with constipation a common associated finding [122,131]. DSRCTs most commonly grow in the peritoneal cavity, with large masses and multiple implants along serosal surfaces, and may metastasize to lymph nodes, liver, lung, bone, spleen, kidney, and pleura. Characteristic computed tomography findings are of bulky intra-abdominal soft tissue masses without a distinct organ of origin [132]. Unusual sites of primary involvement include kidney, lung, bone, and pancreas. Exploratory laparotomy reveals large, firm, multinodular tumors with smooth surfaces. Conventional microscopy reveals sharply demarcated clusters of
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small round tumor cells embedded in a dense desmoplastic stroma. Immunohistochemical staining reveals expression of epithelial (cytokeratin and epithelial membrane antigen), mesenchymal (vimentin), myogenic (desmin), and neural (neuron-specific enolase and CD56) markers [122,133]. WT1 immunoreactivity results from expression of the EWS-WT1 fusion transcript. CD99 (MIC2), commonly used to identify Ewing’s family tumors, is also commonly positive [134]. Nontransplant approaches This tumor is often responsive to chemotherapy such as combinations of doxorubicin, cisplatin, CY, etoposide, and fluorouracil. However, the outcome has been dismal, with median survival of only 17 (range 3–72) months; as of 1996, only seven of 101 reported patients were alive a median of 24 (range 4–48) months from diagnosis [135]. More extensive series have been reported from single institutions. A series of 10 previously untreated patients has been reported from the Memorial SloanKettering Cancer Center. Two of these patients underwent initial gross total resection, and one died early of disease. Chemotherapy consisted of CY, doxorubicin, and vincristine alternating with ifosfamide and etoposide. All seven evaluable patients achieved PR. After second-look surgery, there were seven individuals in CR and two in PR [136]. The Washington Hospital experience included seven patients with intra-abdominal DSRCT, treated aggressively with debulking surgery and intraperitoneal chemotherapy. Two showed objective responses to chemotherapy, including cisplatin/etoposide and dacarbazine as a single agent, and these two patients experienced extended survival of 58 and 102 months from diagnosis, although all seven patients eventually died of progressive disease [123]. A series of 11 patients treated at St. Jude Children’s Research Hospital, Memphis, similarly included only two whose tumors were resectable at presentation, both of whom received adjuvant chemotherapy and are long-term survivors 8 and 10 years from diagnosis. All 11 patients received chemotherapy with the same agents as the Memorial Sloan-Kettering Cancer Center series, and seven of eight evaluable patients responded to chemotherapy [131]. In summary, complete resection of desmoplastic small cell tumors contributes to long-term survival, but these tumors are generally not resectable at initial presentation; they are usually chemosensitive, but the short duration of responses warrants attempts at consolidation with intensified therapy. Hematopoietic cell transplantation There are a few reported cases of HCT as salvage therapy for recurrent or refractory desmoplastic small cell tumor, but none has resulted in long-term survival [135,137,138]. In the Memorial Sloan-Kettering Cancer Center series, the intent was to consolidate first responses of patients with initially unresectable disease with HCT, but two of five patients did not proceed to HCT because of toxicity of initial chemotherapy. Three patients with initially unresectable disease received HCT with thiotepa and carboplatin in first response, two in CR, and one in PR. The patient transplanted in PR progressed at 15 months. The patients transplanted in CR were alive without evidence of disease 13 and 34 months from initiation of therapy [136]. The St. Jude DSRCT series included four patients who underwent autologous HCT. Two patients who received HCT after radiation therapy died of toxicity. Of the two other patients, the one transplanted in CR and treated with radiation after HCT remains alive without disease 16 months after completion of therapy. In an adult population, entered on a protocol with intent to transplant in Milan, only five of 10 patients with DSRCT achieved PR to induction therapy with ifosfamide, vincristine, and etoposide, and proceeded to HCT with a melphalan-based preparative regimen. None of the patients were converted to CR or cleared their EWS-WT1 fusion transcripts after
high-dose melphalan, and all progressed within 12 months [139]. One of these patients subsequently received allogeneic HCT after reducedintensity conditioning, and cleared his fusion transcript after developing grade III acute graft-versus-host disease (GVHD) [139], suggesting the presence of a graft-versus-DSRCT effect. Success has been achieved in small series exploring more intensive alkylator-based approaches. All four patients with DSRCT treated in first PR (n = 2) or CR (n = 2) with busulfan, melphalan, and thiotepa, with or without amifostine, remained alive after 1.1–6.4 (median 3.2) years follow-up, of whom only one had progressive disease [140]. Three patients with DSRCT treated with the VACIME (vincristine, adriamycin, CY, ifosfamide, mesna, and etoposide) regimen with repeated PBPC support became long-term survivors with no evidence of disease 25–66 months from diagnosis [141]. In summary, the role of HCT remains to be defined for patients with DSRCT, but the few long-term survivors have obtained complete surgical resection, and intensified chemotherapy may contribute to survival. Case reports of HCT for pediatric solid tumors Pleuropulmonary blastoma is a rare tumor, approximately 100 cases of which have been reported. The primary therapy is surgical excision, without adjuvant therapy. Because of the histologic resemblance to soft tissue sarcoma and Wilms’ tumor, chemotherapy regimens designed for these entities have been used. Such combination chemotherapy has produced 30–40% DFS [142]. Three patients with pleuropulmonary blastoma have received melphalan-based HCT for progressive disease [143], for chemotherapy-sensitive microscopic residual disease [142], and for refractory metastatic disease [144]. The patients with bulky refractory disease progressed soon after HCT, but the patient with microscopic residual disease was alive in continuous CR at 12 months after HCT. Esthesioneuroblastoma is a rare tumor, arising from olfactory epithelium; of over 240 cases in the literature, only 21% are pediatric. The behavior of this tumor may be more aggressive in childhood. It has been suggested that esthesioneuroblastoma may be a member of the EWST family on the basis of a t(11;22) in two of three cell lines derived from metastatic esthesioneuroblastoma cases. However, unlike Ewing’s tumors, esthesioneuroblastomas do not express MIC2, and primary esthesioneuroblastomas are characterized by trisomy 8, not t(11;22) [145]. In a single-institution series, long-term survival was achieved in three of five adult patients salvaged with CY-containing HCT preparatory regimens versus four of 17 salvaged with conventional chemotherapy [146]. One adolescent was treated for a cervical nodal recurrence with modified radical neck dissection, and consolidation with high-dose carboplatin, etoposide, melphalan, and HCT, and has no evidence of disease 1 year later [147].
Future directions Patterns of failure After HCT for Ewing’s tumors, the majority of relapses are metastatic, most commonly in lung, and secondly in bone [148,149]. A more recent analysis indicates that bony failures often occur in involved bones outside the local radiation field, and in lesions detected by magnetic resonance imaging (MRI) or positron emission tomography (PET) scanning, but not bone scans [150]. Possible solutions to this problem include irradiating the lungs and the entire involved bone system prophylactically and incorporating the use of MRI and/or PET scans in staging before HCT. In contrast, failures after HCT for rhabdomyosarcoma tend to occur early, at a median of 4 months after HCT, almost always (23 of 26 cases)
Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors
in previously known sites [47]. This observation argues for the use of local radiation to all previously known sites of disease.
Regimens Role of TBI For all but the most radiosensitive tumors, the dose of radiation therapy that can be delivered as TBI is inadequate for control of bulky tumors. Doses of adjuvant radiation demonstrated to control the majority of microscopic residual disease (typically 106–108 cells) exceed the maximum tolerable doses of TBI [151]. However, the relationship between the dose of radiation and the percent reduction in risk of recurrence appears to be linear. Extrapolating from these dose–risk data, zero reduction in risk occurs at a dose between 0 and 5 Gy, indicating that there is a low threshold, if any, for tumor cell killing by radiation [152]. Therefore, the modest doses of radiation feasible as TBI may be expected to control very small micrometastases (perhaps 102–104 cells). The question of whether TBI is a necessary or desirable component of the high-dose cytoreductive regimen has been addressed retrospectively by the EBMT. For rhabdomyosarcoma patients, addition of TBI to melphalan appears only to increase toxicity [153]. A retrospective analysis of the German–Austrian Pediatric BMT Group also found no benefit of 12 Gy hyperfractionated TBI added to melphalan, etoposide, and carboplatin [47]. For Ewing’s sarcomas, the EBMTR observed that patients receiving TBI fared worse (n = 30, 19% EFS) than those receiving high-dose chemotherapy alone (n = 33, 34% EFS); the best results were obtained with chemotherapy combinations that included busulfan (51% EFS) [154]. Tandem transplants A substantial number of tandem transplants, in which two or more HCTs are performed with minimal recovery time between transplants, have already been performed in an attempt to improve tumor control. This approach is most likely to be effective if there is more than one active and noncrossresistant cytoreductive regimen, without cumulative toxicity. Even if a single regimen must be used twice, repeated application may, if a similar log cell kill may be obtained in a second course, overcome problems of delivery of drug to the core of a bulky tumor. Whether tandem HCTs as delivered so far have been beneficial is controversial. The results of several institutional series indicate longer responses in patients receiving multiple courses of high-dose therapy [155–157]. However, the cumulative experience of EBMT provides no convincing evidence in favor of double transplants in Ewing’s sarcoma [154]. In addition, at least with carboplatin and etoposide, no patient improved his or her response with a second course [158]. The use of PBPCs instead of bone marrow may improve the efficacy of tandem transplants because faster hematopoietic recovery may allow greater treatment intensity. Two courses of melphalan 100 mg/m2 could consistently be administered with PBPC rescue within 21–34 (median 24) days with no change in pharmacokinetics or pharmacodynamics [159]. A limited institution pilot study demonstrated the feasibility of tandem PBPC transplant for metastatic neuroblastoma or sarcoma, delivering CY 3600 mg/m2, etoposide 2400 mg/m2, and carboplatin 2000 mg/ m2 followed within 28–42 days by melphalan 180 mg/m2 and TBI 12 Gy in 46 of 51 eligible patients [160,161]. Of the five patients who received only one HCT, three were by patient request and two were ineligible to proceed because of liver toxicity. There were four toxic deaths (9%). Three courses of HCT have been administered to 17 of 22 patients, with one toxic death in the acute transplant period [162]. Although short-term toxicity may be acceptable, this pilot trial shows the importance of longterm follow-up, because three patients have developed pancytopenia as
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a late complication, two with graft failure and one with monosomy 7 associated myelodysplastic syndrome. A novel variation is the use of double high-dose therapy with only one HCT procedure [163]. Because the dose-limiting toxicity of melphalan and thiotepa in the setting of hematopoietic cell rescue is mucosal, and the affected tissues recover relatively quickly, two cycles can be given 1 week apart, and patients rescued with a single PBPC product after the second cycle. This strategy allows the safe delivery of thiotepa 1000 mg/m2 and melphalan 280 mg/m2 over 9 days. The indications for HCT were heterogeneous in this phase I–II trial, but 10 of 13 patients entering HCT in CR and six of 13 patients entering HCT with residual disease are alive with no evidence of disease 15–59 (median 35) months after transplantation. Of particular interest is that all three patients with hepatoblastoma who entered HCT with persistent alphafetoprotein elevation achieved long-term CR [163]. Targeted therapy Targeted therapeutics refers to treatments designed to exploit a biologic feature of the tumor to eradicate it. Examples of targeted therapies include tyrosine kinase inhibitors such as imatinib mesylate, and ligandtargeted monoclonal antibodies such as rituximab. There are two significant ongoing applications of targeted therapy for pediatric solid tumors that involve HCT: 131I-metaiodobenzylguanidine for the treatment of neuroblastoma (see Chapter 66) and 153Sm-ethylenediaminetetramethylene phosphonic acid (EDTMP) for the treatment of osteosarcoma. Each of these treatments utilizes a fundamental characteristic of the tumor to direct a radiopharmaceutical agent specifically to the tumor, and ameliorates the myelosuppression with autologous hematopoietic cell support. 153 Sm-EDTMP consists of a radioisotope (153Sm) conjugated to a tetraphosphonate compound that localizes to sites of bone turnover, such as a metastatic bone lesion. Because 153Sm-EDTMP targets bone lesions by a mechanism similar to the radiotracer used in a bone scan, lesions that are visible on bone scan, including both primary osteosarcoma and metastatic deposits, are targeted by this agent. Radioactive decay of the 153Sm yields a medium energy electron (β particle) and a lowenergy photon. The electron has a very short path length (1–2 mm), thus providing exquisitely precise targeting of the cytotoxic energy. The photon can be detected by the same instrument used for a diagnostic bone scan, allowing uptake at a target tumor to be confirmed and the amount of radiation delivered to the tumor to be measured. The half-life of 153Sm is very short (46 hours), facilitating handling. This compound was originally tested in adult patients with bone metastases from carcinomas, such as breast and prostate cancer, and is approved by the United States Food and Drug Administration for palliation of painful metastases. In 2002, Anderson and colleagues published the results of a phase I study of 153Sm-EDTMP for the treatment of high-risk osteosarcoma patients [164]. Thirty patients were treated with 1–30 mCi/kg of 153SmEDTMP. Because this compound significantly depresses bone marrow function, patients were rescued with an infusion of autologous PBPCs 14 days after treatment. Other than low blood counts, the only side-effect was transient hypocalcemia in patients treated with the highest dose. All of the patients began the trial requiring narcotics for pain relief, and every patient experienced a decrease or elimination of their narcotic requirement. In an attempt to improve efficacy, this group subsequently treated patients with gemcitabine, a chemotherapeutic agent that is thought to also act as a radiation sensitizer [165]. Fourteen patients were treated with high-dose 153Sm-EDTMP followed 1 day later by gemcitabine. After 2 weeks, autologous PBPCs were reinfused. At 6–8 weeks of follow-up, there were six partial remissions and two mixed responses, but none of these responses was durable. Thus, 153Sm-EDTMP
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shows promise for treating osteosarcoma, but further work will be required to determine its optimal use. Hematopoietic cell grafts The source of the graft: peripheral blood hematopoietic cells versus bone marrow It is now recognized that mobilizing and collecting PBPCs is feasible in children despite their small size and the intensity of the pediatric chemotherapy regimens, and PBPCs produce faster engraftment [166,167] and immune reconstitution [168] than marrow. Although tumor cells can frequently be detected in mobilized PBPC grafts [169,170], the question of whether the purging of autologous hematopoietic cells can improve outcome remains to be answered. Few data are available regarding the relationship between tumor contamination of the graft with survival outcomes, but long-term DFS can occur in patients with Ewing’s sarcoma who receive PBPC grafts containing detectable fusion transcript [171]. In rhabdomyosarcoma, the major determinant of long-term survival after HCT was whether CR was achieved prior to HCT [153], and 23 relapses occurred in previously known sites, compared with only three in new metastatic sites [47], suggesting that tumor control in the body, not reinfusion of tumor cells in the graft, is the major problem. Allogeneic HCT The role of allogeneic HCT is being explored in pediatric solid tumors. Allogeneic transplantation carries the risk of GVHD, but its potential benefits include the absence of tumor cells in the graft and perhaps an immunologic antitumor effect. For rhabdomyosarcoma, the German– Austrian Pediatric BMT Group found no evidence of benefit from allogeneic HCT, as none of five allogeneic HCT recipients survived [47]. In addition, donor lymphocyte infusion was reported to be ineffective in a case of relapsed rhabdomyosarcoma [172], but since the patient did not have measurable disease at the time of donor lymphocyte infusion and did not develop GVHD, this case did not address the activity of an effective immune response. A CR after reduced-intensity matched sibling peripheral blood HCT for a patient who had failed two prior lines of therapy including autologous HCT suggests benefit from an allogeneic effect [173]. For Ewing’s tumors, there was a trend toward better outcome with allogeneic HCT (50% survival versus 23% survival of the entire group of 27 allografted patients at a median of 56 months) in the singleinstitution experience with (TB)I + melphalan + etoposide with or without carboplatin of Vienna, from 1984 to 1996 [174]. With longer follow-up, incorporating the experience from Düsseldorf, DFS after allogeneic HCT was not better than after autologous HCT for Ewing’s tumors because of higher rates of nonrelapse mortality [31]. However, there are informative case reports of patients with recurrent, metastatic Ewing’s sarcoma who underwent allogeneic HCT and whose disease responded to withdrawal of immune suppression [173] or institution of IL-2 therapy [175] with subsequent development of acute GVHD. However, few data are available regarding allogeneic HCT for pediatric solid tumors. This probably results largely from the role of neuroblastoma as the prototypical poor-prognosis pediatric solid tumor, and the clear consensus that there is no role for allogeneic transplantation in neuroblastoma [176,177]. The lack of benefit from allogeneic HCT would indicate that tumor contamination of autologous grafts is not a major source of relapse, and that alloreactive cytotoxic T-lymphocytes (CTLs) are not substantially effective against high-risk pediatric solid tumors. However, a potentially important biologic difference between neuroblastoma and sarcomas is that, in vitro, neuroblastoma cells are poor targets for CTLs, whereas sarcoma cells are excellent CTL targets [178].
Immunotherapy Rhabdomyosarcoma and Ewing’s tumors are targets both for natural killer cells and for CTLs [179], and tumor-infiltrating lymphocytes isolated from osteosarcomas can lyse allogeneic osteosarcoma cell lines [180]. The possibility that these immunologic effector mechanisms may be clinically important in high-risk Ewing’s tumors was suggested in the pilot trial of hyperfractionated TBI with melphalan and etoposide conducted in Vienna and Düsseldorf. In their initial cohort of 17 patients with very high-risk Ewing’s tumors, three of four allograft recipients were disease-free survivors, along with three of five autograft recipients treated with IL-2 versus two of eight autograft patients without IL-2 [181]. The advantage of IL-2 after autografting persisted in a larger European Intergroup Cooperative Ewing’s Sarcoma Study cohort treated using the same approach: DFS was 52% for patients receiving IL-2 after HCT versus 22% without IL-2 (p < 0.05) [150]. The possible effector mechanisms involved in patients receiving IL-2 after HCT have been explored, and expansion of T and natural killer (NK) cell numbers, but not those of NK/T cells was demonstrated. NK activity was enhanced, in spite of increased activation of the CD94 inhibitor receptor [182]. The National Cancer Institute has conducted a pilot trial of tumorspecific vaccination for Ewing sarcoma and alveolar rhabdomyosarcoma, based on pulsing dendritic cells with the unique EWS-Fli1 and PAX3-FKHR fusion peptides associated with the t(11;22) and t(2;13) translocations characteristic of these tumors [183]. Sixteen patients were enrolled, and 15 received at least one vaccination. Unfortunately, most patients experienced tumor progression during the first 6-week vaccination cycle, so only four patients received two or more cycles of vaccination. One of these four patients had a mixed response, and another had a fusion peptide-specific lymphocyte proliferative response. This study is important not only because of the encouraging suggestion of some immunologic activity against tumor, but also because it identifies several pitfalls of immunotherapy. These patients had severely impaired immunity and large tumor burdens, and the immature circulating dendritic cells used may actually suppress immune responses [183]. Moreover, a single peptide antigen was used, with uncertain human leukocyte antigen restriction and cell surface expression, and the tumor itself may have induced tolerance or anergy in autologous lymphocytes. An alternative strategy of generating anti-tumor CTL cell lines ex vivo has been explored in Ewing’s sarcoma patients. Specific antitumor CTLs were generated from three patients, all of whom had undergone prior autologous HCT, after two or more cycles of stimulation of autologous peripheral blood mononuclear cells with autologous peripheral bloodderived dendritic cells, CD4+ lymphocytes, and irradiated early-passage autologous tumor cells [184]. Although the approach is labor intensive, potential advantages include the response to unknown as well as previously identified tumor antigens, and the reduced potential for escape by loss of antigen expression. The clinical utility of such cell lines has yet to be demonstrated. The development of reduced-intensity allogeneic HCT offers an alternative avenue to immunotherapy for pediatric solid tumors, but novel approaches will be required to demonstrate activity if this approach is beneficial only in minimal residual disease.
Conclusion The prognosis of patients with metastatic, recurrent or refractory pediatric solid tumors is dismal. High-dose preparatory regimens with HCT have demonstrated activity against a broad spectrum of the pediatric solid tumors, and HCT has produced promising results in the treatment of Ewing’s tumors. Prospective randomized trials of HCT versus con-
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ventional chemotherapy are in progress for patients with poor-prognosis Ewing’s tumors and are being considered for high-risk recurrent Wilms’ tumor. For less common indications, phase I and II trials are appropriate.
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Approaches now being investigated to improve the efficacy of HCT for pediatric solid tumors include tandem HCT, targeted therapy, chemoprotection, and immunotherapy, possibly via allogeneic HCT.
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114. Petersen RA, Friend SH, Albert DM. Prolonged survival of a child with metastatic retinoblastoma. J Pediatr Ophthalmol Strabismus 1987; 24: 247– 8. 115. Ekert H, Tiedemann K, Waters KD, Ellis WM. Experience with high dose multiagent chemotherapy and autologous bone marrow rescue in the treatment of twenty-two children with advanced tumors. Austr Paediatr J 1984; 21: 195–201. 116. Dunkel IJ, Aledo A, Kernan NA et al. Successful treatment of metastatic retinoblastoma. Cancer 2000; 89: 2117–21. 117. Rodriguez-Galindo C, Wilson MW, Haik BG et al. Treatment of metastatic retinoblastoma. Ophthalmology 2003; 110: 1237–40. 118. Kremens B, Wieland R, Reinhard H et al. Highdose chemotherapy with autologous stem cell rescue in children with retinoblastoma. Bone Marrow Transplant 2003; 31: 281–4. 119. Matsubara H, Makimoto A, Higa T et al. A multidisciplinary treatment strategy that includes high-dose chemotherapy for metastatic retinoblastoma without CNS involvement. Bone Marrow Transplant 2005; 35: 763–6. 120. Namouni F, Doz F, Tanguy ML et al. High-dose chemotherapy with carboplatin, etoposide and cyclophosphamide followed by a haematopoietic stem cell rescue in patients with high-risk retinoblastoma: a SFOP and SFGM study. Eur J Cancer 1997; 33: 2368–75. 121. Gerald WL, Miller HK, Battifora H, Miettinen M, Silva EG, Rosai J. Intra-abdominal desmoplastic small round-cell tumor. Report of 19 cases of a distinctive type of high-grade polyphenotypic malignancy affecting young individuals. Am J Surg Pathol 1991; 15: 499–513. 122. Lae ME, Roche PC, Jin L, Lloyd RV, Nascimento AG. Desmoplastic small round cell tumor: a clinicopathologic, immunohistochemical, and molecular study of 32 tumors. Am J Surg Pathol 2002; 26: 823–35. 123. Gil A, Gomez Portilla A, Brun EA, Sugarbaker PH. Clinical perspective on desmoplastic small round-cell tumor. Oncology 2004; 67: 231–42. 124. Ladanyi M, Gerald W. Fusion of the EWS and WT1 genes in the desmoplastic small round cell tumor. Cancer Res 1994; 54: 2837–40. 125. Liu J, Nau MM, Yeh JC, Allegra CJ, Chu E, Wright JJ. Molecular heterogeneity and function of EWS-WT1 fusion transcripts in desmoplastic small round cell tumors. Clin Cancer Res 2000; 6: 3522–9. 126. Lee SB, Kolquist KA, Nichols K et al. The EWSWT1 translocation product induces PDGFA in desmoplastic small round-cell tumour. Nat Genet 1997; 17: 309–13. 127. Finkeltov I, Kuhn S, Glaser T et al. Transcriptional regulation of IGF-I receptor gene expression by novel isoforms of the EWS–WT1 fusion protein. Oncogene 2002; 21: 1890–8. 128. Wong JC, Lee SB, Bell MD et al. Induction of the interleukin-2/15 receptor beta-chain by the EWSWT1 translocation product. Oncogene 2002; 21: 2009–19. 129. Palmer RE, Lee SB, Wong JC et al. Induction of BAIAP3 by the EWS–WT1 chimeric fusion implicates regulated exocytosis in tumorigenesis. Cancer Cell 2002; 2: 497–505. 130. Reynolds PA, Smolen GA, Palmer RE et al. Identification of a DNA-binding site and tran-
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John A. Zaia, Amrita Krishnan, & John J. Rossi
Hematopoietic Cell Transplantation for Patients with Human Immunodeficiency Virus Infection
Introduction The acquired immune deficiency syndrome (AIDS) is in many ways analogous to a neoplastic disorder because it is a complex disease with multiple pathogenic features, is treated with modalities that permit at least temporary clinical remission but that fail to cure without eradication of latent virus, and is subject to development of resistance to chemotherapy. Thus, similar to certain cancer therapies, it is not unusual to propose that hematopoietic cell transplantation (HCT) could have a role in the management of AIDS. Several features intrinsic to human immunodeficiency virus type 1 (HIV-1) (Fig. 68.1) infection contribute to the complex pathogenicity of this viral infection. These include an early infiltration and seeding of the hematopoietic tissue with HIV-infected cells, a virus replication cycle that occurs predominantly within this tissue and is coupled to cell activation and function, and virus replication that occurs in multiple cell types and tissues (e.g. thymus, brain, and gut) at all stages of the disease [1]. There is gradual destruction of lymph nodes during the asymptomatic phase of infection and eventual loss of function of the immune system [2]. Confounding the pathogenic features of the disease is the genetic instability of the virus that results in HIV-1 variants in the infected individual [3], a potential constellation of drug-related adverse effects, and the difficulty of adherence to drug regimens that require significant adjustments in lifestyle and daily activity [4]. Two factors, in particular the emergence of drug resistance and the continued replication of drugresistant HIV-1 variants in tissue, undermine the clinical benefit of antiretroviral chemotherapy. In this setting, the potential for cell-based therapy to re-establish an immune system resistant to HIV-1 has been suggested. Because HIV-1 is dependent on a variety of well-described molecular events for either infection or replication, gene-based strategies that could inhibit these events are being assessed as potentially complementary therapies for HIV-1 infection. It is the purpose of this chapter to describe the experience with HCT in AIDS and to view the current status of therapeutic approaches for HIV-1 treatment using gene-based approaches to HIV-1 infection.
Epidemiology and etiology HIV-1 spreads via contact with infectious body fluids and gains entry into susceptible cells following interaction of the viral envelope proteins
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
and a primary cellular receptor, CD4 (Fig. 68.2) [5]. Of hematopoietic interest, chemokine receptors, primarily CXCR4 and CCR5, are coreceptors required for viral entry [6,7]. Chemokines are a diverse family of peptides that bind to receptors and activate immune and/or inflammatory responses [8]. The chemokines with two consecutive cysteines are C-C chemokines, and those with an intervening amino acid are C-X-C chemokines. Natural ligands for CCR5, such as RANTES and macrophage inflammatory protein-1 alpha, can compete with HIV-1 for binding to this receptor and inhibit HIV infection [9]. The ligand for CXCR4 is stromal-derived factor 1, which can also inhibit HIV-1 infection [10]. A meta-analysis of patient cohorts has shown that only the CCR5-Δ32 and the CCR2-64I co-receptor mutations are associated with strong protective effects from progression of HIV-1 infection [11]. As a lentivirus, a subgroup of the retrovirus family, HIV-1 shares major genetic features common to all retroviruses [12]. Based on related findings, a model has emerged that describes the sequence of events leading to HIV-1 entry. Initial virus–cell interaction occurs between the HIV-1 envelope protein (gp120) and the CD4 receptor. The gp120–CD4 complex then engages one of the chemokine receptors, depending on the specific HIV-1 isolate (CCR5 for macrophage-tropic isolates called “R5 strains;” CXCR4 for T lymphocyte tropic or X4 strains) [6,7]. Many other chemokine co-receptors have subsequently been identified that can fulfill this co-receptor function in vitro, but CCR5 and CXCR4 appear to be most critical in vivo [13]. Following formation of the HIV envelope–CD4 receptor–chemokine receptor heterotrimer complex, there is an alteration of the membrane that leads to exposure of a fusion domain contained within the HIV-1 fusion protein (gp41) [14]. The engagement of gp41 with the target cell membrane culminates in penetration of HIV1 into the cell, with subsequent movement into the nucleus. Chemokine receptors have a role in leukocyte trafficking and in inflammatory responses, raising the legitimate concern that therapeutic anti-HIV manipulations, designed to interfere with the receptors’ normal cellular functions, might impinge on cellular homeostasis. However, the findings that these receptors are minimally polymorphic and that individuals with homozygous mutations exhibit no ill effects, yet display relative resistance to HIV-1 infection [10,11,15–18], have fueled the search for strategies that block this very first event in HIV-cell interaction. Based on the crystal structure of HIV envelope [19], specific compounds that inhibit the envelope–CCR5 interaction [20], as well as the gp41-mediated fusion reaction [21], have been designed; one of these fusion inhibitors, enfuvirtide (Fuzeon), has been approved in the United States for use in patients with AIDS. It is likely that additional compounds that inhibit these early steps in the viral life cycle will be designed and developed for clinical applications.
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Nef Ltr
Y
Vpu
Vif
Gag Pol
Vpr
Ltr
Env Tat
Tat Rev
Rev
Fig. 68.1 Schematic structure of human immunodeficiency virus type 1 genome. env, envelope; Gag, group antigen; Ltr, long-terminal repeat; Pol, polymerase; Rev, regulator of expression of viral proteins; Tat, transactivator of transcription; Vif, Vpu, Vpr, Nef, accessory proteins. (Reproduced from Berkhout et al. [150] with permission.)
Chemokine receptor
Infectious virion CD4
Uncoating Viral RNA RT RNaseH Circular DNA
Integrase
Unintegrated linear DNA
Tat/Rev
Integrated proviral DNA
mRNA
Protein synthesis
Genomic RNA Protease
Assembly and budding
Mature virion
Fig. 68.2 Life cycle of human immunodeficiency virus type 1 (HIV-1). HIV-1 binds to susceptible CD4+ T lymphocytes via the cellular CD4 receptor, and enters the cell via a fusion process facilitated by a chemokine receptor. Following penetration, the viral RNA is reverse transcribed by the viral enzyme reverse transcriptase (RT) to an RNA–DNA duplex that is degraded by viral ribonuclease H (RNaseH). Second-strand complementary DNA (cDNA) is completed by the viral RT. The cDNA then migrates to the nucleus, where it integrates into cellular chromosomes. Following activation by either viral regulatory proteins or cellular factors, viral transcription commences, producing Tat and Rev. Under this regulation, an internal circuit exists in which polypeptide precursors are processed by a virus-encoded protease, as shown. The mature peptides and two copies of the genomic RNA eventually migrate to the cell membrane, assemble together, and finally bud from the cell as a mature virion. All of these complex steps are potential targets for gene therapy strategies. mRNA, messenger RNA.
Molecular and clinical biology Lentiviruses, as a group, exhibit genomic complexity far beyond that of a prototype retrovirus, which consists of only the gag, pol, and env genes [12]. Six additional regulatory genes (Fig. 68.1) with a spectrum of biologic function have been identified in HIV-1 (with seven identified in simian immunodeficiency virus, the simian homolog of HIV). Two of these genes, tat and rev, are required for virus replication. The Tat protein is involved early in the virus life cycle, with a primary role in transcriptional activation of the viral promoter, and the HIV requirement
for Tat-induced activation appears to depend on the type and activation state of the infected cell. In contrast, Rev appears to be absolutely required for HIV-1 replication and acts as a temporal gatekeeper later in the replication cycle, allowing transport of late messenger RNAs from the nucleus to the cytoplasm, where they are translated into structural proteins for assembly of progeny virus [22]. There currently are no antiTat and anti-Rev small-molecule compounds in clinical trials, but these have been the target for gene transfer studies. The remaining four unique genes in HIV-1 are the “accessory” genes nef, vif, vpr, and vpu (reviewed Derdeyn et al. in [23]). These genes are
Hematopoietic Cell Transplantation for Patients with Human Immunodeficiency Virus Infection
termed “accessory” based on early studies suggesting that they are dispensable for virus growth in vitro. However, more comprehensive studies performed in vitro or in experimental models that simulate in vivo conditions (e.g. primary T lymphocytes and macrophages infected with clinical HIV-1 isolates) identified several functions for the Nef, Vif, Vpr, and Vpu proteins that are important in HIV-1 replication and pathogenesis. Moreover, each of these regulatory proteins is known to be multifunctional, having distinct roles at different stages of the virus life cycle and in pathogenic events. Replication and T-lymphocyte homeostasis Critical to the understanding of HIV-1 molecular biology and pathogenesis is the mechanism by which CD4+ lymphocyte reduction and eventual loss of immune function occurs. Proposed mechanisms of pathogenesis have evolved as the understanding of HIV-1 infection has increased. Early in the epidemic, much of the immunologic damage of AIDS was proposed to be a manifestation of autoimmune disease [24]. Later studies led to the “sink and drain” model, which uses as its basis the dynamics of HIV-1 RNA levels in the blood (viral load) and CD4+ lymphocyte turnover following the initiation of highly active antiretroviral therapy (HAART) [25,26]. Studies leading to this hypothesis demonstrated that a rise in CD4+ lymphocyte count correlated with a reduction in HIV-1 replication, and that HIV-1 infection appeared to directly destroy the CD4+ T-lymphocyte compartment. The sink and drain model postulated the direct killing (“drain”) of CD4+ lymphocytes and elimination of the CD4+ lymphoid compartment (“sink”) by HIV-1 infection. In this model, a hypothetical hematopoietic “faucet” dynamically replenishes this loss until the putative supply of T cells becomes exhausted, thereby resulting in overt immunodeficiency. Based on this hypothesis, antiviral agents are expected to reverse the loss of CD4+ lymphocytes and restore immune function. However, at the early stages of HIV-1 infection when CD4+ lymphocytes are in decline and the memory CD8+ lymphoid compartment is relatively increasing, both naïve CD4+ and CD8+ T lymphocytes decline at the same rate. Because naïve CD8+ lymphocytes are not usually infected with HIV-1, and naïve CD4+ lymphocytes are relatively resistant to HIV-1 infection, these declining numbers cannot be easily explained by direct killing of cells by HIV-1 [27]. Rather, HIV-1 infection appears to result in a dysregulation of Tlymphocyte homeostasis involving the peripheral pools of these cells and their altered thymic output [28]. These observations have been consolidated into a “redistribution” hypothesis, which postulates that several cytokines are released coincident with active HIV-1 infection and the ensuing lymphoid cell activation [29]. In turn, these cytokines cause trapping of T lymphocytes in peripheral lymphatic sites, where they are efficiently infected by HIV-1 [30,31]. With potent anti-HIV-1 therapy, the viral load is diminished and T lymphocytes are spared from infection. As the levels of the cytokines decline, uninfected cells are released from the lymphatic tissue and enter the peripheral blood, resulting in an increase in CD4+ lymphocytes, albeit a moderate one. This model also predicts that the redistribution of CD4+ and CD8+ lymphocytes will increase as AIDS progresses. This prediction is borne out in clinical studies, demonstrating that the initial response to potent antiHIV-1 chemotherapy is greater in patients with advanced disease compared with that in patients at earlier stages of the disease [32]. Immune reconstitution during potent antiviral therapy With continued chemotherapy and repression of HIV-1, naïve T lymphocytes slowly increase [32]. The degree to which naïve T
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lymphocytes can be restored and their repertoire expanded will most likely determine the success of such treatment. Reports that pretreatment perturbations in the CD4+ lymphocyte immune repertoire are gradually alleviated during potent anti-HIV-1 chemotherapy [33] have not been confirmed in all studies [34]. While it appears that certain recall immune responses lost during HIV-1 disease progression may be reestablished following initiation of anti-HIV-1 chemotherapy, responses to other antigens, particularly those encoded by HIV, do not appear to be reestablished as readily. This variable recovery may be explained in part by the observation that HIV-specific CD4 cells are preferential targets for HIV infection [28]. In order to return to a situation in which T-lymphocyte homeostasis and normal immune function exist, strategies that directly influence immune reconstitution will most likely be required to effectively complement anti-HIV-1 chemotherapy. The ability of HAART to restore antigen-specific immune functions will undoubtedly depend on a number of factors, including the time at which therapy is initiated after HIV-1 infection and the extent and duration of disease progression. Indeed, recent clinical findings indicate that the strength of immune responses to both HIV and non-HIV antigens may be proportional to the CD4+ cell nadir before HAART initiation [35]. Restoration of immune functions will be strongly affected by the ability of the patient to generate new populations of naïve T lymphocytes, a process believed to be largely dependent on the presence of a functional thymus. Thymic function can be affected not only by HIV-1 infection, but also by age, stress, and underlying medical conditions [36,37]. Understanding such person-to-person heterogeneity might shed light on what appear to be conflicting results concerning immune system restoration in different patient populations following potent antiviral therapy [33,34,38,39].
Cellular and anatomic reservoirs of HIV It is now well established that a reservoir of latently infected cells (resting memory CD4+ lymphocytes) persists for prolonged periods of time, even in patients on potent antiviral therapy whose viral loads have decreased to undetectable levels [40–42]. These cells could form a pool from which HIV-1 replication could be rekindled following drug withdrawal or HIV-1 gene activation. In cases where anti-HIV-1 chemotherapy has been suspended during structured treatment interruption or following prolonged use, a rebound of replicating virus has occurred in the majority of patients studied within a few weeks [43,44]. This rapid reappearance of virus indicates that HIV-1 eradication has not occurred and implies that continuous combination therapy must be maintained at all times, possibly for the life of the patient, in order to keep HIV-1 at bay. In contrast to the demonstrated requirement for continuous drug therapy for most patients, a few reports have suggested that, in certain rare circumstances, patients can control viral rebound after withdrawal of the drugs [45,46]. In addition, in rare HIV-infected persons, there is control of HIV viremia for long periods without treatment [47–49]. These persons have CD8-specific adaptive immunity to HIV, which appears to be human leukocyte antigen restricted and is quantitatively different from what is seen in those with progressive HIV infection [50,51]. The confirmation of these observations and the mechanisms responsible for long-term viral suppression in these patients are subjects of intense investigation [43,50]. Encouragingly, a recent report estimates that the half-life of the latent viral reservoir is approximately 4.6 years, which suggests that, if a patient is on continual antiretroviral therapy from the early onset of infection, it may be possible to completely eradicate the latent reservoir in 7.7 years [52].
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Clinical description Antiretroviral therapy Combination antiretroviral therapy using reverse transcriptase and protease inhibitors has resulted in dramatic reductions in HIV-1 blood levels [25,26,53] and in prolonged survival [54]. Current standards of care involve the use of multiple drugs that target both HIV-1 reverse transcriptase and protease proteins [4]. In optimal circumstances, and with careful adherence to the rigid schedule of drug administration demanded by this therapy, many patients achieve dramatic reductions in plasma viral load, often to undetectable levels. It is not clear how long these levels can remain suppressed, but many patients have maintained viral suppression for years while on continuous antiviral therapy. However, these treatments are not without associated complications, which have resulted in current guidelines that these therapies not be initiated in asymptomatic patients until the level of CD4+ cells has declined to 350 cells/mL [4]. Further, the risk of generating drug-resistant variants increases with time on treatment. Because the presence of even low levels of HIV-1 replication in persons receiving anti-HIV-1 treatment is associated with drug resistance [55,56], questions of longterm efficacy of these treatments remain unknown. Nevertheless, as improvements have occurred in antiretroviral chemotherapies, AIDS is becoming more of a chronic disease, with the need to manage the infection in the setting of advancing age [57]. From a hematologic standpoint, with this improved long-term survival, the risk of developing lymphoma is a concern, and an understanding of cancer treatment in the AIDS patient is important. Lymphoma and AIDS The presence of HIV-1 infection and aggressive B-cell lymphomas (specifically Burkitt’s lymphoma, immunoblastic lymphoma, and lymphoma of the central nervous system) was made part of the definition of AIDS in 1985 [58]. The two other cancers associated diagnostically with AIDS are Kaposi’s sarcoma and cervical cancer. A meta-analysis of the incidence of cancer in persons with HIV/AIDS (n = 444,172), compared with iatrogenically immunosuppressed transplant recipients (n = 31,977), found that both immunosuppressed groups share an increased risk for the three types of cancer linked by definition to AIDS, namely Kaposi’s sarcoma, non-Hodgkin’s lymphoma (NHL), and cervical cancer, as well as for all human papilloma virus-related cancers, for Hodgkin’s lymphoma (HL), liver cancer, and stomach cancer [59]. The lymphomas associated with Epstein–Barr virus infection, namely HL and NHL, or with Kaposi’s sarcoma-associated herpesvirus infection, namely primary effusion lymphoma and plasmablastic lymphoma, have a much higher incidence in HIV/AIDS than in other immunosuppressed groups [59]. Thus, the World Health Organization classifies AIDSrelated lymphoma (ARL) into three groups: those lymphomas that occur in immunocompetent patients (e.g. Burrkitt’s lymphoma, diffuse large cell lymphoma of centroblastic or immunoblastic types, HL, T-cell lymphoma, and extranodal mucosa-associated marginal zone B-cell lymphoma), those lymphomas occurring in HIV/AIDS patients (e.g. primary effusion lymphoma and plasmablastic lymphoma), and lymphomas occurring in other immunodeficiency states (e.g. polymorphic B-cell lymphoma) [60]. Effect of HAART on incidence of AIDS-related lymphoma Prior to the availability of HAART, patients with HIV infection had a 60- to 600-fold increased risk of developing NHL compared with the general population and a 7 to 20-fold increased rate of HL [61–63]. With
the availability of HAART, there has been a reduced incidence of opportunistic infections and Kaposi’s sarcoma, increased CD4 counts, and improved overall survival [64,65]. Of the many studies that have assessed the impact of HAART on the incidence of AIDS lymphoma, there are paradoxical changes due to the fact that the proportion of HIV-infected persons progressing to AIDS has declined, the absolute number of ARL cases has decreased, but the relative incidence of some lymphomas has actually increased [66,67]. A meta-analysis of the incidence of ARL in 47,936 HIV-infected persons showed that the incidence rate for NHL declined from 6.2 to 3.6 per 1000 person-years with largest declines in primary central nervous system lymphoma and immunoblastic lymphoma [68]. Yet the occurrence of NHL increased from 3.6% of AIDSdefining illnesses in 1994 to 5.4% in 2000, a change that was significant (p 30 days) resistance to HIV-1 replication [122]. Similarly, human CD34+ blood progenitor cells transduced with RevM10 gave rise to T lymphocytes that exhibited significant resistance to challenge with HIV1 [123]. Based on these and other pivotal studies, clinical studies were undertaken to assess the safety of the transgene and its protective effect on transduced cells measured in cell survival relative to unprotected cells. The first human clinical study examined the potential of RevM10 to prolong the survival of transduced CD4 T lymphocytes in vivo after ex vivo transduction, expansion, and reinfusion into HIV-1-seropositive
subjects [124]. In this study, the duration of engraftment was limited, and transient, but genetically modified cells were detected in one subject up to 2 months after gene transfer, although the recombinant gene was not detectable in two subjects after 2 weeks [124]. In a subsequent attempt to achieve more durable engraftment, a retrovirus delivery vector was used in lieu of plasmid DNA, and, as before, the cells transduced with RevM10 retroviral vectors survived and expressed the recombinant gene for significantly longer time periods than those transduced with a negative control vector in three HIV-positive subjects tested [125]. Intracellular HIV-specific single-chain antibodies (intrabodies; SFv) can sequester or redirect HIV-1 proteins away from their normal subcellular compartment. Combinations of SFv targeted to reverse transcriptase, integrase, and CCR5 have been described by Strayer et al. [126]. Additional transdominant protein transgenes are listed in Table 68.1. Intracellular toxins and conditionally toxic molecules can be used to preferentially eradicate HIV-1 infected cells [127–132]. A major deficiency of this approach is that these gene-modified cells do not possess a selective survival advantage and therefore cannot be enriched relative to nonmodified cells. It is expected that repeated infusions of ex vivomodified cells will be required for therapeutic benefits. Unless an in vivo vector delivery system is available, the aforementioned requirement is likely to render this strategy impractical when the large HIV-infected population is considered. Strategies based on preventing the expression of chemokine coreceptors at the cell surface are particularly attractive primarily because they block the very first step in HIV-1 infection of target cells rather than blocking a viral event that occurs after the establishment of the proviral DNA in the cellular genome. Intrakine-modified cells should also have survival advantages in vivo in HIV-infected individuals because of their resistance to infection by certain HIV-1 isolates. Moreover, a gene-based approach that utilizes a nonpolymorphic human protein is unlikely to induce an immune response against the expressed protein. Such an undesired outcome could lead to clearance by the immune system of transduced cells expressing the foreign protein. Because HIV-1 replication can be influenced by a variety of cellular perturbations, there are many other molecular strategies for inhibiting this virus (Table 68.1), and these strategies will increase as more is known about the biology of HIV-1.
Current application of cell-based gene therapy to AIDS Current gene therapy strategies make extensive use of an ex vivo approach in which cells are taken from a donor, modified with the transgene of interest, expanded, and then returned to the donor. Early studies used a marker gene that allows detection of the modified cells; however, gene transfer has the potential for lethal toxicity, and future gene transfer trials will not be able to ethically use marker genes owing to the potential toxicity of cells expressing foreign proteins [133] or to the induction of integrational mutagenesis [134].
T lymphocytes as targets for gene therapy This approach uses autologous T lymphocytes as the target cells for gene modification, thereby eliminating complications caused by hostversus-graft rejection or graft-versus-host disease that would otherwise occur with grafts from nonidentical individuals. Using this strategy, mature CD4+ lymphocytes have been genetically modified and reinfused into the patients [109,124,135]. A similar strategy, in which mature CD8+ lymphocytes have been modified to kill HIV-infected cells, has also been reported [102,136–138]. In either case, mature T lymphocytes have attributes that make them attractive targets for gene therapy: they
Hematopoietic Cell Transplantation for Patients with Human Immunodeficiency Virus Infection
are easily obtained from the donor’s peripheral blood, and they can be expanded to large numbers in vitro. Indeed, a method of cell expansion that involves cell surface stimulation, using antibodies to CD3 and CD28, promotes the expansion of cells to very high numbers [139]. Because these stimuli also transiently downregulate the expression of the chemokine receptor CCR5 [140], these cells are at a minimum temporarily resistant to infection with macrophage-tropic strains of HIV, adding another advantage for their immediate survival in an HIVinfected host. Targeting mature T lymphocytes for gene therapy has the added advantage that the effect of the therapeutic gene can be rapidly monitored for effects on cell survival, viral load, and other parameters. The transduced cells can also be selected in vitro, using the marker gene included in the vector, before reinfusion in the host, thereby generating a highly enriched population of genetically modified cells. For these reasons, gene therapy approaches that target mature lymphoid populations may be the method of choice for initial evaluation of new gene therapy strategies. Using a lentivirus vector to transfer an antisense RNA target to HIV-1 envelop, CD4 lymphocytes have been safely infused into AIDS patients who were resistant to antiretroviral therapy [102]. A negative aspect of using mature lymphocytes is that they are terminally differentiated and have limited in vivo growth potential and finite life span. Moreover, CD4+ T lymphocytes represent only one of the cell types susceptible to HIV-1 in vivo. In addition, techniques used to expand CD4+ lymphocytes in vitro can affect the expression of various surface markers and result in altered homing properties when the cells are reinfused to the host. Intense efforts are therefore under way to develop gene transfer protocols for HSCs that can give rise to all cells of the lymphoid and myeloid lineages. Conceptually, HSCs transduced with a potent anti-HIV-1 gene should confer anti-HIV-1 protection to all hematopoietic cells lineages, including those susceptible to HIV-1 infection (CD4+ T lymphocytes, monocytes, Langerhans’ cells, dendritic cells, and others) for the life of the infected individual.
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instead of stromal cells [141,142], and the combination of recombinant Flt3-ligand [143,144], thrombopoietin, and stem cell factor [145]. The critical issue is whether the vector can transduce a pluripotent stem cell, and to conclusively demonstrate that transduced CD34+ cells are in fact pluripotent, it is necessary to show that cultures derived from a single transduced cell can differentiate into all hematopoietic lineages (T and B lymphocytes, monocytes, erythrocytes, and others), and can result in reconstitution in vivo of a surrogate host. Studies in non-human primates indicate that lentivirus-based gene transfer, combined with a selection system that is toxic to nontransduced HSCs, can result in high levels of gene-marked blood progeny [146]. The ability of HSCs to engraft in patients who have not been “preconditioned” with cytoreductive or high-dose preparative regimens is an important issue in HSC-based gene therapy. The first successful retrovirus-based gene therapy was performed in patients with severe combined immunodeficiency [147,148]. The initial successful outgrowth of T lymphocytes was demonstrated to contain the transgene and to be functional [148]. This clinical trial provided the first demonstration that gene transfer could cure a genetic disease, and it is a model on which anti-HIV-1 gene therapy using genetically modified HCT has been based. However, the initial successes in patients with severe combined immunodeficiency have yielded retrovirus-related insertional mutagenesis with late T-cell leukemia [149]. Evidence is convincing that this leukemoid reaction was brought about by the interaction of the insertional site and the interleukin γ-chain transgene, and adverse events such as these further define the problems that must be understood before stem cell-based gene therapy can be safe and efficacious. It is arguable that the setting of autologous HCT for AIDS lymphoma is a clinical setting in which these new methods can be ethically evaluated [90]. The comparative value of cytoreduction versus reduced-intensity regimens, the evaluation of new vectors and stability of transgene expression, and the development of selection strategies for expansion of transduced cells in vivo all need to be studied.
Conclusion HSCs as targets of gene transfer While HSCs are an attractive target for gene therapy, they present a unique set of issues that are not encountered when mature T lymphocytes are used as the target cells. In addition to their low frequency, HSCs are quiescent. This relative paucity of cell replication may affect transduction strategies that rely on retroviral vectors, a class of vectors that require cell division for efficient gene transfer (see Chapter 11). As a result, current transduction methods often stimulate cell division, which may compromise the undifferentiated status, functional activity, and subsequent cell lineage commitment of the transduced progenitor cells. Over recent years, newer methods of transduction have resulted in incremental improvements of vector-mediated gene transfer into CD34+ cells. These include the use of recombinant fibronectin fragment CH-296
HCT currently has application to the treatment of relapsed of refractory AIDS-related lymphoma. Patients tolerate dose-intense chemotherapy, and the timing of engraftment is similar to that observed in non-HIV lymphoma patients undergoing the same treatment. Based on this, it has been proposed to use HCT as a delivery method for genetically modified hematopoietic progenitor cells having resistance to HIV-1 infection. Assuming that cell-based gene therapy becomes optimized, specific uses for gene therapy are likely to be identified, and the test-of-concept for this approach could be established. The treatment of relapsed ARL provides the setting for testing these novel therapies and, if gene transfer is ever shown to be effective in this setting, the potential will exist for eventual treatment of the HIV-1 infected person who has no cancer, with a goal of decreasing the virus reservoir and delaying disease progression.
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125. Ranga U, Woffendin C, Verma S et al. Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals. Proc Natl Acad Sci USA 1998; 95: 1201–6. 126. Strayer DS, Branco F, Landre J et al. Combination genetic therapy to inhibit HIV-1. Mol Ther 2002; 5: 33–41. 127. Brady HJ, Miles CG, Pennington DJ et al. Specific ablation of human immunodeficiency virus Tat-expressing cells by conditionally toxic retroviruses. Proc Natl Acad Sci USA 1994; 91: 365– 9. 128. Caruso M. Gene therapy against cancer and HIV infection using the gene encoding herpes simplex virus thymidine kinase. Mol Med Today 1996; 2: 212–17. 129. Caruso M, Klatzmann D. Selective killing of CD4+ cells harboring a human immunodeficiency virus-inducible suicide gene prevents viral spread in an infected cell population. Proc Natl Acad Sci USA 1992; 89: 182–6. 130. Curiel TJ, Cook DR, Wang Y et al. Long-term inhibition of clinical and laboratory human immunodeficiency virus strains in human T-cell lines containing an HIV-regulated diphtheria toxin A chain gene. Hum Gene Ther 1993; 4: 741–7. 131. Dinges MM, Cook DR, King J et al. HIV-regulated diphtheria toxin A chain gene confers longterm protection against HIV type 1 infection in the human promonocytic cell line U937. Hum Gene Ther 1995; 6: 1437–45. 132. Smith SM, Markham RB, Jeang KT. Conditional reduction of human immunodeficiency virus type 1 replication by a gain-of-herpes simplex virus 1 thymidine kinase function. Proc Natl Acad Sci USA 1996; 93: 7955–60. 133. Su L, Lee R, Bonyhadi M et al. Hematopoietic stem cell-based gene therapy for acquired immunodeficiency syndrome: efficient transduction and expression of RevM10 in myeloid cells in vivo and in vitro. Blood 1997; 89: 2283–90. 134. Woods NB, Bottero V, Schmidt M et al. Gene therapy: therapeutic gene causing lymphoma. Nature 2006; 440: 1123. 135. Walker RE. A phase I/II pilot study of the safety of the adoptive transfer of syngeneic gene-modified cytotoxic T lymphocytes in HIV-infected identical twins. Hum Gene Ther 1996; 7: 367–400. 136. Mitsuyasu RT, Anton PA, Deeks SG et al. Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects. Blood 2000; 96: 785–93. 137. Riddell SR, Elliott M, Lewinsohn DA et al. T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nat Med 1996; 2: 216–23. 138. Riddell SR, Greenberg PD, Overell RW et al. Phase I study of cellular adoptive immunotherapy using genetically modified CD8+ HIV-specific T cells for HIV seropositive patients undergoing allogeneic bone marrow transplant. The Fred Hutchinson Cancer Research Center and the University of Washington School of Medicine, Department of Medicine, Division of Oncology. Hum Gene Ther 1992; 3: 319–38. 139. Levine BL, Bernstein W, Craighead N et al. Ex vivo replicative potential of adult human peripheral blood CD4+ T cells. Transplant Proc 1997; 29: 2028.
140. Carroll RG, Riley JL, Levine BL et al. Differential regulation of HIV-1 fusion cofactor expression by CD28 costimulation of CD4+ T cells. Science 1997; 276: 273–6. 141. Hanenberg H, Xiao XL, Dilloo D et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat Med 1996; 2: 876– 82. 142. Moritz T, Dutt P, Xiao X et al. Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors: evidence of direct viral binding to chymotryptic carboxy-terminal fragments. Blood 1996; 88: 855–62. 143. Kiem HP, Andrews RG, Morris J et al. Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor. Blood 1998; 92: 1878–86. 144. Shah AJ, Smogorzewska EM, Hannum C et al. Flt3 ligand induces proliferation of quiescent human bone marrow CD34+CD38– cells and maintains progenitor cells in vitro. Blood 1996; 87: 3563–70. 145. Dao MA, Hannum CH, Kohn DB et al. FLT3 ligand preserves the ability of human CD34+ progenitors to sustain long-term hematopoiesis in immune-deficient mice after ex vivo retroviralmediated transduction. Blood 1997; 89: 446– 56. 146. Neff T, Beard BC, Peterson LJ et al. Polyclonal chemoprotection against temozolomide in a largeanimal model of drug resistance gene therapy. Blood 2005; 105: 997–1002. 147. Aiuti A, Vai S, Mortellaro A et al. Immune reconstitution in ADA-SCID after PBL gene therapy and discontinuation of enzyme replacement. Nat Med 2002; 8: 423–5. 148. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000; 288: 669–72. 149. French gene therapy group reports on the adverse event in a clinical trial of gene therapy for Xlinked severe combined immune deficiency (XSCID). Position statement from the European Society of Gene Therapy. J Gene Med 2003; 5: 82–4. 150. Berkhout B, Silverman RH, Jeang KT. Tat transactivates the human immunodeficiency virus through a nascent RNA target. Cell 1989; 59: 273–82. 151. Krishnan A, Molina A, Zaia J et al. Durable remissions with autologous stem cell transplantation for high-risk HIV-associated lymphomas. Blood 2005; 105: 874–8. 152. Rosenberg SA, Blaese RM, Brenner MK et al. Human gene marker/therapy clinical protocols. Hum Gene Ther 1996; 7: 1621–47. 153. Crooks ET, Moore PL, Richman D et al. Characterizing anti-HIV monoclonal antibodies and immune sera by defining the mechanism of neutralization. Hum Antibodies 2005; 14: 101–13. 154. Bagarazzi ML, Boyer JD, Ayyavoo V et al. Nucleic acid-based vaccines as an approach to immunization against human immunodeficiency virus type-1. Curr Top Microbiol Immunol 1998; 226: 107–43.
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Richard A. Nash
Hematopoietic Cell Transplantation for Autoimmune Diseases
Introduction An autoimmune disease is characterized by organ damage resulting from an immunologic response to self-antigens. Autoimmunity, on the other hand, is an immune response to self-antigens but without the implication that this has resulted in organ damage. Autoimmunity may be present spontaneously in normal individuals or after an infectious or other disease-related event which results in tissue damage (e.g. infarction). Criteria which support a disease having an autoimmune mechanism are: (1) humoral or cellular self-reactivity; (2) disease-related autoantibody or lymphocytic infiltrate in a pathologic lesion without any other identified cause; and (3) autoantibodies or T cells that cause tissue pathology or cell dysfunction by transplancental transmission, adaptive transfer into animals or in vitro experiments. An autoimmune disease develops in approximately 3–8% of the population [1]. Autoimmune diseases may involve single organs or multiple systems and may or may not have defined antigenic targets. Most autoimmune diseases are not life-threatening and can be effectively treated even if not curable. However, some autoimmune diseases may be life-threatening or damage critical organs and become refractory to conventional treatments. This then would be the candidate population for consideration of an approach requiring autologous or allogeneic hematopoietic cell transplantation (HCT).
Proposed mechanisms for autoimmunity and autoimmune diseases To better understand how an approach using HCT might be effective for treatment of an autoimmune disease, an understanding of the pathogenic mechanisms of autoimmune disease is required. There is a marked diversity of the B- and T-cell receptor specificity that results from somatic genome modification in lymphocytes. This allows for an effective immune response to foreign antigens. The unique specificities of these receptors are generated during B- and T-cell differentiation by recombination of the variable (V), diversity (D), and joining (J) genes, and then, later in B cells, somatic hypermutation generates further diversity. These random processes of recombination and somatic hypermutation result in a significant percentage of lymphocytes (up to 50%) with B- and T-cell receptors that have an affinity for self-antigens [2].
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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It has been proposed that there are four cellular strategies to prevent the development of autoimmunity and autoimmune diseases from lymphocytes with self-reactive receptors. First, lymphocytes with highaffinity, self-reactive receptors can be clonally deleted. Clonal deletion occurs in central lymphoid organs during development. Second, a selfreactive receptor can be edited by further V(D)J recombination or somatic hypermutation to display a different receptor that is not selfreactive [3]. Receptor editing induced by self-reactivity can occur in both B and T cells. For B-cell receptor editing, new rearrangements at L chain loci and less commonly at H chain loci are induced. Third, downregulation of cell surface self-reactive receptors and intracellular changes, including those which affect signal pathways, decrease responsiveness to the binding of self-reactive receptors, resulting in clonal anergy [4]. Last, extrinsic factors such as cytokine-dependence for proliferation and regulatory mechanisms, including regulatory T cells, may limit the activity of lymphocytes with self-reactive receptors. In B cells with self-reactive receptors, B-cell activating factor receptor is poorly induced and therefore may be more susceptible to competitive regulation by their requirement for B-cell activating factor than other B cells with non-self-reactive receptors. Some T cells with self-reactive receptors are positively selected in the thymus after T-cell receptor engagement by self-peptide–major histocompatibility complex (MHC) class II complexes. They develop into CD25+CD4+FoxP3+ regulatory T cells which suppress proliferation and the interleukin-2 (IL-2) production of autoreactive CD25−CD4+ and CD8+ T cells [5,6]. Suppression of proliferation by regulatory T cells is contact dependent. Regulatory T cells are IL-2 dependent with constitutively expressed CD25 (IL-2 receptor) and IL-7 independent without CD127 (IL-7 receptor). The transcription factor foxP3 may endow regulatory function to thymic T cells in mice, but this is less clear in humans [7]. Natural killer-T cells and other T-cell subsets producing transforming growth factor-beta and IL-10 have also been associated with immune regulation.
The genetics of autoimmune disease Autoimmune disease can be associated with either single-gene defects or susceptibility genes as part of a complex multigenic trait. Four inherited disorders from single-gene defects have been described which have contributed further insights into autoimmune disease mechanisms in humans (Table 69.1) [8–11]. These include defects in: (1) autoimmune regulator-dependent negative selection in the thymus; (2) cytotoxic T lymphocyte antigen-4-dependent inhibition of T cell function; (3) Fas/
Hematopoietic Cell Transplantation for Autoimmune Diseases Table 69.1 Single-gene defects associated with autoimmunity Autoimmune disease
Gene defect
Mechanisms of autoimmunity
Autoimmune polyglandular syndrome type I [8] Grave’s disease, type I diabetes mellitus, others [9] Autoimmune lymphoproliferative syndrome [10] IPEX [11]
AIRE
Defective expression of thymic self-antigens
CTLA4
Defect in inhibitory signaling
FAS, FASL
Defect in apoptosis
FOXP3
Decreased regulatory T cells
IPEX, immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome.
Table 69.2 Susceptibility genes for common autoimmune diseases
Autoimmune disease
Major histocompatibility complex association (class II)
Candidate susceptibility genes
Multiple sclerosis [15] Rheumatoid arthritis [12]
DR2 DR4
Systemic lupus erythematosus [12] Systemic sclerosis [14] Crohn’s disease [12] Type 1 diabetes mellitus [12]
DR2/DR3
IL-7R, IL-2R PADI4, PDCD-1, PTPN22, FCRL3 PTPN22, PDCD-1
DR11 DRB3*0301 DR3/DR4
PTPN22 NOD2, IBD5 CTLA-4, PTPN22, INS
CTLA-4, cytotoxic T-lymphocyte antigen-4; FCRL3, Fc receptor-like 3; IBD5, inflammatory bowel disease-5 (susceptibility locus on chromosome 5q31); IL-7R, interleukin-7 receptor; INS, insulin (disease-associated polymorphism of the insulin gene); NOD2, nucleotide oligomerization domain containing-2 (also known as caspase recruitment domain family, member 15 [CARD15]); PADI4, peptidyl arginine deiminase type IV; PDCD-1, programmed cell death-1; PTPN22, nonreceptor type 22 protein tyrosine phosphatase.
FasL-dependent apoptosis of B and T cells that recognize self-antigens; and (4) FoxP3-dependent development of regulatory T cells [8–11]. The most common autoimmune diseases are considered to result from alleles of susceptibility genes at multiple loci, environmental triggers, and stochastic events [12,13]. The disease-associated alleles of susceptibility genes have been shown to confer only modest risk for the development of an autoimmune disease and were first identified among the MHC genes (Table 69.2) [12,13]. Most correlations of autoimmune diseases were with MHC class II rather than class I molecules [13]. Other susceptibility genes affect, in general, pathways for activation/ inhibition of T cells (IL-7R, IL-2R, PDCD-1, PTPN22, and CTLA-4) or presentation of self-antigens (insulin, and PADI4) [12,14,15]. It is unclear to what degree environmental factors contribute to risk of developing autoimmune disease. Environmental factors including hydralazine, contaminated rapeseed oil, L-tryptophan, and Borrelia burgdorferi infections in association with specific alleles of the MHC loci have been shown to increase the risk of an autoimmune disease.
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Family or twin studies may provide further information about the contribution of genetic factors to the onset or presentation of autoimmune diseases. In general, the disease concordance rates among monozygotic twins was threefold greater or more than in dizygotic twins for rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type I diabetes mellitus (DM) and multiple sclerosis (MS) [16,17]. The average concordance rates reported for monozygotic twins ranged from 15–30% for DM, MS, SLE, and RA. It has also been reported that there was a higher risk for many autoimmune diseases in other family relatives. Although genetic factors appear to play a role in the development of autoimmune diseases, the relatively low penetrance as noted in the monozygotic twins indicates that nongenetic factors are also important.
Preclinical models of autoimmunity and HCT There are two potential approaches utilizing HCT to consider for the treatment of autoimmune diseases (also see Chapter 20). The first approach is with autologous HCT after high-dose immunosuppressive therapy (HDIT). The therapeutic effects derive from the high-dose cytotoxic immunosuppression. Although it has been proposed, there is no evidence that the infusion of autologous hematopoietic cells has any benefit other than to shorten the period of pancytopenia [18]. Allogeneic HCT, as the second approach, can be done after either high-dose or reduced-intensity conditioning regimens. The therapeutic effect of this approach likely derives from the eradication of autoreactive immune effector cells when complete donor hematopoietic chimerism is established. However, remissions of autoimmune diseases have been reported after establishing only mixed hematopoietic chimerism. Therefore, it is likely that the immunomodulation from establishing the donor graft, including effects on regulatory mechanisms, may have a self-tolerizing effect even if there are persistent host autoreactive immune cells. There are two types of model for autoimmune disease in which studies of autologous and allogeneic HCT have been conducted: (1) a spontaneously developed autoimmune disease, such as a lupus-like systemic condition in NZB mice, or an organ-specific condition, such as DM in NOD mice; and (2) antigen-induced autoimmune diseases, such as adjuvant arthritis and experimental allergic encephalomyelitis (EAE). In the spontaneous autoimmune disease models, high-dose cytotoxic therapy and syngeneic bone marrow transplantation early in life did not prevent the development of the clinical or pathologic autoimmune process, whereas allogeneic marrow transplants from an autoimmune-resistant strain prevented and, when given early after disease onset, ameliorated the autoimmune manifestations (Fig. 69.1) [19,20]. In contrast to the experience in models of spontaneous-onset autoimmune diseases, studies of antigen-induced autoimmune diseases have shown therapeutic responses in recipients after high-dose cytotoxic immunosuppressive treatments and syngeneic or pseudoautologous HCT. Pseudoautologous donors were syngeneic donors with the same disease severity as the recipients at the time of HCT. EAE, an experimental model for MS, may be induced in Buffalo rats and SJL/J mice by active immunization with myelin basic protein. In this model, highdose therapy with total body irradiation (TBI) or cyclophosphamide followed by allogeneic, syngeneic or autologous marrow rescue can prevent or ameliorate disease activity [21,22]. Allogeneic HCT was superior in preventing spontaneous and induced relapses from reimmunization of myelin basic protein. In rats, after immunization with a unique peptide fragment of myelin, the CD4+ T lymphocyte cell initiating EAE is skewed towards overutilization of the Vβ 8.2 T-cell receptor. When analyzed by reverse transcriptase polymerase chain reaction and in situ hybridization, Vβ 8.2 T lymphocytes were not detected in the central nervous system (CNS) after HDIT and syngeneic HCT [23]. Therefore, high-dose therapy was able to eliminate the effector lympho-
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Fig. 69.1 Survival curves of BXSB mice (spontaneous lupus-like syndrome) after treatment with 9.5 Gy of TBI (caesium-137 irradiation, 0.70 Gy/min) and transplantation with T-cell-depleted marrow cells intravenously at 16 weeks of age from BXSB or normal BALB/c donors after the development of the autoimmune disease. The transplant groups were: BALB/c + BXSB → BXSB group (䊐; group I; n = 18), BALB/c → BXSB group (䉭; group II, n = 8), and BXSB → BXSB group (䊊; group III; n = 7). Untreated BXSB mice served as controls (䉬; n = 8). Compared with untreated controls or group III (pseudoautologous), survival was improved in recipients of the allogeneic marrow grafts. (Reproduced from [19], with permission. Copyright [1999], National Academy of Sciences, USA.)
cytes responsible for EAE from the CNS in this model. Similarly, in adjuvant-induced arthritis, the prevention of relapses after HDIT, syngeneic HCT, and repeat immunization is consistent with a tolerizing effect to the inciting antigens (Fig. 69.2). Increased intensity of the highdose cytotoxic immunosuppression resulted in a more effective induction of remissions and fewer relapses [24]. Observations in preclinical models of spontaneous-onset autoimmune diseases would support the use of allogeneic HCT for those human autoimmune diseases related to single-gene disorders with high penetrance (e.g. IPEX syndrome). However, for the other more common human autoimmune diseases, in which environmental or stochastic factors may play a larger role for triggering disease onset, the preclinical data would support the investigation of HDIT and autologous HCT.
Outcomes in patients with autoimmune diseases transplanted for another primary disease Observations in patients with hematologic disorders and a concomitant autoimmune disease gave us our first understanding that the response of human autoimmune diseases to allogeneic HCT could be comparable to the experience in the preclinical models (Table 69.3) [25–42]. Baldwin et al. first reported sustained remissions in patients who were transplanted with severe aplastic anemia but also had a diagnosis of RA. Follow-up was short, and there were early transplant-related deaths. Subsequent case reports of patients transplanted with a concomitant diagnosis of RA confirmed that remissions could be achieved and be
Fig. 69.2 Response of adjuvant-induced arthritis after high-dose immunosuppressive therapy with various doses of cyclophosphamide (CY) and combinations of CY with sublethal total body irradiation (TBI) in Buffalo rats. Adjuvant-induced arthritis is a chronic progressive disease and a preclinical model for rheumatoid arthritis. For this study, arthritis was induced by an intradermal injection of Freund’s complete adjuvant, and the severity of the arthritis was expressed as an arthritic score (increases with increased arthritic severity). Remissions were greater and more durable in recipients which received a more intensive regimen containing TBI, although some benefit was observed with all of the regimens. BMT, bone marrow transplantation. (Reproduced from [24], with permission. Copyright [2000], Macmillan Publishers Ltd.)
sustained for over 20 years [30]. It was also noted that three of the nine patients with RA relapsed after allogeneic HCT. One of the relapses was transient with a subsequent treatment-free remission of 11 years. In one of the two sustained relapses for which data was available, the recipient had complete donor hematopoietic chimerism, but the human leukocyte antigen (HLA)-identical donor was serologically positive for rheumatoid factor but without clinical disease [26]. Sustained remissions and some relapses were also observed in patients with other concomitant autoimmune diseases after allogeneic HCT (Table 69.3). One patient who relapsed with Crohn’s disease had mixed hematopoietic chimerism detected at 3 months after HCT. There were insufficient data from the case reports to develop an understanding of why relapses occurred after allogeneic HCT. In all but one of the cases reporting the use of allogeneic HCT for concomitant autoimmune diseases, the donor was an HLA-identical sibling. Therefore, one possible
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1017
Table 69.3 Outcomes in patients with autoimmune diseases transplanted with an allogeneic hematopoietic cell graft for another primary disease
Autoimmune disease
Hematologic disease
Evaluable patients (total) (n)
Rheumatoid arthritis [25–31]
SAA (n = 8), 1 MM
9
Systemic lupus erythematosus [32] Psoriatic arthritis [29,33–35] Ulcerative colitis [33] Crohn’s disease [36] Multiple sclerosis [37–40] Autoimmune hepatitis [41] Lupus anticoagulant [42]
SAA AML, CML (n = 3) AML CML CML (n = 2), LGL leukemia, AML ALL CML
1 4 1 5 4 1 1
Remission of autoimmune disease (after HCT) (n) 7 (2 relapses, 1 transient relapse) 1* (ANA titer +) 3 (1 relapse) 1 4† 3 1 1
Outcome (alive at last follow-up) (n)
Follow-up
6
2 months–21 years
1 3 1 5 3 1 1
15 years 1, 3, 5, 5 years 4 years 4.5–15.3 years 1, 2, 3, 4 years 4 years 5 years
ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ANA, antinuclear antibody; CML, chronic myelogenous leukemia; HCT, hematopoietic cell transplantation; LGL, large granular lymphocytes; MM, multiple myeloma; SAA, severe aplastic anemia. * Clinical remission but ANA remained positive. † Relapse occurred in patient with mixed chimerism.
explanation for recurrence of the autoimmune diseases may be the presence of shared genetic factors between the related donors and recipients. Another possible explanation is the persistence of host immune cells, resulting in recurrence of disease activity. In the late 1990s, a series of case reports was published on the outcomes of concomitant autoimmune diseases after high-dose cytotoxic therapy and autologous HCT [43,44]. These case reports were of interest since the clinical trials of HDIT and autologous HCT were just being initiated at that time. Most of the reported cases had non-Hodgkin’s lymphoma as the indication for high-dose cytotoxic therapy. Remissions of the concomitant autoimmune diseases were observed in the majority of patients early after treatment, but only five of 15 cases had sustained responses at last follow-up. This experience reflects the risks of progression of autoimmune disease after high-dose cytotoxic therapy. Clinical trials of HDIT and autologous HCT specifically for autoimmune diseases were designed to intensify the immunosuppressive effect compared with standard cytotoxic regimens for treating hematologic malignancies. This was done by depleting T cells from the autologous hematopoietic cell graft and adding other noncytotoxic immunosuppressive agents to the HDIT regimen for in vivo T-cell depletion. The intensity of the HDIT regimen may be important in achieving sustained remissions of autoimmune diseases [45]. These case reports, although small in number, were important in establishing our understanding of the potential role of allogeneic HCT and HDIT with autologous HCT for the treatment of patients with severe autoimmune diseases.
HDIT and autologous HCT for autoimmune diseases: clinical experience HDIT has been performed most commonly for MS, systemic sclerosis (SSc), SLE, RA, and juvenile idiopathic arthritis (JIA) [45]. HDIT regimens which have been investigated have had varying intensities. High-dose single-agent cyclophosphamide has been considered a lowintensity regimen [45]. It is highly immunosuppressive but is not myeloablative. Clinical trials of high-dose cyclophosphamide have been conducted with and without the support of autologous HCT. Regimens which included TBI or high-dose busulfan (BU) were considered high intensity and required support with autologous HCT.
Patient selection Patients eligible for clinical trials of HDIT were generally those with severe autoimmune disease (life-threatening or threatening the function of critical internal organs) and had failed conventional therapy. Although the criteria for selecting patients depended upon the type of autoimmune disease, it was usually required that patients have a poor prognosis based on the activity and severity of the disease.
Collection and processing of the hematopoietic cell graft Although some of the earliest adult cases were transplanted with marrow grafts, most of the later patients were transplanted with hematopoietic cell grafts harvested from the peripheral blood [46]. Marrow was the preferred source of hematopoietic stem cells for many pediatric patients [47]. The optimal dose of peripheral blood CD34+ cells for successful engraftment was 2–5 × 106/kg in patients with hematologic malignancies, and therefore this was the number targeted for collection in patients with autoimmune diseases. Eight of 173 patients (4.6%) failed to mobilize sufficient numbers of CD34+ cells to allow proceeding to HDIT [46]. Five of these eight patients eventually had adequate numbers of cells harvested by either a supplemental marrow harvest (n = 2) or successful second mobilization (n = 3). Early in the clinical trial experience of HDIT for autoimmune diseases, it was observed that some patients had disease flares during mobilization. In one review, five of 56 patients mobilized with granulocyte colony-stimulating factor (G-CSF) alone had exacerbations of the autoimmune disease (MS n = 2; RA n = 3) [46]. There have been other reports of exacerbations during mobilization in MS and RA patients with G-CSF alone [48,49]. Most exacerbations were transient, but one patient with MS died from the development of severe brainstem dysfunction. As a result of these early flares, most patients have now been mobilized with the combination of cyclophosphamide and G-CSF. In 117 patients mobilized with the combination of G-CSF and cyclophosphamide, no exacerbations were observed. In fact, after mobilization with cyclophosphamide, an improvement was noted in the underlying autoimmune disease. The addition of cyclophosphamide to G-CSF also improved the yield of progenitor cells after mobilization [46]. However, there were
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three patient deaths in this report from either cyclophosphamide-related toxicity or infections. In another report of 126 patients from a single center who were mobilized with the combination of G-CSF and cyclophosphamide, there was one patient death from infection [50]. Four mild exacerbations of disease and seven infectious events were also observed during mobilization with a cyclophosphamide-based regimen. Another mobilization strategy that has been used in 22 MS patients without disease exacerbations or infections is the combination of prednisone and G-CSF [51]. G-CSF alone has been used to mobilize 34 patients with SSc without any significant disease exacerbations except for a few cases in which there was an increase in telangiectasias or skin tightness [52]. The risk of exacerbations during mobilization using GCSF alone may be related to the type of autoimmune disease. The use of cyclophosphamide as part of the mobilization regimen should be carefully considered, possibly by the type of autoimmune disease, because of the potential risks associated with the resulting toxicity. Many of the reported clinical trials used T-cell-depleted autologous grafts as a strategy to prevent recurrence of the autoimmune disease. This strategy was based on observations in preclinical models in which the autoimmune disease could be adoptively transferred with T cells. An adoptive transfer of autoimmune disease to human recipients has also been observed after allogeneic HCT [53]. In most of the clinical trials, CD34 selection was performed and resulted in a 3–4-log depletion of T cells for the autologous graft. Further T- and B-cell depletion was done in some clinical trials after the CD34 selection. The importance of Tcell-depletion of the graft was addressed in a retrospective analysis of registry data from the European Group for Blood and Marrow Transplantation (EBMT). No strong beneficial effect or risk of relapse was identified [45]. Although the risk of infection may be increased with T-cell depletion of the autologous graft, the incidence of infectious disease is low if these patients are managed with the same infection prophylaxis strategies used for patients after allogeneic HCT [47,54]. The contribution of T-cell depletion of the autologous hematopoietic cell graft to the prevention of relapse remains to be determined in future clinical trials. HDIT regimens The early rationale for HDIT was to systemically deplete autoreactive immune effector cells. Cytotoxic agents (at conventional doses) had already been observed to have a beneficial effect in most autoimmune diseases, but not all patients had a clinical response and remissions were mostly not sustained. With follow-up of the pilot studies of HDIT with autologous HCT now extending to 7 and 8 years after treatment, it has been observed that there is a high initial response rate, and a significant proportion of patients have achieved sustained remissions [47,52,55– 57]. The clinical response early after HDIT likely resulted from the overall reduction in number of immune effector cells responsible for the autoimmune disease. The sustained response observed after recovery of the lymphocyte counts at 2 years may have resulted from a late immunomodulatory effect of the HDIT regimen [54,58,59]. Randomized clinical trials need to be completed to confirm that the clinical responses after HDIT are an improvement over what might have been expected from conventional management strategies. The intensity of the HDIT regimen was important for the prevention of disease progression. In a report of 473 patients from the EBMT registry, sustained responses were observed in 78% of patients after a regimen with high-intensity conditioning compared with 68% with an intermediate and 30% with a low-intensity conditioning regimen (p = 0.0001) [45]. Regimens that included BU or TBI in combination with cyclophosphamide were placed in the high-intensity conditioning group, and cyclophosphamide alone was placed in the low-intensity conditioning group. All other regimens were considered of intermediate intensity.
The analysis of the registry data had some limitations, including variability of the diagnoses, patient selection criteria, and treatments (as well as having imbalances between diagnosis and type of HDIT regimen), but a dose intensity–response effect on treatment outcomes was evident. One of the most frequently used HDIT regimens was high-dose, single-agent cyclophosphamide (200 mg/kg) followed by autologous HCT for hematopoietic support [45]. A small study compared 100 with 200 mg/kg of cyclophosphamide followed by HCT in patients with RA. Remissions were longer with the higher dose of cyclophosphamide, but all patients relapsed [60]. Relapse rates were also high in a study of early SSc in which patients were treated with high-dose cyclophosphamide (200 mg/kg) alone. Four of 11 patients died by 18 months after treatment (three from progression), and another four patients had progressed requiring secondary treatment [61]. The addition of antithymocyte globulin (ATG) to cyclophosphamide may improve the response rate and duration. Since it is not myeloablative, high-dose cyclophosphamide (200 mg/ kg) as a single agent without autologous HCT has been reported for treatment of autoimmune diseases or immune-mediated diseases such as aplastic anemia [18,62]. The median time to recovery of neutrophils counts was 2–3 days longer without the support of an autologous hematopoietic cell graft, but the upper limit of the range was 7 days longer [18,55]. The relapse rate was also high in SLE patients (n = 14) after high-dose cyclophosphamide without HCT, but a significant proportion (36%) had durable complete remissions. In this small experience, there was no apparent benefit on relapse rate by withholding the infusion of an autologous hematopoietic cell graft. High-dose combination chemotherapy with BCNU, etoposide, cytosine arabinoside, and melphalan (BEAM) with or without ATG was considered an intermediate-intensity regimen [45]. For autoimmune diseases, this regimen has most commonly been used for the treatment of MS [63]. It has been well tolerated in this group of patients, and no significant data exist for other autoimmune diseases. Regimens with either TBI or BU in combination with cyclophosphamide were considered to be high intensity [47,51,52,64–66]. A pilot study of BU in combination with cyclophosphamide has been reported for the treatment of MS [66]. The use of TBI in combination with cyclophosphamide has been reported in clinical trials of HDIT for MS, SSc, and JIA. TBI is highly immunosuppressive and was shown to be effective in preclinical models of autoimmune disease [24,67]. Organs can be shielded with lead during TBI to protect them from the potential adverse effects of this form of cytotoxic therapy, which is not possible with systemic chemotherapy. TBI has the potential for killing cycling and noncycling stem cells as well as immune effector cells. Support with an infusion of autologous hematopoietic stem cells is required after both intermediateand high-intensity HDIT regimens to prevent prolonged pancytopenia or lack of hematopoietic recovery. From the EBMT registry, treatment-related mortality was 14% and 3% in the groups who received the high- and low-dose conditioning intensity regimens, respectively [45]. However, there was no difference in overall survival based on the intensity of the HDIT regimen. Treatment-related mortality resulted primarily from infections and severe organ toxicity. Several fatal cases and one nonfatal case of Epstein–Barr virus-associated posttransplant lymphoproliferative disorder have been reported [65,68]. In patients who have had a high-intensity immunosuppressive regimen, early detection of Epstein–Barr virus reactivation and treatment with rituximab may prevent the clinical manifestations of posttransplant lymphoproliferative disorder. The risks of infectious complications and organ toxicities after HDIT are closely related to the type of autoimmune disease being treated. Patients with MS and RA have had a lower treatment-related mortality than patients with SSc and
Hematopoietic Cell Transplantation for Autoimmune Diseases
1019
SLE who have had significant internal organ involvement at the time of transplant. Prolonged or intensive pretransplant treatment with immunosuppressive agents including corticosteroids, as in the management of patients with severe SLE, likely predisposes to infectious complications after HDIT. Better patient selection and modifications to the treatment regimen appear to have reduced the risks of treatment-related mortality in recent years [47,56,69]. Long-term complications after high-dose cytotoxic therapy include the risk for secondary myelodysplastic syndrome (MDS) and leukemia [52,65]. In one of the three cases of MDS reported, evidence of clonal abnormalities was found in cryopreserved pretransplant hematopoietic cells. The intensity and duration of the pretransplant chemotherapy is known to be an additional risk factor for the development of secondary MDS/leukemia. Secondary autoimmune diseases (other than the one for which patients were transplanted) have been described late after HDIT and autologous HCT [52,65,70]. The type of conditioning agent included in the HDIT regimen may increase the risk of this occurring [70]. Immune reconstitution after an HDIT regimen Natural killer cell counts recovered by 1 month and were followed by recovery of B- and CD8+ T-cell counts by 6–12 months after HDIT. There was a slower recovery of CD4+ T-cell counts, which were lownormal by 2 years [54]. Immune recovery at 2 years after HDIT was associated with increasing thymic-derived naïve CD4+ T cells (Fig. 69.3) [58]. It was also observed that there was an increase in T-cell receptor excision circles (a marker for recent thymic emigrants) in CD4+ T cells at 1 and 2 years after HDIT. There was a steady decrease over time in CD4+ central memory T cells. CD4+ effector memory cells were relatively increased at 6 months after HDIT, likely from homeostatic proliferation, but had recovered to normal levels by 2 years. There were no significant changes in the CD8+ T cell subsets. There was an increase in regulatory T cells, and broader clonal diversity than was present before HDIT (Fig. 69.4) [58,59]. In association with the increased levels of naïve CD4+ T cells, there was hypertrophy of the thymus at 1 and 2 years compared with baseline, especially in the younger patients (less than 43 years of age). This evidence suggested a thymic origin for the recovery of the CD4+ T-cell repertoire after HDIT and autologous HCT. Even though B-cell counts were very low in the first 3 months after HDIT, median serum levels of immunoglobulin G specific for tetanus toxoid, Haemophilus influenzae and Streptococcus pneumoniae, were normal. The clinical responses to HDIT which have persisted for 2 or more years may have resulted from these late immunomodulatory effects, especially evident in the CD4+ T-cell compartment. Specific autoimmune diseases and HCT Phase II clinical trials of HDIT and autologous HCT have been conducted for specific autoimmune diseases, and in some cases the results from long-term follow-up evaluations are available. Multiple sclerosis MS is an autoimmune inflammatory disorder of the CNS manifesting as acute focal demyelination and axonal loss with limited remyelination and focal sclerotic changes. Recurrent inflammatory events result in the development of chronic multiple sclerotic plaques in the brain and spinal cord. This disease has a lifetime risk of 1 in 400, affects 350,000 people in the United States, and is potentially the most common cause of neurologic disability in young adults [71]. Seven of 27 monozygotic pairs (25.9%) and one of 43 dizygotic pairs (2.3%) were concordant for MS, which suggests there is a genetic predisposition for the development of MS with a contribution of environmental factors [17]. Similar to other
Fig. 69.3 Naïve CD4+ T cells increased and central memory CD4+ T cells decreased at 2 years after high-dose immunosuppressive therapy (HDIT) and autologous hematopoietic cell transplantation (HCT) for multiple sclerosis. In a longitudinal analysis of T-cell subsets after HDIT and autologous HCT, a trend towards reduction of naïve (CD45RA+/CD45RO−/CD27+) CD4+ T cells was observed at 6 months, consistent with an increase of effector memory (EM) phenotypes resulting from homeostatic proliferation in a lymphopenic environment. Central memory (CM; CD45RA−/CD45RO+/ CD27+) CD4+ cells decreased steadily during the follow-up. The proportion of EM (CD45RA−/CD45RO+/CD27−) CD4+ T cells rose significantly at 6 months after HSCT and later declined toward baseline levels after the gradual recovery of the absolute CD4+ T-cell numbers. At the 2-year followup, the frequency of naïve CD4+ T cells had increased 118% compared with pretherapy (p = 0.032). Correspondingly, CM CD4+ T cells had decreased 38% at 2 years after therapy (p = 0.008). The frequencies of the EM CD4+ T cells did not change significantly at the 2-year follow-up compared with the baseline. These data support the concept that there is significant immunomodulation at 2 years after HDIT and autologous HCT, and this may explain the observed durable clinical remissions in a significant proportion of patients with autoimmune disease. Tx, treatment. (Reproduced from J Exp Med 2005; 201: 805–16. Copyright [2005], Rockefeller University Press.)
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Chapter 69 (a)
(b)
Fig. 69.4 Recovery of CD4+CD25bright T-cell frequency after high-dose immunosuppressive therapy (HDIT) and autologous hematopoietic cell transplantation (HCT). (a) The relative number of CD4+CD25bright T cells in 25 healthy controls, eight systemic juvenile idiopathic arthritis (JIA) patients on conventional therapy (Syst-co), and 12 children who received autologous stem cell transplantation (ASCT) for refractory JIA was measured by fluorescence-activated cell sorter staining. The patient represented by open triangles suffered a complete relapse 6 months after ASCT. Since it has been shown that the regulatory CD4+ T cells preferentially reside within the CD4+CD25bright population, only the CD4+CD25bright T cells were analyzed. There is a significant increase in CD4+CD25bright T cells after HDIT and autologous HCT (*p = 0.05; **p = 0.06). (b) Mean and standard error of the mean for the absolute number of CD4+CD25bright T cell counts per microliter (and other CD4+ T cell subsets) in 12 children before and after HDIT and autologous HCT for refractory JIA. An increase in the peripheral blood of CD4+CD25bright T cells after HDIT and autologous HCT is consistent with restoration of the immune regulatory network as a possible mechanism for sustained remission of JIA in a significant proportion of patients. Bef, before; CY, cyclophosphamide; PE, phycoerythrin. (Reproduced from [59], with permission. Copyright [2006], American Society of Hematology.)
suspected autoimmune disorders, subjects with MS have an increased frequency of certain HLA haplotypes that code for the class II molecules (particularly for HLA-DR2) involved in antigen recognition by CD4+ T cells. It is postulated that myelin proteins, such as myelin basic protein, proteolipid protein, and perhaps myelin oligodendrocyte glycoprotein, are targeted by pathogenic CD4+ T cells in patients with MS [72]. The pathology shows a predominant T-cell response both in perivascular spaces and in the demyelinated lesion. Axonal injury is evident not only in the acute inflammatory and the chronic nonenhancing lesions, but also in the normal-appearing white matter. The clinical manifestations of the disease may include loss of vision from optic neuritis, diplopia, sensory loss and paresthesias, vertigo, fecal and/or urinary incontinence, impotence, intellectual decline, paroxysmal pain, recurrent infections, loss of coordination or paralysis. Clinical diagnosis depends on two or more attacks involving noncontiguous anatomic regions separated by at least 1 month or progression of symptoms or signs for more than 6 months [71,73]. About 85% of patients with MS present with relapsing-remitting disease, and over 10 years, about 50% of these patients will develop secondary progressive disease
Table 69.4 Types of clinical course for multiple sclerosis (MS) Relapsing-remitting MS
Secondary progressive MS
Primary progressive MS Progressive relapsing MS
Acute episodes of worsening with recovery and stable periods between relapses; loss of neurologic function is associated with relapse and may or may not recover Previous relapsing-remitting disease with gradual neurologic deterioration with or without acute relapses Gradual continuous neurologic deterioration from onset Progressive disability from symptomatic onset but with superimposed relapses
with cumulative disability (Table 69.4). The other 15% of patients have progressive disease from the onset (primary). The standard for measuring outcome in studies of MS is the Kurtzke Expanded Disability Status Scale and, more recently, the Multiple Scle-
Hematopoietic Cell Transplantation for Autoimmune Diseases
rosis Functional Composite. There is little influence of MS on survival until at least 15 years after onset. At 20 years after onset, patients with MS had 85% of the expected survival. Prognostic factors for worse outcome have been identified and include increased age, short intervals between relapses, a high frequency of relapses after disease onset, and early significant disability. Despite responses to immunomodulating agents, no standard therapy is curative or has been demonstrated to prevent development of a progressive clinical course. Agents that have been demonstrated to be effective as disease-modifying therapies in relapsing-remitting MS include interferon-beta 1a (IFN-β1a), IFN-β1b, glatiramer acetate, mitoxantrone, and natalizumab [74]. Treatment reduced clinical relapse rates by 30–68%, with mitoxantrone and natalizumab being more effective than IFN or glatiramer acetate. These agents, however, remain inadequate to completely prevent relapses and progression. There is no effective therapy for primary or secondary progressive MS. The results of eight clinical trials of HDIT and autologous HCT for MS have been reported from North American and European centers (Table 69.5) [51,63–66,75–77]. Although there was variability in the design of each of these clinical trials, they all included patients with advanced MS and the progressive type of the disease. All patients received high-dose combination chemotherapy or TBI and cyclophosphamide. In seven of the eight clinical trials, the hematopoietic cell grafts were mobilized from the peripheral blood, and all but one of the trials included T-cell depletion with CD34 selection. Overall treatmentrelated mortality in these clinical trials was 2.8% (4 of 145) and the progression-free survival or rate of neurologic stability reported in the individual clinical trials ranged from 36% to 95% at 2–3 years after treatment. Five (3.5%) patients died after progression of disease and further loss of neurologic function or other complications. Although no direct comparisons can be made between the clinical trials regarding outcomes, there did not appear to be any advantage to including TBI in the HDIT regimen. In a report by Saccardi et al., 19 MS patients with high levels of disease activity based on magnetic resonance imaging (MRI) of the brain and sustained clinical deterioration were treated with high-dose combination chemotherapy and ATG (Table 69.5) [63]. MRI of the brain was then done on a scheduled basis every 3–6 months for 2 years. There was a marked reduction of gadolinium-enhancing lesions in the brain after the HDIT regimen, which was sustained to 5 years after treatment (Fig. 69.5). Progression-free survival at 6 years was 95%. After treatment, a statistically significant improvement was achieved in measures of quality of life. The overall results of these clinical trials were comparable to what was reported from the EBMT registry in which treatment-related mortality after HDIT and autologous HCT in 178 patients was 5.3%, and neurologic stability or improvement was observed in 64% of patients at a median follow-up of 42 months [56]. Although MRI after transplant showed a marked reduction in gadolinium-enhancing brain lesions in all the groups studied compared with baseline, it is still uncertain if continued loss of neurologic function observed in some of these progressive patients is the result of a degenerative process or a failure to completely control inflammation related to the autoimmune disease. Randomized clinical trials need to be completed to confirm if there is a therapeutic benefit of HDIT and autologous HCT in this patient population of advanced MS. Clinical trials would also be of interest in patients with less-advanced relapsing-remitting MS to determine whether outcomes could be improved and the progressive type of the disease could be prevented. Systemic sclerosis SSc is an uncommon disabling autoimmune disease that is associated with a high mortality. In studies done in different geographic settings,
1021
the prevalence has ranged from 28 to 286 per million, and the incidence has been reported as ranging from 3.7 to 19.2 per million [78]. SSc is characterized by two major clinical features including a non-inflammatory small vessel vasculopathy, and fibrosis of the skin and multiple internal organs [78]. There is evidence of immune dysregulation with SSc-specific autoantibodies, mononuclear cell infiltrates in several affected organs, and elevated cytokine levels in tissue and serum. The anticentromere antibody is found in the group with limited cutaneous SSc, and the antitopoisomerase I antibody (Scl-70) is found in 30–40% of subjects with diffuse cutaneous SSc. Antinuclear antibodies occur in 95% of subjects overall. Diffuse cutaneous SSc is a more severe manifestation of disease than limited cutaneous SSc. It has a higher mortality and is associated with substantial morbidity as a consequence of digital ischemia/skin ulcerations from the vasculopathy, both truncal and acral scleroderma, interstitial lung disease, hypertensive renal crisis, diffuse gastrointestinal disease, and myocardial involvement. In one study of the natural history of SSc, survival was less than 80% at 2 years, less than 50% at 8.5 years, and under 30% at 12 years [79]. Other studies have confirmed the poor survival and poor-risk features of SSc, especially when there is internal organ involvement [80,81]. Two validated tools for evaluating the degree of scleroderma and measuring the effect of disease on overall function are the modified Rodnan Skin Score (mRSS) and the modified Health Assessment Questionnaire Disability Index (mHAQ) [82]. Immunosuppressive therapies investigated for severe SSc have been inadequate or ineffective. A recent study reported that 12 months of daily oral cyclophosphamide was superior to placebo in slowing the rate of progression of SSc lung disease when evaluated at 12 and 24 months after treatment [83]. Patients also had a modest improvement in the mRSS compared with the placebo group. Cyclophosphamide may be considered a standard of care for individuals with early and severe SSc, particularly those with pulmonary involvement, but the therapeutic benefits are very modest. There are no controlled, prospective clinical trials that demonstrate the efficacy of any other immunomodulatory agents. Important supportive care measures for patients with SSc include angiotensin-converting enzyme inhibitors for the management of renal crisis, and bosentan or other agents for the management of pulmonary hypertension. A limited number of clinical trials of HDIT and autologous HCT for SSc have been conducted. Patients selected for these clinical trials had in general a poor prognosis based on the presence of diffuse cutaneous disease and internal organ involvement. In a single-center study of high-dose therapy with a low-intensity regimen consisting of a single cytotoxic agent only (cyclophosphamide n = 10, melphalan n = 1) and autologous HCT, major or partial responses were observed in eight of 11 patients, but at a median of 18 months, eight patients had not responded or had relapsed (Table 69.6) [61]. Four (36%) patients had died by 18 months after HDIT. In a later study of patients (n = 26) who had already survived at least 6 months after high-dose cyclophosphamide only and HCT, survival was 96% and event-free survival was 64% at 5 years [57]. However, about half of the patients included in this study had diffuse cutaneous disease but without internal organ involvement. In a multicenter study of a more intensive HDIT regimen consisting of TBI and cyclophosphamide in 34 patients with diffuse cutaneous disease and internal organ involvement, 17 of 27 (63%) evaluable patients who survived at least 1 year after HDIT had sustained responses (without progression or disease activation) at a median follow-up of 4 years [52]. Patients with sustained responses had required no immunebased treatment after HDIT. There was a major improvement in the degree of scleroderma as measured by the mRSS, and in overall function as measured by the mHAQ at final evaluation (Fig. 69.6). Skin biopsies confirmed that the improvement in skin score was associated with a
14
19
26
21
14
Saiz [76]
Saccardi [63]
Nash [51]
Burt [64]
Samijn [65]
SP (9) RR (5) SP (15) RR (4) SP (17) PP (8) RR (1) SP (14) PR (6) RR (1) SP
SP
SP (19) PP (14) RR (2) SP
6.0 (5.0–6.5)
7.0 (3.0–8.5)
6.5 (5.5–7.5) 6.5 (6.0–7.5) 6.0 (4.5–6.5) 6.5 (5.0–6.5) 7.0 (5.0–8.0)
6.0 (4.5–8.0)
Median EDSS* (range)
CD34 selection
BEAM + ATG TBI/CY + ATG
TBI/CY
TBI/CY + ATG
G-CSF + CY G-CSF + prednisone
G-CSF + CY
None (marrow)
CD34 selection
BCNU/CY + ATG
G-CSF + CY
CD34 selection
None
CD34 selection
CD34 selection + monoclonals
BEAM ± ATG
G-CSF + Cy
CD34 selection
CD34 selection
BU/CY
BEAM + ATG
G-CSF/GM-CSF + CY
T-cell depletion
G-CSF only
High-dose therapy
Mobilization
36 (7–36)
24 (12–60)
27 (2–47)
36 (12–72)
37 (19–55)
8 (1–18)
22 (17–30)
35 (3–67)
Follow-up median months (range)
0/1
0/2
1/1
0/0
0/0
0/0
2/0
1/1†
Treatment/disease-related mortality (n)
PFS 36%
62% stable or improved
PFS 95% (6 years) 76% stable or improved
PFS 86%
90% stable or improved
PFS 40%
PFS 81% at 5 years
Clinical result
ATG, antithymocyte globulin; BEAM, BCNU, etoposide, cytosine arabinoside, melphalan; BU, busulfan; CY, cyclophosphamide; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–macrophage colony-stimulating factor; PFS, progression-free survival; PP, primary progressive; PR, progressive relapsing; RR, relapse-remitting; SP, secondary progressive; TBI, total body irradiation. * In brief, the functional levels of the Expanded Disability Status Scale (EDSS) are graded from 0 to 10 points and include changes in increments of 0.5 points. An EDSS score of 0 indicates a normal neurologic examination in all functional systems. An EDSS score of 10 indicates death from MS. Most of the MS patients who entered these clinical trials of HDIT and autologous HCT had an EDSS score of 5.0–8.0. In general, the function of patients at these different EDSS scores are: 5.0 Ambulant 200 m without aids, difficulty to work full day, functional systems grade 5. 6.0 Intermittent or unilateral walking aid for 100 m. 7.0 Wheelchair-bound (walking 100,000/mm3), and two patients obtained a partial response at a median of 27 (9–41) months after HDIT [107]. An interesting clinical trial was performed in patients with recent-onset DM. Fourteen of 15 patients had prolonged periods of insulin independence after HDIT [108]. The durability of these responses on longer follow-up and safety of the procedure will determine if this approach with its potential for more toxicity could be considered as an acceptable treatment for DM.
Allogeneic HCT for autoimmune diseases: clinical experience Allogeneic HCT may cure severe autoimmune diseases by replacing the “diseased” autoreactive immune compartment with one that is not autoreactive (although potentially alloreactive). The primary risks of allogeneic HSCT are the morbidity and morality associated with delayed immune reconstitution and graft-versus-host disease (GVHD). Although the risk of complications and treatment-related mortality is greater than with autologous HCT, there may be a greater potential for sustained remissions of severe autoimmune diseases after allogeneic HCT, and therefore improved overall outcome. Survival after allogeneic HCT for nonmalignant hematologic disorders or good-risk hematologic malignancies like chronic myelogenous leukemia in the chronic phase has ranged from 85% to 95%. The best outcomes were observed in young patients transplanted from an HLA-matched donor. No clinical trials have yet been conducted of allogeneic HCT for which an autoimmune disease was the primary indication, but a small number of cases have been reported. The largest experience has been in patients with autoimmune cytopenias [109–112]. Of the seven evaluable patients reported from the EBMT registry, five were alive and in remission at a median follow-up of 41
months [109]. One patient with Evan’s syndrome in the report from the EBMT registry had disease progression and died. The second patient died from treatment-related complications after HCT from an HLAhaploidentical donor. Of the three case reports of Evan’s syndrome in the literature, one patient relapsed and died, and the other two died of transplant-related complications in complete remission [110–112]. There are six cases reported of allogeneic HCT for connective tissue diseases such as SSc, overlap syndrome, and RA [113–117]. Two patients with SSc had high-dose conditioning, one of whom was alive at 5 years in disease remission with resolution of the dermal fibrosis and scleroderma lung. Four patients received reduced-intensity conditioning regimens. All patients were alive at last follow-up and in remission. Three of these patients had stable mixed hematopoietic chimerism and no history of GVHD. Regulatory immune mechanisms associated with the establishment of mixed hematopoietic chimerism may be responsible for inducing remission of the autoimmune disease. Conclusions regarding the therapeutic role of allogeneic HCT for severe autoimmune diseases await the results from clinical trials conducted in a carefully selected group of patients.
Autoimmunity after HCT Autoimmune disorders have been observed to occur infrequently after both autologous and allogeneic HCT for hematologic malignancies. This risk needs to be considered for those patients for whom the primary indication for HCT is an autoimmune disease. The mechanisms for autoimmune disorders after HCT are not well understood but may relate to the disruption of immune regulatory pathways as a result of induction to a lymphopenic state with conditioning and then homeostatic expansion of lymphocytes during reconstitution of the immune system [118]. The development of an autoimmune disease after HCT likely depends on predisposing factors and the balance between reconstituting autoreactive and regulatory lymphocytes. The incidence of an autoimmune hemolytic anemia was observed to be 2.3% and 4.4% with onset up to 18 months after allogeneic HCT in two case series of 299 and 272 patients, respectively. There have been additional case reports of autoimmune disorders after both autologous and allogeneic HCT including other cytopenias, thyroid disease, acquired hemophilia, and myasthenia gravis [119]. For patients treated for an existing autoimmune disorder, there may be a predisposition for a new secondary autoimmune disorder to occur [52,70]. After allogeneic HCT, there have also been case reports of adoptive transfer of autoimmune disorders [53].
Conclusion The common autoimmune diseases likely result from a genetic predisposition and an environmental trigger. There is a high rate of disease remissions after HDIT and autologous HCT, and a significant proportion are sustained after 4–5 years. The rate and durability of response may depend on the intensity of the HDIT regimen and the type of autoimmune disease being treated. Randomized clinical trials comparing HDIT and autologous HCT to standard therapy or “best clinical practice” are now being conducted in specific autoimmune diseases. The experience with allogeneic HCT as a treatment for autoimmune diseases is still limited but promises to be highly effective. The risks of increased morbidity and mortality from GVHD have delayed clinical trials of this approach. Carefully selected patients with active autoimmune disease that is life-threatening or threatening critical organs and refractory to standard treatment should be considered as candidates for clinical trials of allogeneic HCT or HDIT followed by autologous HCT.
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70
Vinod Pullarkat & Stephen J. Forman
Hematopoietic Cell Transplantation for Rare Hematologic Malignancies
Introduction In this chapter, we review the results of autologous and allogeneic hematopoietic cell transplantation (HCT) for a variety of rare hematologic malignancies that, in our opinion, would be rarely encountered outside of specialized centers, and for which HCT has been performed (Table 70.1). As would be expected with rare disorders, there is a paucity of prospective well-designed trials, and most published literature is limited to single case reports, small case series or retrospective analyses that include heterogeneous groups of patients. We have attempted to review the published literature and make recommendations based on the preponderance of evidence. Recognizing the bias towards reporting only positive results that is inherent in single case reports, we have used case series and retrospective analysis of registry data rather than single case reports as the basis for our recommendations whenever possible. It is unlikely that prospective studies to test the role of HCT that contain adequate numbers of patients will ever be performed for the extremely rare entities discussed below. However, there is a need for such trials preferably performed by large cooperative groups for the less uncommon malignancies discussed here.
Rare myeloid malignancies Langerhans cell histiocytosis Langerhans cell histiocytosis (LCH) refers to a group of clinical syndromes characterized by clonal Langerhans cell proliferation. These syndromes have historically been designated by different names based on the pattern of organ involvement. These include histiocytosis X, Letterer–Siwe disease, and Hand–Schüller–Christian disease [1]. Although typically a disorder of infants and children, LCH can also occur in adults. Age, pattern of organ system involvement, and organ dysfunction are the most important prognostic factors. In general, children under 2 years of age and adults over 65 years of age do poorly [2–4]. While single-system LCH usually involving the skeleton or skin is an indolent disease, chemotherapy-resistant multisystem LCH (MSLCH) especially involving organs like the bone marrow, liver, spleen, and lungs has an extremely poor prognosis [3,5]. Various case reports have described successful allogeneic HCT for patients with MS-LCH [6–10]. One of the initial reported patients who underwent allogeneic HCT for chemotherapy-resistant disease remained
in complete remission (CR) 12 years later, showing that long-term disease-free survival (DFS) is possible using this approach [11]. The French Langerhans Cell Study Group has reported results of eight patients with refractory MS-LCH transplanted at six institutions. Three patients underwent autologous HCT while five underwent allogeneic HCT. Of the three patients who underwent autologous HCT, two died of relapse and one was alive and in CR 7 years later. Of the five patients who underwent full-intensity allogeneic HCT, two died of regimenrelated toxicity and one died of sepsis 23 months after HCT, while two were alive and in CR 12 years and 21 months, respectively, after HCT, again demonstrating the ability of allogeneic HCT to achieve durable remissions in refractory MS-LCH [12]. The transplant-related mortality of full-intensity allogeneic HCT is high due to the heavy prior exposure of these patients to chemotherapy, as well as involvement of vital organs like the lungs and liver [13]. In an attempt to decrease the treatment-related mortality of allogeneic HCT, Steiner et al. have reported the results of reduced-intensity allogeneic HCT using fludarabine-based conditioning in nine pediatric patients with high-risk LCH. Graft sources included matched siblings, matched unrelated donors, mismatched unrelated donors, and haploidentical parental donors. The three patients who underwent haploidentical HCT underwent second transplants for graft failure. After a median follow-up of 390 days, seven of the nine patients were alive without evidence of disease, which is remarkable for this group of heavily treated high-risk patients [13]. Interestingly, one patient who had engraftment failure with autologous reconstitution after a second allogeneic haploidentical HCT remained in remission 770 days after the first HCT [13], suggesting that autologous HCT may be a viable option for patients who lack suitable donors or are not candidates for allogeneic HCT. Two patients with MS-LCH who underwent successful autologous HCT have been reported. One remained in CR over 4 years and the other at 10 months after HCT [9,14]. Based on the above data and given the poor prognosis of refractory MS-LCH, it seems preferable to perform allogeneic HCT early in the course of disease in patients who have matched donors. Reducedintensity conditioning may be better in patients who have impairment of vital organ function or heavy prior exposure to chemotherapy. Autologous HCT may be an option for patients who lack matched donors. HCT is a promising treatment modality for treatment of MS-LCH, and large clinical trials testing both autologous and allogeneic HCT early in the disease course are warranted. Systemic mastocytosis
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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Systemic mastocytosis (SM) is characterized by infiltration of the bone marrow and other organs by neoplastic mast cells. Activating mutations
Hematopoietic Cell Transplantation for Rare Hematologic Malignancies Table 70.1. Selected rare hematologic malignancies for which HCT has been performed • • • • • • • • • • • • • • • •
Langerhans cell histiocytosis Systemic mastocytosis Hypereosinophilic syndrome Acute megakaryoblastic leukemia Acute erythroleukemia Chronic myelomonocytic leukemia Adult T-cell leukemia/lymphoma Natural killer cell leukemia/lymphoma Mycosis fungoides Sézary syndrome Angioimmunoblastic T-cell lymphoma Primary central nervous system lymphoma Hepatosplenic T-cell lymphoma (gamma/delta T-cell lymphoma) T-cell prolymphocytic leukemia Subcutaneous panniculitic T-cell lymphoma Hematodermic neoplasm (blastic natural killer cell lymphoma)
in the KIT gene (most commonly D816V) are almost invariably present in these neoplastic mast cells. The World Health Organization (WHO) classification recognizes various clinicopathologic SM syndromes, including indolent SM, SM with associated clonal hematologic nonmast cell lineage disease (SM-AHNMD), aggressive SM, and mast cell leukemia (MCL) among others [1,15]. Aggressive SM and MCL have a dismal prognosis [15,16]. While indolent SM has a prolonged clinical course as its name suggests, the clinical course in the case of SMAHNMD is dictated by the underlying malignancy, which most often is acute myeloid leukemia (AML), myelodysplasia or a myeloproliferative disorder [17,18]. A specific association exists between SM and t(8;21) AML [18–20]. The HCT experience in SM is very limited. Nakamura et al. have transplanted three SM patients from human leukocyte antigen (HLA)identical siblings using reduced-intensity conditioning with fludarabine and cyclophosphamide (CY). One patient had MCL, another had SM with myelodysplasia (SM-AHNMD), and the third had aggressive SM. All patients achieved complete T-cell engraftment. Although there was clinical and pathologic evidence of a graft-versus-mast cell effect, this did not translate into durable remissions, and all three patients eventually relapsed. In one patient, there was significant morbidity from mast cell mediator release at day 3 after transplantation and concurrent with flareups of acute graft-versus-host disease [21]. Another report describes a patient with MCL who underwent a matched unrelated donor HCT after standard busulfan and CY (BU/CY) conditioning. The number of residual recipient bone marrow mast cells after HCT could be lowered by administration of a donor lymphocyte infusion (DLI), thereby suggesting a graft-versus-mast cell effect. This patient remained in clinical remission over 3 years after HCT [22]. Patients with SM-AHNMD fare poorly compared with patients with the underlying hematologic disease alone [17]. This is especially true of cases where the associated hematologic disorder is t(8;21) AML, an otherwise low-risk AML subset. However, this is not surprising given the fact that KIT mutations are associated with a poor prognosis in t(8;21) AML [23,24]. Durable remissions have been described in SM– AML with t(8;21) translocation after allogeneic HCT even though recipient bone marrow mast cells carrying t(8;21) were detected a year after allogeneic HCT [20,25]. Prolonged remission after a second allogeneic HCT following high-dose conditioning has also been reported in a case of SM with t(8;21) AML that relapsed after an initial reduced-
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intensity sibling donor HCT [19]. In a reported case of SM associated with myelodysplastic syndrome, mast cell infiltration of bone marrow and liver persisted at 8 months following allogeneic HCT, even after normal hematopoiesis had been restored. This patient also developed venous thrombosis in the post-transplant period, which was attributed to mast cell mediator release [26]. Another patient who was alive 6 years after allogeneic HCT for SM with associated chronic myelomonocytic leukemia (CMML) has also been reported [18]. Given the extremely grim prognosis of patients with aggressive SM and MCL and some evidence of graft-versus-mast cell effect, it appears reasonable to test the efficacy of allogeneic HCT for these subsets of SM, preferably in the context of a clinical trial. Newer kinase inhibitors like dasatinib are capable of inhibiting the D816V KIT and mutation may have a role in conjunction with allogeneic HCT [27]. It is possible that more intense conditioning may also be beneficial [19]. Special approaches may be needed to manage morbidity from mediator release during the peritransplant period in patients with a large mast cell burden [21,26]. For patients with SM and t(8;21), it appears that allogeneic HCT in first CR (CR1) is the best option if a suitable donor is identified based on the limited data available. Recipient-derived bone marrow mastocytosis may persist after allogeneic HCT and does not appear to have clinical significance [20,25]. The efficacy of HCT for SM associated with other hematologic malignancies is unknown at present. It is unlikely that HCT will have a role in indolent SM, especially with development of targeted therapies that inhibit D816V KIT mutation. Hypereosinophilic syndrome Hypereosinophilic syndrome (HES) refers to a heterogeneous group of disorders that are characterized by peripheral blood eosinophilia and a propensity to cause organ damage, especially cardiac dysfunction from endomyocardial fibrosis. Criteria for diagnosis of HES have been defined [28,29]. Myeloproliferative and lymphoproliferative variants of HES are now recognized. A subset of patients, almost exclusively male, with the myeloproliferative variant carry an interstitial deletion resulting in a FIP1L1-PDGFRa (FIP1-like 1–platelet-derived growth factor receptoralpha) fusion and are exquisitely sensitive to imatinib [30–32], while the lymphoproliferative variant is associated with clonal T-cell expansion [33]. The major cause of morbidity and mortality in HES is cardiac dysfunction [28]. The results of allogeneic HCT for HES are limited to case reports. Hence the overall results of allogeneic HCT for this disorder cannot be determined. A number of these reports have been summarized by Cooper et al. [34]. The majority of transplants have been performed from HLA-identical siblings after full-intensity conditioning. A few cases have undergone reduced-intensity conditioning [34,35]. DFS after up to 6 years of follow-up and reversal of myelofibrosis have been reported [34,36]. These reports of long-term remission after allogeneic HCT may be suggestive of the curative potential of allogeneic HCT for this disorder. An interesting observation is that eosinophilia can recur in some cases even when 100% of the bone marrow and peripheral blood cells are of donor origin [34,37,38]. This cannot be considered failure of HCT and usually resolves spontaneously. It is not clear if these cases had the lymphoproliferative variant of HES with clonal T-cell expansion. High interleukin-5 levels were detected in two patients, which suggests that abnormal T cells may persist in lymphoid organs (despite full donor marrow engraftment) and lead to eosinophilia, which probably resolves with complete donor engraftment in lymphoid organs [34,38]. Our understanding of the biology of HES is rapidly evolving. Newer classification based on cytogenetic and molecular abnormalities and
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improved definition of prognostic factors may help better predict patients who are not imatinib responsive and who are more likely to develop end-organ damage. The results of imatinib therapy for FIP1L1PDGFRa-positive patients are excellent, while FIP1L1-PDGFRanegative cases rarely have durable responses to imatinib [39]. Prognosis for FIP1L1-PDGFRa-negative patients who develop end-organ dysfunction is poor, and allogeneic HCT should be strongly considered in suitable patients who have early signs of end-organ damage and have failed prednisone and a second-line agent such as hydroxyurea or alpha-interferon. Although there are reports of improvement in cardiac function after allogeneic HCT in patients with HES and cardiac dysfunction [40], the nonrelapse mortality (NRM) in this situation is likely to be high. Acute megakaryoblastic leukemia Acute megakaryoblastic leukemia (AMKL) accounts for about 1% of AML in adults and about 5–10% of AML in children, with a higher incidence in children with Down’s syndrome [41–43]. The disease has a bimodal age distribution with peaks in early childhood (under 3 years) and adulthood [42]. Bone marrow fibrosis is a prominent feature of this disorder, making AMKL the most common cause of “acute myelofibrosis” [1,41]. AMKL may occur de novo or arise from an underlying myeloproliferative or myelodysplastic syndrome. Although 50% of adult patients achieve a CR with standard AML induction chemotherapy, long-term survival rates are dismal with conventional consolidation chemotherapy [41,44–46]. Hence HCT has been used as consolidation therapy in an attempt to improve the outcome. Single case reports have been published describing reversal of bone marrow fibrosis and durable remissions after allogeneic HCT [47–50]. One of the early reports of allogeneic HCT for AMKL describes a 28year-old woman with “acute malignant myelosclerosis” who was transplanted from her histocompatible brother (Fig. 70.1) [49]. This patient continues to be followed at the City of Hope National Medical Center and remains in remission over 27 years after HCT. A retrospective analysis of the results of HCT for de novo AMKL in CR1 has been performed by the European Group for Blood and Marrow Transplantation (EBMT). At 3 years after allogeneic HCT, the results in children and adults respectively were: leukemia-free survival (LFS) of 66% and 46%; overall survival (OS) of 82% and 43%; and NRM of 0% and 26%. The results of autologous HCT (children and adults respectively) were: LFS of 52% and 27%; OS of 61% and 30%; and NRM of 3% and 8% [51] (Fig. 70.2). Athale et al. have reported their single-institution experience treating 41 patients with childhood AMKL that included six patients with secondary AMKL and six with Down’s syndrome. The 2-year event-free survival (EFS) was 26% for allogeneic HCT compared with 0% for chemotherapy alone. The results of allogeneic HCT were significantly better when performed in remission than with persistent disease (2-year EFS 46% versus 0%). In both of the above-mentioned studies, the outcomes were better for children with Down’s syndrome, who had an estimated 2-year EFS of 83% in the latter study [45]. Based on above data, allogeneic HCT from matched sibling or matched unrelated donors should be the consolidation treatment of choice in adults with AMKL who achieve CR. In children, autologous transplant in CR1 is an option in patients who lack a matched sibling donor, and there are reports of prolonged survival after this approach. The outcome of autologous transplant in adults is very poor. At present, allogeneic HCT cannot be recommended for patients with resistant disease, who should be treated with investigational approaches. There appears to be no advantage for allogeneic HCT over autologous HCT in patients with Down’s syndrome and AMKL.
Acute erythroleukemia Acute erythroleukemia (AEL) is an uncommon form of AML accounting for about 4.5% of all cases of AML [52]. AEL may occur de novo or secondary to an underlying myelodysplastic or myeloproliferative disorder, or after previous chemotherapy [53,54]. AEL has a poor prognosis compared with other AML subtypes, with a median OS of only 9 months in one retrospective study [54]. Like AMKL, AEL has a dismal prognosis with conventional chemotherapy [54,55]. The EBMT has reported the results of 103 autologous and 104 HLAidentical sibling allogeneic HCTs done for de novo AEL in CR1. For allogeneic HCT, the 5-year LFS, relapse incidence, and NRM were 57 ± 5%, 21 ± 5% and 27 ± 5%, respectively, while the corresponding figures for autologous HCT were 26 ± 5%, 70 ± 6% and 13 ± 4%. In multivariate analysis, age remained the most important prognostic factor for both allogeneic and autologous HCT [56]. It must be noted that the median age of patients in this study (40 years for autologous HCT and 36 years for allogeneic HCT) is lower than the median age of patients with AEL, which was 59 years in one series [54]. In a smaller study, Killick et al. have reported the outcome of 27 patients with AEL, which included eight adults with secondary AEL and five children. Among patients with de novo AEL, 95% achieved CR with induction chemotherapy compared with only 57% of patients with secondary AEL. Eighteen of 22 (82%) patients achieving CR underwent consolidation by HCT (15 allogeneic, and three autologous). The median survival of 2.9 years for de novo AEL was not different from that of their matched controls with other forms of AML. The overall relapse rate of only 35% suggests a benefit for HCT consolidation in CR1 [57]. Therefore, allogeneic HCT is presently the treatment of choice for patients with AEL who are in CR1. Autologous HCT may be an option for patients in CR1 who lack matched donors. The results of allogeneic HCT for refractory and relapsed disease remain unknown at the present time.
Chronic myelomonocytic leukemia CMML is a clonal stem cell disorder characterized by peripheral blood monocytosis that shares features with both myelodysplastic and myeloproliferative disorders. The median age at diagnosis is 65–75 years, with a male predominance. Criteria for diagnosis of CMML have been defined in the WHO classification [1]. Since CMML was previously grouped with the myelodysplastic syndromes, patients who underwent allogeneic HCT for CMML were included in reports of HCT results for myelodysplastic syndromes. However, CMML has distinct clinicopathologic features that warrant its consideration as a separate entity. Although CMML includes a heterogeneous group of patients with varying rates of disease progression, the overall prognosis is poor, with a median survival of only 12 months in one study [58]. CMML is not curable with conventional chemotherapy, and the response to targeted therapies is poor except in the small subset of patients who carry a PDGFRb fusion oncogene and are sensitive to imatinib [59]. Recognizing this dismal prognosis, allogeneic HCT has been tried as a potentially curative option. The EBMT has reported the outcome of 50 patients who underwent allogeneic HCT for CMML. Forty-three patients received transplants from related donors, and seven patients received unrelated donor transplants. The median age of the patients was 44 years. Conditioning was total body irradiation (TBI) based in 26 patients and chemotherapy based in the rest. After a median follow-up of 40 months, the 5-year estimated OS and DFS and probability of relapse were 21%, 18%, and 49%, respectively [60]. A report from the Mayo Clinic describes 17 patients
Hematopoietic Cell Transplantation for Rare Hematologic Malignancies
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Fig. 70.1 (a–d) Hematoxylin and eosin stains of bone marrow biopsies from diagnosis and days 15, 100, and 360, respectively, after allogeneic hematopoietic cell transplantation (HCT) in a patient with “acute myelofibrosis.” Progressive restoration of normal hematopoiesis is seen. (e–h) Corresponding reticulin stains show total clearing of reticulin fibrosis after allogeneic HCT. (Reproduced from [49], with permission.)
who underwent allogeneic HCT (14 related and three unrelated) for CMML. Eighteen percent of patients were alive and disease free at a median follow up of 34.5 months, and the NRM at 3 years was 41%. CRs lasting 15 months could be induced with DLI in one relapsed patient and in another with mixed chimerism, suggesting a graft-versusleukemia (GVL) effect [61]. Kerbauy et al. have reported better results for a cohort of 43 patients transplanted at the Fred Hutchinson Cancer Research Center in Seattle. Twenty-two of these patients received HCT from unrelated donors. The median age of the patients was 48 years. BU/CY was the most common conditioning regimen used (15 patients). The estimated 4-year OS and RFS was 41%, and relapse incidence was 23%. Although these authors applied various prognostic-scoring systems, including the MD Anderson prognostic score [58], in an attempt to predict HCT outcome, comorbidity was the only significant predictor for OS (Fig. 70.3) [62]. Laport et al. have recently reported their experience with seven patients with CMML who underwent allogeneic HCT after conditioning with fludarabine and a single 2 Gy dose of TBI [63]. Six of these seven patients have subsequently died of relapsed disease (Laport, personal communication).
Allogeneic HCT from matched siblings or matched unrelated donors should be an early consideration for patients with CMML who are suitable candidates. It must be noted that although the median age of patients in the above studies is much lower than the median age at diagnosis of CMML patients as a whole, the NRM for allogeneic HCT is quite high. Comorbidity is a strong predictor of outcome, and must be an important consideration in selecting patients for allogeneic HCT. The majority of reported patients have undergone full-intensity conditioning, which most likely contributed to the high NRM. Despite the initial discouraging result with reduced-intensity conditioning [63], the optimum conditioning intensity needs to be further explored in order to improve the results of allogeneic HCT as a curative option in CMML. HCT for CMML is also discussed in Chapter 57.
Rare lymphoid malignancies Adult T-cell leukemia/lymphoma Adult T-cell leukemia/lymphoma (ATLL) refers to a specific clinicopathologic syndrome characterized by prominent skin involvement,
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Fig. 70.2 (a) Leukemia-free survival (LFS) after autologous hematopoietic cell transplantation (HCT) in adults and children with de novo acute megakaryoblastic leukemia (AMKL) in first complete remission (CR1). (b) LFS after allogeneic HCT in adults and children with de novo AMKL in CR1. (Reproduced from [51], with permission).
hypercalcemia, and an acute leukemic or lymphomatous presentation. ATLL is specifically associated with infection with type I human T-cell lymphotropic retrovirus (HTLV-1), which is clonally integrated into the genome of the malignant T cells [1]. Although ATLL is endemic in parts of Japan and the Caribbean, cases also occur in the United States, especially in the south-east [1,64]. Chronic or smoldering forms of the disease do occur and have a more indolent course compared with the acute or lymphoma types [1,65]. Not surprisingly, most of the experience in treating ATLL comes from Japan. The results of intensive combination chemotherapy are poor, with a median survival of only 8.5 months in one study and 13 months in another [66,67]. The results of autologous HCT are similarly poor. In a review of eight reported cases, there were no long-term survivors [68]. Hence, allogeneic HCT has been tried with some success. In the largest series reported, Fukushima et al. have reported results of 40 patients with acute or lymphoma types of ATLL who underwent allogeneic HCT at seven institutions in Japan. All but one patient received high-dose conditioning. Twenty-seven patients were transplanted from matched related donors, five from mismatched related donors, and eight from matched unrelated donors. Of the 36 donors whose HTLV-1 status was known, nine were HTLV-1 positive. The estimated 3-year OS, relapse-
Fig. 70.3 (a) Relapse-free survival of 43 patients with chronic myelomonocytic leukemia (CMML) who underwent allogeneic hematopoietic cell transplantation (HCT). Eighteen patients were alive without relapse; five of the 18 had been followed up for 9.6–14.1 years after transplantation and are indicated as censored at 9 years (censored patients are indicated by tick marks). (b) Probability of survival according to the HCTspecific comorbidity index. Patients with scores of 0–2 (solid line) were compared with patients with scores of 3–6 (dotted line). One patient could not be scored because of incomplete data. Four patients with scores of 0–2 were alive at 9.6–14.1 years, and one patient with a score of 3–6 was alive at 12.2 years. Patients indicated as censored at 9 years. (Reproduced from [62], with permission.)
free survival (RFS), and disease relapse rate were 45.3%, 33.8%, and 39.3%, respectively (Fig. 70.4). Of 10 patients that relapsed, three were able to achieve remission by reduction in immunosuppression, suggesting a GVL effect [69]. Another study by Kami et al. with shorter follow up and smaller number of patients showed similar overall results. These investigators performed allogeneic HCT in 11 patients with ATLL. Ten patients received grafts from related donors and one from an unrelated donor. Two donors were seropositive for HTLV-1. Nine patients received highdose conditioning. Four patients remained alive and disease free at a median follow up of 25 months, and the rest succumbed to HCT-related complications. In two patients who had recurrence, CR could be reinduced by withdrawal of immunosuppression and DLI [70]. In another series of 10 cases who underwent high-dose conditioning and allogeneic HCT (nine sibling donors and one unrelated donor), five remained disease free at a median follow-up of 31.5 months [71]. Allogeneic HCT
Hematopoietic Cell Transplantation for Rare Hematologic Malignancies
appears to be the only currently available therapy for ATLL that offers a prospect of long-term survival. Donor-derived ATLL has been reported in a patient who underwent allogeneic HCT from his HLA-matched brother who was an HTLV-1 carrier [72]. Using a tumor-specific polymerase chain reaction technique, it was later shown that the relapse originated from donor T cells that were already infected in the donor [73]. Thus, the immunosuppression of the recipient appears to have facilitated the transformation of HTLV-1-infected donor T cells to frank T-cell leukemia/lymphoma. The precise incidence of donor-derived relapse is not known, and therefore it is not clear at present if HTLV-1-seronegative unrelated donors should be preferred over seropositive matched siblings.
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Fig. 70.4 (a) Estimated 3-year overall survival (OS) of 40 patients who underwent allogeneic hematopoietic cell transplantation (HCT) for adult Tcell leukemia/lymphoma (ATLL). (b) Estimated 3-year relapse-free survival (RFS). (c) Estimate 3-year relapse risk (RR). (Reproduced from [69], with permission.)
Natural killer (NK) cell neoplasms include the extranodal NK/T-cell lymphoma, nasal type, and aggressive NK-cell leukemia subtypes described in the World Health Organization classification. Extranodal NK/T-cell lymphomas have a predilection to arise in the upper aerodigestive tract, but may also involve other sites such as the skin, gastrointestinal tract, and testes. Cases with disseminated disease often have significant comorbidity from cytokine-mediated phenomena like hepatic dysfunction and hemophagocytic syndrome (HPS). Rare cases may have a cytotoxic T-cell phenotype and hence the designation NK/T-cell lymphoma. NK-cell neoplasms are strongly associated with Epstein–Barr virus (EBV), which may have a role in their pathogenesis. There is marked geographic variation in the incidence of NK-cell neoplasms due to their strong predilection for Asian individuals and the indigenous populations of Mexico, Central and South America [1,74]. The so-called blastic NK-cell lymphoma (CD4+CD56+ hematodermic neoplasm) is now known to be a malignancy of plasmacytoid dendritic cells and therefore is not a true NK-cell neoplasm [75]. In the case of extranodal NK/T-cell lymphoma, only about a third of patients with localized disease become long-term survivors with the conventional approach of radiation and combination chemotherapy [76]. Locoregional and systemic failures are common, and patients with relapsed disease or systemic disease at presentation have a dismal outcome [77,78]. The prognosis is even worse for aggressive NK leukemia, with a median survival of only 2 months in one study [79]. Hence, there has been interest in using HCT to improve the poor outcome of NK-cell neoplasms. Despite not being an extremely rare neoplasm in some geographic areas, systematic prospective trials to evaluate the efficacy of HCT for NK-cell neoplasms have not been performed. Anecdotal reports describe success with autologous and allogeneic HCT even for advanced disseminated disease [80–84]. In the largest series of autologous HCTs reported, Au et al. have reported their experience with 18 consecutive cases of nasal type NK/T-cell lymphoma treated at two institutions in Hong Kong. Eight cases were treated in CR1, five in second CR (CR2), and five with refractory disease. Five patients had extranasal disease before autologous HCT. The most common initial treatment used was combination chemotherapy and radiotherapy. Two patients died of treatment-related causes, nine patients relapsed, and seven remained in CR. All patients with active or disseminated disease at time of HCT died early of relapse or disease progression. The actuarial survival for the whole cohort was 39% after 6 months. Disease relapse beyond 6 months was not seen. These results suggest that prolonged survival is possible with autologous HCT in some patients with extranodal NK/T-cell lymphoma if transplanted in CR [85]. The Japanese NK-cell Tumor Study Group has analyzed outcome of 40 patients who underwent HCT from among 228 patients who were diagnosed with NK-cell lymphoma between 1994 and 1998. This was a
Chapter 70
Mycosis fungoides and Sézary syndrome Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma, is a neoplasm of CD4+ T cells that home to the skin. The initial stages of MF are characterized by patch or plaque lesions and typically last for many years. In the late stages, patients develop skin tumors and extracutaneous dissemination to lymph nodes and visceral organs [1,89,90]. Although patients with the early stages of MF often
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heterogeneous patient group including patients with nasal-type NK lymphoma (n = 22), blastic NK-cell lymphoma (n = 11), aggressive NK-cell leukemia (n = 3), and NK-cell precursor acute leukemia (n = 4). Twentyfive patients underwent autologous HCT, and the remaining 15 underwent high-dose conditioning and allogeneic HCT, all except one from matched sibling donors. Overall, there was a highly significant survival advantage for patients who received HCT compared with patients who did not undergo HCT: 40% versus 25% with a median follow up of 51 months and 32 months, respectively. The outcome of HCT was better for patients who were transplanted in CR. More importantly, during the follow-up period, no patient died later than 2 years after HCT, in contrast to patients who did not receive HCT, thereby demonstrating the durability of HCT-induced remissions. The long-term survival and relapse probabilities for autologous versus allogeneic HCT were 47% versus 29% and 17% versus 29%, respectively. However, these differences were not statistically significant. There was significantly higher NRM in allografted patients compared to those who received autologous HCT (48% versus 4%). The OS was better for HCT compared with conventional therapy, both for extranodal NK/T-cell lymphoma and for the other subtypes included in this study. As the authors themselves have cautioned, being a retrospective analysis, it is quite possible that patients with better performance status may have been selected to undergo HCT and thereby account for the better results of HCT [86]. Murashige et al. have reported results of allogeneic HCT for 28 patients with NK-cell neoplasms. The majority (n = 22) had extranodal NK/T-cell lymphoma. Twelve patients were chemosensitive and 16 were chemorefractory at the time of transplant. Twenty-three patients received grafts from matched related donors, and 23 underwent high-dose conditioning. With a median follow-up of 34 months, the 2-year progressionfree survival (PFS) and OS were 34% and 40%, respectively (Fig. 70.5). Patients who did not relapse within 10 months after HCT remained free of disease. In multivariate analysis, hematopoietic cell source (bone marrow versus peripheral blood) and histology (extranodal NK/T-cell lymphoma versus others) were risk factors for PFS (relative risk 3.03 and 3.94, respectively). These results are good considering the fact that 19 of the 28 patients had active disease at the time of HCT [87]. A similar OS rate was seen for 12 patients with NK-cell lymphoma who underwent unrelated donor HCT through the Japan Marrow Donor Program [88]. Based on the above evidence, for extranodal NK/T-cell lymphoma with localized disease, autologous HCT can be recommended as a consolidation therapy for patients who enter CR after up-front radiation therapy. Long-term survival has been reported with this approach, and NRM is expected to be low. Autologous HCT has no role in refractory or disseminated nasal-type extranodal NK/T-lymphoma or aggressive NK-cell leukemia, and these patients should be strongly considered for allogeneic HCT since this is the only therapy known to result in longterm survival in this group of patients. In the case of systemic NK-cell neoplasms, the major obstacle to allogeneic HCT appears to be the comorbidity from paraneoplastic phenomena such as hepatic dysfunction. Therefore, better strategies for the initial management are needed to realize the full curative potential of allogeneic HCT. The existence of a GVL effect after allogeneic HCT has been suggested [80].
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Fig. 70.5 (a) Progression-free survival (PFS) and (b) overall survival (OS) of 28 patients with natural killer cell neoplasms who underwent allogeneic hematopoietic cell transplantation (HCT). PFS was 34% and OS was 40% at 2 years. Median follow-up was 34 months. Broken lines indicate 95% confidence intervals. (Reproduced from [87], with permission.)
respond to current therapies that include retinoids, alpha-interferon, photopheresis, monoclonal antibodies, and chemotherapy among others, these therapies have no curative potential, and the disease ultimately progresses to its more advanced stages [90]. In one study, median survival of stage IV patients was only 13 months from initiation of therapy [91]. Sézary syndrome (SS) is an aggressive variant of MF and is characterized by erythroderma, lymphadenopathy, circulating neoplastic T cells, and a much more aggressive clinical course [1,89,90]. From the limited experience available from two small studies, it can be concluded that autologous HCT is not capable of inducing durable remissions when performed in the advanced stages of MF in heavily pretreated patients. In the first study, Bigler et al. performed autologous HCT in six patients with advanced-stage MF using a variety of conditioning regimens, including total skin electron beam radiation in four patients. Although four of the six patients achieved an initial CR, PFS was short (between 64 days and 1 year), with three of the six patients relapsing within 3 months [92]. In the second study, Olavarria et al. performed T-cell-depleted autologous HCT in nine patients with tumorstage MF. One patient died of sepsis before engraftment, and the remaining eight patients achieved CR. With a median follow-up of 29 months,
Hematopoietic Cell Transplantation for Rare Hematologic Malignancies
all but one patient had relapsed, and the median duration of CR was 7 (range 2–14) months. In the four patients who relapsed, the disease appeared to be more indolent than before HCT [93]. Whether autologous HCT early in the disease course, possibly in combination with posttransplant maintenance therapy with currently available active agents, will produce durable remissions remains to be determined. Given this grim prognosis of patients with advanced stage MF and SS, allogeneic HCT has been attempted to harness a potential GVL effect. Molina et al. have reported the results of a series of eight patients treated at the City of Hope National Medical Center with allogeneic HCT. Three patients had tumor-stage MF, one had erythrodermic MF, and the remaining three had SS. All patients were heavily pretreated and had failed a median of seven prior therapies. Graft source was from a matched sibling in four patients and matched unrelated donor in the other four. Conditioning regimens were fractionated TBI/CY (three patients), BU/CY (one patient), and fludarabine–melphalan (four patients). All patients achieved CR, and their original T-cell clones disappeared from the blood by 60 days after HCT. Although two patients had new T-cell clones detectable in the peripheral blood on follow-up analysis, these did not have any clinical impact, and these patients remained disease free. With a median follow-up of 56 months, six patients remained disease free (Plate 70.1) [94]. Three other reports, containing three patients in each who received HLA-matched sibling HCT for advanced, refractory MF have been published. Guitart et al. transplanted three patients with TBI/CY conditioning. Two of these patients remained in CR at 4.5 years and 15 months, respectively, while the third patient had cutaneous relapse that responded to withdrawal of immunosuppression and DLI [95]. Herbert et al. have reported three patients who received fludarabine–melphalan conditioning. All three patients had relapse or persistent disease after HCT. However, all responded partially to withdrawal of immunosuppression or DLI, and the disease appeared to be more indolent than before HCT [96]. Of the three patients reported by Soligo et al., all patients achieved CR after HCT. While two patients remained in durable CR 18 and 24 months, respectively, after HCT, one died of bacterial sepsis on day 73 post transplantation [97]. Collectively, these reports are evidence for the feasibility as well as the curative potential of allogeneic HCT, even in this group of advancedstage, high-risk patients. Firm conclusions about the optimum conditioning intensity cannot be drawn from the limited data available, although the reduced-intensity regimens are better tolerated. Clear evidence of a potent GVL effect has been demonstrated in MF by the regression of persistent lesions by withdrawal of immunosuppression or DLI [95,96,98]. Viral infections appear to be a major contributor to NRM, no doubt resulting from extensive prior use of therapies with profound immunosuppressive effects. Considering the considerable impact of MF on quality of life and the inability of current treatments to prevent progression, consideration should be given to allogeneic HCT after failure of two or three initial therapies, at least in patients who are under 60 years of age and have matched sibling donors.
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motherapeutic agents, the overall prognosis for AITL is poor [1,99,100]. In one study that analyzed the outcome of 33 patients treated with chemotherapy, the OS at 5 years was 36%. Notably, two of the long-term survivors in this study had received autologous HCT [101]. Although there are some patients with AITL included in various reports of HCT for T-cell lymphomas, two reports have analyzed the outcome of autologous HCT for AITL as a separate cohort. In the first of these reports, Schetelig et al. have analyzed data on 29 patients from 16 EBMT centers. The median age of these patients was 51 years. Twenty-eight patients had stage III or IV disease, and 79% had high intermediate- or high-risk International Prognostic Index scores, clinical features that are typical of patients with AITL. Extranodal disease was present in 17 patients, of whom 12 had bone marrow involvement. Combination chemotherapy was the most common therapy administered prior to HCT. Autologous HCT was part of first-line therapy in 14 patients (48%), and the remaining patients received HCT later in the disease course. Thirteen patients (45%) were in CR at time of HCT, and 22 patients (76%) achieved CR after HCT. The 5-year probabilities of OS and EFS were 44% and 37% respectively. There was a suggestion that patients who received HCT as part of first-line therapy did better than those who received it later. Five patients were in continuous CR for over 5 years, showing that long-term survival is possible with autologous HCT in selected patients [102]. In the second study, the Spanish Lymphoma/Autologous Bone Marrow Transplant Study Group has reported the outcome of 19 patients who underwent autologous HCT. Again, the median age of 46 years of these patients is low for AITL. Eleven patients were in a first or later CR. Fifteen patients (79%) achieved CR after HCT. The OS and PFS at 3 years were 60% and 55% for the whole group (Fig. 70.6). Predictably, bone marrow involvement, refractory disease, and more than one factor of the age-adjusted International Prognostic Index were associated with poor outcome [103]. Although the above two studies undoubtedly represent a selected group of patients who were able to undergo autologous HCT, these results appear to compare favorably with that of conventional therapy. Patients with AITL are at risk for EBV-positive as well as EBV-negative secondary B-cell lymphomas [104,105]. Both EBVpositive and EBV-negative lymphoproliferative disorders can occur after autologous HCT, and these appear to respond to rituximab-based therapy [106,107]. The allogeneic HCT experience in AITL is limited to patients who are included in reports of allogeneic HCT for T-cell lymphomas. In a phase II study of salvage chemotherapy followed by reduced-intensity allogeneic HCT for relapsed peripheral T-cell lymphomas, four patients (age range 39–50 years) with AITL were included. Three of these patients who were in PR at time of HCT were alive at 12.8, 6.8, and 60 months after allogeneic HCT, while one patient died early of sepsis [108]. The advanced age of patients with AITL and the significant tumorassociated comorbidities are significant hurdles for successful allogeneic HCT. However, allogeneic HCT should be considered in suitable patients who have primary or relapsed refractory disease since these patients do not benefit from autologous HCT.
Angioimmunoblastic T-cell lymphoma Angioimmunoblastic T-cell lymphoma (AITL) is a rare lymphoma accounting for 1–2% of non-Hodgkin’s lymphomas. AITL is strongly associated with EBV, which can be detected within the B cells in involved tissues in the majority of cases. Systemic symptoms, skin rash, immunodeficiency, and autoimmune phenomena such as hemolytic anemia are characteristic features of AITL. Patients with AITL are usually middle-aged or elderly and often present with advanced disease. Although anecdotal reports describe responses to a variety of agents including immunosuppressants, biologic response modifiers, and che-
Primary central nervous system lymphoma Primary central nervous system lymphoma (PCNSL) comprises about 1–2% of non-Hodgkin’s lymphoma cases. Its incidence is rising both in immunocompromised and immunocompetent individuals [109]. PCNSL typically presents as unifocal or multifocal periventricular parenchymal masses. Ocular involvement, either clinical or asymptomatic, may be present in 10–20% of patients at diagnosis. Other systemic involvement is extremely rare at presentation. The median age at diagnosis is 60 years. Over 90% of PCNSLs have a diffuse large B-cell histology, while
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1.0 0.9
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0.8 0.7 0.6 OS at 3 yrs: 60% (37–38)
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Fig. 70.6 (a) Overall survival (OS) and (b) progression-free survival (PFS) of 19 patients who underwent autologous hematopoietic cell transplantation (HCT) for angioimmunoblastic T-cell lymphoma (AITL). (Reproduced from [103], with permission.)
2–4% of cases are of T-cell origin. Unlike the case in immunocompromised patients, EBV does not play a role in the pathogenesis of PCNSL in immunocompetent patients [110]. Although data from randomized trials are lacking, current therapy for PCNSL involves chemotherapy with high-dose methotrexate (MTX), alone or in combination, followed by whole-brain radiotherapy (WBRT). The long-term outcome of PCNSL is poor. Although a 2-year PFS survival of 57% and OS of 67% were reported in a recent study [111], the 10-year OS of patients under 60 years of age in another trial was only 32% [112]. Notably, in the latter trial, there were no long-term survivors among patients who were over 60 years of age at diagnosis. Neurotoxicity resulting from WBRT and chemotherapy is a major cause of morbidity in survivors, especially in those above 50 years of age who comprise the majority of PCNSL patients [113]. Therefore, high-dose chemotherapy with autologous hematopoietic cell support after initial chemotherapy has been tried as a strategy to minimize neurotoxicity by eliminating or reducing WBRT dose and improve the long-term outcome in PCNSL. The small sizes of reported studies as well as the marked variations in the initial and high-dose chemotherapy regimens make it difficult to draw firm conclusions regarding the efficacy of the autologous HCT approach as front-line therapy of PCNSL. In general, these studies have selected chemotherapeutic agents for conditioning based on their ability to cross the blood–brain barrier. Illerhaus et al. have reported results of 30 patients younger than 65 years of age with PCNSL who were enrolled
in a trial of initial chemotherapy with three cycles of high-dose MTX followed by a cycle of cytarabine and thiotepa, following which peripheral blood stem cells were collected. High-dose therapy consisted of carmustine and thiotepa. Fractionated radiotherapy (45 Gy, given as two doses of 1 Gy/day) was given as consolidation. Twenty-three patients were able to undergo autologous HCT, 15 of whom achieved CR and eight achieved partial remission. All 21 patients who underwent WBRT achieved CR. With a median follow-up of 63 months, the 5-year OS probability was 69% for the whole group and 87% for patients who received HCT. Leukoencephalopathy developed in 17% of patients, all of whom had received WBRT [114]. In another recently reported phase II study, Colombat et al. treated 25 patients below 60 years of age with two cycles of high-dose MTX-based chemotherapy. Seventeen of the responding patients then received high-dose therapy with carmustine, etoposide, cytarabine, and melphalan (BEAM) and autologous HCT followed by 30 Gy of WBRT. With a median follow-up of 34 months, the estimated 4-year OS and EFS were 64% and 46%, respectively. Although follow-up is short, no leukoencephalopathy has developed in evaluable patients who received the whole planned therapy [115]. In another small study of six patients treated with the same regimen as the above study, four patients were alive without evidence of disease with a median follow-up of 41.5 months [116]. WBRT was eliminated from two studies of high-dose therapy for newly diagnosed PCNSL. In the larger study, 28 patients received induction with high-dose MTX and cytarabine followed by high-dose therapy with BEAM in chemosensitive patients. Fourteen patients underwent autologous HCT. With a median follow-up of 28 months, median EFS was 5.6 months for all patients, and 9.3 months for the 14 patients who underwent HCT. Six (43%) of these 14 patients were disease free at the time of the report [117]. Cheng et al. treated seven patients with highdose MTX induction followed by high-dose therapy with thiotepa, BU, and CY. Two patients in this study received autologous HCT as sole therapy for progressive disease after initial chemotherapy. Five of the seven patients remained disease free at 5–42 months from diagnosis, including the two patients with progressive disease after initial chemotherapy. These authors attributed their good result in these poorprognosis patients to the use of agents like thiotepa and BU that have better CNS penetration than the BEAM regimen [118]. No neurotoxicity was observed in these two studies during the short follow-up. High-dose chemotherapy with autologous hematopoietic cell support has also been tried as salvage therapy for patients with refractory or recurrent PCNSL who had chemosensitive disease or intraocular lymphoma that was refractory to high-dose MTX. Soussain et al. have reported results for 22 such patients, 20 of whom received autologous HCT after high-dose therapy with thiotepa, BU, and CY. Sixteen of these 20 patients achieved CR after autologous HCT, and 14 remained alive with a median follow up of 41.5 months. The 3-year OS probability was 63.7%. Seven patients suffered neurologic adverse events, which were lethal in two patients [119]. A single case report describes a patient with refractory PCNSL who underwent an allogeneic sibling HCT after reduced-intensity conditioning. This patient remained disease free 30 months after HCT, suggesting that a graft-versus-lymphoma effect may be operational in the CNS [120]. In summary, high-dose chemotherapy with autologous HCT is feasible as front-line therapy for chemosensitive PCNSL and carries a low treatment-related mortality, at least in patients less than 65 years of age. A reduced dose of WBRT can be given after autologous HCT, with low short-term neurotoxicity, although it is not clear if high-dose chemotherapy can allow for complete elimination of WBRT. There is a suggestion that using agents such as thiotepa and BU that have better CNS penetration for high-dose therapy is superior to conventional lymphoma
Hematopoietic Cell Transplantation for Rare Hematologic Malignancies
regimens like BEAM. Autologous HCT may have a role in salvaging patients who are refractory to initial chemotherapy or who have relapsed after chemoradiotherapy, although neurotoxicity appears to be high in this setting. Other rare lymphoid malignancies Hepatosplenic T-cell lymphoma is an aggressive T-cell lymphoma characterized by marked hepatosplenomegaly and almost invariable involvement of the bone marrow. The vast majority are of the gamma/delta T-cell type (WHO). Various reports describe durable remissions after allogeneic HCT for hepatosplenic T-cell lymphoma even in chemotherapy-refractory cases [121–124]. T-cell prolymphocytic leukemia (T-PLL) is an aggressive neoplasm with a poor response to chemotherapy [1,125]. De Lavallade et al. have reported a patient who had relapsed after an autologous HCT but continued to remain in CR at 38 months after matched sibling HCT with reduced-intensity conditioning, thereby suggesting a GVL effect [126]. Kruspe et al. have recently reviewed the literature on allogeneic HCT for T-PLL. Among 13 published case reports, including a case of their own, there were 10 patients who remained in CR for periods ranging from 2 to 48 months after HCT. Notably, only four of these 13 were in CR at the time of HCT [127]. Subcutaneous panniculitic T-cell lymphoma (SPTL) mostly affects young adults, who present with subcutaneous nodules. A hemophago cytic syndrome may occur [1]. In a review of 21 patients treated with a variety of modalities, including five patients who received HCT, the OS from diagnosis was only 15 months [128]. Alpha/beta (AB) and gamma/ delta (GD) subtypes (the latter also called cutaneous gamma/delta T-cell lymphoma) of SPTL are now recognized and have distinct clinical features and markedly different prognoses. In a study of 83 patients with SPTL, the 5-year OS was 82% for the AB subtype versus only 11% for the GD subtype. Among patients with SPTL-AB, the presence of HPS was the most important prognostic factor, with a 5-year OS of only 46% for patients with HPS compared with 91% for those without [129]. There are single case reports of prolonged survival after autologous HCT even in patients who have relapsed disease and other adverse prognostic factors, such as elevated lactate dehydrogenase, HPS, and bone marrow involvement [130–134]. Allogeneic sibling HCT has been reported in a patient with progressive disease who remained in CR 31 months after HCT [135].
1039
CD4+/CD56+ hematodermic neoplasm (termed blastic NK-cell lymphoma in the initial WHO classification) [1] is a malignancy of plasmacytoid dendritic cells with a marked predilection for skin involvement. Bone marrow involvement is common [75,136]. Although there are some reports of durable CR after combination chemotherapy [137], the overall outcome with conventional combination chemotherapy alone is considered poor [75]. Eleven cases of hematodermic neoplasm have been included in a report of HCT outcome for NK-cell neoplasms. Of the six patients who underwent autologous HCT, three achieved durable CR, while among the five patients treated with allogeneic HCT, two were in CR at 11 and 43 months after HCT, suggesting benefit for both approaches in selected patients [86]. There are single case reports of durable CR after autologous HCT in patients with widely disseminated disease, including bone marrow involvement [138,139]. One patient had progressive disease on conventional chemotherapy prior to autologous HCT, thereby suggesting a benefit for dose intensification of chemotherapy in this disorder [138]. Yoshimasu et al. have reported a case of relapsed hematodermic neoplasm that was successfully treated with unrelated cord blood allogeneic HCT. This case and other reports of durable CR after allogeneic HCT (reviewed by these authors) suggest a role for allogeneic HCT in the treatment of this disorder [140,141]. HCT should be considered early in the treatment plan for the four extremely rare neoplasms discussed in this section. For hepatosplenic T-cell lymphoma and T-PLL, allogeneic HCT up front appears to be the treatment of choice, preferably after inducing remission with chemotherapy or antibody-based therapy. There is currently no evidence to suggest a benefit for autologous HCT for these two disorders. In the case of SPTL and hematodermic neoplasm, it appears reasonable to perform autologous HCT to consolidate an initial CR or at chemosensitive relapse. Allogeneic HCT could be reserved for primary refractory disease or relapsed cases that are chemorefractory, and for patients who have poor prognostic factors like extensive bone marrow involvement. Although no conclusions about the overall effectiveness of HCT can be drawn from the reported cases, at present HCT is the only therapy known to produce durable remissions in these aggressive lymphoid malignancies. Exceptions may be hematodermic neoplasm, where the curative potential of modern intensive chemotherapy regimens remains to be fully defined, and indolent cases of SPTL-AB without HPS, for which local treatments, steroids or immunosuppressive therapy may suffice [129].
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Brenda M. Sandmaier & Rainer Storb
Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies
Introduction Allogeneic hematopoietic cell transplantation (HCT) has an important role in the treatment of malignant and nonmalignant hematological diseases. Successful allogeneic HCT must overcome two immunologic barriers: graft-versus-host (GVH) and host-versus-graft (HVG) reactions. The conventional strategy to overcome this bidirectional barrier has relied upon three elements. First, intensive conditioning regimens are delivered with the dual purposes of immunoablation and disease eradication, which consist of otherwise supralethal doses of irradiation and/or chemotherapy. Second, donor hematopoietic cells are given to rescue patients from lethal myeloablation. Third, T-cell depletion or post-grafting immunosuppression is used to control GVH disease (GVHD) and establish long-term graft–host tolerance. Toxicities related to the conditioning regimens are a major limitation in the application of high-dose HCT. Toxicities include pancytopenia, which sets the stage for life-threatening infections, and damage to the liver, kidneys, and lungs, which may impair the ability to deliver the doses of immunosuppression necessary for control of GVHD. For these reasons, high-dose allogeneic HCTs have been carried out in specialized hospital wards and their use restricted to relatively young patients at most transplant centers. The concept that conditioning dose intensification was the only approach for eradicating malignancy came under question early in the history of clinical HCT. Findings in the late 1970s and early 1980s drew attention to graft-versus-tumor (GVT) effects, as evidenced by better relapse-free survival in patients with acute or chronic GVHD [1–6]. Additional evidence supporting GVT effects included lower relapse rates among patients who received unmodified allogeneic HCT compared with T-cell-depleted allografts [6], autologous grafts [7,8] or syngeneic grafts [9], and the induction of durable remissions with donor lymphocyte infusions (DLIs) in patients who had relapsed after allogeneic HCT [10–15]. These observations led to the hypothesis that GVT effects might be exploited in patients thought to be too old or medically infirm to tolerate high-dose conditioning regimens. Accordingly, several groups of investigators have developed reduced-intensity regimens that minimize the regimen-related toxicities and rely on GVT effects to treat the underlying malignancies.
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
The least intensive regimens rely on pre- and post-transplant immunosuppression to establish the allografts. It is important to remember that both HVG and GVH reactions are mediated by T cells of host and donor origin, respectively, when transplants are carried out across minor histocompatibility barriers. From these facts follows the hypothesis that HVG reactions can, to some extent, be controlled through novel and effective agents that at the same time serve to control GVHD. Consequently, much of the toxic high-dose pretransplant therapy previously employed for HVG control can be replaced by relatively nontoxic immunosuppression. Once established, grafts would create marrow space through subclinical GVH reactions, and cytotoxic and myeloablative doses of total body irradiation (TBI) or busulfan (BU) would not be needed for that purpose. Ideally, control of HVG and GVH reactions results in mutual graft–host tolerance and initial mixed donor–host hematopoietic chimerism. While mixed chimerism is likely to correct phenotypic expression of certain genetic diseases, in patients with hematologic malignancies full donor chimerism may be required to control disease through GVT effects. Thus, novel transplantation programs can be designed that are safer than high-dose regimens and have the potential to be administered in the ambulatory care setting rather than on intensive care wards. This chapter discusses the results of clinical studies with reduced-intensity conditioning for hematological malignancies, including the preclinical studies that served as their basis.
Preclinical studies Murine studies A number of successful preclinical studies have involved inbred strains of mice. Most have in vivo depletion of host T cells using antibodies, followed by TBI at high but, at least for mice, sublethal doses and dose rates. In other cases, TBI was delivered along with thymic irradiation and postgrafting cyclosporine (CSP) or high-dose cyclophosphamide (CY). A murine study by Cobbold et al. [16] combined pretransplant treatment with anti-CD4 and anti-CD8 monoclonal antibodies (mAbs) for in vivo T-cell depletion and 6–8.5 Gy TBI delivered at 0.35 Gy/min. In other studies, nonobese diabetic mice received ≥7.5 Gy TBI followed by infusion of very large numbers of allogeneic marrow cells, ≥30 × 106/mouse (≥1.5 × 109/kg) [17,18]. In one report, mice were conditioned for successful allografts with 5 Gy TBI and given additional immunosuppression with 200 mg/kg CY after transplantation [19]. Other investigators combined anti-CD4 and anti-CD8 mAbs with 3 Gy TBI and 7 Gy thymic irradiation to ensure engraftment [20]. They reported that the need for thymic irradiation could be avoided by anti-
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CD4 and anti-CD8 mAb injections after transplantation, thereby depleting host T cells [21,22]. The need for TBI could be eliminated by pretreatment with anti-CD4 and anti-CD8 mAbs, thymic irradiation, and the injection of the equivalent of 8.7 × 109 marrow cells/kg, suggesting that TBI provided immunosuppression [23]. Yet another study, using A/JÆB10 and B10.AÆB10 murine marrow grafts, showed persistent chimerism when recipients were given anti-CD4 and anti-CD8 mAbs and 3 Gy TBI administered at 1 Gy/min before and antibodies against natural killer (NK) cells after transplantation for up to 16 weeks [24]. Thymic irradiation could be replaced by the injection of one co-stimulatory blocker (anti-CD154 mAb or cytotoxic T lymphocyte antigen-4 [CTLA-4] immunoglobulin [Ig]) [25], whereas both thymic irradiation and host T-cell-depleting mAbs could be eliminated by single injections each of anti-CD154 mAb and CTLA-4 Ig [26]. Koch & Korngold [27] obtained prolonged chimerism in major histocompatibility complex (MHC)-mismatched murine allograft recipients when a synthetic CD4–CDR3 peptide analog was used for immunosuppression in combination with TBI. In mice sensitized to donor cells, treatment with the peptide was more effective than anti-CD4 mAb therapy for enhancing donor chimerism, suggesting this approach might be valuable in obviating rejection after MHC-mismatched HCT. Seledtsov et al. [28] used 200 mg/kg CY to condition mice bearing P815 leukemia for allografts and observed mixed chimerism and prolonged survival compared with syngeneic controls, raising the possibility of using nonlethal cytoreductive therapy in combination with allogeneic cells for a GVT effect. Slavin et al. used fractionated irradiation (34 Gy) targeted to the lymphoid tissues, including the spleen, lymph nodes, and thymus (total lymphoid irradiation) to condition mice for bone marrow transplants from H-2-disparate donors. The mice became chimeras, and none of the recipients developed clinical evidence of GVHD, with 80% of mice surviving beyond 110 days after HCT [29]. Subsequent studies in the same MHC-disparate model using TBI demonstrated that depletion of NK1.1+ TCRαβ+ cells from the marrow inoculum resulted in development of lethal GVHD, suggesting a “suppressor” function of these NK T cells [30]. More recent work showed that the cells that suppressed GVHD after total lymphoid irradiation and H-2-incompatible HCT were CD4+CD25+ “naturally occurring” regulatory T cells [31]. Canine studies MHC-matched grafts A clinically successful protocol employing 2 Gy TBI before and immunosuppression with mycophenolate mofetil (MMF)/CSP after HCT has been developed in a canine model. The following describes the development of the regimen. Marrow toxicity of TBI While previous studies have shown 2 Gy TBI (given at 0.07 Gy/min) to be sublethal and 4 Gy to be uniformly lethal [32], more recent studies, using more intensive antibiotics, parenteral fluid, and transfusion support, showed that dogs given 7 Gy TBI survived with endogenous marrow recovery, while 8 Gy was lethal. TBI doses needed for marrow engraftment Table 71.1 shows results of unmodified dog leukocyte antigen-identical marrow allografts following increasing single doses of TBI delivered at 0.07 Gy/min [33]. At a dose of 4.5 Gy, only 13% of dogs showed alldonor-type engraftment, and 28% were stable mixed donor–host chimeras, while 59% of dogs rejected those grafts and either died of marrow aplasia (23%) or survived with eventual autologous recovery of hematopoiesis (36%). Survival of the latter dogs was probably because of the
extended hematopoietic support provided by the transient allograft. Host immunosuppression sufficient for sustained engraftment of 95% of dogs required 9.2 Gy TBI. Low-dose TBI before and immunosuppressive drug treatment after marrow transplantation Two drugs that had been widely used in GVHD prevention, CSP and prednisone, were evaluated in dogs conditioned with 4.5 Gy TBI [34]. Postgrafting immunosuppression in this and subsequent experiments was discontinued at the latest by day 35. Prednisone, administered in extremely high doses patterned along a regimen used by Kernan et al. [35] along with antithymocyte globulin (ATG) to condition patients for second marrow grafts, failed to enhance engraftment after 4.5 Gy TBI. By comparison, all dogs given CSP were stably engrafted and none developed GVHD. This result was significantly better than that seen in controls and consistent with the theory that postgrafting immunosuppression could control both HVG and GVH reactions. A comparable rate of engraftment in the absence of CSP was seen only after conditioning with 9.2 Gy (Table 71.1). It follows that the immunosuppression accomplished by postgrafting CSP was similar to that achieved by 4.7 Gy TBI. In subsequent experiments, the TBI dose was decreased to 2 Gy [36]. CSP alone proved ineffective: all four dogs treated rejected their allografts after 4 weeks but survived with autologous recovery. Six dogs were given methotrexate along with CSP, a drug combination previously shown to be synergistic in preventing GVHD both in dogs and humans [37,38]. Three of five evaluable dogs became stable mixed chimeras, and two rejected their grafts. MMF, whose metabolite, mycophenolic acid, blocks de novo purine synthesis by binding to inosine monophosphate dehydrogenase and interferes with de novo purine synthesis required for lymphocyte replication, was next combined with CSP. The two drugs had been found to be synergistic and superior to methotrexate/CSP for GVHD prevention in the canine model [39]. Only one of 12 dogs rejected the allograft at week 12, while 11 dogs remained stable mixed chimeras for up to 130 weeks after HCT without clinical evidence of GVHD. Further reducing the TBI dose to 1 Gy resulted in only transient engraftment in MMF/CSP-treated dogs, suggesting a delicate balance of host and donor immunities. Similar results were seen by combining rapamycin and CSP [40]. Marrow space versus immunosuppression In order to clarify whether the major role of low-dose TBI before transplantation was to create marrow space or provide host immunosuppression, six canine recipients were given 4.5 Gy (2 Gy/min) pretransplant irradiation to the cervical, thoracic, and upper abdominal lymph node
Table 71.1 Doses of total body irradiation (TBI) needed for the engraftment of dog leukocyte antigen (DLA) identical marrow in the absence of postgrafting immunosuppression
TBI dose (Gy)* 9.2 8 – myeloablative and supralethal 7 6 4.5
Number of dogs studied
% with sustained engraftment
21 5
95 80
0 0
5 23 39
60 52 41
0 17 36
* Delivered at 0.07 Gy/min.
% with autologous recovery
Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies
1045
CD45, respectively, could be substituted for low-dose TBI. α-Emitters were an attractive alternative to β-emitting radionuclides for targeting hematopoietic cells given that their high energy is deposited over only a few cell diameters (40–90 μm), sparing surrounding normal tissue. 213 Bi has a half-life of 46 minutes and delivers a short-range high-energy radiation, leading to a high relative biologic effectiveness. Studies showed that 213Bi labeled anti-TCRαβ and anti-CD45 mAb resulted in stable engraftment of dog leukocyte antigen-identical littermate marrow [44,45]. Selectively ablating T cells or ablating all nucleated hematopoietic cells in this way allowed transplantation with very low toxicity, making it suitable both for patients with nonmalignant and with malignant hematologic disorders. Further, mAbs against the panhematopoietic marker CD45 would be useful in patients with MHCnonidentical donors where additional host immune cells, such as NK cells, play a major part in graft rejection. Based on results in both the murine and large animal models, clinical studies with β-emitting radionuclides have been initiated in patients. Data from ongoing preclinical studies with α-emitters will allow us to refine future approaches for our patients.
chain. This was followed by dog leukocyte antigen-identical littermate marrow grafts and MMF/CSP post transplant [41]. In all six, mixed chimerism was present in peripheral blood granulocytes, T cells and monocytes, and nonirradiated marrow and lymph node spaces. Two of the six rejected their grafts after 8 and 18 weeks, and four remained stable mixed chimeras. When, more than 1 year after HCT, T cells from the marrow donors were infused which had been sensitized to recipient minor antigens, conversion to all donor chimerism occurred. While other explanations were possible, results seemed consistent with the hypothesis that pretransplant irradiation provided host immunosuppression and grafts could create their own marrow space. Therefore, conceivably, the small doses of either lymphoid radiation or TBI currently used for establishing mixed chimerism could ultimately be replaced by more specific and less toxic means of inducing HVG anergy, including mAbs to T-cell surface determinants. These efforts were prompted by concerns that even low doses of radiation or chemotherapy might increase the risk of secondary malignancies. Subsequent studies showed that the equivalent of 1 Gy TBI could be replaced by co-stimulatory signal blockade with either CTLA-4 Ig or a mAb directed against CD154 [42,43]. Recipient T cells were activated with intravenous injections of donor peripheral blood mononuclear cells before 1 Gy TBI, with concurrent administrations of either the fusion peptide CTLA-4 Ig, which blocks T-cell co-stimulation through the B7: CD28 signal pathway, or the mAb against CD154, which blocks the signal from CD40 to CD154. Most dogs so treated showed mixed chimerism lasting for the observation periods of 2 years. Control dogs not experiencing T-cell co-stimulatory blockade showed transient mixed chimerism lasting for 3–12 weeks.
Clinical results Several groups of investigators have explored the feasibility of reducedintensity HCT with a variety of regimens to minimize regimen-related toxicities while exploiting GVT effects. Figure 71.1 outlines the spectrum of commonly used regimens relative to their hypothetical immunosuppressive and myelosuppressive effects. In some cases, drugs were chosen with proven activities against the targeted malignancies for disease control while waiting for GVT effects to occur. With the minimally myelosuppressive regimens, engraftment was ensured by innovative postgrafting immunosuppression that also controlled GVHD, and disease control was more dependent on GVT effects. Other differences
Radiolabeled mAb In other studies, we investigated whether radioimmunotherapy with an α-emitting radionuclide, bismuth-213 (213Bi) and mAbs to TCRαβ and
Conditioning regimens Reduced intensity
Myeloablative Cy120/TBI 12 Gy Cy120/TBI 5.5 Gy Bu16/Cy120
Cy200/ATG
HLA-matched unrelated
HLA-identical sibling
Immunosuppression
Fig. 71.1 Commonly used nonmyeloablative or reduced-intensity conditioning regimens in relation to their immunosuppressive and myelosuppressive properties. Ale, alemtuzumab; ATG, antithymocyte globulin; Bu8, busulfan 8 mg/kg; Bu16, busulfan 16 mg/kg; Cy, cyclophosphamide; Cy120, cyclophosphamide 120 mg/kg; Cy200, cyclophosphamide 200 mg/kg; F, fludarabine; FlagIda, fludarabine/cytosine arabinoside/idarubicin; M, melphalan; M 140, melphalan 140 mg/m2; M 180, melphalan 180 mg/m2; TBI, total body irradiation; TLI, total lymphoid irradiation; TT, thiotepa, (Adapted from [113], with permission. Copyright, American Society of Hematology.)
Genetic disparity
HLA-haploid related/ T-cell depleted
F/Cy/TBI 2 Gy
Ale/F/M 140
F/M 180
F/TBI 2 Gy
Bu8/F/ATG
F/M 140 TLI ATG
F/Cy
Flag-Ida TBI 2 Gy
TT-Cy
Myelosuppression Aggressiveness of malignancy
F/Bu16
1046
Chapter 71
among the reduced-intensity regimens included the degrees of marrow aplasia and the tempo of establishing complete donor chimerism. In the reduced-intensity regimens that have more myelosuppressive doses of chemotherapy, recipients uniformly become severely hypoplastic before graft function occurs. Complete donor engraftment occurs rapidly. By contrast, recipients of a more immunosuppressive regimen show only moderate declines of peripheral blood cell counts before recoveries are seen, and all patients are initially mixed donor–host hematopoietic chimeras. In these patients, it may take 6–12 months before donor engraftment is complete. The comparative benefit of one over another reduced-intensity regimen may depend on the underlying diseases, stage of diseases, and clinical status of the patients. In diseases presumed to be more sensitive to GVT effects, such as chronic myelogenous leukemia, chronic lymphocytic leukemia, and low-grade lymphomas, immunosuppressive regimens may be sufficient to ensure engraftment because there is time for GVT effects to become operative. In rapidly progressive diseases or diseases where GVT is less potent or less able to keep ahead of growth of the underlying malignancy, such as high-grade lymphomas, Hodgkin’s lymphoma, multiple myeloma, and acute myeloid leukemia (AML) beyond first remission, a certain amount of cytoreduction may be necessary to minimize residual disease. In this chapter, we discuss strategies of reduced-intensity transplants, with a review of the initial results and current approaches, and with a focus on minimally myelosuppressive TBI-based regimens. HLA-matched related HCT While many studies of reduced-intensity hematopoietic transplants have been performed in elderly and medically infirm patients unable to tolerate a high-dose preparative regimen, some studies have included younger patients who were otherwise eligible for high-dose transplants with standard eligibility criteria. These differences in patient selection generally preclude a meaningful comparison among regimens. Table 71.2 outlines the initial publications regarding reduced conditioning regimens currently being investigated. Initial studies at the MD Anderson Cancer Center in Houston, Texas, utilized purine nucleoside analog-based regimens for treatment of both myeloid and lymphoid malignancies. Giralt et al. [46] combined fludarabine with idarubicin and cytarabine or melphalan, or cladribine and cytarabine before HCT from HLA-identical or single HLA antigenmismatched sibling donors for treatment of AML or myelodysplastic syndromes. Of 15 patients treated, four failed to engraft, and one treatment-related death occurred before HCT. With a median follow-up of 100 days, six patients were alive and two were disease-free. Khouri et al. [47], also at MD Anderson, treated 15 patients with lymphoid malignancies using a regimen of fludarabine/CY or fludarabine/cytarabine/cisplatin. Eleven of 15 patients attained durable engraftment, and eight of 11 engrafted patients achieved complete remissions. Three nonrelapse deaths occurred. With a median follow-up of 180 days, five of six patients with chemosensitive disease were alive compared with two of nine with refractory or untreated malignancies. Giralt et al. [48] subsequently reported on a study combining purine analogs (fludarabine or cladribine) with melphalan in patients with hematologic malignancies. It was hypothesized that these drug combinations would have enhanced abilities to achieve remissions given the antitumor effects of high-dose melphalan and the observation that purine analogs have been shown to inhibit DNA repair after alkylator-induced damage. Seventy-eight of 86 patients received fludarabine in combination with high-dose melphalan, and eight received cladribine/melphalan. The day 100 nonrelapse mortality (NRM) rate was 37% for the fludarabine/melphalan combination and 88% for the cladribine/melphalan com-
bination. Both regimens were sufficiently immunosuppressive to allow for engraftment of both HLA-matched unrelated and related, and one HLA antigen-mismatched related donor grafts. Fifty-seven percent of patients achieved complete remissions, illustrating the beneficial effect of the cytoreductive conditioning therapy. Two-year overall and diseasefree survivals were 28% and 23%, respectively. Slavin et al. [49] at Hadassah University in Israel used a regimen that consisted of fludarabine, BU (8 mg/kg) and ATG in a younger group of patients (median 34 years, range 1–61 years) with both hematologic malignancies and genetic diseases. All patients achieved either partial or complete donor chimerism with this regimen, with four patients developing moderate to severe hepatic sinusoidal obstructive syndrome, suggesting that this regimen was still quite toxic. This same regimen was used in another group of heavily treated high-risk lymphoma patients with grafts from HLA-matched related and unrelated donors [50]. Again, there was consistent engraftment with disease-free survival of 40% at 37 months. Childs et al. [51] at the National Cancer Institute combined CY and fludarabine to treat patients both with hematologic malignancies and with solid tumors. One of 15 patients rejected the graft, and two patients died of transplant-related causes. Ten of 14 patients surviving more than 30 days had disease regression, suggesting GVT effects. The observation was made that full donor T-cell chimerism preceded both acute GVHD and disease regression. Investigators at the Massachusetts General Hospital in Boston evaluated the use of CY, ATG, and thymic irradiation (among patients without previous mediastinal radiotherapy) in patients given HLA-matched transplants [52]. Eighteen of 20 evaluable patients developed persistent mixed hematopoietic chimerism. Ten of the 20 received prophylactic DLI to convert mixed to full donor chimerism and to optimize GVL effects. Six of eight patients receiving prophylactic DLI converted to full donor chimerism. Transplant complications included CY-induced cardiac toxicity. One patient died from transplant complications and one patient died from GVHD after two prophylactic DLIs. Overall, 11 of 21 patients survived; four had continued complete responses with a median follow-up of 14.6 months. In an attempt to reduce the incidence of GVHD, Campath (anti-CD52 mAb) was added to fludarabine and melphalan conditioning, in recipients of both HLA-matched sibling and unrelated donor grafts [53]. Forty-two of 43 evaluable patients had sustained engraftment. Eighteen of 31 patients studied were full donor chimeras, while 13 patients were mixed chimeras. The regimen prevented grade III–IV acute GVHD, and only two patients developed grade II GVHD. At a median follow-up of 9 months, 33 of the 44 patients remained alive either in complete remission or without disease progression. While the chemotherapy-based reduced-intensity regimens had significantly fewer toxicities compared with conventional high-dose allogeneic HCT, patients still experienced profound pancytopenias, and most required hospitalizations of durations that were similar to those for high-dose transplants. In order to further reduce the regimen-related toxicities and thereby perform allogeneic HCT in the outpatient setting, the low-dose (2 Gy) TBI conditioning regimen developed in the dog model was evaluated in a multi-institutional clinical study [54]. TBI of 2 Gy was given as a single fraction on day 0, with postgrafting immunosuppression of CSP from day −1 to day 35, and MMF given twice a day from day 0 to 27. Granulocyte colony-stimulating factor-mobilized peripheral blood hematopoietic cells (PBHCs) collected over 2 days from HLA-identical related donors were used as sources of hematopoietic cells. Trial eligibility criteria required patients to be either too old to be eligible for a high-dose HCT, or, if younger, to have medical contraindications to high-dose HCT. Overall, the HCT regimen was well tolerated, with the majority of eligible patients having their transplants in the outpatient clinic setting. Most patients did not develop severe
50 (23–68)
44 (22–62)
15
86
26
23
15
21
426
37
44
19
5
68
Houston [46]
Houston [47]
Houston [48]
Jerusalem [49]
Jerusalem [50]
NIH [51]
Boston [52]
Seattle [112]
Stanford [57]
London [53]
Freiburg [65]
Boston [58]
Baltimore and Seattle [61]
46 (1–71)
30 (20–51)
64 (60–70)
41 (18–56)
52 (28–66)
55 (9–74)
41 (13–63)
34 (1–61)
52 (22–70)
55 (47–71)
59 (27–71)
15
Transplant center [Reference]
Median age in years (range)
Number of patients studied
Haplo
Haplo
MRD MURD
MRD MURD
MRD MURD
MRD
MRD
MRD
MRD
MURD MRD 5/6 RD MRD
MRD
MRD 5/6 RD
Donor
BM
BM
PBHC BM
PBHC BM
PBHC
PBHC
BM
PBHC
PBHC
PBHC
BM PBHC
PBHC BM
PBHC BM
Stem cell source
4
CSP
CSP + MMF T + MMF CSP + MMF
CSP ± MTX
CSP + MMF
CY + ATG ± TI 2 Gy TBI ± F TLI + ATG
F+M+ Campath F + BCNU +M+ ATG CY + ATG ± TI
HM
HM
NHL
AML MDS
HM
HM
HM
CY + F + 2 Gy TBI
Cy + T + MMF
CSP
24
CSP
F + CY
HM ST
13
0
0
2
16
7
0
0
CSP
F+B+ ATG F+B+ ATG
L
HM GD
HM
2
27
±T ± MTX
T + MTX T + MP CSP + MP CSP
27
CSP + MP
F+I+A F+I+M 2-CDA + A F + CY F+C+A
Rejections (%)
Postgraft immunosuppression
Conditioning regimen
F+M 2-CDA + M
CLL NHL
AML MDS
Diagnosis
Table 71.2 Nonmyeloablative conditioning regimens for patients with hematologic malignancies
35
100
59
5
3
47
29
60
35
38
40
7
20
Acute grade II–IV
GVHD* (%)
Cy1: 25% Cy2: 5%
NA
65
2
27
50
NA
27
9
27
24
13
0
Chronic
OS: 40% DFS: 20% Median F/U: 103 days 2-year OS: 36% 2-year PFS: 27%
OS: 40% DFS: 13% Median F/U: 100 days OS: 47% DFS: 33% Median F/U: 180 days 2-year OS: 28% 2-year DFS: 23% 2-year NRM: 45% OS: 85% DFS: 81% Median F/U: 240 days OS: 43% DFS: 43% Median F/U: 675 days OS: 53% PFS: 53% Median F/U: 200 days OS: 52% DFS: 33% Median F/U: 445 days 3-year OS: 51% 3-year PFS: 38% OS: 73% DFS: 62% Median F/U: 446 days OS: 82% PFS: 75% Median F/U: 270 days 1-year OS: 68% 1-year PFS: 61%
Outcome(s)
Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies
1047
16
42
47
52
89
103
Transplant center [Reference]
Jerusalem [62]
Dresden [63]
London [64]
Leipzig [67]
Seattle [68]
Seattle [69]
54 (17–70)
53 (4–69)
48 (6–65)
44 (18–62)
47 (16–65)
17 (8–48)
Median age in years (range)
MURD
MURD
MURD MMURD MURD MMURD
MURD MMURD
MURD
Donor
PBHC
BM PBHC
BM PBHC
BM
PBHC BM
BM
Stem cell source
HM
HM
HM
HM
HM
HM
Diagnosis
CSP + MMF
CSP + MMF
2 Gy TBI + F
5
21
12
CSP + MMF
2 Gy TBI + F
4
CSP
21
CSP + MMF CSP ± MTX
F+M+ Campath 2 Gy TBI + F
0
CSP
F+B+ ATG F+B+ ATG
Rejections (%)
Postgraft immunosuppression
Conditioning regimen
53
52
63
21
26
44
Acute grade II–V
GVHD* (%)
49
37
25
6
38
0
Chronic
3-year OS: 75% 3-year DFS: 60% OS: 36% DFS: 26% Median F/U: 390 days 1-year OS: 75.5% 1-year PFS: 61.5% OS: 35% DFS: 25% Median F/U: 570 days 1-year OS: PBHC: 57%; BM: 33% 1-year PFS: PBHC: 44%; BM: 17% 2-year OS: 58% 2-year PFS: 49%
Outcome(s)
* GVHD before donor lymphocyte infusion. 2-CDA, cladribine; A, ara-C; AML, acute myeloid leukemia; ATG, antithymocyte globulin; B, busulfan; BCNU, carmustine; BM, bone marrow; C, cisplatin; Campath, Alemtuzumab; CLL, chronic lymphocytic leukemia; CSP, cyclosporine; CY, cyclophosphamide; DFS, disease-free survival; F, fludarabine; F/U, follow-up; GD, genetic diseases; Haplo, haploidentical related donor; HM, hematologic malignancies; I, idarubicin; L, lymphoma; M, melphalan; MDS, myelodysplastic syndrome; MMF, mycophenolate mofetil; MMURD, mismatched unrelated donor; MP, methylprednisolone; MRD, matched related donor; MTX, methotrexate; MURD, matched unrelated donor; NA, not available; NHL, non-Hodgkin’s lymphoma; NRM, nonrelapse mortality; OS, overall survival; PBHC, peripheral blood hematopoietic cells; PFS, progression-free survival; RD, related donor; ST, solid tumors; T, tacrolimus; TBI, total body irradiation; TI, thymic irradiation; TLI, total lymphoid irradiation.
Number of patients studied
Table 71.2 (Continued)
1048 Chapter 71
Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies n = 212
100,000
Neutrophils/μL
10,000
Max Median
1000
tion of regulatory NK/T cells [56], consisted of total lymph node irradiation (8 Gy total lymphoid irradiation) and ATG (Thymoglobulin, 7.5 mg/kg total dose) in combination with MMF and CSP for postgrafting immunosuppression. Of the first 37 patients reported [57], two developed acute GVHD, and nine of 33 evaluable developed chronic GVHD.
Min
100
HLA-mismatched related donor allografting
10
1 0
10
20
30
40
50
60
10,000
Platelets × 1000/μL
1049
1000 Max 100
Median
Min
10
1 0
10
20
30
40
50
60
Days after HCT
Fig. 71.2 Neutrophil and platelet changes among 212 patients given 2 Gy total body irradiation ± fludarabine before and mycophenolate mofetil/ cyclosporine after human leukocyte antigen-matched related hematopoietic cell transplantation (HCT). (Reproduced from [114], with permission. Copyright, American Society of Clinical Oncology.)
neutropenia or thrombocytopenia (Fig. 71.2.) While all patients had initial donor engraftment, nine (20%) of the first 45 patients experienced graft rejection between 2 and 4 months after transplant, a timing that coincided with discontinuation of the MMF/CSP immunosuppression [54]. Lack of intensive preceding chemotherapy before HCT predicted rejections, and rejecting patients had low-level donor T-cell chimerism on day 28. The rejections were nonfatal, and all patients had autologous reconstitution of peripheral blood counts. To prevent graft rejection, the conditioning regimen was modified by adding 30 mg/m2/day fludarabine on days −4, −3, and −2 to the 2 Gy TBI, with a subsequent reduction of rejection rate to 3%. The outcomes of different durations of CSP administration were analyzed in 185 patients who received grafts from HLA-matched donors and CSP for 56, 77 or 180 days after HCT in combination with MMF for 28 days. While the duration of CSP prophylaxis did not significantly influence the overall rate of acute GVHD (grade II–IV), chronic GVHD, or NRM, prolonged administration of CSP (180 days) was associated with a significantly decreased hazard of grades III–IV acute GVHD and an increased likelihood of discontinuing all systemic immunosuppression when compared with the shortest course of CSP (35 days) [55]. Only 3.7% of patients receiving CSP through day 180 developed grades III–IV acute GVHD. The Stanford group took a different approach to reduce the incidence of GVHD. The reduced-intensity regimen, which was adapted from murine studies that were designed to favor the presence of a high propor-
In a report by Sykes et al. [58], five patients with refractory non-Hodgkin’s lymphoma underwent marrow transplantation from HLA-haploidentical related donors, sharing at least one HLA-A, B or DR allele on the mismatched haplotypes after conditioning with high-dose CY (200 mg/kg), 7 Gy thymic irradiation, and ATG (Table 71.2). Four evaluable patients showed engraftment with predominantly donor lymphoid cells and varying degrees of myeloid donor chimerism. All five patients developed grades II–III acute GVHD, with three patients dying between days 12 and 108 from pulmonary hemorrhage, progressive lymphoma, and aspergillosis. At the time of the report, one patient was alive at day 103 with a partial response, and one was alive at day 416 in continuous complete remission. The Johns Hopkins’ group developed a conditioning regimen for HLA-haploidentical related recipients based on a murine model whereby high-dose CY administered after HCT inhibited both graft rejection and GVHD [59]. The regimen for patients consisted of pretransplant CY 29 mg/kg, fludarabine 150 mg/m2, and TBI 2 Gy followed by an infusion of marrow and post-transplant CY 50–100 mg/kg, and tacrolimus and MMF (three times a day). A recent study expanded on the initial observations [60] in two groups of patients at two centers (Johns Hopkins and Fred Hutchinson Cancer Research Center [FHCRC]), which differed by the regimens of post-transplantation immunoprophylaxis [61]. On day 3 (Seattle group) or days 3 and 4 (Hopkins group), 50 mg/kg/day Cy was administered. All patients received tacrolimus and MMF beginning on the day after the last dose of Cy. Sixty-eight patients received these regimens, of whom 67 had hematologic malignancies and one had paroxysmal nocturnal hemoglobinuria. Thirteen percent of evaluable patients had graft failure, 35% and 6% of patients had grades II–IV and III–IV acute GVHD, respectively. The only difference seen between the two groups of patients was that the rate of extensive chronic GVHD in the group that received one dose of post-transplant Cy was 25% compared with 5% in the group that received two doses post transplant (p = 0.05). The overall and event free survivals at 2 years after HCT were 36% and 27%, respectively. HLA-matched and mismatched unrelated donor allografting Nagler et al. [62] reported the results of HLA-matched unrelated marrow transplantation following conditioning with fludarabine and BU (8 mg/ kg) and ATG. Fifteen of 16 patients achieved 100% donor chimerism, and one patient became a mixed chimera. Seven patients developed grades II–IV acute GVHD, with one patient dying from complications related to acute GVHD. The overall and disease-free survival estimates at 36 months were 75% and 60%, respectively, with this reduced-intensity conditioning regimen in a relatively young population of patients (median age 17 years, range 8–48 years). Using a similar regimen of fludarabine, BU, and ATG, Bornhauser et al. [63] treated 42 patients with hematologic malignancies with HLAmatched or single HLA antigen-mismatched unrelated grafts. In this older population group (median 47 years, range 16–65 years), diseasefree survival was 64% for patients with lymphoid malignancies, 38% for patients with standard-risk leukemias, and 14% for patients with
Chapter 71
high-risk diseases. Nine patients (21%) had either primary or secondary graft failures. In patients with stable engraftment, the probability of acute GVHD grades II–IV was 32%, with one patient dying from grade IV GVHD. In a similarly aged group of patients with hematologic malignancies using a different conditioning regimen consisting of Campath mAb, fludarabine, and melphalan [64], Chakraverty et al. described results in 47 patients who received HLA-matched or mismatched unrelated donor transplants. Primary graft failure occurred in two patients, and three patients developed grades III–IV acute GVHD. The overall and progression-free survivals at 1 year were 75.5% and 61.5%, respectively. Bertz et al. reported on 19 patients (ages 60–70 years) with myeloid malignancies who received conditioning with fludarabine, melphalan, and carmustine before HLA-matched related (n = 7) or unrelated (n = 12) HCT [65]. With a median follow-up of 825 days, the 1-year NRM and survival rates were 22% and 68%, respectively. These results were confirmed in a subsequent update from this group on 34 patients receiving unrelated HCT using this regimen [66]. Using the low-dose TBI (2 Gy) and fludarabine (90 mg/m2) conditioning regimen, Niederwieser et al. [67] reported on 52 patients (median age 48 years, range 6–65 years) with advanced hematologic malignancies given unrelated grafts. Seventy-one percent were HLA matched at the antigen (serologic) level, and 29% were one HLA antigen mismatched. Additionally, 15 patients had allele level mismatches for class I HLA antigens. Durable donor engraftment was attained in 88% of patients. Grades II–IV acute GVHD occurred in 63% of patients, with 9% of patients having fatal GVHD. With a median follow-up of 19 months, 35% of the patients were alive and 25% were in remissions of their underlying malignancies. In a subsequent study, Maris et al. [68] reported on 89 patients with unrelated donor grafts. Early data on 22 of the patients were included in the report by Niederwieser et al. [67]. Eligibility criteria for this trial included serological matching for HLA-A, B, and C, and allele-level matching for HLA-DRB1 and DQB1. The median patient age was 53 (range 5–69) years. While the preferred source of stem cells was PBHCs, these were available in only 71 patients, while 18 patients received marrow. Initial donor T-cell engraftment was documented in 87% of patients, with median donor T-cell chimerisms on days 28, 56, and 84 being higher in PBHC recipients than marrow recipients (p = 0.02, 0.01, and 0.08, respectively). The peripheral blood granulocyte and marrow donor chimerism values were not significantly different between the two groups of recipients at the various time points. Sustained donor engraftment occurred in 79% of patients. By multivariate analysis, graft rejection was more frequent in marrow recipients (p = 0.003) and in patients without preceding chemotherapy (p = 0.003). Donor T-cell chimerism values of less than 50% on day 28 were highly associated with eventual graft rejection (p = 0.001). Preceding conventional HCT or preceding chemotherapy reduced the risk of rejection, presumably because the prior cytotoxic therapy increased pretransplant immunosuppression of the host. Of the 85 patient–donor pairs with complete 10/10 HLA antigen sequencing, HLA class I allele-level mismatch (16 of 85 pairs) was associated with a trend towards increased risk of graft rejection (p = 0.13). With a median follow-up of 13 (range 0.6–28) months, the cumulative probabilities of NRM were 11% at 100 days and 16% at 1 year. The Kaplan–Meier estimates for 1-year survival were 57% versus 33%, and for progression-free survival were 44% versus 17% for recipients of PBHCs versus marrow, respectively. A high degree of day 28 CD3 chimerism (>50%) was associated with less relapse (p = 0.05) and a trend towards improved progression-free survival (p = 0.10). By multivariate analysis, better survival was conferred to patients with low-risk diseases (p = 0.04), with no prior transfusions (p = 0.003), with less than 5% blast cells in the marrow before HCT
100
Donor chimerism (%)
1050
CSP MMF
80
60
40 CD3 Gran BM
20
0 0
50
100
150
Days after HCT
Fig. 71.3 Donor chimerism (fluorescence in situ hybridization) in a patient with Philadelphia chromosome-negative myeloproliferative disorder after unrelated hematopoietic cell transplantation (HCT). BM, bone marrow; CSP, cyclosporine; Gran, granulocytes; MMF, mycophenolate mofetil.
(p = 0.002), with HCT from a female donor (p = 0.01), and with CD3+ cell dose greater than or equal to the median (p = 0.0006). Better progression-free survival was identified in a multivariate analysis in patients with less than 5% blasts in the marrow prior to HCT (p = 0.0001) and those who received PBHCs as a hematopoietic stem cell source (p = 0.006). Figure 71.3 illustrates the donor chimerism changes in a 65-year-old patient with Philadelphia chromosome-negative myeloproliferative disorder whose disease was characterized by high white blood cell counts, very high platelet counts (>1 million/mL), and high basophil counts. The patient’s T-cell chimerism increased to 80% by day 84, whereas the marrow and granulocyte donor chimerism declined from 40–45% on day 28 to 10% in the marrow on day 84. The patient’s platelet and basophil counts increased to 900,000/mL and 1500/mL, respectively, consistent with disease progression after an initial response. CSP was rapidly tapered to induce a GVT response. The marrow and granulocyte donor chimerism rose to nearly 100% by day 150, and both basophilia and thrombocytosis resolved. The patient also developed mild signs of chronic GVHD which responded to CSP. The patient is surviving over 7 years after HCT with complete donor engraftment and no evidence of recurrent myeloproliferative disorder. This clinical case illustrates the relationship between postgrafting immunosuppression, GVHD, and GVT effects. It also emphasizes the importance of lineage-specific chimerism evaluations in monitoring patients. The study suggested that PBHCs were the preferred source of grafted hematopoietic cells in unrelated transplantation with a fludarabine/2 Gy TBI conditioning regimen. Given the importance of postgrafting immunosuppression to both facilitate engraftment and reduce the risk of GVHD, our attention was drawn to another finding of the study, the short (3.5-hour) serum half-life of the active metabolite of MMF, mycophenolic acid. This finding resulted in a modification of the post-transplant immunosuppression: an increase in the dosing of MMF to three times daily in the hope of minimizing the risk of late graft rejections. Among 103 patients so treated, graft rejections occurred in 5%, while acute GVHD remained at 53%, with 2-year NRM and overall and progressionfree survival of 19%, 58%, and 49%, respectively [69]. Because three times daily compared with twice-daily MMF diminished the incidence of graft rejection, MMF should be administered three times a day in unrelated donor recipients receiving this regimen. In a subsequent study of 71 patients who received an extended course of MMF until day 150 and tapered through day 180, and a shortened course of CSP through
Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies
day 80, there was an incidence of 77% acute and 47% chronic GVHD [70] compared with 52% (p = 0.03) and 49% (p = 0.43), respectively, in patients who had MMF until day 40 with a taper-through day 96 and CSP until day 100 with a taper-through day 180 [69]. Chimerism and engraftment kinetics Donor chimerism after allogeneic HCT has been evaluated by several means in the past. Currently, fluorescent in situ hybridization of sex chromosomes and amplified fragment length polymorphism analysis are the most commonly used methods (reviewed in [71] and see Chapter 25). Amplified fragment length polymorphism-based techniques utilize the polymerase chain reaction for analysis of polymorphic DNA sequences such as variable number of tandem repeats or short tandem repeats. Additionally, real-time polymerase chain reaction of single nucleotide polymorphisms is being evaluated for this purpose. Reduced-intensity conditioning usually leads to an initial state of mixed chimerism [51,72–74], although some patients given grafts after more myelosuppressive regimens achieve full donor chimerism in cellular subsets as early as day 14 after HCT [75]. Childs et al. [51] evaluated the kinetics of engraftment after conditioning with fludarabine (125 mg/m2) and CY (120 mg/kg). Most frequently after that regimen, full donor chimerism was achieved earlier in peripheral blood T cells than in myeloid cells. The NK-cell chimerism correlated with the T-cell chimerism, whereas the B-cell chimerism was distinct from the T-cell and myeloid lineages. Bornhauser et al. observed that patients with NK chimerism below 75% in the first month after unrelated donor HCT had a higher risk of graft failure after conditioning with fludarabine 150 mg/ m2, BU 6.6 mg/kg intravenously, and ATG [63]. The kinetics of chimerism was also evaluated in patients who received conditioning with 2 Gy TBI with or without fludarabine (90 mg/m2) [74]. Most patients remained mixed donor–host chimeras for up to 3–6 months after HCT. Patients given preceding chemotherapy or peripheral blood stem cell grafts had the highest degrees of donor chimerism. High levels of donor T- or NKcell chimerism (>50%) 28 days after HCT have each been associated with a reduced risk of graft rejection [54,74]. There was also an association with higher donor T-cell chimerism and an increased risk of grade II–IV acute GVHD [74]. An association between the kinetics of engraftment and GHVD was observed by Childs et al. [51]. The achievement of full donor T-cell chimerism preceded the development of grades II–IV GVHD in patients who received conditioning with fludarabine and CY. The studies by Mattsson et al. [76], by contrast, found that 82% of patients had mixed donor–host T-cell chimerism at the time of the development of acute GVHD. This is similar to the results observed after conditioning with TBI with or without fludarabine, in which 83% of the patients were mixed donor–host T-cell chimeras at the time of the development of GVHD [74]. However, when chimerism was analyzed as a continuous linear variable, patients with higher levels of donor T-cell chimerism on day 28 after HCT were at higher risk for grade II–IV acute GVHD [54,68,74], with a corresponding reduced risk of relapse in those patients who achieved full donor T-cell chimerism [74]. GVHD and GVT effects Reconstitution of donor-derived immunity after reduced-intensity conditioning differed from what occurred after high-dose conditioning. First, as mentioned above, there was an initial state of mixed donor–host chimerism in the recipients of reduced-intensity conditioning that changed the balance of both GVH and HVG tolerance, which may affect the onset of GVHD. Second, high-dose conditioning regimens may contribute to the pathophysiology of GVHD via tissue damage causing the release of cytokines, the so-called “cytokine storm” [77]. However,
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the number of host-derived antigen-presenting cells may be higher after reduced-intensity conditioning, thus contributing to acute GVHD given their putative role in the initiation of GVHD [78]. In order to address the relationship between conditioning intensity and GVHD, several retrospective studies have been carried out. Most have shown lower incidences of acute and similar or lower incidence of chronic GVHD [79–82]. One age-matched retrospective analysis was carried out comparing the incidence of GVHD in recipients of reducedintensity versus high-dose conditioning. The cumulative incidence of grades II–IV acute GVHD was lower in reduced-intensity HCT (64% versus 85%; p = 0.001), resulting in the use of less systemic immunosuppression during the first 3 months after transplant [79]. There was no difference in the cumulative incidence of chronic GVHD requiring treatment. Of note, reduced-intensity HCT was associated with a syndrome of acute GVHD occurring beyond day 100 in a subset of the related recipients, and this should be taken into consideration in the design of prospective studies comparing reduced-intensity and high-dose HCT. The prognostic relevance of early-onset GVHD was evaluated in 395 patients who had HCT from HLA-matched related (n = 297) or unrelated (n = 98) donors after conditioning with fludarabine and 2 Gy TBI [83]. The cumulative incidence of grades II–IV acute GVHD and chronic GVHD were 45% and 47%, respectively, with related donors, and 68% and 68%, respectively, with unrelated donors. The median days of highdose corticosteroid use for treatment of acute or chronic GVHD (used as a marker for the onset of clinically relevant GVHD) were 79 (range 8–799) days and 30 (range 5–333) days after HCT for related and unrelated recipients, respectively. The cumulative incidence of NRM among patients with GVHD was 55% at 4 years when corticosteroids were started before day 50 as compared with 29% when started after day 50 in related recipients, suggesting that early-onset GVHD may benefit from intensified primary immunosuppressive therapy. There was no association of NRM with early-onset GVHD seen in unrelated recipients. Historically, the occurrence of GVHD was associated with GVT effects in patients receiving high-dose conditioning regimens [1,2]. Several retrospective analyses have been carried out to evaluate the impact of GVT on outcomes after reduced-intensity conditioning regimens since the regimens themselves have less of a role in disease control. Martino et al. demonstrated a reduced risk of relapse in patients with AML and myelodysplastic syndrome who experienced acute and/or chronic GVHD compared with those who did not [84]. Kroger et al. found that while there was no impact of acute GVHD on the risk of relapse in patients with multiple myeloma, there was a significantly lower risk of relapse in patients who had chronic GVHD [85]. These outcomes were also observed in an analysis of multiple myeloma patients by Crawley et al. for the European Group for Blood and Marrow Transplantation [86]. Blaise et al. performed a landmark analysis starting on day 100 in patients with AML and observed a lower risk of relapse and higher leukemia-free survival in the patients that developed chronic GVHD [87]. GVT effects were analyzed in a cohort of 322 patients (related donor n = 192, unrelated donor n = 130) with hematologic malignancies following conditioning with fludarabine and TBI [88]. Fifty-seven percent of patients with measurable disease (n = 221) achieved complete (n = 98) or partial (n = 28) remission. The achievement of full donor chimerism was associated with a decreased risk of relapse/progression. Grades II–IV GVHD did not impact progression/relapse but were associated with an increase in NRM and decreased progression-free survival. Conversely, chronic GVHD was associated with a decrease in progression/ relapse and an increase in progression-free survival without an increase in NRM. This suggests that protocols designed to reduce acute GVHD might improve survival while allowing GVT to occur later.
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Toxicities In a retrospective cohort study, recipients of reduced-intensity conditioning had significantly reduced transfusion requirements compared with conventional HCT recipients, with only 23% of the patients requiring platelet transfusions and 63% requiring red blood cell transfusions compared with 100% of high-dose HCT recipients (p ≤ 0.0001). Recipients of reduced-intensity HCT who did require transfusion used significantly fewer units of platelets and red blood cells (p ≤ 0.0001) [89]. Recipients of reduced-intensity conditioning had shorter periods of neutropenia than recipients of myeloablative conditioning (p < 0.0001), and this was associated with fewer episodes of bacteremia during the first 30 days (p = 0.01) [90]. Similarly, there was a trend to less cytomegalovirus antigenemia, viremia, and disease in reduced-intensity HCT recipients compared with recipients of myeloablative conditioning, with a significant reduction when more serious manifestations (cytomegalovirus viremia and disease) were combined during the first 100 days (p = 0.01) [91]. The cumulative incidence of proven or probable invasive mold infections in the first year after reduced-intensity HCT was 15%, which was similar to that for myeloablated recipients [90,92]. Severe acute GVHD and cytomegalovirus disease were the primary risk factors associated with invasive mold infections after reduced-intensity conditioning, with high-dose corticosteroid therapy on diagnosis of invasive mold infections being associated with an increased risk for death related to this cause. The incidence of idiopathic pneumonia syndrome was significantly lower following reduced-intensity conditioning compared with “conventional” conditioning regimens (2.2% versus 8.4%; p = 0.003) [93]. Liver injury is a frequent, serious complication of high-dose preparative regimens. The frequency and severity of hepatic injury after reduced-intensity conditioning was evaluated in 193 consecutive patients [94]. No patients developed sinusoidal obstructive syndrome. Twentysix percent of patients developed hyperbilirubinemia of 4 mg/dL or more, most commonly resulting from cholestasis due to GVHD or sepsis. Overall survival was superior for patients who had maximum bilirubin in the normal or minimally elevated (1.3–3.9 mg/dL) ranges compared with those in the elevated (≥4 mg/dL) ranges. Incidences of acute renal failure (ARF) among 140 recipients of highdose and 129 of reduced-intensity conditioning were compared [95]. Severity of ARF was classified into four grades based on serum creatinine increases in the first 100 days after HCT. Recipients of reducedintensity conditioning were significantly older and had greater pretransplant comorbidities. Despite this, high-dose patients had greater incidences of severe ARF (73% versus 47%; p < 0.001) and dialysis (12% versus 3%; p < 0.001) compared with those receiving reducedintensity regimens. NRM was also greater in the high-dose group at 100 days and 1 year. The incidences of chronic kidney disease after reducedintensity HCT were analyzed in 122 patients from three centers [96]. Multivariate analysis revealed that ARF in the first 100 days was associated with chronic kidney disease. Previous autologous HCT, long-term calcineurin use, and chronic GVHD were independently associated with chronic kidney disease. Future research should focus on preventing ARF and decreasing the use of calcineurin inhibitors.
Comparing morbidity and mortality: influence of pretransplant comorbidities Until recently, allogeneic HCT has been reserved for young patients in good medical condition. Comorbidities, defined as any additional clinical entity in a patient with an index disease, affect therapeutic plans and outcomes after treatment for the index disease. Many patients with hematologic malignancies either have pre-existing comorbidities or
develop them during the course of therapy for their malignancies. In clinical trials in hematology and oncology, patients with comorbidities are often excluded if it is thought that a specific comorbidity may dramatically impact outcomes. This is also true in clinical trials of HCT. With the advent of reduced-intensity conditioning regimens, HCT is being carried out in older patients and those with considerable comorbidities. Objective measures of patients’ underlying health are required in order to study and disseminate transplantation approaches in an older and medically less fit population. While efforts to analyze the impact of comorbidities on outcomes after HCT are still in the early stages, initial data have suggested that comorbidity indices are useful tools in predicting outcomes that can be used in the design of inclusion/exclusion criteria for clinical trials. One of the indices used was the Charlson Comorbidity Index (CCI), which was originally developed in a cohort of patients at a general medical center [97]. The CCI was applied to patients with hematologic malignancies after HCT following both high-dose and reduced-intensity conditioning regimens. In both related [98] and unrelated [99] recipients, the adapted CCI successfully predicted NRM. Yet the CCI had limitations. Some comorbidities were rarely found among HCT recipients due to existing exclusion criteria, particularly in patients conditioned with highdose regimens, and the CCI did not capture other frequent comorbidities. Further, the CCI lacked sensitivity. Therefore, a study was designed to better define previously identified comorbidities by utilizing pretransplant laboratory data and, additionally, investigating additional HCTrelated comorbidities so as to develop a scoring system more suited to HCT. Data were collected from 1055 patients and randomly divided into training and validation sets [99]. Based on the prognostic significance, various weights were assigned to the individual comorbidities and then validated. The new HCT-Comorbidity Index (HCT-CI) was more sensitive than the CCI since it captured more than 62% of patients with a score of over 0 compared with 12%, respectively. The HCT-CI additionally showed a better survival prediction than the CCI (Fig. 71.4). Pretransplant score assignments with the index were simple to accomplish and should be highly objective. This tool has been validated in patients with myelodysplasia or AML, where similar outcomes have been found after highdose and reduced-intensity conditioning after stratification of patients into low- and high-risk disease categories and low and high HCT-CI scores [100]. This suggests that patients with low HCT-CI scores could be candidates for randomized trials comparing conditioning intensity. Currently, studies are ongoing comparing outcomes using this scoring system in patients treated at other transplant centers, and early results suggest similar utility in predicting outcome [101]. The development of the HCT-CI sets the stage for prospective studies such as a riskstratification system that could discriminate which patients could be enrolled in prospective randomized studies comparing high-dose and reduced-intensity regimens to evaluate which patients benefit from either kind of conditioning. Relapse risk for hematologic malignancies Reduced-intensity allogeneic HCT relies on GVT effects for the eradication of malignancy. An analysis was carried out in 834 patients who had received conditioning with 2 Gy TBI with or without fludarabine to get an estimate of relapse risk according to disease characteristics [102]. Median patient age was 55 (range 5–74) years, and recipients received either related (n = 498) or unrelated (n = 336) donor grafts. Relapse rates per patient–year at risk for the first 2 years after HCT were calculated for 29 different diagnosis and disease stages. Excluding multiple myeloma patients who had a planned autologous transplant followed by an allogeneic HCT, there was no evidence that a history of
Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies (a)
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Fig 71.4 Nonrelapse mortality (NRM) as stratified by the HCT-Comorbidity Index (HCT-CI) (a) compared with the Charlson Comorbidity Index (CCI) (b), and Kaplan–Meier estimates of survival as stratified by the HCT-CI (c) and CCI (d). HCT, hematopoietic cell transplantation.
a failed autologous transplant was associated with a higher risk of relapse. The overall risk of relapse per patient–year was 0.36 and was 0.35 among patients with related donors, and 0.37 among those with unrelated donors. Patients were grouped into low-, standard-, and highrisk groups (Table 71.3). The survivals, cumulative relapse rates, and cumulative NRM rates according to risk groups are shown in Fig. 71.5. Patients with low-grade lymphoproliferative diseases experienced the lowest relapse rates, whereas patients with advanced myeloid and lymphoid malignancies had the highest relapse rates after reduced-intensity HCT. In order to improve patient outcomes in the high-risk group, the option of allografting should be considered earlier in the disease course when the tumor burden is lower. Alternatively, the use of debulking chemotherapy prior to HCT, or targeted therapies with limited systemic toxicities, should be added to the conditioning. Consolidative reduced-intensity allografts following planned autografts In patients with aggressive malignancies, the GVT effect following a reduced-intensity allograft may not be sufficiently fast enough to eradicate large-volume disease. Carella et al. adopted the strategy of using debulking autologous HCT followed by reduced-intensity allogeneic HCT in patients with refractory Hodgkin’s and non-Hodgkin’s lymphomas [103]. Patients (n = 15) were first given high-dose therapy with carmustine, etoposide, cytarabine, and melphalan, with reinfusion of autologous PBHCs. At a median of 61 days after engraftment, patients were given fludarabine 90 mg/m2 and CY 900 mg/m2 followed by PBHCs from HLA-matched related donors. With a median follow-up of 337 days, 10 patients were alive, with five patients in remission. This approach was evaluated in 17 patients with multiple myeloma by Kroger et al. [104]. After autografting with melphalan (200 mg/m2), the patients
received a dose-reduced regimen consisting of fludarabine (180 mg/m2), melphalan (100 mg/m2), and ATG (3 × 10 mg/kg) followed by allografting. After a median follow-up of 13 months after allogeneic HCT, 13 patients were alive, 12 of whom were free of relapse or progression. A similar approach was evaluated by Maloney et al. in patients with multiple myeloma (n = 54) who were given a cytoreductive autograft with melphalan conditioning (200 mg/m2) followed by an allograft from an HLA-identical sibling after conditioning with 2 Gy TBI [105]. One patient died before day 100 following the allograft because of disease progression. With a median follow-up of 550 days after allograft in surviving patients, the overall survival rate was 78%. The overall disease response rate was 83%, with 57% of patients achieving complete remission and 26% partial remission. Based on this study, a large phase III trial comparing tandem autologous transplant with tandem autologous/ allogeneic transplant has been carried out by the Bone Marrow Transplant–Clinical Trials Network. All patients have been enrolled and are currently in follow-up. Another trial based on this approach has been recently reported by Bruno et al., comparing allografting with autografting for newly diagnosed multiple myeloma [106]. Patients (n = 162) were enrolled at the time of diagnosis and treated with vincristine, doxorubicin, and dexamethasone followed by melphalan and autologous PBHC infusion. Patients with an HLA-identical sibling then received 2 Gy TBI and PBHCs from the sibling donor, whereas those without an HLA-identical sibling received a second autograft. The median overall survival was longer in the patients who had an HLA-identical sibling than those who did not (80 versus 54 months, respectively). Among patients who completed their treatment protocols, the disease-related mortality was significantly higher in the tandem autologous group (43%) compared with the autologous–allogeneic group (7%), without a significant difference in NRM.
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Table 71.3 Relapse rates in 29 diagnosis and disease stage groups Number of patients Low risk CLL NHL – low grade NHL – low grade Waldenström’s macroglobulinemia MM NHL – mantle cell NHL – mantle cell MPD NHL – high grade ALL Standard risk CLL MM MDS AML CML AML High risk MDS AML NHL – high grade HD MDS HD AML CML CML Renal cell ALL ALL CMML
CR Not in CR CR
Relapse rate per patient–year
7 34 9 9 29 16 25 19 26 19
8.2 40.8 11.1 10.8 42.8 15.7 30.4 18.6 31.0 21.0
0.00 0.15 0.18 0.19 0.19 0.19 0.20 0.21 0.23 0.24
75 136 20 80 26 59
89.0 174.8 18.2 78.3 32.8 62.6
0.26 0.27 0.33 0.33 0.34 0.37
23 42 36 13 18 38 13 14 7 18 8 3 12
19.2 29.1 26.3 9.6 14.4 30.6 9.2 10.1 3.8 10.6 4.6 2.2 6.3
0.52 0.55 0.57 0.62 0.70 0.72 0.87 0.99 1.05 1.23 1.29 1.35 1.42
CR† CR Not in CR CR 1st CR Not in CR Not in CR RA/RARS 1st CR 1st CP ≥2nd CR
Patient–years of follow-up*
RAEB/RAEB-t Evolved from MDS Not in CR CR Secondary Not in CR Not in CR AP/BC 2nd CP ≥2nd CR Not in CR
ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; AP, accelerated phase; BC, blast crisis; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; CP, chronic phase; CR, complete remission; HD, Hodgkin’s disease; MDS, myelodysplastic syndrome; MM, multiple myeloma; MPD, myeloproliferative disease; NHL, non-Hodgkin’s lymphoma; RA, refractory anemia; RAEB, refractory anemia with excess blasts; RAEB-t, refractory anemia with excess blasts in transformation; RARS, refractory anemia with ringed sideroblasts. * Patient–years of follow-up are the total number of person–years of observation time from transplant until death, relapse/progression, last contact, or 2 years. † The criteria for complete remission from multiple myeloma were absence of monoclonal immunoglobulin and of discernible light chains in the urine by standard electrophoresis, absence of visible monoclonal bands on immunofixation, fewer than 1% plasma cells in marrow aspirates, absence of evidence of clonal disease according to flow cytometry of marrow cells, and absence of an increase in the size or number of osteolytic lesions. Adapted with permission from [102]. Copyright, American Society of Hematology.
Reduced-intensity allografting after failed high-dose transplants Use of conventional high-dose conditioning for allografts following failed autologous or allogeneic HCT has resulted in poor outcomes in adult patients, primarily related to early fatal regimen-related toxicities [107]. A number of investigators have therefore evaluated reducedintensity regimens to condition patients for second HCT. Investigators in Jerusalem conditioned patients with fludarabine, BU, and ATG, with CSP as GVHD prophylaxis [108]. Among 12 relatively young patients transplanted (median age 33 years, range 8–63 years), one nonrelapse death occurred and six patients were disease-free with a median followup of 23 months. Actuarial overall and disease-free survivals were 56% and 50%, respectively, at 34 months. Investigators at Massachusetts
General Hospital described their results with 13 patients (median age 38 years) who relapsed following autologous HCT and received HLAmatched related donor allografts following conditioning with CY and ATG with or without thymic irradiation [109]. DLI was administered at 5–6 weeks post transplant to facilitate full donor chimerism. One nonrelapse death occurred, and 2-year estimates of overall and disease-free survivals were 45% and 38%, respectively. Investigators at the City of Hope National Medical Center have described their results in 28 patients who failed previous autologous transplants or developed myelodysplastic syndrome after autografts [110]. The median interval from the failed autologous HCT and allogeneic HCT was 15 months. The median age of the patients was 47 years, and patients were treated with either fludarabine plus melphalan (n = 24) or fludarabine plus 2 Gy TBI (n = 4). Patients received HCT from
Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies (a)
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Using the 2 Gy TBI-based regimen (with or without fludarabine), the Seattle Consortium reported on 147 patients (median age 46 years, range 9–73 years) who failed conventional autologous (n = 135), allogeneic (n = 10) or syngeneic (n = 2) HCT and subsequently received HLAmatched related (n = 62) or unrelated (n = 85) allografts [111]. The 3year NRM was 32% in related recipients and 28% in unrelated recipients with a median follow-up of 813 (range 84–1925) days. The Kaplan– Meier estimates of relapse and overall survival were 48% and 32%, respectively, for related recipients, and 44% and 44%, respectively, in unrelated recipients. The best outcomes were observed in patients with non-Hodgkin’s lymphoma, whereas patients with multiple myeloma and Hodgkin’s lymphoma had the poorest outcomes due to high incidences of relapse and progression. A lower risk of relapse or progression was seen in patients in partial or complete remission at the time of HCT and in patients who developed chronic GVHD. Factors associated with better overall survival were the partial or complete remission of underlying diseases and lack of comorbidity at the time of HCT. Thus, allogeneic GVT effects can be used with some success in patients who have exhausted other treatment options, including high-dose therapy with stem cell rescue (either autologous or allogeneic). Outcomes using related or unrelated donors were comparable.
Conclusion
(c)
Fig 71.5 (a) Overall survival rates, (b) cumulative relapse rates, and (c) cumulative nonrelapse mortality rates according to risk groups. HCT, hematopoietic cell transplantation.
either HLA-matched related (n = 14) or unrelated (n = 14) donors. The day 100 mortality and NRM were 25% and 21%, respectively. With a median follow-up of 24 months in surviving patients, the 2-year probabilities of overall survival, event-free survival, and relapse rate were 56.5%, 41%, and 41.9%, respectively.
The use of reduced-intensity or minimally myelosuppressive preparative regimens has resulted in durable donor engraftment and the development of GVT effects in the setting of underlying malignant diseases. The regimens have been relatively nontoxic in older patients and have been successfully applied to younger patients with comorbid conditions that had excluded them from high-dose HCT. The optimal regimen may ultimately be defined by the nature of the diseases being treated. In aggressive lymphomas, acute leukemias not in remission, and other fastgrowing malignancies, cytoreduction by either preceding chemotherapy with or without autologous stem cell rescue, or the use of a reducedintensity regimen with disease-specific chemotherapy may be necessary to allow time for adequate GVT responses to develop. For low-grade lymphomas, acute leukemias in first complete remission, chronic leukemias, and other more indolent diseases, minimal reduced-intensity regimens may be sufficient for successful outcomes by allowing the donor T cells to generate sufficient GVT responses and eradicate the underlying malignancies. The role of DLI for the achievement of full donor chimerism and specific GVT effects needs to be explored, and current ongoing research is aimed toward those goals. Other current issues include minimizing GVHD, which may result in prolonged immunosuppression, thereby placing patients at risk for infections that, in turn, contribute significantly to NRM in all regimens. Additionally, prolonged immunosuppression may blunt optimal GVT effects. Ultimately, reduced-intensity HCT might be used to establish mixed donor–host chimerism that can serve as a platform for adoptive immunotherapy with the infusion of tumor-specific cytotoxic T cells that would exert GVT effects without causing GVHD. In the future, reduced-intensity transplants may become the procedure of choice also for younger patients with either malignant or nonmalignant diseases. Towards that end, phase III studies are needed to determine both immediate- and long-term outcomes in different disease categories and age groups.
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67. Niederwieser D, Maris M, Shizuru JA et al. Lowdose total body irradiation (TBI) and fludarabine followed by hematopoietic cell transplantation (HCT) from HLA-matched or mismatched unrelated donors and postgrafting immunosuppression with cyclosporine and mycophenolate mofetil (MMF) can induce durable complete chimerism and sustained remissions in patients with hematological diseases. Blood 2003; 101: 1620–9. 68. Maris MB, Niederwieser D, Sandmaier BM et al. HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with hematologic malignancies. Blood 2003; 102: 2021–30. 69. Maris MB, Sandmaier BM, Storer BE et al. Unrelated donor granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell transplantation after nonmyeloablative conditioning: the effect of postgrafting mycophenolate mofetil dosing. Biol Blood Marrow Transplant 2006; 12: 454–65. 70. Baron F, Sandmaier BM, Storer BE et al. Extended mycophenolate mofetil and shortened cyclosporine failed to reduce graft-versus-host disease after unrelated hematopoietic cell transplantation with nonmyeloablative conditioning. Biol Blood Marrow Transplant 2007; 13: 1041–8. 71. Baron F, Sandmaier BM. Chimerism and outcomes after allogeneic hematopoietic cell transplantation following nonmyeloablative conditioning [Review]. Leukemia 2006; 20: 1690–700. 72. Dey BR, McAfee S, Colby C et al. Impact of prophylactic donor leukocyte infusions on mixed chimerism, graft-versus-host disease, and antitumor response in patients with advanced hematologic malignancies treated with nonmyeloablative conditioning and allogeneic bone marrow transplantation. Biol Blood Marrow Transplant 2003; 9: 320–9. 73. Carvallo C, Geller N, Kurlander R et al. Prior chemotherapy and allograft CD34+ dose impact donor engraftment following nonmyeloablative allogeneic stem cell transplantation in patients with solid tumors. Blood 2004; 103: 1560–3. 74. Baron F, Baker JE, Storb R et al. Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 2004; 104: 2254–62. 75. Ueno NT, Cheng YC, Rondon G et al. Rapid induction of complete donor chimerism by the use of a reduced-intensity conditioning regimen composed of fludarabine and melphalan in allogeneic stem cell transplantation for metastatic solid tumors. Blood 2003; 102: 3829–36. 76. Mattsson J, Uzunel M, Brune M et al. Mixed chimaerism is common at the time of acute graftversus-host disease and disease response in patients receiving non-myeloablative conditioning and allogeneic stem cell transplantation. Br J Haematol 2001; 115: 935–44. 77. Mohty M, Blaise D, Faucher C et al. Inflammatory cytokines and acute graft-versus-host disease after reduced-intensity conditioning allogeneic stem cell transplantation. Blood 2005; 106: 4407– 11. 78. Shlomchik WD, Couzens MS, Tang CB et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 1999; 285: 412–15.
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79. Mielcarek M, Martin PJ, Leisenring W et al. Graft-versus-host disease after nonmyeloablative versus conventional hematopoietic stem cell transplantation. Blood 2003; 102: 756–62. 80. Couriel DR, Saliba RM, Giralt S et al. Acute and chronic graft-versus-host disease after ablative and nonmyeloablative conditioning for allogeneic hematopoietic transplantation. Biol Blood Marrow Transplant 2004; 10: 178–85. 81. Aoudjhane M, Labopin M, Gorin NC et al. Comparative outcome of reduced intensity and myeloablative conditioning regimen in HLA identical sibling allogeneic haematopoietic stem cell transplantation for patients older than 50 years of age with acute myeloblastic leukaemia: a retrospective survey from the Acute Leukemia Working Party (ALWP) of the European group for Blood and Marrow Transplantation (EBMT). Leukemia 2005; 19: 2304–12. 82. Scott BL, Sandmaier BM, Storer B et al. Myeloablative vs nonmyeloablative allogeneic transplantation for patients with myelodysplastic syndrome or acute myelogenous leukemia with multilineage dysplasia: a retrospective analysis. Leukemia 2006; 20: 128–35. 83. Mielcarek M, Burroughs L, Leisenring W et al. Prognostic relevance of “early-onset” graftversus-host disease following nonmyeloablative hematopoietic cell transplantation. Br J Haematol 2005; 129: 381–91. 84. Martino R, Caballero MD, Simón JA et al. Evidence for a graft-versus-leukemia effect after allogeneic peripheral blood stem cell transplantation with reduced-intensity conditioning in acute myelogenous leukemia and myelodysplastic syndromes. Blood 2002; 100: 2243–5. 85. Kroger N, Perez-Simon JA, Myint H et al. Relapse to prior autograft and chronic graft-versus-host disease are the strongest prognostic factors for outcome of melphalan/fludarabine-based dosereduced allogeneic stem cell transplantation in patients with multiple myeloma. Biol Blood Marrow Transplant 2004; 10: 698–708. 86. Crawley C, Lalancette M, Szydlo R et al. Outcomes for reduced-intensity allogeneic transplantation for multiple myeloma: an analysis of prognostic factors from the Chronic Leukemia Working Party of the EBMT. Blood 2005; 105: 4532–9. 87. Blaise DP, Boiron JM, Faucher C et al. Reduced intensity conditioning prior to allogeneic stem cell transplantation for patients with acute myeloblastic leukemia as a first-line treatment. Cancer 2005; 104: 1931–8. 88. Baron F, Maris MB, Sandmaier BM et al. Graftversus-tumor effects after allogeneic hematopoietic cell transplantation with nonmyeloablative conditioning. J Clin Oncol 2005; 23: 1993– 2003. 89. Weissinger F, Sandmaier BM, Maloney DG, Bensinger WI, Gooley T, Storb R. Decreased transfusion requirements for patients receiving nonmyeloablative compared with conventional peripheral blood stem cell transplants from HLAidentical siblings. Blood 2001; 98: 3584–8. 90. Junghanss C, Marr KA, Carter RA et al. Incidence and outcome of bacterial and fungal infections
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Ginna G. Laport & Robert S. Negrin
Management of Relapse after Hematopoietic Cell Transplantation
Introduction Disease relapse is an ominous clinical event following either autologous or allogeneic hematopoietic cell transplantation (HCT). Following autologous HCT, relapse is the major cause of treatment failure. Although disease recurrence is less common after allogeneic HCT, it remains a frequent clinical event. In both settings, patients are highly sensitized to the risk of return of the underlying malignancy. As such, great care is necessary when discussing and investigating signs and symptoms of potential disease that requires radiographic and often pathologic investigation before a specific diagnosis is made. In this chapter, we will review efforts to reduce the risk of disease recurrence and the management of patients who suffer a relapse following HCT.
Management of relapse after allogeneic HCT Allogeneic HCT is an accepted curative treatment modality for patients with malignant and nonmalignant diseases. In contrast to autologous HCT, which relies solely on chemotherapy and/or total body irradiation (TBI) to eradicate disease, allogeneic HCT is associated with a graftversus-tumor (GVT) effect mediated by alloreactive donor T and B cells. Despite this added benefit of adoptive immunotherapy, relapse remains one of the leading causes of treatment failure after allogeneic HCT and typically portends a very poor prognosis. Historically, salvage chemotherapy, withdrawal of immunosuppression, and/or a second HCT were the only available options. However, additional options are now available that include various forms of cellular adoptive immunotherapy such as donor leukocyte infusions (DLIs), cytokine-induced killer (CIK) cells, natural killer (NK) cells, and antigen-specific cytotoxic T lymphocytes (CTLs). These interventions attempt to harness the powerful therapeutic benefits of the donor’s immune system to eliminate residual host tumor. For patients with relapsed chronic myelogenous leukemia (CML), additional options include the tyrosine kinase inhibitors such as imatinib to reinduce remissions or to eradicate persistent disease following allogeneic HCT. The following section of this chapter will discuss the above-mentioned approaches, with an emphasis on the utility of adoptive immunotherapy.
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
Withdrawal of immunosuppressive therapy Withdrawal of immunosuppressive medications is usually the initial step taken when relapse occurs after allogeneic HCT, and typically precedes subsequent interventions such as DLI or palliative chemotherapy. Immunosuppression withdrawal is intended to allow for maximal exertion of the GVT effect to induce remission, which unfortunately is rarely successful. In addition, graft-versus-host disease (GVHD) remains a serious complication. Scattered case reports in almost all hematologic malignancies have been published, with occasional durable remissions seen. The highest response rates have been observed in patients with CML and indolent lymphomas, with less favorable results in the acute leukemias and aggressive lymphomas. The more rapid proliferation rates of these diseases may simply outpace the slower kinetics of the potential GVT effects. The largest series comes from Germany, in which cyclosporine was withdrawn from 42 patients with CML and acute leukemia [1]. The 4-year probability of achieving a durable remission was 84% among the CML patients, 10% for the patients with acute myelogenous leukemia (AML) at 3 years, and 0% for those with advanced CML or acute lymphoblastic leukemia (ALL). These results underscore the marked sensitivity of early-phase CML to the GVT effect, which appears to be much weaker against the acute leukemias, especially ALL. In most cases of relapse, discontinuation of immunosuppressive agents is a reasonable initial intervention in the absence of active GVHD, but unfortunately rarely induces remissions except in cases of CML. Donor leukocyte infusions DLI confers a direct graft-versus-malignancy effect by infusion of alloreactive donor lymphocytes. Purposes of DLI include conversion of mixed donor chimerism to full donor chimerism after HCT, as preemptive therapy to prevent relapse or for the treatment of relapse. Table 72.1 summarizes published trials highlighting the clinical results of DLI in selected reports. The use of DLI was first reported by Kolb et al. in 1990, in which three patients with relapsed CML achieved long-term remissions with the use of interferon (IFN) and donor buffy coat cells [2]. Since then, numerous reports with related and unrelated donors have confirmed the efficacy of DLI in reinducing remissions in patients with hematologic malignancies, with the highest responses observed in patients with CML [3–6]. Disease status at the time of DLI and disease type (acute versus chronic leukemia) appear to be the most important predictors of response. The application of DLI, however, is not without toxicity and carries a mortality rate of 3–10%, with acute GVHD and marrow aplasia being
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Table 72.1 Selected studies of the use of donor leukocyte infusions for relapse after allogeneic hematopoietic cell transplantation
Group/year
n
Disease
Donor
Complete remission
EBMT, 2007 [25] European, 2006 [23] Hopkins, 2006 [3]
171 63 83
AML MM Various
MRD/URD MRD/URD MRD
EBMT, 2002 [11] Hammersmith, 2000 [6]
298 66
CML CML
MRD/URD MRD/URD
34% 19% CML 71% AML 31% Others or =60 years old with
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relapsed or refractory B-cell lymphoma. J Clin Oncol 2007; 25: 1396–402. Gopal AK, Rajendran JG, Petersdorf SH et al. High-dose chemo-radioimmunotherapy with autologous stem cell support for relapsed mantle cell lymphoma. Blood 2002; 99: 3158–62. Nademanee A, Forman S, Molina A et al. A phase 1/2 trial of high-dose yttrium-90-ibritumomab tiuxetan in combination with high-dose etoposide and cyclophosphamide followed by autologous stem cell transplantation in patients with poor-risk or relapsed non-Hodgkin lymphoma. Blood 2005; 106: 2896–902. Vose JM, Bierman PJ, Loberiza FR Jr., Bociek RG, Matso D, Armitage JO. Phase I trial of (90)Yibritumomab tiuxetan in patients with relapsed B-cell non-Hodgkin’s lymphoma following highdose chemotherapy and autologous stem cell transplantation. Leuk Lymphoma 2007; 48: 683– 90. Jacobs SA, Vidnovic N, Joyce J, McCook B, Torok F, Avril N. Full-dose 90Y ibritumomab tiuxetan therapy is safe in patients with prior myeloablative chemotherapy. Clin Cancer Res 2005; 11: 7146s–50s. Mundt AJ, Williams SF, Hallahan D. High dose chemotherapy and stem cell rescue for aggressive non-Hodgkin’s lymphoma: pattern of failure and implications for involved-field radiotherapy. Int J Radiat Oncol Biol Phys 1997; 39: 617– 25. Mundt AJ, Sibley G, Williams S, Hallahan D, Nautiyal J, Weichselbaum RR. Patterns of failure following high-dose chemotherapy and autologous bone marrow transplantation with involved field radiotherapy for relapsed/refractory Hodgkin’s disease. Int J Radiat Oncol Biol Phys 1995; 33: 261–70. Wendland MM, Smith DC, Boucher KM et al. The impact of involved field radiation therapy in the treatment of relapsed or refractory nonHodgkin lymphoma with high-dose chemotherapy followed by hematopoietic progenitor cell transplant. Am J Clin Oncol 2007; 30: 156–62. Poen JC, Hoppe RT, Horning SJ. High-dose therapy and autologous bone marrow transplantation for relapsed/refractory Hodgkin’s disease: the impact of involved field radiotherapy on patterns of failure and survival. Int J Radiat Oncol Biol Phys 1996; 36: 3–12. Kahn ST, Flowers CR, Lechowicz MJ, Hollenbach K, Johnstone PA. Refractory or relapsed Hodgkin’s disease and non-Hodgkin’s lymphoma: optimizing involved-field radiotherapy in transplant patients. Cancer J 2005; 11: 425–31. Freedman AS, Neuberg D, Mauch P et al. Longterm follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma. Blood 1999; 94: 3325–33. van Besien K, Loberiza FR Jr., Bajorunaite R et al. Comparison of autologous and allogeneic hematopoietic stem cell transplantation for follicular lymphoma. Blood 2003; 102: 3521–9. Galimberti S, Guerrini F, Morabito F et al. Quantitative molecular evaluation in autotransplant programs for follicular lymphoma: efficacy of in vivo purging by rituximab. Bone Marrow Transplant 2003; 32: 57–63. Gianni AM, Magni M, Martelli M et al. Longterm remission in mantle cell lymphoma following high-dose sequential chemotherapy and in
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to double autologous transplantation in multiple myeloma: results of a multicenter randomized clinical trial. Blood 2008; 111: 1805–10. Miller JS, Tessmer-Tuck J, Pierson BA et al. Low dose subcutaneous interleukin-2 after autologous transplantation generates sustained in vivo natural killer cell activity. Biol Blood Marrow Transplant 1997; 3: 34–44. Fierro MT, Liao XS, Lusso P et al. In vitro and in vivo susceptibility of human leukemic cells to lymphokine activated killer activity. Leukemia 1988; 2: 50–4. Stein AS, O’Donnell MR, Slovak ML et al. Interleukin-2 after autologous stem-cell transplantation for adult patients with acute myeloid leukemia in first complete remission. J Clin Oncol 2003; 21: 615–23. Attal M, Blaise D, Marit G et al. Consolidation treatment of adult acute lymphoblastic leukemia: a prospective, randomized trial comparing allogeneic versus autologous bone marrow transplantation and testing the impact of recombinant interleukin-2 after autologous bone marrow transplantation. BGMT Group. Blood 1995; 86: 1619– 28. Burns LJ, Weisdorf DJ, DeFor TE et al. IL-2based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release: a phase I/II trial. Bone Marrow Transplant 2003; 32: 177–86. Thompson JA, Fisher RI, LeBlanc ML et al. Total body irradiation, etoposide, cyclophosphamide and autologous peripheral blood stem cell transplantation followed by randomization to therapy with interleukin-2 versus observation for patients with non-Hodgkin’s lymphoma: results of a phase III randomized trial by the Southwest Oncology Group (SWOG 9438). Blood 2006; 108; 326a. Goldberg GL, Zakrzewski JL, Perales MA, van den Brink MR. Clinical strategies to enhance T cell reconstitution. Semin Immunol 2007; 19: 289–96. Alvarnas JC, Linn YC, Hope EG, Negrin RS. Expansion of cytotoxic CD3+ CD56+ cells from peripheral blood progenitor cells of patients undergoing autologous hematopoietic cell transplantation. Biol Blood Marrow Transplant 2001; 7: 216–22. Leemhuis T, Wells S, Scheffold C, Edinger M, Negrin RS. A phase I trial of autologous cytokineinduced killer cells for the treatment of relapsed Hodgkin disease and non-Hodgkin lymphoma. Biol Blood Marrow Transplant 2005; 11: 181–7. Takayama T, Sekine T, Makuuchi M et al. Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial. Lancet 2000; 356: 802–7. Petvises S, Pakakasama S, Wongkajornsilp A, Sirireung S, Panthangkool W, Hongeng S. Ex vivo generation of cytokine-induced killer cells (CD3+ CD56+) from post-stem cell transplant pediatric patients against autologous-Epstein–Barr virustransformed lymphoblastoid cell lines. Pediatr Transplant 2007; 11: 511–17. Rosenberg SA, Lotze MT, Muul LM et al. A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Engl J Med 1987; 316: 889– 97.
134. Klebanoff CA, Khong HT, Antony PA, Palmer DC, Restifo NP. Sinks, suppressors and antigen presenters: how lymphodepletion enhances T cellmediated tumor immunotherapy. Trends Immunol 2005; 26: 111–17. 135. Barao I, Hanash AM, Hallett W et al. Suppression of natural killer cell-mediated bone marrow cell rejection by CD4+CD25+ regulatory T cells. Proc Natl Acad Sci U S A 2006; 103: 5460–5. 136. Riddell SR, Watanabe KS, Goodrich JM, Li CR, Agha ME, Greenberg PD. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 1992; 257: 238–41. 137. Gottschalk S, Heslop HE, Roon CM. Treatment of Epstein–Barr virus-associated malignancies with specific T cells. Adv Cancer Res 2002; 84: 175–201. 138. Rooney CM, Smith CA, Ng CY et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein–Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 1998; 92: 1549–55. 139. Kennedy-Nasser AA, Bollard CM, Rooney CM. Adoptive immunotherapy for Hodgkin’s lymphoma. Int J Hematol 2006; 83: 385–90. 140. Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 1997; 90: 3245–87. 141. Bollard CM, Rossig C, Calonge MJ et al. Adapting a transforming growth factor beta-related tumor protection strategy to enhance antitumor immunity. Blood 2002; 99: 3179–87. 142. Krance RA, Kuehnle I, Rill DR et al. Hematopoietic and immunomodulatory effects of lytic CD45 monoclonal antibodies in patients with hematologic malignancy. Biol Blood Marrow Transplant 2003; 9: 273–81. 143. Laport GG, Levine BL, Stadtmauer EA et al. Adoptive transfer of costimulated T cells induces lymphocytosis in patients with relapsed/refractory non-Hodgkin lymphoma following CD34+selected hematopoietic cell transplantation. Blood 2003; 102: 2004–13. 144. Rapoport AP, Stadtmauer EA, Aqui N et al. Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer. Nat Med 2005; 11: 1230–7. 145. Wen YJ, Barlogie B, Yi Q. Idiotype-specific cytotoxic T lymphocytes in multiple myeloma: evidence for their capacity to lyse autologous primary tumor cells. Blood 2001; 97: 1750–5. 146. Liso A, Stockerl-Goldstein KE, AuffermannGretzinger S et al. Idiotype vaccination using dendritic cells after autologous peripheral blood progenitor cell transplantation for multiple myeloma. Biol Blood Marrow Transplant 2000; 6: 621–7. 147. Davis TA, Hsu FJ, Caspar CB et al. Idiotype vaccination following ABMT can stimulate specific anti-idiotype immune responses in patients with B-cell lymphoma. Biol Blood Marrow Transplant 2001; 7: 517–22. 148. Mitsuyasu RT, Anton PA, Deeks SG et al. Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects. Blood 2000; 96: 785–93. 149. Jensen M. Strategies to enhance the therapeutic efficacy of autologous hematopoietic stem cell
Management of Relapse after Hematopoietic Cell Transplantation transplantation by posttransplantation adoptive transfer of T cells with engineered graft-versustumor activity. Biol Blood Marrow Transplant 2005; 11: 34–9. 150. Cooper LJ, Topp MS, Serrano LM et al. T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-Blineage leukemia effect. Blood 2003; 101: 1637– 44. 151. Kowolik CM, Topp MS, Gonzalez S et al. CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persis-
tence and antitumor efficacy of adoptively transferred T cells. Cancer Res 2006; 66: 10995–1004. 152. Singh H, Serrano LM, Pfeiffer T et al. Combining adoptive cellular and immunocytokine therapies to improve treatment of B-lineage malignancy. Cancer Res 2007; 67: 2872–80. 153. Todisco E, Castagna L, Sarina B et al. Reducedintensity allogeneic transplantation in patients with refractory or progressive Hodgkin’s disease after high-dose chemotherapy and autologous stem cell infusion. Eur J Haematol 2007; 78: 322–9.
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154. Martino R, Caballero MD, de la Serna J et al. Low transplant-related mortality after second allogeneic peripheral blood stem cell transplant with reduced-intensity conditioning in adult patients who have failed a prior autologous transplant. Bone Marrow Transplant 2002; 30: 63– 8. 155. Branson K, Chopra R, Kottaridis PD et al. Role of nonmyeloablative allogeneic stem-cell transplantation after failure of autologous transplantation in patients with lymphoproliferative malignancies. J Clin Oncol 2002; 20: 4022–31.
Section 6 Hematopoietic Cell Transplantation for Inherited Diseases
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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Guido Lucarelli & Javid Gaziev
Hematopoietic Cell Transplantation for Thalassemia
Introduction The thalassemias refer to a diverse group of hemoglobin disorders characterized by a reduced synthesis of one or more of the globin chains (α, β, γ, δβ, γδβ, δ, and εγδβ) and are the most common monogenic disorders to cause a major public health problem worldwide [1]. Globally, it is estimated that there are 270 million carriers of hemoglobin disorders, of which 80 million are carriers of β-thalassemia. In most countries where the transfusion and iron chelation treatment is given irregularly, mainly due to the high cost, the majority of patients with homozygous β-thalassemia die in childhood from chronic anemia and its complications, and from the organ iron overload. Currently recommended nontransplant therapy is available in most countries, and consists of transfusions to maintain hemoglobin levels between 9 and 10 g/dL, together with chelation therapy aimed at preventing iron accumulation as a consequence of the transfusion therapy. To be effective, chelation therapy should consist of continuous subcutaneous infusion for at least 8–12 hours daily. The successful implementation of this treatment regimen has improved life expectancy up to and beyond the second decade of life, and has dramatically improved the quality of life for children with thalassemia. Future treatment of the thalassemic patient may be easier and improved if orally administered iron chelators become available. Two potentially useful oral chelators are now available, but their efficacy compared with desferrioxamine (DFO) in the long term has not yet been determined. Hematopoietic cell transplantation (HCT) has been used in attempts at curing thalassemia. The first successful marrow transplant for thalassemia was reported in 1982 by Thomas and his colleagues [2], and the first reports of series of transplants were by Lucarelli et al. [3,4]. Some discussion of the epidemiology, molecular biology, pathophysiology, clinical features, and supportive therapy is necessary in an attempt to define how, when, and for whom to pursue bone marrow transplantation (BMT) for thalassemia.
Epidemiology and etiology Thalassemia is a growing global public health problem worldwide. Even though the relationship has not been proven conclusively for β-thalas-
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
semia, the geographic distribution coincides historically with malarial areas, and there is more convincing evidence for β-thalassemia and sickle cell disease. Because of a selective advantage of heterozygotes against the severe form of malaria, the frequencies of β-thalassemia are particularly high in the malarial tropical and subtropical regions of the Mediterranean area, the Indian subcontinent, Africa, and South and East Asia [1]. It is rare in people of Northern European origin, around one in 1000 being carriers, while in the Northern Mediterranean countries (south Europe) the carrier prevalence is 1–19%. In the Arab world, the carriage rate is around 3%, while in central Asia the prevalence is 4– 10%. From the Indian subcontinent to South-east Asia, β-thalassemia coexists with hemoglobin E (HbE) in carrier rates that range from 1% to 40% [5]. Worldwide, about 60,000 children with a major thalassaemia and 250,000 with a sickle cell disorder are born annually, giving a rate of more than 2.4 affected children per 1000 births [6]. The epidemiology of the disease is changing due to recent population migrations. In Europe, the United States, and Australasia, thalassemia has become an important part of clinical practice.
Molecular and clinical biology More than 200 mutations affecting the β-globin gene are known to result in a phenotype of β-thalassemia. With the exception of the 619 base pair deletion, which accounts for 20% of the β-thalassemias in Asian Indians [7], the common mutations of β-thalassemias are caused by point mutations as single-base substitutions, minor insertions or deletions of a few bases within the gene or its immediate flanking sequences, and are classified according to the mechanism by which they affect gene regulation: transcription, RNA processing or RNA translation [8]. These genetic defects lead to a variable reduction in β-globin production ranging from a minimal deficit (β+-thalassemia) to complete absence (β0-thalassemia). Mutations affecting transcription generally result in a mild-to-minimal deficit of β globin and can be silent in carriers. Mutations that affect RNA processing can involve the splice junction, in which case normal splicing is completely abolished, with resulting β0-thalassemia phenotype, or can occur within the consensus sequences at the splice junction, reducing the efficacy of normal splicing to varying degrees and producing a β+-thalassemia phenotype that ranges from mild to severe. Approximately half of the β-thalassemia alleles affect the different stages of RNA translation, which leads to a lack of β-globin production, resulting in β0-thalassemia. Most of these defects result from the introduction of premature termination codons due to frameshifts or nonsense mutations [8]. The pathophysiology of β-thalassemia relates to a quantifiable deficiency of functional β globin, which leads to imbalanced globin chain
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production and an excess of α-globin chains [1,9]. The latter are not able to form viable tetramers and instead precipitate in the red cell precursors, forming inclusion bodies that cause mechanical damage and the premature destruction of red cell precursors in the bone marrow, leading to ineffective erythropoiesis. The red cells that survive to reach the peripheral circulation are prematurely destroyed in the spleen, which becomes enlarged, eventually leading to hypersplenism. Thus, anemia in β-thalassemia results from a combination of ineffective erythropoiesis, peripheral hemolysis, and an overall reduction in hemoglobin synthesis, leading to intense proliferation and expansion of the bone marrow, with the resulting skeletal deformities. These secondary complications of bone disease, splenomegaly, and endocrine and cardiac damage can be related to the severity of anemia and the iron loading that results from increased gastrointestinal iron absorption and blood transfusions.
Clinical features The clinical manifestations of β-thalassemia are extremely diverse, ranging from the transfusion-dependent state of thalassemia major to the slightly less severe transfusion-dependent state of thalassemia intermedia or to the asymptomatic state of thalassemia trait. The common βthalassemia alleles that are prevalent in malarial regions are inherited typically in a Mendelian recessive manner, although some forms of βthalassemia are dominantly inherited. The most severe form of disease is characterized by the complete absence of β-globin production and results from the inheritance of two β0-thalassemia alleles, homozygous or compound heterozygous states. These combinations usually result in β-thalassemia major, and patients present within 6 months of birth with severe anemia; if not treated with regular blood transfusions, they die within the first 2 years of life. The anemia is due to a combination of ineffective erythropoiesis, excessive peripheral red blood cell hemolysis, and progressive splenomegaly. The red cells are microcytic with marked anisochromasia. The bone marrow shows marked erythroid hyperplasia, and the serum ferritin level is elevated. The hemoglobin pattern of patients with β-thalassemia major consists of a variable increase in HbF, which can account for 8–90% of the total hemoglobin concentration and show a variable quantity of HbA2 up to 6% [1]. In children and young adults, the expansion of hyperplastic bone marrow results in skeletal deformities and bone disease. Because of chronic anemia and iron overload, endocrinopathies such as hypopituitarism, hypothyroidism, hypoparathyroidism, diabetes mellitus, cardiomyopathy, and testicular and ovarian failure become common as the child with thalassemia grows older [1,10]. Chronic lung disease and pulmonary hypertension are other complications that are being increasingly recognized in older β-thalassemic patients, and are thought to be related to the increased tendency for thrombosis and small pulmonary emboli [11].
Nontransplant approaches The current conventional treatment of β-thalassemia major consists of lifelong regular blood transfusions combined with daily subcutaneous iron chelation therapy with DFO. In 1964, Wolman reported that patients maintained at a higher baseline hemoglobin level had less severe complications and were in better health than those maintained at lower hemoglobin levels [12]. Stimulated by this observation, Piomelli and his colleagues studied a transfusion regimen designed to prevent the baseline hemoglobin from falling below 10 g/dL. When this hypertransfusion regimen was instituted in infancy and rigorously implemented, cardiomegaly and bone malformation were greatly reduced, although normalization of growth was not achieved and the development of hypersplenism was not prevented [13]. The target hemoglobin level of
10 g/dL was designed to depress endogenous erythropoiesis and thereby avoid marrow hypertrophy with accompanying marrow space extension. This objective was partially achieved, but complete suppression of endogenous red cell production requires that the hemoglobin be maintained at levels greater than 13 g/dL [14]. There are major obstacles to achieving such levels. Raising the baseline hemoglobin level from 10 to 11 g/dL requires a 20% increase in requirements for red cell transfusion [15], and there are many reasons for minimizing transfusion in patients with thalassemia. Hypertransfusion regimens have a major impact on iron traffic and distribution in patients with severe hemolytic anemia. The complications of iron overload, such as endocrine deficiency, hepatic and pancreatic damage, and heart disease, are the principal causes of morbidity and mortality in patients with thalassemia receiving this form of therapy. The transfusion of 165 mL of packed red cells per kilogram per year is associated with an annual iron intake of approximately 180 mg/kg [16]. More recently, clinical experience suggested that a moderate transfusion regimen maintaining a hemoglobin level of 9–10 g/dL throughout life may reduce iron loading in patients with β-thalassemia major without producing an excessive expansion of erythropoiesis [17]. Iron accumulation resulting from both repeated blood transfusions and increased gastrointestinal absorption in β-thalassemia major patients may cause serious organ damage and ultimately death. Effective removal of this excess iron can improve the survival of these patients. In the last 30 years, DFO was the only effective iron-chelating drug in patients with β-thalassemia major. DFO has a strong and selective affinity for iron and does not appear to have any major side-effects. However, DFO displays poor bioavailability when given orally and has a rapid plasma clearance. Therefore, DFO is efficient only when administered parenterally over several hours in order to keep a constant serum level. Since the maintenance of steady plasma levels of this agent is essential for the effective management of iron traffic, it must be continuously administered either intravenously or subcutaneously. This has led to the now standard therapeutic approach, that is, administration of DFO by slow infusion overnight (8–12 hours for 5–7 nights) using a special pump. Intensive long-term chelation with DFO has proved beneficial for a large number of patients, as evidenced by the gradual decrease of their serum ferritin and liver iron, with improved survival [18]. DFO therapy, when rigorously adhered to, substantially reduces, but does not eliminate, the iron overload of patients on hypertransfusion therapy. There are two common reasons for the failure of chelation therapy with DFO. In emerging countries where thalassemia is a substantial public health problem, the high cost of the drug and of the supplies for its administration make it unavailable for most patients. Despite the wide availability of DFO in Western countries, some patients are unable to comply sufficiently with the prescribed treatment because the effective chelation is unpleasant and cumbersome, requiring daily subcutaneous or intravenous administration, which leads to poor compliance with therapy. It is therefore obvious that there is a pressing need for an orally administered chelator. Two oral iron chelators are currently available for clinical use. Deferiprone or L1 (DFP) is a bidentate chelator which forms a chelator–iron complex excreted mainly in the urine [19]. At a standard dose (75 mg/ kg/day in three doses), iron stores may decrease in some patients, remain stable in others, and increase in still others [20]. DFP is approved in Europe and many other countries, often as a second-line treatment for iron overload. In general, DFP is less effective in removing liver iron, although great individual variations occur. Toxic effects include agranulocytosis, neutropenia, and arthropathy, which demand close monitoring. Studies in patients treated with DFP have shown significant improvement in cardiac magnetic resonance imaging, consistent with a reduction in cardiac iron overload and improved cardiac function, in
Hematopoietic Cell Transplantation for Thalassemia
comparison with DFO, supporting a potential cardioprotective role of DFP [21,22]. Another oral chelator is now available – deferasirox (ICL670), which is a member of a new class of tridentate iron chelators [23]. It is orally bioavailable with a terminal elimination half-life between 8 and 16 hours, allowing for once-daily administration. Deferasirox–iron complexes are excreted in the stools. A recent phase III randomized study comparing deferasirox with DFO in pediatric and adult patients with β-thalassemia major receiving regular blood transfusions showed that, at 20–30 mg/kg/day, deferasirox can keep most but not all patients in even or negative iron balance in rough equivalence to moderate doses of DFO. The most common adverse events included rash, gastrointestinal disturbances, and mild nonprogressive increases in serum creatinine [24]. While the efficacy and limits of DFO therapy in transfusion-dependent β-thalassemia major patients are well known, long-term data on these two new oral chelators in treating iron overload are not yet available, and they should be investigated in large-scale prospective randomized studies. The beneficial effect of high levels of HbF in β-thalassemia and sickle cell anemia has been recognized for many years. Pharmacologically, three classes of agents have been shown to be capable of inducing HbF to therapeutic levels: erythropoietins, short-chain fatty acid derivatives, and chemotherapeutic agents. However, the sustained pharmacologic induction of HbF to therapeutic levels in β-thalassemia has been disappointing. Despite addressing the ineffective erythropoiesis, correction of the anemia has been difficult because of suppression of erythropoiesis by the currently available pharmacologic agents. Gene therapy for β-thalassemia requires gene transfer into hematopoietic stem cells (HSCs) using integrating vectors that direct the regulated expression of β globin at therapeutic levels. Early attempts using conventional oncoretroviral vectors carrying the human β-globin gene and portions of the locus control region have suffered from problems of vector instability, low titers, and variable expression [25]. Recently, human immunodeficiency virus-based lentiviral vectors have been shown to stably transmit the human β-globin gene and a large locus control region element, resulting in correction of the mouse thalassemia intermedia phenotype, with variable levels of β-globin expression [26]. The levels of β-globin expression achieved from insulated self-inactivating lentiviral vectors were sufficient to phenotypically correct the thalassemia phenotype from four patients with thalassemia major in vitro, and this correction persisted long term for up to 4 months in xenotransplanted mice in vivo [27]. Despite promising results of gene therapy in animal models, its clinical potential remains uncertain, and the safety of these vectors to use for gene therapy of hemoglobinopathies remains to be seen. Programs for prevention of thalassemia are presently based on prospective carrier screening and prenatal diagnosis. Although traditional prenatal diagnosis of β-thalassemia has been highly effective in some countries, the fact that affected pregnancies must be terminated to avoid the birth of affected children makes it unacceptable to most people. Recently developed preimplantation genetic diagnosis with in vitro fertilization has already become an alternative to traditional prenatal diagnosis, allowing the establishment of only unaffected pregnancies and avoiding the risk for pregnancy termination [28]. Preimplantation genetic diagnosis completed with human leukocyte antigen (HLA) typing allows selection of healthy and compatible embryos in families with affected children with the certainty that a source of stem cells will be available for transplantation. However, this technique, apart from raising bioethical concerns due to selecting the “HLA-compatible and healthy” embryo and discarding other (equally not sick) embryos just because they are not HLA compatible with the affected child, requires a high degree of technical expertise and is expensive, making it difficult for large application.
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Bone marrow transplantation At present, allogeneic HCT is the only rational therapeutic modality for the eradication of β-thalassemia major [29]. It is a form of gene therapy that uses allogeneic stem cells as vectors for genes essential for normal hematopoiesis. Eventually, the vector may well be autologous stem cells transformed by the insertion of normal genes, but there is no indication that this approach will be a clinical option in the foreseeable future. All of the transplant procedures in Pesaro discussed in this chapter were performed with bone marrow, and the term “bone marrow transplantation” is used for these procedures. There are no controlled trials of HCT versus medical treatment for thalassemia major, and few studies comparing quality of life. Regular blood transfusion and iron chelation have improved both the survival and quality of life of patients with thalassemia, and have changed a previously fatal disease with early death to a chronic, although progressive, disease compatible with prolonged survival [6,30]. Despite the prolonged life expectancy, a recent study from the UK Thalassemia Registry has shown a steady decline in survival starting from the second decade, with fewer than 50% of patients remaining alive beyond 35 years, mainly because of poor compliance with chelation therapy [31]. In the developing world, where thalassemia is more common, most children die before the age of 20 years because of the unavailability of safe blood products and/or expensive iron-chelating drugs. The two recently developed oral iron chelators could allow better compliance with chelation, and therefore could have a favorable impact on the survival of patients with thalassemia. However, even an ideal iron chelator with rigorous adherence only substantially reduces, but does not eliminate, the iron overload of patients on lifelong transfusions. The acute toxicities of transplantation are challenged by the observation that, in the developed world, patients with thalassemia are well served by medical treatment, and in some countries patients receiving regular blood transfusion and chelation could have a better survival rate [18,32]. Unfortunately, these same patients, with increasing age, currently experience poor outcome on conventional treatment, even in well-resourced countries with universal access to good medical treatment [10,31]. The first two transplant procedures for the treatment of thalassemia with marrow from matched related donors were performed in December 1981, in Seattle, WA, and in Pesaro, Italy. The Seattle approach was based on the assumption that the risks associated with BMT would be increased by the iron overload and by sensitization to HLAs induced by hypertransfusion. Therefore, it was decided that early clinical studies would be conducted in very young patients who had received very few transfusions. On December 3, 1981 a 14-month-old child with β-thalassemia major who had been transfused with a total of 250 mL of packed red blood cells received BMT from his HLA-identical sister in Seattle [2]. The treatment was completely successful. The Pesaro approach was based on an assessment that restricting transplants to untransfused patients was impracticable. On December 17, 1981, the Pesaro team performed a transplant in a 16-year-old thalassemic patient who had received 150 red blood cell transfusions, using marrow from his HLA-identical brother. This patient rejected the graft and was the first of an extensive series of transplants for thalassemia that provides most of the data supporting this chapter. Preparatory regimens Preparatory regimens for HCT in patients with diseases other than aplastic anemia must achieve two objectives. One is elimination of the (disordered) marrow, and the other is establishment of a tolerant environment that will permit transplanted marrow to survive and thrive. Total body irradiation (TBI) can accomplish both these objectives, but there are many reasons to avoid the use of this marrow-ablative modality. These
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Chapter 73
include the known growth-retarding effects of TBI in young children and the increased risk of secondary malignancies, which has been reported in patients treated for leukemia [33], lymphoma, and aplastic anemia [34,35]. These hazards are particularly objectionable in very young patients with potential for a long life span. The risk of these toxicities has not yet been fully explored for cytotoxic regimens that do not involve TBI. There is a considerable body of experience with the use of busulfan (BU) and its derivatives in ablating marrow in patients undergoing HCT for the treatment of nonmalignant conditions such as the Wiskott–Aldrich syndrome [36,37] and inborn errors of metabolism [38]. Cyclophosphamide (CY) is an agent that is well established as providing immunosuppression adequate for allogeneic engraftment of patients with aplastic anemia [39,40]. Experience in the use of chemotherapy-only transplant regimens for the treatment of malignancy [41– 46] has been pivotal in developing regimens appropriate for the treatment of thalassemia by transplantation. BU is an alkylating agent with exquisite specificity for the most primitive precursors of the myeloid–erythroid axis. It has been used in low doses for more than 30 years for the treatment of patients with chronic myeloid leukemia, and its toxicity and effectiveness have been well documented in that setting. Studies in rodents demonstrated that marrow-lethal doses of BU have minor toxicity for the lymphoid system and cause little immunosuppression [47]. Canine studies of transplants between dog leukocyte antigen-identical littermates demonstrated 50% engraftment after BU alone and 95% engraftment when antithymocyte serum was added to the conditioning regimen [48]. Clinical experience with the use of BU in very high doses was delayed due to the lack of an acceptable preparation suitable for intravenous use. Santos et al. reported the first clinical trials of very high-dose BU in the context of BMT [43]. In these studies, patients with acute myeloid leukemia received allogeneic marrow transplants after immunosuppression with CY (200 mg/kg over 4 days), and oral BU (16 mg/kg over 4 days) was administered as additional antitumor therapy. Early results with this therapy were encouraging, and in successful attempts to reduce early transplant-related toxicity, Tutschka et al. reduced the CY dose to 120 mg/kg over 2 days [49]. Further, CY has been a component of most conditioning regimens for transplanting patients with hematologic malignancies. Santos et al. reported on its use in high doses (200 mg/kg over 4 days) as the sole antitumor agent in patients receiving allogeneic transplants for leukemia and demonstrated that it was sufficiently immunosuppressive to permit sustained allogeneic engraftment [41]. Also, CY is most commonly employed for the treatment of lymphoid malignancies and solid tumors, and it has been used as a component of combination chemotherapy for the treatment of acute leukemia. It is not considered a highly effective agent against myeloid malignancies, although single-drug studies are not available in this context. The dose-limiting toxicity of CY is to the heart and not to the marrow. Mice, monkeys, and humans recover hematopoiesis promptly after the highest doses of CY because CY does not eliminate HSCs. Therefore, CY alone is not an appropriate conditioning modality for BMT for the treatment of thalassemia because the thalassemic marrow would recover rapidly. On the other hand, BU is an agent that has a good possibility of eradicating a diseased erythron but when used alone is not likely to be sufficiently immunosuppressive to permit sustained allogeneic engraftment. In summary, a combination of BU and CY can eradicate the thalassemia and facilitate sustained allogeneic engraftment.
marrow hyperplasia with aggressive extension of a rapidly proliferating erythron into intra- and extramedullary areas not usually occupied by marrow. This results in major bone remodeling together with marked hepatomegaly and splenomegaly. By analogy with the behavior of malignant tissue, it might be hypothesized that this large mass of rapidly proliferating hematopoietic tissue would be more difficult to eradicate than normal hematopoietic tissue, and more likely to recur after transplantation. Although post-transplant thalassemic recurrence is a problem, it occurs in circumstances that differ from those usually observed with leukemic relapse. The most common presentation of leukemic relapse is a return of host-type leukemia in the presence of a persisting immune system of donor origin. In contrast, the recurrence of thalassemia usually occurs in the context of a return of host-type immune and hematologic reconstitution. Thus, this event has aspects of both relapse and rejection, and it is customary to speak of this phenomenon as rejection. Engraftment Most patients undergoing transplantation for the treatment of thalassemia have been transfused repeatedly. Experience with BMT for the treatment of aplastic anemia in heavily transfused patients demonstrated that a history of many pretransplant transfusions increased the probability of graft rejection [50,51]. There is a substantial incidence of graft rejection in thalassemia patients after most preparatory regimens for marrow grafting, and this seems to be related to the stage of disease at the time of transplant. As discussed above, in most cases of graft rejection the thalassemic marrow will regrow and subsequent survival will be long (albeit with thalassemia). Occasionally, patients will reject grafts without recurrence of thalassemia. Unless rescued by a second transplant, such patients will die from the consequences of marrow aplasia. The treatment of patients after graft rejection will differ depending on whether the rejection is accompanied by regeneration of host-type hematopoiesis or by marrow aplasia. The Pesaro experience with this situation is discussed below. Transplant-related morbidity and mortality HCT is a dangerous undertaking. Regimens capable of eradicating a diseased marrow and facilitating persistent engraftment are necessarily toxic, and the consequences of successful allogeneic marrow engraftment include acute and chronic graft-versus-host disease (GVHD), syndromes associated with severe immune incompetence. Both prophylaxis against GVHD and methods for its treatment are immunosuppressive. Transplant-associated toxicity may be aggravated by GVHD and by measures aimed at the prevention of this complication. Such toxicity can be categorized either as regimen-related toxicity or as GVHD. In studies of HCT for the treatment of hematologic malignancies, regimen-related toxicity from the preparative regimens has been well described [52,53]. The lungs and liver are the organs most at risk for toxicity induced by TBI and BU, while the heart is the main site of CYinduced damage. Increasing patient age, previous exposure to cytotoxic agents, and the presence of latent viruses such as hepatitis C and cytomegalovirus adversely influence these toxicities. Patients transplanted for the treatment of thalassemia derive benefit from the fact that they are usually young and without prior exposure to cytotoxic agents. However, because of previous intensive transfusion therapy, they will have a high probability of carrying harmful viruses and have organ damage induced by extreme iron overload. Clinical transplant experience
Disease eradication
The Pesaro experience
Many of the hematopoietic manifestations of thalassemia resemble those of hematopoietic malignancy. The disease is characterized by extreme
By far the largest body of experience in the treatment of thalassemia with BMT has been accumulated in Pesaro, Italy. From December 1981
Hematopoietic Cell Transplantation for Thalassemia
to June 2001, 915 patients with homozygous β-thalassemia received marrow transplants. The thalassemia-free survival of the entire group of patients (aged 1 through 35 years at the time of the transplantation) is 69%. For 880 patients, the donors were HLA identical (853 siblings and 27 parents), 29 were HLA partially matched relatives, and six were HLA-identical unrelated donors. Most patients were treated with one of two types of regimen: one, used for 547 patients, prescribed BU 14 mg/ kg and CY 200 mg/kg, and the other used BU with a lower dose of CY for patients believed to be especially susceptible to the toxic complications associated with this high dose of CY. In early experience, the best results were obtained in younger patients [4,54,55]. Of the first six patients older than 16 years, four died of GVHD-related causes within the first 100 days, one died of infection on day 235, and one had recurrence of thalassemia on day 48 and died of consequent cardiac damage more than 6 years after transplantation. In view of this experience, early studies concentrated on patients under the age of 17 years.
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had all three, and class 2 patients had one or two adverse risk factors. This analysis confirmed the prognostic significance for transplant outcome of risk class and the strong influence of the quality of chelation therapy. By the end of December 2003, 524 patients under 17 years of age had received HLA-identical transplants using regimens containing BU 14 mg/kg and CY 200 mg/kg. There were 145 class 1 patients, 333 class 2 patients, and 46 class 3 patients. The probabilities of survival, thalassemia-free survival, rejection, and nonrejection mortality for class 1 patients were 90%, 87%, 3%, and 10%, respectively, and for class 2 patients they were 87%, 85%, 3%, and 13%, respectively (Table 73.1). The probabilities of thalassemia-free survival for class 1 and for class 2 patients are reported in Fig. 73.1. The probabilities of survival, thalassemia-free survival, rejection, and nonrejection mortality for the 46 patients in class 3 were 53%, 51%, 7%, and 42%, respectively, calculated at 15 years. In an attempt to improve results in class 3 patients, new treatment regimens were devised using BU 14 mg/kg and lower doses of CY (160 or 120 mg/kg). These regimens improved the probability of survival in class 3 younger (age 3 months = 66.67% n = 51
Proportion
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Fig. 75.1 Overall survival at 15 years of SBA−E−-mismatched T-celldepleted bone marrow transplantation for severe combined immunodeficiency syndrome performed 1980–2006 at the Memorial Sloan Kettering Cancer Center (n = 71) on the basis of age at transplant (≤3 months or >3 months).
BMT for the treatment of SCID have been reported from other centers, including the University of Ulm [95], the University of California Medical Center at San Francisco [96], and the Duke University Medical Center, NC [66]. In each of these series, patients did not receive any post-transplant drug prophylaxis against GVHD. In contrast, HLA-mismatched related HCT depleted of T cells by Erosetting alone or by treatment with Campath-1M have been administered together with post-transplant prophylaxis with cyclosporine. Such transplants have achieved long-term survival rates ranging from 52% to 70% [67,11]. Acute GVHD requiring therapy has occurred in 35–40% of cases and has been the major predictor of mortality [67]. For example, in a report of the European Group for Blood and Marrow Transplantation and European Society for Immunodeficiencies, summarizing results for 267 patients with SCID who had received HLA-disparate, TCD grafts after cytoreduction with, busulfan (BU; 8 mg/kg) and cyclophosphamide (CY; 200 mg/kg) [67], outcomes further improved to a 70% survival for transplants administered from 1996 to 1999, a majority of which were depleted of T cells by positive selection of CD34+ progenitor cells. With this approach, doses of T cells could be reduced from a 5 × 105 T cells/ kg upper limit selected by the European Society for Immunodeficiencies in 1986 to 1 × 104 T cells/kg body weight. Transplant outcomes are also significantly influenced by the status of the patient at the time of the procedure. In the European series, a multivariate analysis revealed that infants with B-cell deficient variants of SCID and those with an antecedent pulmonary infection were at significant risk for inferior outcome [67]. As noted in the European Group for Blood and Marrow Transplantation report [67], centers with greater experience in the use of HLAnonidentical TCD grafts also achieved significantly better outcomes (57% versus 43%). In fact, the Hospital Necker-Enfants Malades in Paris has reported a 70% survival for children with SCID who received transplants of marrow depleted by E-rosetting alone [11]. Similarly, of 17 infants with SCID at the Royal Victoria Infirmary in Newcastle, UK, between 1986 and 1998 who received HLA-disparate grafts depleted of T cells by treatment with Campath-1M and complement, 11 (64%) achieved long-term survival with reconstitution [93]. Recently, several studies have evaluated the results of unmodified HLA-matched and TCD HLA-haploidentical HCT in the rarer forms of SCID. Results from these small series underscore the differences in graft
resistance exhibited by specific variants of SCID. For example, Roberts et al. [97] recently reported results of HCT in 10 patients with JAK-3deficient SCID. Two of these patients engrafted following an HLAmatched, unmodified graft without conditioning. Of eight patients who received an SBA−E− BMT from an HLA-haploidentical related donor without conditioning, six achieved engraftment and functional reconstitution of donor T cells. However, no patient showed engraftment of donor B cells. Furthermore, two patients failed to engraft and reconstitute despite multiple SBA−E− BMT; one of these patients was ultimately reconstituted following high-dose conditioning and a cord blood graft. In contrast, in a series of 16 patients with SCID associated with an IL7Rx deficiency, Giliani et al. [98] reported that each of six recipients of TCD, HLA-haplotype-disparate HCT achieved engraftment, reconstitution and long-term survival when cytoreduced pre transplant with BU and CY. However, despite conditioning, B-cell engraftment was not detected in these patients and was only detected in two of nine recipients of HLA-matched marrow grafts, of whom three had received pretransplant conditioning. Overall, 13 of 16 patients in this series survive with reconstitution. For patients with SCID associated with either Omenn’s syndrome or reticular dysgenesis, cumulative evidence indicates that high-dose conditioning, or at least reduced-intensity conditioning, prior to transplant is likely indicated to secure engraftment and functional reconstitution following a TCD, HLA-haploidentical, related HCT [99]. In patients with Omenn’s syndrome, early transplants administered without cytoreduction were associated with a high incidence of graft failure. In contrast, such patients, when transplanted with HLA-haplotype-disparate, TCD HCT after cytoreduction with BU and CY, have ultimately achieved full engraftment and reconstitution in seven of nine patients reported [100]. Thus, such cytoreduction can overcome the graft resistance thought to be mediated by the small numbers of autoactivated T cells and NK cells consistently detected in these patients. Similarly, in a series of 10 patients with reticular dysgenesis transplanted without high doses of BU and CY, none survived with sustained engraftment, while each of five patients treated with this high-dose combination engrafted, three of whom are long-term survivors [101]. There are only a limited number of studies published on the specific use of TCD peripheral blood hematopoietic cells transplantation for the treatment of SCID. Lanfranchi et al. [94] evaluated the use of frozen TCD peripheral blood hematopoietic cells plus TCD bone marrow in nine patients with primary immunodeficiency disease, including four with SCID, three with Omenn’s syndrome, and two with CID. This approach was undertaken primarily to determine whether high cell doses could overcome the increased incidence of graft failure observed in patients with CID, who possess abnormally low but residual T-cell function. Patients were cytoreduced with BU 16 mg/kg, CY 200 mg/kg, and thiotepa 10 mg/kg. In the majority of cases, the bone marrow was T-cell depleted by Campath-1M, and peripheral blood hematopoietic cells by CD34 selection followed by E-rosetting. Although the follow-up is short (mean 7.5+ months), six of the seven patients transplanted for SCID or Omenn’s syndrome survive. Although all four patients with SCID engrafted, one died of a disseminated CMV infection. Dupuis-Girod et al. [102] recently reported successful transplantation of HLA-mismatched, maternal, TCD peripheral blood HCT (PBHCT) in a 12-month-old child with ectodermal dysplasia and SCID, following cytoreduction with BU 5 mg/kg per day for 4 days, followed by 4 days of CY 50 mg/kg/day. This patient engrafted on day 25 after transplantation, had no evidence of GVHD, and survives over 7 years post HCT, with full chimerism and a normal T-cell response to phytohemagglutinin, tetanus, and polio, as well as antibody responses to immunization with tetanus and Haemophilus influenzae vaccines. Other than recurrent plantar warts, the child is clinically well.
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Hematopoietic Cell Transplantation for Immunodeficiency Diseases 1.0
(a) 10000
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0– 3m 3– 6m 6– 9m 9– 12 12 m –1 8 18 m –2 4 24 m –3 6 36 m –4 8 48 m –6 0 60 m –7 2m 0– 3m 3– 6m 6– 9m 9– 12 12 m –1 8 18 m –2 4 24 m –3 6 36 m –4 8 48 m –6 0 60 m –7 2m
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3 CD4:CD8 ratio
Fig. 75.2 Overall survival of CD34+ peripheral blood hematopoietic cell transplantation for severe combined immunodeficiency syndrome, with (䉬) or without (䊏) conditioning, performed in Ulm, Germany from 1995 to 2004 (n = 53).
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From 1995 to 2006, 53 patients received a CD34-selected, rigorously TCD mismatched, parental peripheral blood hematopoietic cells graft at the University of Ulm for the treatment of ADA-SCID (n = 2), B−T− SCID (n = 13), Omenn’s syndrome (n = 3), B+ SCID (n = 29), B+T+ (n = 3), bare lymphocyte syndrome (n = 2), and reticular dysgenesis (n = 1). The majority of patients received cytoreduction (41 of 53 patients), primarily with BU/CY. Again, no drug prophylaxis has been administered post transplant to prevent GVHD. To date, 38 (72%) patients are alive and well, at a median of 80 (range 14–144) months post TCD PBHCT, none of whom has GVHD. There were two graft failures, one in a patient with an Artemis mutation, the other in one with B+T+ SCID/ CID. Neither child was given pre-transplant cytoreduction. There were 15 deaths, all due to infection, one following graft failure. Neither de novo acute nor chronic GVHD was observed in any of the surviving patients. The overall survival rate in this group of SCID patients following TCD, mismatched, CD34+-selected transplants (Fig. 75.2) is similar to the experience in SCID patients who have received rigorously TCD BMT. As with marrow grafts, complete engraftment and reconstitution by donor cells remained highly dependent on prior use of myelosuppression. Infusion of purified CD34+ cells in significant numbers did not facilitate full donor chimerism. Time to develop effective T-cell immunity post TCD, mismatched PBHCT ranged from 3 to 6 months and was similar to that observed after TCD BMT. Thus, the use of purified blood CD34 cells in high numbers does not appear to accelerate the functional maturation of donor T cells. Following an SBA−E− mismatched, related BMT, the majority of patients will develop normal numbers of CD3 (Fig. 75.3(a)), CD8, and CD4 T cells by 3–6 months post HCT, irrespective of whether or not cytoreduction is employed [1,66,103]. Approximately 50% of patients will have a normal phytohemagglutinin response by 6 months post transplant, and 80% will have normal T-cell mitogen and antigenspecific responses by 6–12 months (reviewed in [1,66,67]). In a large European study of mismatched, TCD HCT recipients using a variety of TCD techniques, the median time to develop normal T-cell proliferative responses to tetanus toxoid was 8.7 months [103]. Of the 116 patients in this study who survived at least 6 months post HCT, only 43% of those with absent T-cell function at 6 months (n = 21) ultimately developed normal T-cell responses, compared with 71% of those patients with normal or near-normal T-cell function 6 months post transplant [103].
Months post TCD MM related BMT for SCID, 2007
Fig. 75.3 (a) 10%, 50%, and 90% of CD3 recovery following SBA−E−-Tcell-depleted (TCD) human leukocyte antigen (hla)-mismatched bone marrow transplantation (BMT) for severe combined immunodeficiency disease (SCID) with and without cytoreduction. (b) Median CD4 : CD8 ratio following SBA−E−- CD HLA-mismatched (MM) BMT for SCID on the basis of cytoreduction.
The quality and kinetics of T-cell reconstitution in the European series correlated with SCID phenotype, with a higher percentage of patients with B+ SCIDs exhibiting normal T-cell function at all time points later than 6 months post HCT [103] compared with patients with B− SCID. Several studies have evaluated factors that influence long-term survival and immune reconstitution following TCD, mismatched, related HCT for SCID. Younger age at the time of transplant has been associated with a better outcome, with particularly good results achieved in patients transplanted in the neonatal period [66,67,75,104–108]. In a study by Myers et al. [75], long-term survival was observed in 21 of 22 infants transplanted within the first 28 days of life, compared with 51 of 67 children transplanted at later time points. As seen in earlier studies [108], interstitial pneumonia [66,67,103,108], particularly antecedent CMV or adenoviral infections, was associated with an increased mortality. Failure to develop T-cell function by 6 months post transplant or the presence of chronic GVHD for more than 6 months post HCT also confers an increased risk of mortality [103]. In the combined MSKCC and Ulm series (reviewed in [1]), NK+ SCID patients who did not receive pretransplant cytoreduction had a poorer outcome primarily due to graft failure. Although not all studies have shown an association between NK function and graft resistance [66], this may relate to differences in the genetic basis giving rise to NK+ SCID in the transplant population evaluated.
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Chapter 75
In comparative studies of the quality and kinetics of immune reconstruction, Muller et al. [104] did not find any differences in the pattern of naïve CD4+CD45RA+ T-cell reconstitution in patients who received an unmodified HLA-matched sibling graft versus TCD, mismatched, related HCT for SCID. Furthermore, in patients who received no pretransplant cytoreduction prior to SBA−E−, mismatched, parental HCT, there was a dramatic increase in the percentage and absolute numbers of CD3+CD45RA, TREC-bearing T cells within the first year following HCT [104]. Myers et al. [75] also demonstrated that infants who receive an SBA−E−, mismatched, parental BMT within 28 days of birth develop higher numbers of CD45RA+ and TREC+ T cells, and more robust T-cell proliferative responses within the first 3 years post transplant, than infants transplanted at later time points. Recently, evaluations of patients with SCID 10–15 years after successful HLA-haplotype-disparate TCD grafts have focused attention on those characteristics of host and HCT that sustain thymic output and normal T-cell function. Initially Patel et al. [105] showed that the average number of naïve T cells and T-cell mitogen responses decreased with time after such transplants. Data from the MSKCC (Fig. 75.3(b)) also demonstrate that, in the absence of cytoreduction, the CD4 : CD8 ratio inverts in a percentage of patients following TCD SBA−E− BMT, paralleling a decline in phytohemagglutinin response in this subset of patients. However, these alterations are not seen in cytoreduced transplant recipients. Similarly, Borghans et al. [106] evaluated predictors of long-term T-cell reconstitution in a heterogeneous group of 19 SCID patients evaluated 5–32 years post HCT. Unlike the patients in the study of Patel et al. [105], who received SBA−E− HCT without prior conditioning, 74% of patients in this study were cytoreduced, and 14 of 19 patients received transplants that were T-cell depleted by E-rosetting and concomitant GVHD prophylaxis. In 11 of 19 patients, there was no evidence of impaired T-cell reconstitution, naïve T cell production or reduced telomere length post HCT when compared with age-matched controls. In the eight patients with impaired T-cell reconstitution late after transplant, these parameters were already abnormal early after HCT, suggesting that poor engraftment rather than reduced thymic output with time was the etiology of the poor T-cell reconstitution. However, two more recent studies have shown that those patients with SCID that have received haplotype-disparate, TCD HCT who have donor cells in their peripheral myeloid lineages or among the CD34+ progenitor cells in the marrow late after transplant also have sustained normal levels of TREC+ T cells without evidence of a decline in T-cell number or function [107,108]. The study of Friedrich et al. [108] is particularly informative, since 25 of the 31 patients studied had received HLA-haplotype-disparate, SBA−E− TCD marrow grafts, either with (n = 13) or without (n = 12) pretransplant cytoreduction. Sustained engraftment of donor-type CD34+ progenitor cells was regularly observed in the cytoreduced group and was directly correlated with persistently normal numbers of TREC+ and CD45RA+ T cells. Donor CD34+ cells were not detected in the noncytoreduced group. These patients also exhibited significant reduction of CD45RA+ T cells 15 years post transplant. Although these studies demonstrate the ability of the epithelium of the SCID thymus to support robust thymopoiesis in most patients, the declining generation of naïve thymus-dependent T cells observed in some patients, particularly those who received no cytoreduction prior to transplant, remains of concern [105]. Thymopoiesis may not be sustained if long-term engraftment of hematopoietic stem cells capable of continuously providing thymic precursors is not achieved, a scenario that is common in noncytoreduced patients [107,108]. Unfortunately, in patients with poor T-cell reconstitution, Slatter and colleagues demonstrated that boosts of TCD HCT following initial transplant, in the absence of cytoreduction, were not associated with improved function [109].
Reconstitution of specific antibody formation following SBA−E−, mismatched, related HCT, as with other TCD techniques, is generally observed only in those patients given pretransplant cytoreduction, and is correlated with the degree of donor B-cell engraftment (reviewed in [1,66,67,109]). In the MSKCC series, normal antibody responses following immunization were observed in all patients engrafted with donor B cells, compared with less than 10% of patients with exclusively host B cells (reviewed in [1]). Forty-five of 72 infants transplanted at Duke University Medical Center [66] required prolonged gammaglobulin therapy, the majority of whom received an SBA−E− TCD, mismatched, related marrow graft without prior cytoreduction. This contrasts to the smaller study by Dror et al. [96] which showed that, despite absence of donor B cells, six patients developed specific antibody responses 1.5–10.0 years following an SBA−E−, mismatched, related BMT. In a single-center study by Haddad et al. [110], donor B-cell engraftment was documented in four of five and three of 17 recipients of unmodified HLA-matched or TCD, mismatched, related BMT, respectively. Ten of 18 patients with host B cells remained on intravenous Ig more than 2 years post BMT. In this study, in contrast to the combined MSKCC–ULM experience, cytoreduction with BU 8 mg/kg and CY 200 mg/kg did not promote donor B-cell engraftment or the development of specific antibody production [110]. In patients with B− Artemis mutations, B-cell engraftment was observed only if autologous precursor B cells in the bone marrow were eradicated [111]. Despite the success of TCD, mismatched, related HCT for patients lacking an HLA-matched sibling donor, several problems clearly remain, namely: 1 graft failure in some series in those patients with NK-cell function or minimal T-cell function (reviewed in [1]); 2 GVHD following less rigorous TCD techniques [67,90,91]; 3 EBV-LPD, due to uncontrolled proliferation of transformed B cells prior to T-cell development [112,113]; 4 lack of donor NK-cell engraftment, which has been associated with epidermodysplasia verruciformis in both MSKCC and other series [114,115]; 5 autoimmune disorders due to dysregulated B-cell function [116,117]; 6 hypogammaglobulinemia, despite successful T-cell reconstitution in patients not given cytoreduction [66,68,110] who fail to develop donor B cells; such patients require regular infusions of Ig to avert recurrent sinopulmonary infections, bronchiectasis, and respiratory insufficiency; 7 diminished long-term capacity to produce naïve T cell populations [104,105]. An ongoing debate at many centers performing alternative donor transplants for SCID is whether all clinically stable children should receive pretransplant cytoreduction, even in the absence of factors known to inhibit T-cell engraftment. Universal cytoreduction would increase the proportion of children who engraft not only donor T cells, but also donor B and NK cells, thus avoiding continued need for intravenous Ig, risk of recurrent sinopulmonary infections despite intravenous Ig, the potential of diminished thymic output with time, and complications associated with lack of NK cells [114,115]. However, adoption of this approach obviously needs to be weighted against the long-term side-effects of cytoreduction in sick infants, particularly in newborns diagnosed antenatally or with newborn screening techniques. Furthermore, while most infants will develop at least partial donor B-cell chimerism and specific B-cell function with reduced doses of BU and CY, this is not true in all patients [110]. It is possible that reduced-intensity conditioning, for example with BU combined with fludarabine, followed by TCD HCT, will be more readily tolerated than BU/CY and will still allow the devel-
Hematopoietic Cell Transplantation for Immunodeficiency Diseases
opment of donor-derived T- and B-cell reconstitution. This is currently being explored.
1.0 0.9 0.8
Unrelated HCT for the treatment of SCID was first performed in 1973 by O’Reilly and colleagues [118]. The stem cell source was bone marrow derived from a one-antigen HLA class I-disparate female donor and administered to an HLA-A1-homozygous male infant. After multiple transplants from this donor, lymphoid engraftment and full immunologic reconstitution were achieved, complicated by the development of extensive chronic GVHD. This patient ultimately died 9 years post HCT of metastatic squamous cell carcinoma of the skin. In 1992, Filipovich et al. [119] reported on eight children who received unmodified, unrelated HCT. All but one child was cytoreduced with BU/CY and antithymocyte globulin (ATG). All patients received GVHD prophylaxis. Six of eight children survived with immunologic reconstitution between 1.0 and more than 3.8 years after BMT. Only one patient developed significant GVHD. In 1996, an update of unrelated BMT for SCID (n = 24) was published [120]. In this series, children with X-linked or ADA+ SCID were not given cytoreduction. Of those given cytoreduction, most received BU, CY, ATG, and GVHD prophylaxis with cyclosporine (CSP) and methotrexate. Four patients died less than 28 days post HCT and could not be evaluated. Of the 20 remaining evaluable patients, graft failure occurred in two. The estimated 3-year survival was 61%, with grade II–IV GVHD occurring in 17% of engrafted patients [120]. Nine children with SCID received an unmodified, unrelated BMT at the Hospital for Sick Children at Toronto, Canada, between December 1987 and August 1997 [121] following cytoreduction with BU/CY. CSP, methylprednisolone, with or without short-course methotrexate, was given to prevent GVHD. Donors were identified at a mean of 4.7 (range 1–13) months. Despite antecedent pneumonia in four of the nine SCID patients, six survive a median of 54.5 (range 18–72) months post unrelated transplant. Death in the other three patients was due to graft failure (n = 1) or grade III GVHD (n = 2). T-cell reconstitution occurred in all six patients. B-cell reconstitution was documented in four patients. Grunebaum et al. [81] reported on the results of unrelated HCT performed at the University of Brescia in Italy and the Hospital for Sick Children in Canada between 1990 and 2004. Thirty-three of 41 (80.5%) patients who received an unmodified unrelated graft survive. Acute GVHD was observed in 71% of patients. In the 2003 update of the European experience of HCT for SCID, the 3-year survival was 63% in the 28 recipients of an unrelated HCT [67]. Results of unrelated HCT (bone marrow or peripheral blood) reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) from 1990 through 2004 are shown in Fig. 75.4. The probability of overall survival after unrelated allogeneic donor HCT for SCID at 5 year (including unrelated bone marrow and peripheral blood) is 60% (95% confidence interval [CI] 52–67%). Reduced-intensity conditioning and HCT for SCID A subset of infants with SCID present with comorbidities which prohibit the use of high-dose conditioning. Several groups have explored the use of reduced-intensity conditioning for such patients [122,123]. Rao et al. [122] compared survival in 13 patients with SCID, of whom seven received BU (16 mg/kg) and CY (200 mg/kg), and six received reducedintensity conditioning with fludarabine, melphalan, and either alemtuzumab or ATG. The former group received unrelated bone marrow depleted of T cells with Campath and donor complement. Recipients of reduced-intensity conditioning received unmodified bone marrow. Five of seven patients with SCID who received high-dose conditioning sur-
Overall survival, 61% (95% CI 52-70) @ 5 years
0.7 Probability
Unmodified, unrelated BMT for SCID
1111
0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
1
2
3
4
5
Years
Fig. 75.4 Probability of overall 5-year survival following unrelated bone marrow or peripheral blood transplantation for patients with severe combined immunodeficiency disease performed from 1990 to 2004, as reported to the Center for International Blood and Marrow Transplant Research (n = 129). (Reproduced with permission from the CIBMTR.)
vived, compared to five of six patients who received reduced-intensity conditioning [122]. Umbilical cord blood cell transplants for SCID In 2000, Knutsen and Wall [124] described eight infants with severe T-cell immunodeficiency disease who received an unrelated cord blood cell transplant, seven of whom engrafted. Graft failure occurred in one infant with reticular dysgenesis given BU/CY who subsequently engrafted following a cord blood transplant from a second unrelated donor after total body irradiation conditioning. Despite significant HLA mismatching between the cord blood donor and recipient in this group of eight patients, five patients developed grade I acute GVHD, and only one patient developed severe GVHD involving the skin and gut. Tand B-cell immunity recovered at 60–100 days post-transplant [123]. Buckley et al. have successfully used unrelated cord blood transplantation to rescue three children with X-linked SCID who rejected a TCD, mismatched, related BMT given in the absence of cytoreduction. In two of these cases, BU/CY was given prior to cord blood transplantation [66]. Successful correction of Omenn’s syndrome (n = 1) and X-linked SCID (n = 2) by unrelated cord blood cell transplant have been reported, as well as correction of T−B−NK+ SCID by HLA-matched sibling cord blood cell transplant [124–128]. Bhattacharya and colleagues transplanted eight patients with SCID with unrelated (n = 7) or related (n = 1) cord blood transplant [128]. Infants were treated for ADA deficiency (n = 4), reticular dysgenesis (n = 1), T−B+NK+ SCID (n = 2), and one undefined SCID syndrome. Conditioning consisted of BU (16 mg/kg)/ CY (200 mg/kg) in four patients, and no conditioning in three of four patients with ADA deficiency, one of whom received a matched sibling cord blood transplant. Seven of eight patients engrafted, and six of eight survive. One patient developed severe sinusoidal obstruction syndrome, which was successfully treated with defibrotide. Two deaths occurred. One patient with ADA deficiency died of respiratory failure secondary to parainfluenza-3 which antedated transplant. The other death was due to multiorgan failure. The results of a CIBMTR analysis of 77 cord blood transplants performed for SCID between 1990 and 2004 are shown in Fig. 75.5. The probability of overall survival at 5 years was 57% (95% CI 44–69%) [129].
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1.0 0.9 0.8
Overall survival, 57% (95% CI 44-69) @ 5 years
Probability
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
1
2
3
4
5
Years
Fig.75.5 Probability of overall 5-year survival following unrelated donor cord blood transplants, 1990–2004, reported to the Center for International Blood and Marrow Transplant Research (n = 77). (Reproduced with permission from the CIBMTR.)
In utero HCT for SCID In 1996, Flake et al. reported the first successful correction of SCID by a TCD, haploidentical parental marrow graft administered by in utero transplantation [130]. This report was followed closely by similar case reports of Wengler et al. in 1996 [131], and Gil and colleagues in 1999 [132]. The patient reported by Flake et al. had X-linked SCID that was diagnosed during the first trimester [130]. This patient received three infusions of TCD bone marrow cells containing 114.0, 8.9, and 6.2 × 106/kg CD34+ cells and less than 5.2 105 CD3+ cells/kg fetal weight. The cells were injected into the abdomen of the fetus between 15.0 and 18.5 weeks gestation. Circulating donor T cells were observed at birth and at 3 and 6 months thereafter. However, B cells, NK cells, monocytes, and granulocytes remained of host origin. The engrafted paternal T cells were tolerant to the patient and demonstrated normal in vitro T-cell proliferative responses. Subsequent analysis of this patient has demonstrated persistent engraftment of donor T cells with specific T- and B-cell function 4 years post HCT [130,133,134]. Muench [135] summarized the results of in utero transplants in 10 patients performed between 1996 and 2005 to correct γc (n = 5) or IL7Rα deficient (n = 1) SCID, Omenn’s syndrome (n = 1) or genetically uncharacterized T−B+ (n = 1), T−B− (n = 1) or T−B+NK+ (n = 1) SCID. Of the 10 patients with SCID and absent T cells, nine received TCD bone marrow or peripheral blood; one patient received fetal liver and thymus antenatally and additional fetal liver and thymus, as well as TCD HCT, postnatally. This latter patient lived for 9 years post HCT, but succumbed after a liver transplant performed at 9.5 years of age. Engraftment of T lymphocytes with or without donor NK cells was observed in eight of ten patients. Although these cases demonstrate the feasibility of in utero transplants to induce T-cell reconstitution, comparison of this treatment with other modalities is difficult due to the small numbers of such transplants. To date, B-cell reconstitution has been achieved in only one patient [136]. Conclusion As summarized above, the results of related HLA-disparate, TCD HCT, which date from the early 1980s and are applicable to all patients with SCID, continue to improve and are now associated with a low risk of graft failure and a very low incidence of severe GVHD [1,67]. Centers with large series of patients so treated report disease-free survival rates
with sustained engraftment and reconstitution of T-cell immunity in over 70% of cases [1,66,80,93,95,96]. Similar survival rates are now also being achieved with unmodified grafts from HLA-matched unrelated donors, albeit with higher rates of both acute and chronic GVHD [81,119]. While a European retrospective multicenter analysis has reported significantly better overall results with unrelated transplants, analysis of patients transplanted since 1987, when unrelated donor identification became practicable, did not indicate a significant advantage [67]. A more recent retrospective comparison of transplant results in Brescia and Toronto [81] also suggested an advantage to the use of a matched unrelated donor. However, in that study, the vast majority of clinically unstable patients referred for transplant received a TCD HLAhaplotype-disparate parental graft. This analysis also did not account for the clinical consequences of the excess time required to identify an HLA-identical unrelated donor. More importantly, the survival rates recorded for recipients of unrelated transplants were comparable to those for recipients of HLA-nonidentical TCD grafts but markedly inferior to those currently reported from other large centers that regularly employ HLA-haplotype-disparate TCD grafts [1,66,90,93,95,96]. At this time, only a prospective study comparing outcomes of alternative donor transplants in terms of survival, graft failure, GVHD, infectious complications, and long-term immune reconstitution is likely to determine whether and to what degree one or the other approach is more beneficial. Further, from a clinical prospective, the optimal alternative donor, stem cell source, and cytoreductive regimen to be selected is unlikely to be uniform for this heterogeneous population. The clinical status of the affected infant, the variant of SCID requiring correction, the availability of a suitable HLA-matched related or unrelated donor, and the T-cell-depletion techniques available to an individual transplant center all need to be considered to determine how best to transplant the child. Many children with SCID who lack a matched sibling donor present with severe pneumonias caused by viruses or other pathogens and are too ill to be transferred to a transplant center experienced in the use of TCD, HLA-haplotype-disparate, parental grafts for SCID. While donor marrow grafts can be depleted of T cells at a specialized center and transported back for transplantation into such patients, this approach is at present logistically difficult. In such circumstances, an unmodified, HLA-matched marrow or cord blood graft could be the better option, but only if the identification and procurement of such a transplant can be achieved promptly. If the time needed to identify a suitable unrelated donor exceeds 3–4 weeks, the added infectious morbidity and risk of fatality imposed on an infant with SCID is not justified. Conversely, a matched unrelated marrow or cord blood graft would be preferred if a graft from a haplotype-matched parental or unrelated donor would impose an undue risk of infection, such as active CMV or hepatitis B or C infection. Transplantation for other CIDs and lethal congenital disorders The CIDs, including Nezelof syndrome [137], include a heterogeneous group of rare diseases in which patients present with recurrent severe acute and chronic infections associated with profound but not absolute deficiencies of T- and B-cell function (reviewed in [6,7,137]). In these disorders, T-cell numbers may be normal or reduced. Although antigenspecific responses to pathogens are not detectable, proliferative responses to nonspecific mitogens and allogeneic cells, while markedly abnormal, are not absolutely deficient as they are in patients with SCID. In several CID syndromes, B cells are prominent and Ig classes may be normal or increased. However, specific antibody production is usually absent (reviewed in [6,7,137,138]).
Hematopoietic Cell Transplantation for Immunodeficiency Diseases
The genetic mutations responsible for many of the CID syndromes are known, permitting definitive diagnosis. Genetic analysis is also important to distinguish these patients from a subset of patients with variants of SCID including Omenn’s syndrome [14,15], ADA deficiency [27], CD3 invariant chain mutations [51], and defects of DNA ligase IV [22–24], and Cernnunos [39] expression who may present with partial rather than complete absence of T- and B-cell function. A second set of CID syndromes are the result of defects in cytokines or their receptors or lack of cell surface molecules involved in cell contact, antigen presentation, activation, and/or proliferation. These include: • defective cytokine production or responsiveness; • defective IL-1 responsiveness [139]; • defective transcription of multiple cytokine genes [140]; • abnormal IL-2 production [141,142]; • nucleoside phosphorylase deficiency [143,144]; • CD8 deficiency caused by mutations in ZAP-70 [58]; • bare lymphocyte syndromes [6,7,145–147], a series of genetic disorders resulting in defective transcription and expression of HLA class I and/or class II determinants on the surface of lymphoid and hematopoietic cells; • deficiency of leukocyte function-associated antigen-1 (LFA-1) [148,149]; • CD40 ligand and CD40 deficiency [150–154]; • common variable immunodeficiency disease [6,7,155]. A third set of disorders for which transplantation may be curative includes a series of genetically defined diseases that cause either lymphoproliferative disease or severe and uncontrolled autoimmune disorder. These include: • X-linked lymphoproliferative syndrome [156,157]; • abnormalities of T cell regulation: IPEX, autoimmune lymphoproliferative syndrome (ALPS: ALPS1a, ALPS1b, ALPS2, and ALPS3) (reviewed in [6,7,158]). Each of these disorders of immunity can be corrected by a marrow allograft. However, because these disorders are so rare, experience with allogeneic HCT in these diseases has been largely limited to transplants of from one to three patients at any given center. In aggregate, the results of transplants applied to patients with these disorders have been distinctly inferior to those reported for transplants applied to patients with SCID or WAS. For example, in the European experience of HCT for non-SCID patients with T-cell immunodeficiency performed between 1968 and 1993, 3-year overall survival was 63% for 47 recipients of an HLA-matched sibling graft, and only 35% in 72 recipients of an HLAmismatched, TCD graft [67]. For comparison, survival rates for patients with SCID receiving such grafts were 81% and 54%, respectively. The inferior results achieved in these disorders reflect in part the higher incidence of graft rejections in these patients, indicating the need for effective immunoablation, and possibly myeloablation, to achieve consistent engraftment. They also reflect the increase in transplant-related mortality due to toxicity and infection in these children, who are often chronically infected and debilitated at the time of diagnosis and referral for transplantation. However, based on analysis of recent experience in certain more common CID diseases, over the last decade, an improved diagnosis of these disorders, recognition of the need for effective pretransplant cytoreduction, and improvements in the identification of compatible donors and in the development of less toxic cytoreductive regimens for transplantation are likely resulting in better outcomes [67,98,159–169]. Recent results of transplants applied to patients with hyper IgM syndrome caused by CD40 ligand deficiency are a case in point. For example, Gennery et al. [167] updated the European experience of transplants for X-linked hyper IgM syndrome (n = 38) performed from
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1993 through 2002. Donors included an HLA-identical sibling (n = 14), a phenotypic HLA-matched family member (n = 4), and a volunteer unrelated donor (n = 22). Thirty-four of thirty-eight patients engrafted, and 26 patients survived. Twenty-two patients developed normal T-cell function, and 18 developed specific antibody following vaccination. One patient who engrafted but subsequently developed impaired T-cell reconstitution had autologous reconstitution, 22 (58%) were cured, and one other patient engrafted but had poor T-cell immune reconstitution. Twelve patients (32%) died from infection, which was associated with grade II–IV GVHD in six. Poor prognostic factors in this study included pre-existing liver and pulmonary disease. Recently, however, Tsuji et al. [165] reported the results of eight consecutive patients with hyper IgM syndrome transplanted from 1998 to 2004. The median age of the patients was 12.75 (range 3.9–19.9) years. Six patients received a transplant from an HLA-matched sibling, and two patients received an unrelated donor transplant. Of the eight patients, six received conditioning with BU/CY. Six patients remain alive at 350–2190 days post HCT, including both recipients of an unrelated HCT. Two patients died, one of sepsis and one of an Aspergillus infection. In 2004, Tomizawa et al. [166] reported the outcome of seven patients with hyper IgM syndrome, four of whom received an HLA-matched sibling BMT and three unrelated HCT. The median age of these patients was 11.3 (range 3–19) years. Cytoreduction consisted of BU/CY in six of seven patients with CSP and methotrexate for GVHD prophylaxis. Five of the seven patients are alive with normal expression of CD40L, four no longer remain on intravenous Ig. Two patients with hyper IgM syndrome received unmodified, HLA-matched sibling HCT following cytoreduction with a reduced-intensity regimen consisting of low-dose BU, fludarabine, ATG, and CSP [167]. Both children had significant liver disease documented by liver biopsy prior to transplant, which resolved after HCT. Both children are alive more than 2 years post HCT, one a mixed chimera, the other a full chimera. Normal T-cell function and freedom from intravenous Ig have been achieved in both patients. The impact of reduced-intensity conditioning, initially applied to other combined immune deficiencies by Amrolia et al. [159], is increasingly reflected in recent results from several centers. Rao et al. [122], in an update of the Great Ormond Street series, reported a 1-year survival rate of 95% for 27 patients with non-SCID congenital immune deficiencies who received unmodified, HLA-matched or one-alleledisparate transplants from unrelated donors after conditioning with fludarabine, melphalan, and either alemtuzumab or ATG. Reduced-intensity conditioning was also used by Cohen et al. [169] in eight patients with various CID syndromes who developed an EBVLPD pre HCT. This study included patients transplanted from an unrelated donor (n = 6), an HLA-matched sibling (n = 1), and a phenotypically HLA-matched family member (n = 1). All patients received rituximab prior to transplant. Patients were transplanted for intractable diarrhea of infancy (n = 1), Chediak–Higachi syndrome (n = 1), ALPS-like syndrome (n = 2), undefined CID (n = 2), X-linked lymphoproliferative syndrome (n = 1), and WAS (n = 1). Seven of eight patients were cytoreduced with fludarabine (150 mg/m2 over 5 days), melphalan (140 mg/m2), and alemtuzumab (1 mg/m2 in five divided doses). Patients received CSP alone (n = 5) or combined with mycophenolic acid (n = 3). All patients are currently alive without evidence of EBV-LPD between 1 and more than 7 years post HCT. Reduced-intensity conditioning has also been used successfully as preparation for successful transplants for patients with purine nucleoside phosphorylase deficiency [163] and IPEX syndrome [164]. While the results of transplants from matched related, unrelated, and HLA-disparate related donors have continued to improve in the treatment of most CID syndromes, patients with HLA class II deficiency
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remain a major challenge. In the initial European survey of transplants for immunodeficiencies conducted between 1968 and 1985 [161], only one of six patients with bare lymphocyte syndrome, who received an HLA-matched transplant, and none of four recipients of HLAnonidentical grafts survived reconstitution. However, despite advances in transplantation medicine and improvements in diagnosis and supportive care, recent reports from centers with the largest experience with such patients attest to continuing major impediments to the success of transplants in this patient group [162]. Of particular concern are the high incidence of graft rejection despite use of high-dose conditioning and the strikingly high mortality resulting from viral infections, of which the majority have antedated the transplant period. In the series from London [167], two of four recipients of HLAmatched and the one recipient of an HLA-nonidentical partial graft rejected their transplants despite preparative cytoreduction with BU and CY or melphalan and fludarabine. Similarly, in the Paris experience [162], one of six recipients of unmodified, HLA-matched and eight of 12 recipients of HLA-disparate, TCD transplants rejected their transplants despite preparative cytoreduction with BU and CY with or without anti LFA-1. In both series, viral infections were a major cause of morbidity and mortality. In aggregate, only four of the 11 recipients of HLA grafts and seven of 14 recipients of HLA-nonidentical transplants survive with reconstitution in these two series. In part, the complications observed may reflect the activation of quiescent and partially resistant host lymphocytes responding to HLA alloantigen newly presented by transplanted donor cells as well as the inability of donor T cells to interact effectively with infected host tissue that do not express HLA [170,171]. Both series indicate that the probability of a successful transplant in these disorders is greatly impaired by the presence of an active viral infection.
Wiskott–Aldrich syndrome WAS is a rare X-linked disorder characterized by thrombocytopenia, immunodeficiency, and eczema that is estimated to occur in between 1 and 10 per million live male births [3,4,172–175]. Affected individuals also have an increased risk of lymphomas, particularly EBV-LPD [176,177], and autoimmune disorders [178,179]. The mutated gene responsible for WAS and the milder disorders of X-linked thrombocytopenia and X-linked neutropenia was discovered in 1994 by Derry and colleagues [179]. The normal gene, which maps to Xp11.22–p11.23, encodes a 501 amino acid, 53 kDa intracellular protein (WAS protein or WASp), expressed in most hematopoietic cells [179]. WASp is involved in cytoskeletal organization (reviewed in [174]) and plays an essential role in T- and NK-cell activation [174,180–182]. Derry and colleagues identified three separate mutations in four patients with WAS, two of whom were related [174]. Since then, over 150 unique mutations in more than 300 families worldwide have been described [174,183–185]. In 124 patients with WAS and 116 patients with X-linked thrombocytopenia, the most common mutations found in the former group were deletions (30 of 124 patients). In a recent study by Proust et al. [184], 28 new mutations were reported using multiplex polymerase chain reaction [185]. In 2006, the mutation responsible for WAS in the three brothers described by Wiskott in 1937 was identified – a deletion of two nucleotides at positions 73 and 74 in exon 1 of the WAS gene – which resulted in a frameshift mutation [186]. Recently, several females with WAS have also been described (reviewed in [174]). Although all patients with WAS are thrombocytopenic, the level of T- and B-cell immunodeficiency, and the subsequent risk of autoimmune phenomena (nephropathy and vasculitis) and lymphoid malignancies can be quite variable. Scherbina et al. [187] demonstrated that the
clinical heterogeneity observed in WAS patients is due to differences in the expression of WASp in lymphoid cells. Patients could be divided into four clinical groups on the basis of WASp expression, ranging from group A – patients with mild clinical disease associated with low levels of WASp in their peripheral blood mononuclear cells and higher levels in EBV-immortalized B-lymphoblastoid cell lines, together with near normal levels of WASp RNA – to group D, patients who manifested severe disease in which peripheral blood mononuclear cells and EBVimmortalized B-lymphoblastoid cell lines lacked WASp expression. Knowledge of the pattern of WASp expression in individual patients [187–189] is used to determine which patients lacking an HLA-matched, related donor should be treated with supportive care alone [175], and which should be early candidates for HCT from alternative donors or, potentially, gene therapy [190,191, reviewed in 192]. Several other studies have shown a strong genotype–phenotype correlation in this disorder. Imai et al. studied the outcome of 50 Japanese patients with WAS/XLT from 43 unrelated families [188]. All patients with missense mutations had detectable levels of WASp. In contrast, patients with nonsense mutations, large deletions, small deletions or small insertions were WASp negative and had more severe disease. A separate study evaluated the correlation of genotype with phenotype in 262 patients from 227 unrelated families with WAS/XLT followed in two centers, one in Seattle, the other in Italy [189]. As in the previous study, the best predictor of clinical manifestations was determined by WASp expression. The majority of patients in this study who had WASpnegative lymphoid cells had deletions or insertions which resulted in a frameshift, nonsense or splice-site mutations. Seventy-four percent of patients in this study with XLT had missense mutations. HCT for WAS In 1968, Bach et al. [193] reported correction of the immunologic abnormalities associated with WAS following HLA-matched sibling transplantation. Despite the use of high-dose CY, this patient did not demonstrate engraftment of hematopoietic elements and remained thrombocytopenic. In 1978, Parkman et al. [194] corrected the immunologic and hematologic defects in a child with WAS by administering an HLA-matched marrow graft following myelosuppressive and immunosuppressive cytoreduction consisting of total body irradiation, procarbazine, and ATG. These authors hypothesized that myeloablation as well as immunosuppression was required to correct disorders involving nonlymphoid lineages by providing “space” in which progenitor cells could proliferate and develop. Subsequently, Kapoor et al. [195] demonstrated that BU/CY provided a myelosuppressive and immunosuppressive regimen adequate to insure full hematopoietic chimerism, correcting both the lymphoid and platelet defects in a series of children with WAS. A worldwide review from 1968 through 1997 indicated that 57 of 65 children with WAS survived in the long term after HLA-matched BMT [196]. Single-center studies of HLA-identical, related BMT from Brochstein et al. [197], Ozsahin et al. [198], Rimm and Rappeport [199], and Pai [200] have recorded long-term survival rates of 80% (eight of 10), 91% (10 of 11), 88% (seven of eight), and 100% (four of four), respectively. In the study by Imai et al., all of the 11 patients with WAS who received HLA-matched sibling HCT are surviving [188]. Nine of 11 recipients of HLA-matched, related HCT reported by Kobayashi et al. are surviving, compared with five of 10 mismatched related, and 17 of 21 unrelated, transplant recipients for WAS [201]. For patients without an HLA-matched sibling, transplants from closely matched, unrelated donors have provided excellent results. Of 30 patients with WAS who received unrelated, unmodified BMT prior to July of 1994, the overall actuarial survival was 67% [202]. Analysis
Hematopoietic Cell Transplantation for Immunodeficiency Diseases (a)
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of these transplantation results demonstrated that children under 2 years of age at the time of transplant had a much better prognosis, with 21 of 23 surviving (actuarial survival at 3 years 91%). Acute grade II–IV GVHD occurred in 50% of patients, with the highest incidence and severity observed in patients over 5 years of age at HCT. All four children who received an unmodified, unrelated BMT reported by Lenarsky et al. [203] survived between 3 to over 17 months post BMT. In 2001, Filipovich et al. [204] summarized and compared the results of 170 transplants reported to the International Bone Marrow Transplant Registry and/or the National Marrow Donor Registry (1968–96), including 58 patients transplanted from HLA-matched siblings, 48 from other relatives, and 67 patients from unrelated donors. Transplant outcome differed according to donor type, with a probability of 5-year survival of 87% (74–93%) with an HLA-identical sibling donor, 52% (37–65%) with partially compatible related donor, and 71% (58–80%) with unrelated donors [204]. For recipients of unrelated HCT, the best outcomes were observed in patients transplanted at less than 5 years of age. Pai reviewed 16 unrelated cases of HCT for WAS performed at the University of Brescia from 1990 through 2005 [200]. All patients received cytoreduction with oral BU (16 mg/kg) and intravenous CY. Thirteen patients were treated with ATG. All patients received unmodified bone marrow. Seven patients received additional CD34+-selected bone marrow. Donors were 6/6, 8/8 or 10/10 HLA-matched donors in 2, 5, and 4 cases, respectively. Donors for four patients were HLA mismatched at one (n = 3) or two (n = 1) loci. In one case, HLA typing was not stated. GVHD prophylaxis consisted of oral CSP 5 mg/kg twice daily. Of the 16 patients, 13 (81.2%) are surviving a median of 6 (range 0.6–9) years post HCT. Cause of death has been EBV-LPD and CMV (n = 1), Pneumocystis carinii pneumonia (n = 1), and postsplenectomy infection (n = 1). The Committee for Stem Cell Transplantation of the Japanese Society of Pediatric Haematology reported that 80% of WAS patients who received an unrelated HCT from an adult volunteer donor (n = 21) have survived [205]. Prognosis was poorer in patients who were transplanted at over 5 years of age and in those who received cytoreduction with a regimen other than BU/CY. Of the 156 unrelated BMTs or PBHCTs from unrelated donors reported to the CIBMTR from 1999 through 2004 [129], the overall survival at 5 years was 65% (95% CI 57–73%) (Fig. 75.6(a)). In the last 5 years, the results of cord blood transplantation for WAS have been reported from several centers, including results of double cord blood transplants [201,206,207]. The majority of patients received cytoreduction with BU and CY with or without ATG. Of the 21 unrelated cord blood transplants performed, the majority in Japan [201], 17 resulted in long-term survival. Figure 75.6(b) demonstrates the results of 54 cord blood HCTs reported to the CIBMTR from 1990 through 2004, showing an overall survival of 70% (95% CI 56–82%) at 5 years. For patients who lack a suitable cord blood or unrelated donor, TCD HCT has been utilized with variable success. In a review of 15 recipients of TCD HCT, only six patients have become long-term survivors [195]. In the initial MSKCC series, only one of six patients survived long term. In the recent update of the European experience, 3-year survival for WAS was 45% in 43 recipients of an HLA-mismatched, related HCT compared with 81% in 32 recipients of an HLA genotypically matched transplant [67]. Failures in these series were primarily due to EBV-LPD and GVHD [194]. However, with the recent development of therapies such as rituximab and EBV-specific T cells that are effective in treating or preventing EBV-LPD in these patients [208–215], the success of such transplants may significantly improve. For example, two patients with WAS have recently been successfully transplanted at MSKCC from their TCD, HLA-mismatched mothers following cytoreduction with hyperfraction-
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Fig. 75.6 (a) Probability of overall 5-year survival following unrelated donor bone marrow or peripheral blood transplants, 1990–2004, for Wiskott– Aldrich syndrome (WAS) reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). (b) Probability of overall 5-year survival following unrelated donor cord blood transplantation, 1990–2004, for WAS reported to the CIBMTR. (Reproduced with permission from the CIBMTR.)
ated total body irradiation, thiotepa, and fludarabine followed by a CD34+E− PBHCT. At 96 and 56.9 months post HCT, both children are alive and fully reconstituted with complete donor chimerism and without GVHD. Both children received post-transplant rituximab to prevent EBV-LPD. One child required prolonged treatment due to recurrence of an autoimmune hemolytic anemia post HCT. However, late autoimmune disorders have been noted in up to 20% of patients transplanted for WAS, particularly in patients with sustained mixed chimerism [216].
Gene therapy for SCID and other lethal disorders of immunity The identification and subsequent cloning of genes responsible for many of the lethal disorders of hematopoiesis and immunity, coupled with the development of increasingly efficient methods for the isolation of hematopoietic stem cells, and the genetic modification of hematopoietic stem cells derived from a diseased subject to express a normal gene product, both in vitro and in animal models, in vivo, has spawned a series of clinical trials exploring the potential of gene therapy to correct these diseases. The severe combined immunodeficiency caused by ADA [25,217] was initially selected as a target for clinical trials of gene therapy for several reasons [218]. First, it was recognized that this disorder, as well as other variants of SCID, could be corrected by HLA-matched, related marrow grafts, and, further, that T-cell chimerism alone regularly induced
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full functional reconstitution [219]. Second, in vitro studies [220] as well as small clinical trials had demonstrated that transfusions of normal red cells [221] or treatment with polyethylene glycol-conjugated ADA (PEG-ADA) [222,223] could provide sufficient ADA activity to reduce toxic levels of deoxyadenosine triphosphate in lymphocytes, and thereby to permit the generation of T cells and at least a partial correction of functional deficiencies in immunity [224]. Finally, the group of Bordignon [225] established that human ADA− lymphocytes, transduced to express the normal ADA gene, exhibited a long-term survival advantage over ADA cells and developed significant immune reactivity after adaptive transfer into immunodeficient mice. Initially, it was not expected that the introduction of a normal gene into hematopoietic progenitors bearing one of the mutations causing other lethal immunodeficiencies would also confer a survival advantage to the genetically modified cells. However, over the past 13 years, several groups have reported patients with immunodeficiencies who have developed spontaneous somatic mutations in clones of lymphoid cells resulting in correction of the underlying defect, followed by preferential expansion and long-term survival of these normal, functional clones [226–233]. Such spontaneous lymphoid revertants have now been demonstrated in rare cases of the ADA-deficient and X-linked γc deficient variants of SCID [226–230], the Nemo variant of combined immunodeficiency [231], type 1 leukocyte adhesion deficiency [232], and a significant proportion of patients with WAS [233]. Such cases have provided compelling evidence supporting the possibility that in vivo transfer of a limited population of genetically corrected lymphoid progenitor cells could also result in significant and sustained partial or complete reconstitutions of immunity in patients with these disorders. The evolution of gene therapy as a clinically feasible therapeutic strategy for these immune deficiencies derives from a series of advances in the design of vector constructs inducing sustained high-level constitutive expression of the normal human gene in transduced cells [234–239], the isolation of hematopoietic progenitor cells targeted for genetic modification [240], and the development of systems for efficient transduction and integration of the normal gene constructs into hematopoietic progenitor cells [241,242]. So far, the clinical trials that have been conducted have employed replication of incompetent retroviruses as the vectors because of their high efficiency for gene transfer, their capacity to integrate into the genomic DNA of dividing somatic cells, and their anticipated safety [243]. The vectors most frequently used are constructed from a replication- incompetent Moloney leukemia virus lacking gag, pol, and env sequences but retaining the packaging signal (psi), the viral long tandem repeat (LTR), and sequences essential for reverse transcription and integration. The normal human gene construct is inserted under the transcriptional control of the viral LTR, which acts as the promoter/enhancer for gene expression [234,235,243–245]. In order to generate infectious virus, these constructs are introduced into packaging cells which generate gag, pol, and env proteins from independent gene inserts to produce infectious virus in response to the vector construct’s packaging signal, psi [239,246]. Cells targeted for genetic correction have included patient-derived peripheral blood lymphocytes and CD34+ hematopoietic progenitor cells isolated from the bone marrow or cytokine-mobilized peripheral blood hematopoietic cells, and cord blood cells. Following infection of cells with the retroviral vector, the vector RNA is reverse-transcribed and forms a preintegration complex which can only be transferred into the nucleus and integrated into the DNA of cells during mitosis [247,248]. Activated human T lymphocytes, which divide at significant frequency, are readily transduced at high efficiency with retroviral vectors. In contrast, self-renewing human hematopoietic stem cells, which normally divide at very low frequency, are markedly less susceptible to the successful transduction and integration of genes when
retroviral vectors are employed. As a consequence, although 30–40% of the CD34+ cells infected may express vector-encoded genes, the proportion of stem cells capable of multilineage differentiation that are transduced may represent only 0.1–1% of the population. Thus, in the absence of a selective growth or survival advantage, such cells would be expected to develop into no more than a minor population of the host’s hematopoietic progenitor pool. Because of this limitation, the initial trial of gene therapy for the treatment of ADA-deficient SCID [249,250] explored the therapeutic potential of peripheral blood T lymphocytes transduced with the Moloney leukemia virusbased vector LASN, which contained the human ADA complementary DNA under the transcriptional control of the retroviral LTR and a neomycin phosphotransferase gene controlled by an SV40 promoter for the selection of transduced cells. The two patients in this trial received 11 and 12 infusions, respectively, of vector-treated T cells, providing, in aggregate, approximately 1011 total lymphocytes, of which 18–53% (patient 1) and 0.9–3% (patient 2), respectively, expressed the vector. Both patients were also treated with PEG-ADA both prior to and continuously following infusion with transduced cells. Concurrent treatment with PEG-ADA has made ascertainment of the effects of gene transfer on immune function difficult, and likely reduced or eliminated the potential growth advantage of the transduced cells. Nevertheless, patient 1 still has a significant proportion of vector-modified lymphocytes over 16 years post initiation of therapy [250,251]. Subsequently, Bordignon et al. [252,253] treated two patients with deteriorating immune function despite treatment with PEG-ADA with infusions of autologous T cells and bone marrow progenitors that were transduced with separate ADA gene-containing vectors. This study again demonstrated long-term survival of transduced cells, but also showed that the proportion of marrow-derived transduced T cells greatly exceeded that of the T cells derived from transduced peripheral blood at times later than 1 year post treatment. At 16 months post infusion, 2–4.8% of circulating T cells and 17.25% of clonable marrow cells expressed the vector genes. Both patients have exhibited sustained improvements in T-cell function following these infusions. Again, however, coadministration of PEG-ADA in both patients has limited accurate estimates of the contribution of gene therapy to the functional reconstitution of immunity observed. Because hematopoietic stem cells in human cord blood replicate at significant frequency and are capable of self-renewal [254], Kohn et al. [255] used the LASN vector to transduce autologous cord blood-derived CD34+ cells from three neonates with ADA− SCID and evaluated their growth after reinfusion. Initially, each of these patients achieved sustained but low levels of the genetically modified peripheral blood lymphocytes (0.01–0.3%) in the blood, and higher frequencies of clonogenic myeloid progenitors (4–6% colony-forming myeloid cells) in the marrow. These patients were also being treated with PEG-ADA, which again prevented assessment of the effects of gene therapy on immune function. Nevertheless, over the subsequent 3 years of observation, the proportion of gene-transduced T cells gradually increased in each patient to 1–10% of the circulating T-lymphocytes. To test whether these ADA+ T cells would preferentially expand in an ADA-deficient host, PEGADA treatment was stopped in one of these patients. Although the proportion of transduced ADA+ T cells in the blood increased, progressive lymphopenia and impaired T-cell function necessitated resumption of PEG-ADA [256]. Taken together, these early trials demonstrated that peripheral blood T cells or CD34+ bone marrow progenitor transduced to express a normal human ADA gene could be safely transferred into patients with ADA− SCID, and that populations of such cells could survive and continuously express the transduced gene for periods extending to 16 years post infusion without deleterious complications. However, the proportional
Hematopoietic Cell Transplantation for Immunodeficiency Diseases
representation of transduced marrow progenitor cells and peripheral blood T cells remained low, and their contribution to the overall immune system of the host uncertain. This could reflect either the lack of a growth or survival advantage for ADA+ over ADA− marrow stem cells or a diminished capacity of ADA+ lymphoid progenitors to compete with ADA− progenitors in the presence of normal tissue levels of ADA provided by concurrent treatment of the patients with PEG-ADA. Recently, Aiuti et al. [257] directly addressed both of these possibilities in a trial in which children with ADA-deficient SCID were treated with transduced autologous marrow CD34+ cells containing 25% cells expressing the GIADA-1 vector encoding human ADA, after preparatory myelosuppression with BU 2 mg/kg/day for 2 days. Of critical importance, these patients received no PEG-ADA prior to or following this treatment. The two patients initially reported exhibited sustained full (patient 1) and partial (patient 2) reconstitution of normal T-, B-, and NK-cell counts, with 70–100% of circulating T cells expressing vectorencoded ADA. Expression of the vector in B lymphocytes, and in 20% of myeloid, megakaryocytic, and erythroid progenitors, suggested durable engraftment of transduced stem cells. Increases of TREC+ and CD45RA T lymphocytes in the circulation indicated effective thymopoiesis. The ADA+ T cells generated responded to mitogens and antigens normally. Further, Ig levels increased to normal and antibody responses to vaccination were recorded. Since that report, an additional 11 patients have been treated on this trial. Of the 13 evaluable cases, 12 have achieved full functional reconstitution of T- and NK-cell populations, of which 70–100% contain the vector. B-cell numbers and IgM levels have normalized. In 11 patients now off intravenous Ig, IgG levels are also sustained; five of these patients have generated antibody responses following tetanus vaccination. All patients are surviving in good health (Aiuti, personal communication). Confirmation of the potential of this approach has also been provided by Gaspar et al. [258], who treated a patient with ADA-deficient SCID with autologous CD34+ marrow cells transduced with a γ retroviral vector (SFUDA/W) containing the human ADA gene under the transcriptional control of a spleen focus-forming virus LTR after myelodepletion with melphalan 140 mg/m2/day for 1 day. This patient had previously received PEG-ADA, but this treatment had been stopped 1 month prior to cytoreduction and the infusion of transduced cells. Following a 2–3-week period of melphalan-induced pancytopenia, the patient experienced recovery of hematopoiesis followed by rapid recovery of CD4+ and CD8+ T-cell populations, expansion of TREC+ naïve T cells, and recovery of cell-mediated immune function. These latter studies demonstrated the potential of hematopoietic cells transduced to express ADA to repopulate a myelodepleted ADA− SCID and for ADA+ lymphoid cells to preferentially expand and restore immune function in an ADA-deficient host. While these studies do not formally establish a need to induce myelodepletion to secure a high level of engraftment of transduced progenitors, emerging data from a trial in which transduced marrow has been administered without either myelosuppressive or PEG-ADA treatment suggest that, in the absence of such preparative cytoreduction, lymphopenia persists and lymphoid reconstitution is more limited [259].
X-linked γc-deficient SCID In 2000, Cavazzana-Calvo et al. [260] reported the first clear demonstrations of the potential of gene therapy to correct a lethal congenital immunodeficiency. They used an MFG retroviral vector containing the normal human gene encoding the IL-2 γ chain under the transcriptional control of the viral LTR to transduce autologous purified CD34+ marrow cells from two children with X-linked γc-deficient SCID. These trans-
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duced progenitors were then administered to each patient without preparatory conditioning. Each patient achieved rapid reconstitution of γc+ T-cell populations bearing the vector, with full recovery of proliferative responses to mitogens and antigens and T-cell receptor diversity. In these patients, approximately 0.1% of the myeloid cells and 1% of the B cells tested over extensive follow-up have also demonstrated that they contain the vector, indicating durable engraftment of transduced pluripotent stem cells and again demonstrating the growth advantage of transduced immunocompetent T cells in these T-lymphopenic hosts [261,262]. At 8–9 months post infusion, and 5–6 months following cessation of monthly intravenous gammaglobulin, Ig levels had increased and the patients generated responses to tetanus diphtheria and polio virus vaccinations. Since this report, a total of 10 patients with X-linked SCID have been accrued to this trial [262,263]. Of these, eight have achieved reconstitution of T cells and their function, as well as at least partial recovery of B-cell function. While the number of NK cells has transiently increased early after treatment, long-term reconstitution of NK cells and their function has been poor. The two patients who failed to achieve sustained recovery of T cells and their functions had received the lowest doses of transduced cells (1 × 106/kg versus >3 ×106/kg), suggesting that a high level of engraftment of the transduced stem cells may be required to sustain normal levels of lymphopoiesis [263]. Subsequently, in a separate trial, Gaspar et al. [264] treated another four children with X-linked γc− SCID with autologous CD34+ marrow cells transduced with the same γc gene-containing vector construct packaged in a gibbon ape leukemia virus envelope. Three of the four patients subsequently achieved full, and one patient a partial, reconstitution of γc+ T-cell populations. All patients exhibit normal T-cell proliferative responses to mitogen, antigens, and allogeneic cells. Two patients who were taken off intravenous Ig replacement generated antibodies to polio and H. influenzae B vaccinations. However, as also observed in the series from Paris, recovery of NK-cell populations and functions has been poor. Since this report, an additional six patients have been treated successfully by this group using the same approach, but the level of reconstitution achieved has not been reported. Taken together, these two series have clearly demonstrated the potential of lymphoid cells derived from progenitor cells transduced to express a normal IL-2R γ chain to provide sustained and complete corrections of the T-cell defects associated with X-linked SCID and to induce recovery of B cells capable of producing antibodies in response to microbial immunogens. Whether the restoration of humoral immune responses observed reflects the activity of the small number of transduced B cells detected or unmodified host B cells responding to transduced γc+ T helper cells, however, is as yet unclear. Similarly, the basis for the failure of this approach to induce sustained corrections of NK cells and their function remains to be determined. Nevertheless, the sustained clinical responses of these children and their normal responses to infection have been striking. Recently, however, the success of this approach has been counterbalanced by the late (>30 months) development of T-cell acute lymphoblastic leukemia (ALL) in four of the original 10 children with X-linked SCID treated (Fischer, personal communication) [265]. Although these patients exhibited a prompt response to chemotherapy and achieved remissions, one subsequently relapsed and died of leukemia. In each case, the cloned leukemia cells have exhibited a mature T-cell phenotype not normally seen in children. Molecular analyses have demonstrated vector sequences in each of these leukemias. In each case, the transduced provirus has been found to be inserted within or in proximity to a known proto-oncogene involved in the control of early T-cell differentiation, specifically the LMO-2 gene in three cases and the cyclin D2 gene in one patient (Fischer, personal
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communication) [262,265]. Subsequent studies have demonstrated that, contrary to initial expectations, retroviral vector insertions are not random, but tend to cluster in transcription start sites of genes highly expressed in the targeted CD34+ cells [266–272]. Further, a comparison of insertion sites in CD34+ cells in nine patients of the original series, both immediately after transduction and in the marrow of patients late after treatment, demonstrated a different and highly skewed spectrum of insertion sites in CD34+ cells isolated from the patients many months after treatment, strongly suggesting that cells with these insertions may have a selective growth advantage [270]. Of the common insertion sites detected in clones of the CD34+ cells, insertions in LMO-2 and CCND2 were significantly increased in frequency [270]. A significant role for vector insertion in the promoter region or first intron of LMO-2 in the pathogenesis of T-cell ALL in three of these patients is inferred from the fact that a translocation affecting LMO-2 is associated with one variant of T-cell ALL affecting children [273,274], and, further, that its aberrant expression in mice is associated with late development of a T-cell leukemia [275]. However, it remains unclear in these patients whether altered expression of LMO-2 is causal or whether the T-cell ALL develops preferentially in T-lymphocyte progenitors possessing the selective growth advantage provided by altered LMO-2 expression. In the clinical experience reported to date, none of the ADA-deficient SCID patients who have received retroviral vector-transduced peripheral blood lymphocytes of marrow CD34+ cells and remain engrafted have developed a leukemia over up to 16 years of follow-up. Further, while an evaluation of vector insertion sites in transduced CD34+ cells in ADA-deficient patients has exhibited a pattern of common insertion sites similar to that exhibited in the initial series of X-linked SCID patients treated, clonal selection of cells bearing these inserts has not been observed, nor have the cells bearing LMO-2 inserts exhibited increased expression of LMO-2 [271]. This finding, coupled with studies in γc knockout mice [275], has raised the possibility that the γc-deficient Xlinked SCID may be particularly prone to retrovirally mediated mutagenesis. However, it is also striking that none of the 10 X-linked SCID patients treated by Gaspar et al. [264] have developed a leukemia, suggesting the possibility that other factors, such as the vector used or the conditions used for marrow cell transduction, may also play an important or determining role. However, longer follow-up will be required before the risk of leukemogenesis in the second trial can be assessed, since the leukemias have developed more than 30 months after treatment with transduced cells, and most of the patients in the second series have yet to reach this milestone. Because of these serious adverse events, all trials evaluating gene therapy for X-linked SCID were initially put on hold. However, trials incorporating the gibbon ape leukemia virus packaging vector employed by Gasper et al. [264] have recently been reopened for patients lacking suitably matched related or unrelated donors. Further, trials employing self-inactivating vectors that delete the retroviral LTR [276] and vectors that include insulation sequences to reduce activation of genes neighboring insertion sites [277,278] are being developed.
Other lethal immunodeficiencies The striking level of immune reconstitution achieved in recent trials of gene therapy in the treatment of ADA-deficient and X-linked γcdeficient SCID has stimulated extensive preclinical exploration of this approach in the treatment of other lethal congenital immunodeficiencies, particularly those disorders which, unlike X-linked SCID, require preparation with high-dose chemotherapy to secure consistent engraftment of an HLA-matched related graft, or a transplant from a matched, unrelated or HLA-haplotype-disparate, related donor.
In murine models, infusions of marrow progenitors transduced with retroviral vectors encoding a normal gene have corrected the immune deficiencies observed in JAK-3[279], RAG-2−/− [280], Artemis−/− [281] mice, and Wiskott–Aldrich−/− mice [282–285]. In vitro corrections of the phenotypic and functional deficiencies of human T cells generated from patients with ZAP-70 deficiency [286] and WAS [282–285] have also been achieved by transducing lymphocytes from those patients with retroviral or lentiviral vectors encoding the normal human gene. Clinical trials evaluating the effects of CD34+ cells transduced with a retroviral vector encoding a normal WASp gene, when administered to patients with high risk factors for WAS after low-dose conditioning, have been initiated (Klein, personal communication). However, these studies are too early to permit evaluation.
Conclusion Allogeneic HCT transplantation from a normal, matched related donor remains the treatment of choice for each of the different genetic variants of SCID. Currently, HLA-matched, unmodified grafts from unrelated donors administered with post-transplant drug prophylaxis to prevent GVHD and rigorously TCD, HLA-haplotype-disparate, related transplants administered without post-transplant GVHD prophylaxis yield equivalent results, with long-term disease-free survival rates of approximately 70% overall and approximately 80% for patients transplanted in the first 3 months of life. The relative advantages of each approach will require careful prospective analyses of large series. Certain types of SCID, particularly Omenn’s syndrome, ADA-deficient SCID and NK+ variants, exhibit graft resistance and require preparative cytoreduction, particularly when the donors are other than HLA-matched siblings. Preparatory cytoreduction with myelosuppressive agents such as BU, thiotepa or melphalan may also be preferable to secure consistent engraftment of donor B cells and NK cells, as well as T cells, in all variants with reconstitution of both cell-mediated and humoral immunity. For patients with other combined immunodeficiency diseases and WAS, preparative cytoreduction with a myelosuppressive agent together with immunoablation with CY or fludarabine is required to achieve consistent engraftment and reconstitution. For these indications, transplants of unmodified, HLA-matched, related and unrelated HCT have thus far been associated with better outcomes than TCD, HLA-disparate grafts. Certain of these disorders, particularly the bare lymphocyte syndrome associated with HLA class II deficiency, exhibit excessive rates of graft failure despite use of these regimens. For the future, the development of well-tolerated preparative regimens and transplant approaches which secure consistent engraftment and full chimerism is a high priority. In the future, introduction of selected cytokines or specific cell therapies at the time of transplant may foster full engraftment and enhance recovery of immune function. While treatment with genetically modified autologous hematopoietic stem cells has recently shown great promise in the treatment of children with ADA-deficient SCID when administered after myelosuppression, the proportion of patients achieving full reconstitution is thus far not different from that already achievable with matched, unrelated or TCD, HLA-nonidentical grafts. Longer follow-up will be required to determine whether and to what degree gene therapy will supplant HCT as a treatment of choice for this disease. For patients with X-linked γcdeficient SCID, gene-modified autologous HCT can reconstitute both T- and B-cell immunity without preparative cytoreduction. However, improved vectors are needed to reduce or eliminate the risk of late leukemic transformation of transduced cells in this patient group.
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76
Peter F. Coccia
Hematopoietic Cell Transplantation for Osteopetrosis
Introduction Osteopetrosis is a rare inherited disorder characterized by generalized skeletal sclerosis that occurs in various mammals including man [1–3]. Osteopetrosis is a result of the dysfunction of osteoclasts, the multinucleated giant cells that resorb bone and mineralized cartilage [4,5]. The osteoclast is a specialized macrophage polykaryon derived from the hematopoietic stem cell (HSC) [4–6]. Some variants of osteopetrosis in laboratory animals and man can be corrected by hematopoietic cell transplantation (HCT) [1,3,7–9]. In the past several years, remarkable advances have been made in the understanding of osteoclast biology, in the elucidation of the osteoclast resorption defects that result in the various clinical phenotypes of human osteopetrosis, and in the identification of multiple genetic mutations in patients with osteopetrosis. In this chapter, the origin and function of the osteoclast will be described briefly. Studies of HCT in laboratory animals with congenital osteopetrotic mutations and transgenic mutations will be reviewed. The clinical and mutational classification of the various types of osteopetrosis in humans will be detailed. The role of HCT and other therapeutic modalities in the treatment of infantile malignant osteopetrosis will be discussed, and future directions will be considered.
The osteoclast and bone resorption There is extensive information in the literature concerning the origin, structure, and function of the osteoclast and its role in bone resorption [4,5,10]. A brief overview will be provided to clarify issues important to an understanding of the pathophysiology of osteopetrosis and the role of HCT in its correction. The cell of origin of the osteoclast is the pluripotent HSC. The colonyforming unit-granulocyte–macrophage is likely the committed progenitor cell of origin of the osteoclast, as well as the circulating monocyte, the tissue macrophage, and the foreign body giant cell [5,11]. Osteoclasts are thought to form from repeated asynchronous fusions of postmitotic mononuclear precursors that migrate to skeletal sites via vascular pathways. Bone-resorbing osteoclasts have from one to over 20 nuclei. Differentiation, fusion, maturation, activation, inhibition, and apoptosis are controlled by multiple lymphokines, monokines, and hormones.
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
Macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B (NFκB) ligand (RANKL) promote differentiation of bone marrow macrophages into multinucleated mature osteoclasts. A soluble protein, osteoprotegrin, inhibits bone resorption by binding RANKL. Osteoprotegrin appears to inhibit osteoclast maturation. Both osteoblasts, the bone-forming cells of mesenchymal origin that synthesize bone matrix, and marrow stromal cells express M-CSF and RANKL on their cell surface and have important roles in the development, activation, and regulation of osteoclasts. Bone formation and remodeling are intimately linked and interdependent. Degradation of bone mineral complexes and collagenous bone matrix requires activation of the osteoclast. Activation involves elaboration of cytoplasmic infoldings of the plasma membrane next to the bone surface that is known as the ruffled border. Once the ruffled border is formed, plasma and lysosomal membranes fuse, and lysosomal contents including cathepsin K and other proteases, carbonic anhydrase isoenzyme II (CAII), and acid hydrolases are released to the extracellular space. Acidification requires the influx of hydrogen ions produced by CAII by the vacuolar H+ ATPase proton pump and the exchange of chloride/ bicarbonate mediated by a chlorine channel pump (ClCN7). Essentially, all reported mutations described in human osteopetrosis are a result of defects in acidification: T-cell immune regulator-1 (TCIRG1), which is also known as ATP6i; ClCN7; osteopetrosis transmembrane protein-1 (OSTM1), or gl/gl; and CAII (see below). Bone resorption is confined to a small area of osteoclast–bone interface by a circumferential seal of the plasma membrane that is defined as the clear zone. Hydroxyapatite crystals are first solubilized by hydrochloric acid at a pH of 4.5, and then the organic matrix is digested by the proteases. The products of resorption are then endocytosed for additional intracellular processing.
Osteopetrotic mutations in laboratory animals Congenital osteopetrosis is a well-known mutation in a variety of mammalian species [1,3,7]. There are eight well-studied mutations in common laboratory animals, including the mouse, rat, and rabbit (Table 76.1). In all variants, the mode of inheritance is autosomal recessive. All variants demonstrate osteoclast hypofunction, generalized skeletal sclerosis, absent or poorly developed marrow cavities, delayed tooth eruption, reduced size of osseous foramina, defective bone modeling and remodeling, and weak bone susceptible to pathologic fracture. All mutants studied are resistant to the hypercalcemic effects of parathyroid hormone and 1,25 dihydroxyvitamin D, and all have increased blood levels of 1,25 dihydroxyvitamin D.
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Table 76.1 Mammalian osteopetrosis. Osteoclasts Mutation
Symbol
Gene identified
Number
Size
Ruffled borders
Survival
Effective treatment
Mouse Grey lethal Microphthalmia Osteosclerosis Osteopetrosis
gl/gl mi/mi oc/oc op/op
OSTM1 ? TCIRG1 M-CSF
N, ↑ N ↑ ↓↓
↑ ↓↓ ↓ ↓
± − − +
Lethal Normal Lethal Normal
HCT HCT HCT M-CSF
Rat Incisors absent Osteopetrosis Toothless
ia/ia op/op tl/tl
PLEKHM1 ? M-CSF
↑↑ ↓ ↓↓
N ↑↑ ↓
− ± ±
Normal Lethal Reduced
HCT HCT M-CSF
Rabbit Osteopetrosis
os/os
?
↓
N
−
Lethal
None
Modified, expanded, and updated from numerous literature reports and reviews [1,3,7]. Bibliography available upon request. Increases or decreases with respect to normal littermates/control subjects are indicated by arrows. N, same as normal littermate/control; + and −, presence or absence of ruffled borders; ?, not reported/unknown. HCT, hematopoietic cell transplantation; M-CSF, macrophage colony-stimulating factor.
An additional mutation in the Norway rat, microphalmia blanc (mib/ mib), has been characterized [12,13]. Affected mutants have a mild transient osteopetrosis at birth with reduced osteoclast numbers. Skeletal sclerosis gradually resolves, osteoclast numbers increase, and survival is normal. Gene mapping reveals a mutation at 4q34–q41 that produces a nonfunctional microphthalmia-associated transcription factor gene. The major differences among the variants that have been described in the current literature are compiled in Table 76.1. Included are remarkable differences in osteoclast numbers from virtually absent in the tl/tl rat to significant hyperplasia in the ia/ia rat; differences in osteoclast size from primarily mononuclear forms in the mi/mi mouse to very large with many nuclei in the op/op rat; and differences in the appearance of the ruffled border at the osteoclast–bone interface from absent to normal in the various mutants. Reported gene mutations are included for mutants if they have been elucidated. Disease severity as measured by survival past infancy and known effective treatment are also detailed. The development of osteopetrosis is an in utero event. The defect may be intrinsic to the osteoclast or in the microenvironment supporting the development and activation of the osteoclasts. In all cases, the failure of the osteoclast to resorb calcified cartilage during bone development leads to persistent primary spongiosa characterized by cores of calcified cartilage within the bone. This resorption failure prevents or delays development of the bone marrow cavity and is responsible for the distinctive radiographic appearance of homogeneously dense bones. The severity of disease and the potential therapeutic interventions depend on the etiology of the osteoclast dysfunction in the various osteopetrotic mutations. In addition to spontaneous mutations, various induced mutations have produced transgenic mice by targeted disruptions of proto-oncogenes [1,3,7]. These loss of function, or “knockout,” experiments have resulted in abnormalities in either the production or function of osteoclasts, and the mutant mice usually have severe osteopetrosis. Examples of induced mutations that result in abnormal osteoclast production or maturation include the targeted disruption of c-fos that yields mice with abundant bone marrow macrophages but no osteoclasts, and PU.1, a hematopoietic transcription factor, that produces mice deficient in both macrophages and osteoclasts that die within 24–48 hours
of birth of septicemia. Additional mutants that fail to generate osteoclasts include those with deficient NFκB1 plus NFκB2 and with deficient RANKL. Mutations that result in abnormal osteoclast function include the disruption of c-src, a tyrosine kinase, yielding mice with normal osteoclast numbers, absent ruffled borders, and defective bone resorption. Mice produced with a deficiency in tumor necrosis factor receptor-associated factor 6 have impaired osteoclast function. Transgenic mice with specific resorption defects have also been created. Cathepsin K-deficient mice lack a cysteine proteinase necessary to degrade bone matrix. TClRG1 (Atp6i) deficient mice have an extracellular acidification defect and cannot solubilize bone mineral [14]. HCT in animal models In a series of elegant experiments, Walker [15] demonstrated that osteopetrosis can be cured in the gl/gl and mi/mi mouse mutants by either temporary parabiosis or HCT. Infusions of marrow or spleen cells from normal littermates into lethally irradiated osteopetrotic mice resulted in complete correction of osteopetrosis and normal survival. Conversely, infusion of spleen cells from osteopetrotic mice to lethally irradiated normal littermates led to the development of osteopetrosis [16]. Similar experiments have demonstrated complete correction after HCT in the ia/ia [17] and op/op [18] rat mutations. Correction or induction of osteopetrosis in these four strains by HCT suggests defects intrinsic to the osteoclast progenitor or the osteoclast itself. Studies in the mi/mi mouse which demonstrate that the bone resorbing osteoclasts are of donor origin after HCT support this viewpoint [19,20]. Beige mice, which have giant lysosomes in their hematopoietic cells, were used as hematopoietic cell donors for HCT into irradiated mi/mi mice. After HCT, giant lysosomes were present in the functional osteoclasts of the recipients. Experimental HCT in animal models has been very consistent in its approach. In most reported studies, the preparative regimen has utilized a single fraction of total body irradiation (TBI) of 600 cGy for recipient osteopetrotic mutants or 900 cGy for recipient normal littermates delivered by a cobalt-60 source. This dose is considered marrow lethal for each group. Marrow or spleen cells at 10 × 106–50 × 106 nucleated
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cells/animal are given intravenously or intraperitoneally 2 hours later. Donors are highly inbred littermates, full engraftment is routine, and graft-versus-host disease (GVHD) has not been reported. HCT from normal littermates has also been reported to cure osteopetrosis in transgenic mutants [1,3,7]. Complete correction has been reported in the c-src, c-fos, NFκB1 plus NFκB2, and PU.1 variants. In the newborn PU.1– mutant, HCT is performed soon after birth utilizing 4-week-old PU.1+ donor animals in germ-free environments. HCT should be successful in all mutants that have osteopetrosis secondary to an induced mutation that results in a defect in the HSC, but not in mutants that have defects in the microenvironment. In three other spontaneous mutants (op/op mouse, tl/tl rat, and os/os rabbit), HCT is not able to correct osteopetrosis. This observation suggests that the defects in these mutants are not intrinsic to the osteoclast progenitor or the osteoclast. The lethal mutation in the os/os rabbit has not been identified [7]. Both the op/op mouse and the tl/tl rat have been found to be severely deficient in M-CSF, and daily treatment with recombinant human MCSF remarkably improves bone resorption in both mutants [21]. The op/op mouse has a nonlethal point mutation within the coding region of the M-CSF gene, absent M-CSF, and markedly reduced monocytes, macrophages, and osteoclasts. The osteopetrosis progressively corrects over time. Treatment with M-CSF results in normalization of monocytes, macrophages, and osteoclasts, and rapid correction of osteopetrosis. Granulocyte–macrophage colony-stimulating factor treatment restores monocytes, and macrophages, but fails to stimulate osteoclast development or correct osteopetrosis. The tl/tl rat also has decreased monocytes, macrophages, and osteoclasts, but does not demonstrate skeletal improvement with age and has a reduced life span. Bioassay reveals low M-CSF activity and treatment with M-CSF improves osteopetrosis and prolongs survival. The genetic defect in the oc/oc mouse spontaneous mutation has been identified [14,22]. The defective gene encodes an H+ ATPase necessary for acidification, and appears to be a defect similar to that of the TCIRG1 (Atp6i)-deficient transgenic mouse model [22]. While this lethal mutation is intrinsic to the osteoclast, successful HCT had not been reported until recently. The oc/oc mouse has been studied extensively as it has the identical mutation found in 50–60% of children with infantile malignant osteopetrosis (see below). Previous attempts to transplant oc/oc mice have been unsuccessful as the animals have only a 3-week life expectancy. HCT in 1-day-old oc/oc mice results in long-term survival [23]. The same group also has reported successful HSC-targeted gene therapy with oc/oc fetal liver cells transduced with a retroviral vector expressing TCIRG1 in irradiated neonatal oc/oc mice [3,24]. A cell line derived from oc/oc mice has been developed which differentiates into murine osteoclasts but lacks resorption activity [25].
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order rarely have dense bones. Most patients are asymptomatic and have a normal life expectancy. Minimal disability has been reported, secondary to an increased susceptibility to fractures, and occasionally, bone pain, osteomyelitis of the mandible, and cranial nerve palsies [28]. This autosomal dominant form has been extensively studied, and two distinct types (types I and II) have been described based on clinical, radiologic, histologic, and biochemical criteria. Type I is rarer; fractures are uncommon, bones are uniformly dense, and osteoclasts are reduced in number and size. Recently, the defect in autosomal dominant type I disease has been shown to be due to an osteoblast defect, a mutation in low-density lipoprotein receptor-related protein-5 (LRP5), which mediates increased bone formation [29,30]. As the LRP5 gain-of-function mutations do not cause an osteoclastic resorption defect, most authors suggest excluding this diagnosis from the osteopetrosis family of disorders [1–3]. More families with type II disease have been reported. Fractures are more frequent, bone density is more variable, and osteoclasts are increased in number, large, and highly multinucleated. Type II autosomal dominant osteopetrosis has been mapped to chromosome 16p13.3 [31]. Heterozygous mutations of ClCN7 have been found in almost all patients with type II disease [32–37]. Clinical manifestations have ranged from entirely asymptomatic in one-third of subjects to the more typical misshaped, brittle bones with increased fractures and occasionally other manifestations of osteopetrosis. There is no genotypic–phenotypic correlation, and families frequently have both unaffected gene carriers and even severely affected individuals [35–37]. A number of variants with autosomal recessive inheritance in addition to the infantile malignant variant, which is the subject of this chapter, have been described. CAII deficiency, a syndrome with mild osteopetrosis, renal tubular acidosis, and cerebral calcification, has been documented in multiple kindreds [38,39]. Patients have mutations of the CAII gene resulting in a profound deficiency of CAII. Transient infantile osteopetrosis has been reported [40–42] and is speculated to be similar to the mib/mib mutation in the Norway rat [12,13]. Intermediate autosomal recessive and mild autosomal recessive osteopetrosis continues to be reported [2,33,35,43,44]. Usually, it represents patients with dense bones in the first year of life without significant hematologic problems, but authors have presented variable findings to define the condition. Likely, it most frequently represents early onset of type II autosomal dominant disease with ClCN7 mutations, compound heterozygous mutations of ClCN7 or mild phenotypes of autosomal recessive osteopetrosis with homozygous ClCN7 mutations. Other obscure variants of osteopetrosis [1,2,6] and other disorders that cause increased skeletal mass [6,26,45] continue to be described. Infantile malignant osteopetrosis
Osteopetrosis in humans Historically, two major forms of osteopetrosis have been described. The benign or “adult” form is inherited in an autosomal dominant pattern, and is associated with minimal disability and a normal life expectancy. The malignant or “infantile” form is inherited in an autosomal recessive pattern and has widespread systemic manifestations. However, both forms have been found to be clinically heterogeneous, and other specific variants of osteopetrosis have been reported [2,6,26]. The adult benign autosomal dominant form was first described by the German radiologist Albers-Schönberg in 1904 [27] by roentgenographic demonstration of the characteristic dense, radiopaque bones (AlbersSchönberg syndrome or marble bone disease). Diagnosis is usually made in late childhood or adulthood by roentgenographic demonstration of diffuse skeletal sclerosis that increases over time. Infants with this dis-
The classic form of infantile malignant osteopetrosis is characterized by autosomal recessive inheritance and is almost always diagnosed in early infancy [2,8,35,46–48]. The inability to resorb and remodel bone due to osteoclast dysfunction, in the presence of normal bone formation by osteoblasts, results in the deposition of excessive mineralized osteoid and cartilage. All bones are uniformly dense, sclerotic, and radiopaque. Medullary cavities are absent on long bone radiographs [45,49]. Bone biopsies reveal encroachment of medullary cavities by bone and mineralized cartilage, thick trabeculae, and decreased medullary spaces. The residual medullary cavities are occupied with large numbers of nonfunctional osteoclasts, and there is usually increased marrow fibrosis [47,50]. Osteoclasts are reported to be normal in size in some patients and increased in size in others, with the large osteoclasts having increased numbers of nuclei. Electron microscopy of osteoclasts has been reported
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to demonstrate normal ruffled borders at the bone junction in some cases and decreased or complete absence of ruffled borders in other cases. Encroachment of marrow spaces leads to extramedullary hematopoiesis, progressive hepatosplenomegaly, and hypersplenism. The result is anemia with reticulocytosis, leukoerythroblastosis, and thrombocytopenia. Encroachment of cranial nerve foramina leads to retinal atrophy which progresses to blindness [48,51,52], auditory nerve damage [53], and oculomotor and facial nerve palsies [48]. Defective bone resorption also leads to progressive macrocephaly, frontal bossing, hypertelorism, exophthalmos, and other craniofacial abnormalities. Hydrocephalus, ventricular enlargement, increased intracranial pressure, cerebral vascular occlusive complications and seizures are also reported [47,48]. Nasal stuffiness is a common presenting symptom that is secondary to progressive narrowing and obstruction of the nasal airways [46]. Obstructive sleep apnea has also been described. Excessive tearing may occur secondary to nasolacrimal duct stenosis. Seizures and tetany in the neonatal period secondary to hypocalcemia have been reported in a few cases [47]. Rarely, diagnosis may be obscured in the neonate secondary to maternal vitamin D deficiency. Other reported complications are osteomyelitis of the mandible and maxilla, retarded tooth eruption [54], and rampant caries. Sclerotic bones are brittle, and pathologic fractures occur frequently. Linear growth is retarded, with dwarfism reported in survivors to the second decade. Neuropsychologic and developmental evaluation reveals a wide range of cognitive, adaptive, and language skills with routinely delayed gross motor development [48]. Infections are common in children with infantile malignant osteopetrosis. Various subtle defects in neutrophil and monocyte function have been reported. Included are defects in phagocytosis, decreased intracellular killing, decreased natural killer cell function, abnormal nitro blue tetrazolium reduction by both neutrophils and monocytes, and decreased response to stimuli of neutrophil activation. The children become progressively severely debilitated, and in the past survival beyond the first decade was not reported. Infection, bleeding, and severe anemia were the usual reported causes of death. Improvements in supportive care, antibiotics, and a ready availability of blood components have prolonged survival [47]. Affected children are at high risk for anesthetic morbidity and mortality [55]. A variant of infantile malignant osteopetrosis has been described that is associated with neuronal storage of ceroid lipofuscin. Children with this disorder have features of infantile malignant osteopetrosis but, in addition, are severely retarded and demonstrate chorioretinal degeneration and progressive generalized neurodegeneration. Findings include cerebral atrophy, ventricular dilatation, hypotonia or hypertonia, central apnea, and seizures. The disorder is invariably fatal before 2 years of age, and after HCT, while bone changes are improved, the disease progresses [47,48,56–60]. Gerritsen and co-workers [47] reported on 33 patients in 26 sibships seen in Paris, France and Leiden, Holland between 1972 and 1988, and summarized the major findings of an additional 59 cases reported in the literature to 1990. About 30% of children survived to 6 years of age, with survivors to the second or third decade having poor quality of life. About 75% developed visual impairment secondary to optic atrophy in the first year of life, most in the first 3 months. About 75% also developed hematologic abnormalities in the first year of life, which was an unfavorable prognostic sign, especially when associated with early visual impairment. The eight children with both visual and hematologic impairment before the age of 3 months died before 1 year of age. An important caveat to consider in evaluating the above descriptions of complications in patients with infantile malignant osteopetrosis is that some reported series may include patients with the variant with neuronal
storage disease, patients with CA II deficiency, and patients with the autosomal dominant and intermediate forms of osteopetrosis. Molecular genetics of osteopetrosis Until the advent of widespread molecular genetic analysis [32–37,56– 59,61–65], the etiology of the bone resorption defect in infantile malignant osteopetrosis was a subject of speculation. The finding of increased numbers of multinucleated osteoclasts in essentially all patients studied suggested the disorder was not a defect in stem cell differentiation, maturation or cell fusion. Osteoclast hyperplasia suggested a disorder of function supported by the observed increase in humoral stimulators of bone resorption. Possible etiologies were speculated to include defective activation due to abnormal cell surface receptors, defective recognition of effete bone, and digestive incompetence secondary to abnormal intracellular enzymes. Utilization of gene-linkage analysis and mutational analysis has resulted in the identification of five mutations that explain the etiology of about 80% of the cases of autosomal recessive osteopetrosis. Gene mapping in two consanguineous Bedouin kindreds to 11q13 [66] correlated to the position predicted by comparative mapping of the oc/oc murine mutation [14,22]. The Israeli team identified 13 children from four related families belonging to one Bedouin tribe and performed HCT on nine patients [67]. Prenatal linkage analysis identified three fetuses with osteopetrosis, and two received HCT as infants [68]. Concurrent reports [69,70] described mutations in the a3 subunit of the vacuolar H+ ATPase and defects in the ATP6i subunit of the vacuolar protein pump – the same defect that is now designated TCIRG1. This is the same protein pump mutation reported in the oc/oc mouse [14,22]. Subsequent studies [33,61,63,71] have demonstrated that 50–60% of all patients with infantile malignant osteopetrosis have mutations in TCIRG1. TCIRG1 mutations appear only to cause an acidification defect in osteoclasts and not cause defects in other cell types. ClCN7 [32–37] mutations also cause acidification defects. Homozygous mutations result in severe osteopetrosis similar to the phenotype seen with TCIRG1. ClCN7 mutations are found in about 15% of all patients with infantile malignant osteopetrosis. A ClCN7 knockout model in mice demonstrated the expected severe osteopetrosis, but in addition neural degeneration and retinal degradation secondary to deficient ClCN7 expression in the central nervous system [72]. The TCIRG1 natural mutation in the oc/oc mouse and a TCIRG1 knockout did not demonstrate neural or retinal defects [72]. It is speculated that children with ClCN7 homozygous mutations may be at an increased risk for retinal problems in addition to the expected optic nerve compression [3,48]. OSTM1 mutations [56–60] are responsible for the osteopetrotic variant with neuronal storage of ceroid lipofuscin [47,48]. The mutation is also present in the gl/gl mouse, and is rapidly fatal in both mouse and man. OSTM1 protein is thought to function as a subunit of ClCN7 and regulate hydrochloric acid secretion. OSTM1 mutations have been identified in eight patients [60] and represent 1–3% of autosomal recessive osteopetrosis. The fourth mutation was recently reported [64] as a pleckstrin homology domain-containing family M member-1 (PLEKHM1), which is present in the ia/ia rat model. PLEKHM1 protein is essential for bone resorption. Two siblings were identified with a mild form of osteopetrosis with dense deformed bones and no other symptoms. The carrier parents were related. The most recently described variant is the first human variant with a phenotype that does not represent a resorption defect [65,73]. It is described as osteoclast-poor human osteopetrosis due to RANKL deficiency. Patients who were negative for the above four mutations and
Hematopoietic Cell Transplantation for Osteopetrosis
who on bone biopsy had decreased or absent osteoclasts were screened for other mutations. Six affected children were identified, and three had correction of bone resorption by cultured osteoclasts when exposed to RANKL, as previously shown in the mouse RANKL knockout model. The other three children had genetic testing after HCT and could not be studied in vitro. Occasional patients have been reported previously with osteoclast-poor osteopetrosis [2,62,74]. The above five defects and CAII deficiency account for about 80% of human autosomal recessive patients. Potentially, the remaining patients may have a few or many more mutations. Most patients who have no currently identifiable mutations are phenotypically similar to infants with TCIRG1 and ClCN7 mutations. It is speculated [2,3] that most will also have acidification defects, as other mutations would likely involve many other cell types and lead to embryonic lethality. In addition to the remarkable progress in molecular genetics that the above studies represent, advances in cell culture have allowed the defects to be studied and elucidated in vitro. Investigators had attempted to culture functional osteoclasts for over 20 years from bone marrow and blood cells [8], but succeeded only in producing multinucleated giant cells which did not resorb bone. Methods have been developed to generate large numbers of both normal and defective osteoclasts in vitro in both laboratory animals [75] and humans [76–78]. Osteoclasts can now be obtained in large numbers from cultures of peripheral blood monocytes (enriched CD14 cells) and bone marrow macrophages stimulated with both M-CSF and RANKL. Bone resorption can be studied in vitro, as can the various gene products involved in acidification and matrix degradation [4,25,30,34–36,62,64,65].
HCT for infantile malignant osteopetrosis Historically, treatment of infantile malignant osteopetrosis consisted of supportive care measures and attempts to control mineral intake. Anemia and thrombocytopenia were treated with transfusions and occasionally splenectomy. Dietary manipulations to reduce calcium intake, increase phosphate intake or both were employed in an attempt to mobilize bone calcium without success. Attempts to induce bone resorption with infusions of parathyroid hormone, vitamin D, and calcitonin were also unsuccessful. Treatment with high-dose corticosteroids resulted in decreased hepatosplenomegaly, decreased leukoerythroblastosis, increased hemoglobin and platelet counts, and decreased need for transfusion. However, there was no significant effect on the underlying process, and patients became profoundly Cushingoid. When corticosteroids were tapered or discontinued, the patients’ condition deteriorated. Other than HCT, no other curative therapies have been reported for infantile malignant osteopetrosis. Treatment with high-dose calcitriol (1,25 dihydroxyvitamin D) may stabilize disease findings [79]. Recombinant human interferon-gamma (IFN-γ) [79] has been utilized to treat children with infantile osteopetrosis. IFN-γ increases superoxide production that is reduced in granulocytes, transformed B lymphocytes, and osteoclasts of osteopetrotic patients. Patients treated for 6–18 months demonstrate increases in hemoglobin concentration and platelet counts, and transfusion requirements decrease. However, no changes in bone density are observed on radiographs. All patients have biochemical evidence of increased bone resorption, and bacterial infections decrease. Two patients receiving IFN-γ have subsequently been reported to develop fatal acute respiratory distress syndrome after receiving a platelet transfusion [80]. A subsequent clinical trial of IFN-γ1b plus calcitriol demonstrated that patients with infantile osteopetrosis experienced fewer infections and had a 50% reduction in bone mass, and increases in the size of both the optic nerve foramina and auditory canals [79]. While children receiving calcitriol and IFN-γ
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demonstrate clinical improvement, the underlying disease process remains unchanged. Treatment of infantile malignant osteopetrosis with M-CSF has been suggested in view of the observations in the op/op mouse [21,81] and tl/tl rat [21,82]. Measurement of M-CSF in 13 patients with osteopetrosis demonstrated normal-to-increased levels in all patients [83]. Further, in six patients studied, the M-CSF present was biologically active [83]. In patients undergoing evaluation for HCT, treatment with combinations including low-dose corticosteroids, high-dose calcitriol, IFN-γ, and possibly M-CSF may be beneficial, especially if there are delays in finding a suitable donor [79,83]. Reports [15,17,18] of successful correction of osteopetrosis in mouse and rat mutants in the mid 1970s by HCT led to attempts to utilize allogeneic HCT to treat children with infantile malignant osteopetrosis. In 1977, Ballet et al. [84] reported the first HCT procedure for osteopetrosis. A 3-month-old girl was transplanted without immunosuppression with marrow from a human leukocyte antigen (HLA)-identical 2-yearold sister. Although durable engraftment was not demonstrated, radiologic and other evidence for significant bone resorption was documented. Of interest, the family was highly inbred and later found to be a CAII deficiency kindred [38]. In 1980, Coccia et al. [8] reported a 5-month-old girl who received an HCT from her HLA-identical, mixed leukocyte culture-compatible brother after preparation with cyclophosphamide (CY; 200 mg/kg) and modified TBI (400 cGy with head and lung shielding). Engraftment was documented by chromosomal analysis. Anemia, thrombocytopenia, leukoerythroblastosis, and metabolic abnormalities corrected within 12 weeks of HCT. Comparison of bone biopsies before HCT and at 13 weeks after HCT revealed complete correction (Fig. 76.1). Serial X-rays demonstrated bony remodeling and new nonsclerotic bone formation (Fig. 76.2). Fluorescent Y-body analysis after HCT showed that nuclei were of donor origin (male) in osteoclasts (Fig. 76.3), but nuclei in osteoblasts remained of recipient origin (female). Subsequent follow-up showed progressive loss of her graft. Dense new bone formation can be appreciated on the last three panels of Fig. 76.2 but with preservation of bone marrow cavities. Since 18 months after HCT, no male karyotypes have been detected in her peripheral blood or marrow cells. The recipient is now more than 29 years after HCT. Her bones are extremely sclerotic and she is short, but no other evidence of osteopetrosis remains. While her visual acuity is diminished, she has normal hearing, intelligence, and pubertal development. She is a college graduate with a Master’s degree and is gainfully employed. She has no hepatosplenomegaly, and blood counts and chemistries are normal. In this patient, it appears that once marrow cavities and foramina were remodeled, the correction was permanent in spite of sclerotic new bone formation. Fifty percent of the patients treated by our HCT group are long-term survivors who have engrafted promptly, have normal bones, and are fully chimeric; some are blind, but all have normal development and intelligence. Over 100 case reports and small series of patients detailing HCT for osteopetrosis have been published between 1981 and 2007. Most patients are represented in the large studies which are reviewed below, or their data are in HCT databanks. Analysis of these individual reports is beyond the scope of this chapter. Gerritsen et al. [9] for the European Group for Blood and Marrow Transplantation (EBMT) have reported a detailed summary of the outcome of 69 patients receiving HCT for osteopetrosis between 1976 and 1994 in Europe, Saudi Arabia or Costa Rica. The 69 children received 83 HCTs at 17 centers. Median age was 3 (range 1–81) months. Four patients had HCT without conditioning. Of the remaining 65 patients, 19 had matched sibling donors, nine had five- or six-antigen
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Fig. 76.1 Bone histology before bone marrow transplantation (BMT) (left) and 13 weeks after BMT (right). The biopsy specimen studied before BMT had the characteristic features of osteopetrosis. The trabecular matrix mass was markedly increased and composed of mineralized cartilage (C) as well as bone (white arrow). Numerous nonresorbing osteoclasts (black arrows) were present in the residual marrow space and contained few hematopoietic precursors (undecalcified; modified Masson stain, magnification: ×100). The biopsy specimen obtained 13 weeks after BMT revealed virtual normalization of the bone and marrow. The trabecular matrix mass was markedly reduced, and there were few residual cartilaginous bars. The marrow space contained abundant normal hematopoietic precursors, and the osteoclasts, the number of which was markedly reduced, were actively resorbing bone, as evidenced by their presence in resorption bays (insert) (undecalcified; modified Masson stain, magnification: ×100; insert, ×450). (Reproduced from [8], with permission.)
Fig. 76.2 Representative roentgenograms of the left leg obtained before bone marrow transplantation (BMT) and 10–69 weeks after BMT. Before BMT, the bones were very dense. The fraying of the ends of the long bones is typical of rachitic change. Ten weeks after transplantation, metaphyseal and periosteal remodeling and new bone formation of nearly normal density were seen. The rachitic changes were resolving. The provisional zone of calcification was reestablished. At 18 and 29 weeks, there was a marked increase in the thickness of normal-appearing new bone as a result of longitudinal and appositional growth. Metaphyses and epiphyses had a normal appearance. No evidence of rickets was seen. Dense remnants of the original bone were seen within the normal-appearing new bone. At 40, 49, and 69 weeks, new bone formation is again of increased density, suggesting recurrence of the osteopetrotic process. Marrow cavities have not been replaced by dense bone. The small round hole seen in the proximal tibia within the dense remnant of original bone is at the site of a trephine needle biopsy performed 6 weeks after BMT.
HLA-matched family donors, seven had six-antigen HLA-matched unrelated donors, and the remaining 30 had family donors with two or more nonidentical HLA antigens. Most patients were prepared with busulfan (BU) and CY. Many of the patients with HLA-disparate donors received additional immunosuppression. All 19 children with matched sibling donors engrafted, while only 30 of 42 with other donors engrafted. Engraftment was not evaluable in the remaining patients because of early death. In engrafted patients who were evaluable, persistent osteoclast function was docu-
mented by serial radiographs in 37 of 41 cases. Severe hypercalcemia developed after HCT in seven of 29 evaluable recipients (discussed below). Six of seven with hypercalcemia underwent HCT after 2 years of age. At the time of the report, 39 of 69 patients reported had died. Of the 30 survivors, 25 have osteoclast function, and most are healthy aside from visual disability in some. The authors conclude that HCT is the treatment of choice in children with matched sibling donors, that early HCT is important if vision is to be preserved [47], and that hypercalcemia is a worrisome complication,
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Fig. 76.3 Osteoclasts with fluorescent Y bodies after bone marrow transplantation (BMT). The osteoclasts were stained with quinacrine dihydrochloride. Arrows indicate the Y bodies in nuclei that are seen in this focal plane (fluorescence microscopy, magnification: ×100; right panel enlarged for clarity). (Reproduced from [8], with permission.)
especially in older recipients. The authors express concern with the poor outcome (13%) in patients with family donors with two or more disparate antigens and recommend search for an unrelated phenotypically HLA-identical donor. However, in children with early hematologic and visual impairment [47], they recommend HCT from the best available family donor as these children may not survive the search process to identify a matched unrelated donor. A German team [85] reported five of seven infants alive with complete resolution of osteopetrosis at a median of 4 years after HCT utilizing purified CD34+ T-cell depleted, mobilized peripheral blood from HLA-haploid identical parents. This encouraging result is a remarkable improvement in the outcomes reported by the EBMT group [9] and the Center for International Blood and Marrow Transplant Research (CIBMTR) [86] for HLA-haplotype mismatched HCT. The EBMT report [9] has been updated and expanded [87]. From 1980 to January 2001, 122 patients received 141 HCTs for autosomal recessive osteopetrosis in 20 centers, including the 65 conditioned patients in the previous report [9]. Median age was 6 (range 1–105) months. Visual impairment was severe in 42% and mild in 23%. Forty had matched sibling donors (group A); 20 had matched unrelated donors (two cord blood) (group B); 21 had six- or five-antigen HLA-matched family donors (group C); and 41 had family donors with two or more nonidentical HLA antigens (group D). Engraftment was documented in 95%, 79%, 52%, and 61% in groups A–D respectively. Survival with osteoclast function is show in Fig. 76.4. Actuarial 5-year disease-free survival were 73%, 40%, 43%, and 24% for groups A–D respectively. Group D patients included the seven cases previously reported [85]. Six of 19 patients retransplanted engrafted. At last evaluation, 56 of 122 patients (46%) were alive with osteoclast function at a median of 5 (range 0.5–16.5) years. Six survived with persistent osteopetrosis, and 62 died. Most deaths were related to graft failure or myeloablative conditioning toxicity. In 116 evaluable patients, 17 developed acute GVHD, four with grade III–IV, and six developed chronic GVHD. Conservation of vision was better if HCT occurred before age of 3 months of age. There were 272 patients registered through 2006 with CIBMTR, who had HCT for osteopetrosis between 1978 and 2006. Year of HCT was between 1978 and 1989 in 37, 1990–1999 in 137, and 2000–2006 in 98. The group includes 155 males and 118 females. Age at HCT was 1–136 (median 6) months. Donors were 36% matched siblings, 24% other related, and 40% unrelated. Of the 109 unrelated donor HCTs, 47 were umbilical cord blood grafts. Most patients (66%) were prepared with BU- and CY-containing regimens. Three-year probability of survival was 55 ± 9% for matched siblings, 49 ± 12% for alternate related donors,
Fig. 76.4 Survival with osteoclast function. Kaplan–Meier curves of diseasefree survival after first or subsequent hematopoietic stem cell transplantation (HSCT) in relation to human leukocyte antigen (HLA) compatibility with the HSC donor. A, genotypically HLA-identical sibling donor; B, matched unrelated donor; C, related donor phenotypically HLA identical or with one nonidentical HLA antigen; D, related donor with two or more nonidentical HLA antigens (haploidentical). Survival of group A is significantly better compared with that of groups B, C, and D (all p < 0.03). The differences between B, C, and D are not significant. (Reproduced from [87], with permission of Nature Publishing Group; Copyright © 2003, Nature Publishing Group.)
and 44 ± 9% for unrelated donors. When the CIBMTR registry data were reviewed in 2001, the 3-year probabilities were 54%, 47%, and 35%, respectively. (It should be noted that the data presented here are preliminary and were obtained from the Statistical Center of the CIBMTR. The analysis has not been reviewed or approved by the Advisory or Scientific Committee of the CIBMTR.) Recently, Tolar et al. [88] reported HCT for osteopetrosis using a reduced-intensity conditioning regimen including BU, fludarabine, and total lymphoid irradiation. Six patients received bone marrow or peripheral stem cell grafts, and all engrafted with over 75% chimerism. All five recipients of umbilical cord blood first grafts failed to develop donor chimerism. Four of 11 survive with donor engraftment at 1 year.
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Transplantation issues In general, HCT for osteopetrosis is similar to HCT for other nonmalignant inherited disorders detailed in this book. However, there are certain unique issues that should be understood to select appropriate patients and donors and to improve outcomes. The typical infant with osteopetrosis will present with anemia and thrombocytopenia. Transfusion may be necessary, and red cell and platelet survival may be short secondary to hypersplenism. A course of corticosteroid therapy may stabilize the patient hematologically and temporarily reduce transfusion requirements. Some patients will present with, or develop, severe bone pain, progressive debilitation, and poor oral intake, requiring narcotic analgesics and hyperalimentation prior to HCT. Evaluation for optic atrophy [48], including fundoscopic examination, visual evoked-response electroencephalograms, and optic foramina imaging, should be completed quickly as early optic nerve decompression may preserve vision [51,52,89]. Early correction of hypocalcemia may prevent or resolve seizures [47]. Baseline imaging studies should include long bone radiographs, measurements of liver and spleen size, and cranial computed tomography or magnetic resonance imaging scans [45,48,49]. It is recommended that an open-wedge bone biopsy from the anterior or posterior iliac crest be obtained to confirm the diagnosis [8]. Trephine needle biopsies may cause fractures, and are usually not adequate for evaluation due to crush artifact. Marrow aspiration is rarely successful. Light microscopic analysis of osteoclast number, size, nucleation, and morphology should be performed on touch preparations, and histologic sections examined to detect the rare osteoclast-poor phenotype [38,62,74]. Marrow cellularity and the degree of myelofibrosis should be determined. In patients over 2 years of age, fewer osteoclasts are identified due to increased fibrosis. Recipient and donor cytogenetic or molecular studies to assess the degree of hematopoietic chimerism after HCT are essential. Molecular genetic studies should be sent to a laboratory that can screen for known and potential mutations. Infants with early neurologic degeneration may have the syndrome of osteopetrosis associated with neuronal storage of ceroid lipofuscin [2,3,47,48,56–60]. They should not be considered as candidates for HCT as the storage disease progresses in spite of correction of the osteopetrosis. HCT in other variants of osteopetrosis is controversial. Dini and coworkers [43] report HCT in two children at 5 and 12 years of age with a variant of mild autosomal recessive osteopetrosis. Both had severe optic atrophy and dense bones but no significant hematologic or other complications of osteopetrosis. They each engrafted, developed severe hypercalcemia, had subsequent normalization of bone density but remained blind without catch-up growth, and are alive and well 5 and 6 years after HCT, respectively. They may represent one of the variants of ClCN7 mutations [44]. CAII deficiency syndrome [38,39] produces mild osteopetrosis, renal tubular acidosis, cerebral calcification, mental retardation, growth failure, and dental complications. Hematologic complications are not reported, and cranial nerve compression is usually mild. While HCT should correct bone manifestations, it will not improve renal insufficiency or central nervous system complications [38]. McMahon et al. [90] recently reported HCT in two children aged 9 months and 4 months from related Irish families with severe progressive visual and hearing loss. Both successfully engrafted, normalized bone density, and stabilized vision and hearing. HCT did not correct the metabolic acidosis, the renal lesions or the developmental delays. HCT may have delayed the onset of cerebral calcifications. In both mild and CAII osteopetrosis, it remains questionable whether correction of only the skeletal problems warrants the risks and morbidity of HCT.
An HLA-matched nonaffected sibling is the preferred donor. If a matched sibling is unavailable, it is possible that a matched or minimally mismatched parental or extended family donor can be identified. Obligate heterozygote relatives are suitable donors. Since progression of the disease is relatively slow in many cases, unrelated donor search and unrelated marrow or cord blood (see Chapters 39 and 47) HCT are reasonable options. However, if infants have early visual and hematologic impairment, rapid identification of a donor is essential [9,47,87]. Cord blood HCT with high-dose conditioning [88] and haploid-identical parent donors are reasonable options if a closely matched family donor is unavailable. Failure of engraftment [47,87] is a serious concern with mismatched donors, T-cell-depleted grafts, and reduced-intensity conditioning followed by cord blood HCT [88]. Additional immunosuppression is probably advisable in these situations. Second HCT is frequently necessary [47,87]. Serial re-evaluations to document engraftment and bone resorption are important to assess efficacy of therapy. Studies to assess the extent of hematopoietic chimerism and, in cases of mixed chimerism, to document sustained engraftment or late graft failure are vital for long-term patient management. Marrow aspirations and biopsies at 3–6 months after HCT are valuable to assess bone resorption, marrow cavity formation, and marrow cellularity. Serial long bone radiographs are the best indicators of sustained correction of osteopetrosis in most cases. The Ulm transplant team [91] reported that seven of 11 children after HCT between 1996 and 2001 developed sinusoidal obstructive syndrome (SOS). Three cases were severe, and one child died. Between 2001 and 2005, nine additional patients had HCT with defibrotide prophylaxis. One of the nine developed moderate SOS. Two groups have reported a high incidence of pulmonary hypertension after HCT for osteopetrosis. Steward et al. [92] for the EBMT reported that eight of 28 patients who received HCT between 1996 and 2002 at three institutions developed severe pulmonary hypertension at day 2 before HCT to day 89 after. Six required ventilator support, and five died. Three children had a sustained improvement after prostacyclin administration. Kasow et al. [93] reported five of 12 children at four institutions from 1997 to 2003 who developed primary pulmonary hypertension. Four died, and one dramatically improved. An extensive literature review has not revealed other reports with excessive morbidity or mortality from either SOS or pulmonary hypertension. An unusual but not necessarily surprising complication after HCT, referable to osteopetrosis, is hypercalcemia secondary to excessive donor osteoclast resorptive activity after HCT. A 30-month-old male child (Gluckman, personal communication) received a marrow graft in 1980 from his HLA-identical sister after preparation with CY and 400 cGy modified TBI. He engrafted rapidly and developed mild acute GVHD. The child demonstrated both histologic and radiologic improvement and grew 5 cm in the first 2 months after HCT. Severe hypercalcemia developed on day 60 after HCT, and was refractory to saline diuresis, Lasix, corticosteroids, calcitonin, and dialysis. Death with hypercalcemia occurred on day 165 after HCT. In the recent EBMT update, Driessen et al. [87] report severe hypercalcemia in eight of 50 evaluable patients. Two of 40 patients aged 2 years or less, and six of 10 older than 2, developed the complication. The current availability of agents that selectively inhibit calcium resorption from bone should provide effective options to control this serious complication. Bisphosphonate derivatives [94] selectively inhibit osteoclast-mediated bone resorption. These agents also can induce osteoclast apoptosis in murine osteoclasts [5]. Side-effects of concern are myelosuppression and nausea. Titration of these agents should allow controlled resorption of sclerotic bone without severe life-threatening hypercalcemia.
Hematopoietic Cell Transplantation for Osteopetrosis
An illustrative example is a 9-month-old male (Chang and Coccia, personal observation) who received a graft from an HLA-matched sibling donor for infantile osteopetrosis, rapidly developed complete hematopoietic chimerism without GVHD, and promptly demonstrated new bone formation of normal density. At 6 months after HCT, his serum calcium was 12.7 mg/dL. He responded poorly to saline diuresis and furosemide, and 4 weeks later his serum calcium was 13.8 mg/dL. He received pamidronate at 0.5 mg/kg intravenously, with a rapid decline to 12.3 mg/dL. Four weeks later, his serum calcium was 14.8 mg/ dL, and after 0.75 mg/kg intravenous pamidronate, it was 10.8 mg/dL within 3 days. Four weeks later, he received 1.5 mg/kg intravenous pamitronate for a serum calcium of 14.0 mg/dL. All subsequent serum calcium determinations have been normal. The child remains fully chimeric with complete resolution of all manifestations of osteopetrosis 8 years after HCT.
Future directions In July 1978, a 3-month-old female infant was seen in a St Paul, Minnesota emergency room with a stuffy nose and fever. On examination, she had impressive hepatosplenomegaly. Her complete blood count revealed anemia, marked leukoerythroblastosis, and thrombocytopenia. A chest X-ray was reported as normal but with dense bones consistent with “osteopetrosis.” In 1978, there were two types of osteopetrosis – the “good” autosomal dominant and the “bad” autosomal recessive. Donald Walker (1925–79) thought it would be exciting to translate his mouse experiments to a human. The patient and her only sibling, a 5-year-old brother, matched at the A and B loci and were nonreactive in mixed lymphocyte culture. Osteoclasts were the huge cells with all the nuclei rarely seen in marrow aspirates. Thus began the odyssey.
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Today, there are numerous subtypes of osteopetrosis, and the gene defects have been characterized in mouse and man for almost all patients with dominant and about 80% with recessive osteopetrosis. Acidification defects at the osteoclast–bone interface is likely the etiology of over 90% of inherited osteopetrotic disorders in humans. After more than two decades of failed attempts to culture osteoclasts, abundant osteoclasts can now be cultured in vitro by stimulating CD14+ cells from patients and controls with M-CSF and RANKL. This breakthrough allows the study of digestion and indigestion of bone fragments. Yet, only 50% of infants are cured with a toxic, expensive, and frequently unreliable therapy. This is about the same percentage reported in the mid-1980s [95]. No other effective therapies have yet been developed. Certainly, the rapidly increasing understanding of osteoclast bone biology is being translated into improved therapies for osteoporosis, Paget’s disease of bone, periodontal disease, abnormal fracture healing, and other disorders of excessive bone resorption. The development of bisphosphonates [94], which mediate osteoclasts apoptosis, have led to improved therapy for osteoporosis and multiple myeloma. The remarkable progress that continues to be made in our understanding of the osteopetrotic syndromes, the immunodeficiencies, storage diseases, and other inherited disorders of the HSC will eventually lead to in utero HCT (see Chapter 40), gene therapy (see Chapter 10) [3,24], reduced-intensity conditioning [88], and other strategies to increase the effectiveness and decrease the toxicity of HCT. Antiresorptive medications based on gene products are in clinical trials, and other gene products identified in the molecular biologic studies will potentially have a role in the development of therapeutic agents for various subtypes of osteopetrosis [3]. Many of the children cured of infantile malignant osteopetrosis by HCT are productive, contributing members of society.
References 1. Tolar J, Teitelbaum SL, Orchard PJ. Osteopetrosis. N Engl J Med 2004; 351: 2839–49. 2. Balemans W, Van Wesenbeeck L, Van Hul W. A clinical and molecular overview of the human osteopetroses. Calcif Tissue Int 2005; 77: 263–74. 3. Askmyr MK, Fasth A, Richter J. Towards a better understanding and new therapeutics of osteopetrosis. Br J Haematol 2008; 140: 597–609. 4. Abu-Amer Y. Advances in osteoclast differentiation and function. Curr Drug Targets Immune Endocr Metabol Disord 2005; 5: 347–55. 5. Teitelbaum SL. Osteoclasts: what do they do and how do they do it? Am J Pathol 2007; 170: 427– 35. 6. Helfrich MH. Osteoclast diseases. Microsc Res Tech 2003; 61: 514–32. 7. Van Wesenbeeck L, Van Hul W. Lessons from osteopetrotic mutations in animals: impact on our current understanding of osteoclast biology. Crit Rev Eukaryot Gene Expr 2005; 15: 133–62. 8. Coccia PF, Krivit W, Cervenka J et al. Successful bone-marrow transplantation for infantile malignant osteopetrosis. N Engl J Med 1980; 302: 701– 8. 9. Gerritsen EJ, Vossen JM, Fasth A et al. Bone marrow transplantation for autosomal recessive osteopetrosis. A report from the Working Party on Inborn Errors of the European Bone Marrow Transplantation Group. J Pediatr 1994; 125: 896–902. 10. Asagiri M, Takayanagi H. The molecular understanding of osteoclast differentiation. Bone 2007; 40: 251–64.
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26. Cohen MM Jr. The new bone biology: pathologic, molecular, and clinical correlates. Am J Med Genet A 2006; 140: 2646–706. 27. Albers-Schönberg H. Roentgenbilder einer seltenen Knochener-krankung. Münch Med Wochenschr 1904; 51: 365–8. 28. Johnston CC Jr., Lavy N, Lord T, Vellios F, Merritt AD, Deiss WP. Osteopetrosis: a clinical, genetic, metabolic, and morphologic study of the dominantly inherited, benign form. Medicine 1968; 47: 149–67. 29. Van Wesenbeeck L, Cleiren E, Gram J et al. Six novel missense mutations in the LDL receptorrelated protein 5 (LRP5) gene in different conditions with an increased bone density. Am J Hum Genet 2003; 72: 763–71. 30. Henriksen K, Gram J, Hoegh-Andersen P et al. Osteoclasts from patients with autosomal dominant osteopetrosis type I caused by a T253I mutation in low-density lipoprotein receptor-related protein 5 are normal in vitro, but have decreased resorption capacity in vivo. Am J Pathol 2005; 167: 1341– 8. 31. Benichou O, Cleiren E, Gram J, Bollerslev J, de Vernejoul M-C, Van Hul W. Mapping of autosomal dominant osteopetrosis type II (AlbersSchönberg Disease) to chromosome 16p13.3. Am J Hum Genet 2001; 69: 647–55. 32. Kornak U, Kasper D, Bosl MR et al. Loss of the CIC-7 chloride channel leads to osteopetrosis in mice and man. Cell 2001; 104: 205–15. 33. Frattini A, Pangrazio A, Susani L et al. Chloride channel ClCN7 mutations are responsible for severe recessive, dominant, and intermediate osteopetrosis. J Bone Miner Res 2003; 18: 1740–7. 34. Henriksen K, Gram J, Schaller S et al. Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II. Am J Pathol 2004; 164: 1537–45. 35. Del Fattore A, Peruzzi B, Rucci N et al. Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment. J Med Genet 2006; 43: 315–25. 36. Chu K, Snyder R, Econs MJ. Disease status in autosomal dominant osteopetrosis type 2 is determined by osteoclastic properties. J Bone Miner Res 2006; 21: 1089–97. 37. Waguespack SG, Hui SL, DiMeglio LA, Econs MJ. Autosomal dominant osteopetrosis: clinical severity and natural history of 94 subjects with a chloride channel 7 gene mutation. J Clin Endocrinol Metab 2007; 92: 771–8. 38. Sly WS, Shah GN. The carbonic anhydrase II deficiency syndrome: osteopetrosis with renal tubular acidosis and cerebral calcification. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill; 2001. pp. 5331–43. 39. Shah GN, Bonapace G, Hu PY, Strisciuglio P, Sly WS. Carbonic anhydrase II deficiency syndrome (osteopetrosis with renal tubular acidosis and brain calcification): novel mutations in CA2 identified by direct sequencing expand the opportunity for genotype-phenotype correlation. Hum Mutat 2004; 24: 272–81. 40. Monaghan BA, Kaplan FS, August CS, Fallon MD, Flannery DB. Transient infantile osteopetrosis. J Pediatr 1991; 118: 252–6. 41. Iacobini M, Migliaccio S, Roggini M et al. Apparent cure of a newborn with malignant osteopetrosis
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60. Alroy J, Pfannl R, Ucci A, Lefranc G, Frattini A, Megarbane A. Electron microscopic findings in skin biopsies from patients with infantile osteopetrosis and neuronal storage disease. Ultrastruct Pathol 2007; 31: 333–8. 61. Scimeca JC, Quincey D, Parrinello H et al. Novel mutations in the TCIRG1 gene encoding the a3 subunit of the vacuolar proton pump in patients affected by infantile malignant osteopetrosis. Hum Mutat 2003; 21: 151–7. 62. Blair HC, Borysenko CW, Villa A et al. In vitro differentiation of CD14 cells from osteopetrotic subjects: contrasting phenotypes with TCIRG1, CLCN7, and attachment defects. J Bone Miner Res 2004; 19: 1329–38. 63. Ogbureke KU, Zhao Q, Li YP. Human osteopetroses and the osteoclast V-H+-ATPase enzyme system. Front Biosci 2005; 10: 2940–54. 64. Van Wesenbeeck L, Odgren PR, Coxon FP et al. Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans. J Clin Invest 2007; 117: 919– 30. 65. Frattini A, Vezzoni P, Villa A, Sobacchi C. The dissection of human autosomal recessive osteopetrosis identifies an osteoclast-poor form due to RANKL deficiency. Cell Cycle 2007; 6: 3027–33. 66. Heaney C, Shalev H, Elbedour K et al. Human autosomal recessive osteopetrosis maps to 11q13, a position predicted by comparative mapping of the murine osteosclerosis (oc) mutation. Hum Mol Genet 1998; 7: 1407–10. 67. Kapelushnik J, Shalev C, Yaniv I et al. Osteopetrosis: a single centre experience of stem cell transplantation and prenatal diagnosis. Bone Marrow Transplant 2001; 27: 129–32. 68. Shalev H, Mishori-Dery A, Kapelushnik J et al. Prenatal diagnosis of malignant osteopetrosis in Bedouin families by linkage analysis. Prenat Diagn 2001; 21: 183–6. 69. Kornak U, Schulz A, Friedrich W et al. Mutations in the a3 subunit of the vacuolar H(+)-ATPase cause infantile malignant osteopetrosis. Hum Mol Genet 2000; 9: 2059–63. 70. Frattini A, Orchard PJ, Sobacchi C et al. Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nat Genet 2000; 25: 343–6. 71. Sobacchi C, Frattini A, Orchard P et al. The mutational spectrum of human malignant autosomal recessive osteopetrosis. Hum Mol Genet 2001; 10: 1767–73. 72. Kasper D, Planells-Cases R, Fuhrmann JC et al. Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J 2005; 24: 1079–91. 73. Sobacchi C, Frattini A, Guerrini MM et al. Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL. Nat Genet 2007; 39: 960–2. 74. Nicholls BM, Bredius RG, Hamdy NA et al. Limited rescue of osteoclast-poor osteopetrosis after successful engraftment by cord blood from an unrelated donor. J Bone Miner Res 2005; 20: 2264– 70. 75. David JP, Neff LCY, Rincon M, Horne WC, Baron R. A new method to isolate large numbers of rabbit osteoclasts and osteoclast-like cells; application to the characterization of serum response element binding proteins during osteoclast differentiation. J Bone Miner Res 1998; 13: 1730–8.
Hematopoietic Cell Transplantation for Osteopetrosis 76. Teti A, Migliaccio S, Taranta A et al. Mechanisms of osteoclast dysfunction in human osteopetrosis: abnormal osteoclastogenesis and lack of osteoclastspecific adhesion structures. J Bone Miner Res 1999; 14: 2107–17. 77. Flanagan AM, Sarma U, Steward CG, Vellodi A, Horton MA. Study of the nonresorptive phenotype of osteoclast-like cells from patients with malignant osteopetrosis: a new approach to investigating pathogenesis. J Bone Miner Res 2000; 15: 352– 60. 78. Helfrich MH, Gerritsen EJA. Formation of nonresorbing osteoclasts from peripheral blood mononuclear cells of patients with malignant juvenile osteopetrosis. Br J Haematol 2001; 112: 64– 8. 79. Key LLJ, Ries WL. Osteopetrosis. Principles of Bone Biology. San Diego: Academic Press; 2002. 80. Madyastha PR, Jeter EK, Key LL Jr. Cytophilic immunoglobulin G binding on neutrophils from a child with malignant osteopetrosis who developed fatal acute respiratory distress mimicking transfusion-related acute lung injury. Am J Hematol 1996; 53: 196–200. 81. Umeda S, Takahashi K, Naito M, Shultz LD, Takagi K. Neonatal changes of osteoclasts in osteopetrosis (op/op) mice defective in production of functional macrophages colony-stimulating factor
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reduced intensity conditioning regimen. Bone Marrow Transplant 2006; 38: 783–7. Hwang JM, Kim IO, Wang KC. Complete visual recovery in osteopetrosis by early optic nerve decompression. Pediatr Neurosurg 2000; 33: 328– 32. McMahon C, Will A, Hu P, Shah GN, Sly WS, Smith OP. Bone marrow transplantation corrects osteopetrosis in the carbonic anhydrase II deficiency syndrome. Blood 2001; 97: 1947–50. Corbacioglu S, Honig M, Lahr G et al. Stem cell transplantation in children with infantile osteopetrosis is associated with a high incidence of VOD, which could be prevented with defibrotide. Bone Marrow Transplant 2006; 38: 547–53. Steward CG, Pellier I, Mahajan A et al. Severe pulmonary hypertension: a frequent complication of stem cell transplantation for malignant infantile osteopetrosis. Br J Haematol 2004; 124: 63–71. Kasow KA, Bonfim C, Asch J et al. Malignant infantile osteopetrosis and primary pulmonary hypertension: a new combination? Pediatr Blood Cancer 2004; 42: 190–4. Whyte MP, Wenkert D, Clements KL, McAlister WH, Mumm S. Bisphosphonate-induced osteopetrosis. N Engl J Med 2003; 349: 457–63. Coccia PF. Cells that resorb bone. N Engl J Med 1984; 310: 456–8.
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Charles Peters
Hematopoietic Cell Transplantation for Storage Diseases
Introduction The storage diseases are a diverse group of disorders arising from singlegene defects that often involve a lysosomal hydrolytic enzyme or vital peroxisomal function, causing devastating systemic diseases that can affect the brain (Tables 77.1–77.3). Progressive losses of neurodevelopmental milestones and neurologic function are common. Cardiopulmonary disease and compromise lead to shortened life expectancies. Diseases with onset during infancy or childhood are characterized by rapid deterioration. Treatment recommendations and clinical decision making must take account of the disorder, age at onset, rate of progression, presence of clinical signs and symptoms, family values, and risks and benefits associated with available therapies, which include hematopoietic cell transplantation (HCT), enzyme replacement therapy (ERT), substrate depletion, and gene transfer. Lysosomes are cellular organelles that contain hydrolytic enzymes; peroxisomes are subcellular organelles catalyzing metabolic functions primarily related to lipid metabolism. In 1968, Fratantoni and Neufeld established the concept of transferable lysosomal enzymes through metabolic cross-correction of defects in co-cultured fibroblasts of patients with Hurler and Hunter syndromes [1]. Metabolic correction of lysosomal storage diseases is due to the mannose-6-phosphate receptor-mediated endocytosis of secreted enzyme, or by direct transfer of enzyme from adjacent cells [2–4]. It is likely that both mechanisms occur following HCT (Fig. 77.1). Variability in receptor-mediated enzyme endocytosis in various cells and tissues could affect hydrolase uptake after HCT [5]. For example, monocytes and macrophages have receptors for N-acetylglucosamine and mannose; glial cells have receptors for sialic acid [6]. X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder of very long chain fatty acid (VLCFA) β-oxidation. The likely mechanism by which HCT halts cerebral demyelination is perhaps threefold: immunosuppression, replacement with metabolically normal cell populations leading to decreased perivascular inflammation, and metabolic correction. The impact of normalizing VLCFAs in plasma is unclear. A critical question in HCT for storage diseases with central nervous system (CNS) involvement is whether donor-derived cells enter the CNS and achieve metabolic correction or halt inflammation. Microglia are mononuclear phagocytes in the CNS, which are hematopoietically derived [7,8]. They account for 5–10% of non-neuronal cells in brain. Activated microglia participate in antigen presentation, inflammatory
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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responses, infection, and CNS injury [9,10]. In humans, repopulation with donor-derived microglia can take a year. The kinetics of microglial replacement after HCT is slower than that of other tissue macrophages (e.g. alveolar macrophages or Kupffer cells) [11]. This observation explains, in part, the inability of HCT to stabilize effectively the CNS and neurologic aspects of a rapidly progressing disorder.
HCT for storage diseases History On April 8, 1980, a 9-month-old boy received the first bone marrow transplant for Hurler syndrome [12]. Today, fully engrafted with maternal marrow, he uses a computer as a self-reliant employee. Despite genotype analysis revealing homozygous W402X mutations indicative of severe Hurler syndrome (mucopolysaccharidosis IH [MPS IH]) phenotype, he has stable, normal intelligence [12]. In 1982, the first child with Maroteaux–Lamy syndrome, a 13-year-old girl, was successfully transplanted from her human leukocyte antigen (HLA)-matched, enzymatically normal sister, and experienced resolution of hepatosplenomegaly and normalization of cardiopulmonary function [13]; she survives and functions independently. In the late 1980s and into the 1990s, successful transplants were performed for each of the leukodystrophies, including cerebral X-ALD [14], globoid cell leukodystrophy (GLD) [15], and metachromatic leukodystrophy (MLD) [16]. In the mid-1990s to the mid-2000s, the first large multicenter reports for HCT were published on Hurler syndrome [17–19] and cerebral X-ALD [20]. Sources of hematopoietic cells for transplantation included bone marrow [15,16,20], peripheral blood [21], and umbilical cord blood (UCB) [20,22,23]. In addition to high-dose regimens, reduced-intensity conditioning regimens were employed effectively [21]. Successful use of combination therapy, ERT, and HCT for patients with Hurler syndrome has been described [24,25]. Finally, participants in the field have recognized the importance of a comprehensive approach to caring for these patients, which is described below. General considerations The following elements are critical to the success of HCT in treating patients with storage disorders: a multidisciplinary and multispecialty approach to evaluation and follow-up; careful attention to optimizing HLA typing, and donor and hematopoietic cell selection; the risks and benefits of high-dose versus reduced-intensity conditioning regimens, supportive care measures, and long-term post-HCT evaluations. Several sets of evaluation and follow-up guidelines are provided later in this chapter (Tables 77.4 and 77.5).
Autosomal recessive Autosomal recessive Autosomal recessive
Autosomal recessive 248/242 2111/726 422/285
β-Glucuronidase Phosphotransferase
128/169 235/230 1407/593 1056/514 201/206
162/136,000
111/148
Galactose 6-sulfatase β-Galactosidase Arylsulfatase B
Heparan-N-sulfatase N-acetylglucosaminidase AcetylCoA : N-acetyltransferase N-acetylglucosamine 6-sulfatase
Iduronate-2-sulfatase
X-linked
Autosomal recessive
α-L-Iduronidase
Enzyme/protein
Autosomal recessive
Genetics
ERT, enzyme replacement therapy; GT, gene therapy; HCT, hematopoietic cell transplantation; SD, substrate depletion. Data from [170].
Mucolipidoses (ML) ML-II (I-cell) ML-III (pseudo-Hurler polydystrophy)
Sly (MPS VII)
Mucopolysaccharidoses (MPS) MPS I Hurler (MPS IH) Hurler/Scheie (MPS IH/S) Scheie (MPS IS) Hunter MPS II Severe (MPS IIA) Attenuated (MPS IIB) Sanfilippo (MPS III) MPS IIIA MPS IIIB MPS IIIC MPS IIID Morquio (MPS IV) MPS IV A MPS IV B Maroteaux–Lamy (MPS VI)
Disorder
Incidence in thousands/ carrier frequency (estimates)
/ / / /
− − − − / / / /
− − − − / / / /
− − − −
HCT in a very limited number of a cases Significant clinical challenges to performing HCT primarily due to airway and pulmonary issues
+/−/−/−
ERT is the preferred therapy; however, HCT can be considered
Severe skeletal deformities preclude HCT
No evidence of neurocognitive efficacy following successful, timely HCT
ERT is currently offered to patients with Hunter
HCT or HCT + ERT ERT is considered the standard therapy
Comments
+/−/−/−
−/−/−/− −/−/−/− +/+/−/−
− − − −
−/+/−/− −/+/−/−
+/+/−/− +/+/−/− +/+/−/−
Treatment options: HCT/ERT/SD/GT
Table 77.1 The mucopolysaccharidoses and mucolipidoses: mode of inheritance, enzyme/protein deficiency, incidence and carrier frequencies, treatment options, and comments
Hematopoietic Cell Transplantation for Storage Diseases
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Autosomal recessive
Autosomal recessive X-linked Autosomal recessive
Galactocerebrosidase
Autosomal recessive Autosomal recessive
Proteolipid protein Mutations in translation initiation factor eIF2B and possibly others Several, including accumulation of VLCFA, marked deficiency of plasmalogens
Arylsulfatase-A
ALD protein (ALDP)
Enzyme/protein
X-linked
Genetics
Long-term follow-up of infants transplanted with UCB is needed The extended time to CNS stabilization and the inability of HCT to favorably affect the PNS dampens enthusiasm regarding quality of life outcomes in late-onset patients. ERT may be effective for treating the PNS; however, further study is needed
+/−/−/− +/+/−/−
−/−/−/− −/−/−/−
−/−/−/−
?/?
−/−/−/−
HCT is the only effective long-term therapy offering the possibility of stabilization of the cerebral disease course. Lorenzo’s oil appears to decrease the likelihood of developing cerebral disease in selected boys with biochemically diagnosed X-ALD
+/−/−/+ +/−/−/− +/−/−/− ?/−/−/? −/−/−/−
?/? ?/?
?/?
121/152
201/188
19/19,000
Comments
ERT, Enzyme replacement therapy; GT, gene therapy; PNS, peripheral nervous system; SD, substrate depletion; ?, unknown. See text for other abbreviations. Data from [170].
Zellweger syndrome
Pelizaeus–Merzbacher disease Vanishing white matter disease
Alexander
Metachromatic leukodystrophy
Leukodystrophies and other white matter diseases X-ALD phenotypes Childhood-onset cerebral X-ALD Adolescent-onset cerebral X-ALD Adult-onset cerebral X-ALD AMN with or without cerebral X-ALD Addison disease only Globoid-cell leukodystrophy
Disorder
Incidence in thousands/ carrier frequency Treatment options: (estimates) HCT/ERT/SD/GT
Table 77.2 The leukodystrophies: mode of inheritance, enzyme/protein deficiency, incidence and carrier frequencies, treatment options, and comments [170]
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Table 77.3 The glycoprotein disorders and miscellaneous lysosomal storage diseases and inborn errors of metabolism: mode of inheritance, enzyme/protein deficiency, incidence and carrier frequencies, treatment options, and comments [170]
Disorder
Genetics
Enzyme/protein
Fucosidosis
Autosomal recessive Autosomal recessive
Fucosidase
Gaucher disease I (non-neuronopathic) II (acute neuronopathic) III (subacute neuronopathic)
α-Mannosidosis Aspartylglucosaminuria Cerebrotendinous xanthomatosis Fabry disease Farber lipogranulomatous disease Gangliosidoses (GM1 and GM2 including Tay–Sachs, Sandhoff, GM2 activator deficiency) GM1 gangliosidoses GM2 gangliosidoses – Tay–Sachs – Sandhoff – GM2 activator deficiency Glycogen storage disease II (Pompe) Neuronal ceroid lipofuscinosis NCL1 NCL2
Glucocerebrosidase
Aspartylglucosaminidase
2111/726
+/−/−/−
Autosomal recessive Autosomal recessive
Ceramidase
Autosomal recessive Autosomal recessive
117/117,000
−/+/+/−
ERT is the preferred therapy
?/?
+/−/−/−
β-Galactosidase Hexosaminidase A Hexosaminidase A and B
422/310 222/224 422/310
?/−/−/− −/−/+/−
No clear evidence of efficacy for HCT in any of these disorders
α-Glucosidase
201/191
−/+/−/−
ERT: preferred therapy
?/?
+/−/−/−
Palmitoyl protein thioesterase Tripeptidyl peptidase I
Acid lipase
Canavan disease
Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive or autosomal dominant
Aspartoacylase
Hypophosphatasia
ERT and/or SD: preferred therapies Long-term favorable outcomes have been seen with HCT, particularly in patients with Norrbottnian form Impressive, favorable results with HCT
−/−/−/−
27-Hydroxylase
Autosomal recessive
Multiple sulfatase deficiency
+/+/+/+ −/+/+/− +/+/+/−
+/−/−/−
Wolman syndrome
Sialidosis
59/119
1056/514
α-Galactosidase
Comments
+/−/−/−
α-Mannosidase
Autosomal recessive
Sialic acid storage disease
Treatment options: HCT/ERT/SD/GT
Autosomal recessive Autosomal recessive Autosomal recessive X-linked
Niemann–Pick disease A B C
Cystinosis
Incidence in thousands/ carrier frequency (estimates)
Acid sphingomyelinase Acid sphingomyelinase NPC1 (cholesterol trafficking)
A and B: 264/249 211/230
−/+/−/− +/+/−/− +/−/−/−
704/363
+/−/−/− −/−/−/−
Very limited clinical use of HCT and rapid disease course make it difficult to reach a conclusion ERT may have a role in type A and B patients. HCT may be effective in type B and C patients; however, experience remains very limited HCT: extremely high morbidity and mortality Increased in Ashkenazi Jews
Cystine transporter
281/219
−/−/+/−
Sialic acid transporter
603/363
?/−/−/−
HCT: role is unknown
Neuraminidase
4222/1027
?/−/−/−
HCT: role is unknown
Multiple sulfatase factor, all sulfatases Tissue nonspecific alkaline phosphatase
1407/593
+/−/−/−
HCT: extremely limited clinical experience HCT: extremely limited clinical experience
+/−/−/−
ERT, enzyme replacement therapy; GT, gene therapy; HCT, hematopoietic cell transplantation; SD, substrate depletion; ?, unknown. Data from [170].
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Fig. 77.1 Transport of a lysosomal enzyme in a normal cell and correction of storage in the cell of a patient with mucopolysaccharidosis. The mannose-6phosphate-recognition signal is added to the lysosomal enzyme precursor in the late Golgi compartments, where enzyme modified by mannose-6-phosphate binds to mannose-6-phosphate receptors. The enzyme–receptor complex is packaged into a transport carrier vesicle and delivered to early endosomes, in which low pH promotes the dissociation of the enzyme from the receptor. The enzyme is then delivered to the mature lysosome, and the mannose-6-phosphate receptor is recycled to the Golgi apparatus. A small amount of the mannose-6-phosphate-modified enzyme escapes capture by the mannose-6-phosphate receptors and is released into the extracellular space. This enzyme can be recaptured by binding to a mannose-6-phosphate receptor in a clathrin-coated pit on the cell surface. In a patient who has undergone hematopoietic stem cell transplantation, enzyme released from a donor-derived cell can be taken up by a mucopolysaccharidosis cell, which corrects aberrant glycosaminoglycan storage. (Reproduced from Muenzer J, Fisher A. Advances in the treatment of mucopolysaccharidosis type I. N Engl J Med 2004; 350: 1960–9, with permission.)
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Table 77.4 Guidelines for the pre-hematopoietic cell transplantation (HCT) evaluation (section A) and post-HCT (section B) follow-up of patients with mucopolysaccharide, mucolipidosis, and glycoprotein metabolic disorders Section A
Pre HCT
Neurology Neuropsychology
Examination including assessment for signs or symptoms of hydrocephalus in patients with Hurler syndrome Test of cognitive ability according to age and developmental level, e.g. Mullen Scales of Early Learning to obtain Early Learning composite or WISC III Language assessment to obtain receptive and expressive language scores Vineland Adaptive Behavior Scales MRI of brain and upper cervical spine with attention to the odontoid process in patients with Hurler syndrome Echocardiogram, EKG Chest X-ray (posteroanterior and lateral) Polysomnogram ENT as needed for placement of tubes Brainstem auditory evoked response if requested by audiology department Routine evaluation and if possible ocular pressures in patients with Hurler syndrome
Neuroradiology Cardiology Pulmonology Audiology Ophthalmology Genetic counseling Neurophysiology Occupational, physical, and speech and language therapy Donor studies Molecular biology studies Lumbar puncture
Electrophysiology studies (EMG) with surface electrodes to evaluate for carpal tunnel syndrome if clinically indicated in patients with Hurler syndrome Assessment and therapies as needed, of particular importance in children with Hurler syndrome Donor enzyme activity level, if possible, in addition to infectious disease markers On admission to hospital, consider obtaining blood samples for DNA (e.g. mutation analysis, pharmacogenomics) If there are clinical concerns about possible, significant increased intracranial pressure, a lumbar puncture with determination of opening and closing pressures may be performed with determinations of cerebrospinal fluid glucose and protein in patients with Hurler syndrome
Section B
Post HCT
Blood and marrow transplant (BMT) team
BMT physician, neurologist, neuropsychologist or developmental pediatrician, cardiologist, pulmonologist/sleep disorder specialist, endocrinologist, audiologist, ophthalmologist, occupational and physical therapist, speech and language therapist, advanced practice nurse, nurse coordinator, social worker; others as needed, including occupational and physical therapists Examination Test of cognitive ability according to age and developmental level, i.e. Mullen Scales of Early Learning (Early Learning Composite), or Stanford Binet Intelligence Scale (Composite Score), or Wechsler Preschool and Primary Scale of Intelligence (Full Scale IQ) Language assessment to obtain receptive and expressive language scores Vineland Adaptive Behavior Scales MRI of brain and upper cervical spine (attention to odontoid process) Echocardiogram EKG Chest X-ray Consider follow-up polysomnogram as clinically indicated ENT as needed for placement of tubes Brainstem auditory evoked response if requested by audiology department Electroretinogram Orthopedic evaluation of spine, hips, and knees, and hand surgery assessment for carpal tunnel syndrome Growth hormone stimulation test Free thyroxine Thyroid-stimulating hormone levels Dual-energy X-ray absorptiometry scan Bone age X-ray Electrophysiology studies (EMG) with surface electrodes to evaluate for carpal tunnel syndrome Assessment and therapies as needed
Neurology Neuropsychology
Neuroradiology Cardiology Pulmonology Audiology Ophthalmology Orthopedic surgery Endocrinology
Neurophysiology Occupational, physical, and speech and language therapy Engraftment and chimerism studies
Variable number of tandem repeat or XY fluoresence in situ hybridization studies and enzyme level (days +21–30, +60, +100, +6 months, +1 year, and yearly post HCT thereafter as needed
EKG, electrocardiogram; EMG, electromyelogram; ENT, ear, nose, and throat; MRI, magnetic resonance imaging.
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Table 77.5 Guidelines for the pre-hematopoietic cell transplantation (HCT) evaluation (section A) and post HCT (section B) follow-up of patients with leukodystrophies Section A
Pre HCT
Neurology Neuropsychology
Examination Age-appropriate battery of neuropsychologic tests, i.e. Wechsler Preschool and Primary Scale of Intelligence (WPPSIrevised) and Wechsler Intelligence Scale for Children III (WISC-III) Wechsler Adult Intelligence Scale III to obtain: • Full Scale IQ Score • Verbal IQ Score • Performance IQ Score Other testing to measure school achievement, language, visual perception, attention, memory, motor function, executive function. and behavior Vineland Adaptive Behavior Scales MRI of brain including contrast agent (gadolinium) with severity score determinations (Loes Score [80]); Magnetic resonance spectroscopy if available and deemed to be potentially informative MRI of brain
Neuroradiology – cerebral X-ALD Neuroradiology – GLD [15] and metachromatic leukodystrophy CSF Pulmonology Ophthalmology Endocrine Neurophysiology Occupational, physical, and speech and language therapy
Examination of CSF (including CSD protein): may be informative in patients with advanced disease at HCT to evaluate status of blood–brain barrier disruptions (i.e. degree of CSF protein elevation; e.g., in infants with GLD, CSF protein levels of 500–600 mg/dL can be seen) Chest X-ray Assessment as needed To assess treatment of adrenal insufficiency (i.e. dosing of hydrocortisone) in boys with X-ALD; routine assessment of all patients for thyroid function, progress toward puberty, growth, and development Electrophysiology studies (EMG): a long-term evaluation in boys transplanted for cerebral X-ALD who are reaching the age at which they are at risk for developing AMN, i.e. approximately ≥20 years of age. The question of whether such men will develop AMN is unanswered Assessment and therapies as needed
Section B
Post HCT
Blood and marrow transplant (BMT) team Neurology Neuropsychology
BMT physician, neurologist, advanced practice nurse, nurse coordinator, social worker; others as needed, including occupational and physical therapists Examination Age appropriate battery of neuropsychologic tests, i.e. Wechsler Preschool and Primary Scale of Intelligence (WPPSIrevised) and Wechsler Intelligence Scale for Children III (WISC-III) Wechsler Adult Intelligence Scale III to obtain: • Full Scale IQ Score • Verbal IQ Score • Performance IQ Score Other testing to measure school achievement, language, visual perception, attention, memory, motor function, executive function, and behavior Vineland Adaptive Behavior Scales MRI of brain including contrast agent (gadolinium) with severity score determinations (MRI severity or Loes Score [80]); recommended minimum number of examinations – approximately 2–3 months post HCT to evaluate for resolution of gadolinium enhancement (hallmark of active demyelination in cerebral X-ALD) and at 1 year post HCT to evaluate severity score at time when stabilization has been achieved [81]. Subsequent brain MRIs as clinically indicated Magnetic resonance spectroscopy if available and deemed to be potentially informative MRI of brain at 1 year post HCT and annually, if clinically indicated, until stable myelination is demonstrated Examination of CSF (including CSD protein): may be informative in patients with advanced disease at HCT to evaluate status of blood–brain barrier disruptions (i.e. degree of CSF protein elevation; e.g. in infants with GLD, CSF protein levels of 500–600 mg/dL can be seen) Chest X-ray Assessment as needed To assess treatment of adrenal insufficiency (i.e. dosing of hydrocortisone) in boys with X-ALD; routine assessment of all patients for thyroid function, progress toward puberty, growth, and development Electrophysiology studies (EMG): a long-term evaluation in boys transplanted for cerebral X-ALD who are reaching the age at which they are at risk for developing AMN, i.e. approximately ≥20 years of age. The question of whether such men will develop AMN is unanswered Patient’s variable number of tandem repeats and appropriate enzyme levels (days +21–30, +60, +100, +6 months, +1 year, and yearly post HCT thereafter); follow-up evaluation of VLCFA has been discontinued as published studies showed a 55% reduction in these levels after HCT but not complete normalization [85]. VLCFA levels do not appear to have any clinical significance or correlation with disease activity or clinical status
Neuroradiology – cerebral X-ALD
Neuroradiology – GLD and MLD CSF Pulmonology Ophthalmology Endocrine Neurophysiology Engraftment and chimerism studies
AMN, adrenomyeloneuropathy; CSF, cerebrospinal fluid; EMG, electromyogram; GLD, globoid cell leukodystrophy; ML, metachromatic leukodystrophy; MRI, magnetic resonance imaging; VLCFA, very long chain fatty acid; X-ALD, X-linked adrenoleukodystrophy.
Hematopoietic Cell Transplantation for Storage Diseases
MPSs and mucolipidoses In this section, MPS disorders (Hurler, Hurler/Scheie, Scheie, Hunter, Sanfilippo, Morquio, Maroteaux-Lamy, and Sly) and mucolipidoses (MLs; I-cell disease and pseudo-Hurler polydystrophy) will be presented and discussed (Table 77.1). Epidemiology and etiology The MPS diseases are a group of lysosomal storage disorders caused by deficiency of degradative enzymes of glycosaminoglycans (GAGs) [26], which include heparan, dermatan, keratan, and chondroitin sulfates, that individually or in combination accumulate intracellularly, leading to cellular dysfunction. These GAGs are usually excreted in urine and can be detected by initial diagnostic screening tests. Genes and their complementary DNAs (cDNAs) encoding most of the enzymes have been cloned, leading to characterization of their primary structures, production of recombinant enzymes, and elucidation of disease-causing mutations. MPS disorders, their genetics and respective enzymes, incidence and carrier frequencies, and treatment options are presented in Table 77.1. All of the MPS and ML disorders are autosomal recessive in their inheritance except for Hunter syndrome (X-linked). ML II (I-cell) and ML III (pseudo-Hurler polydystrophy) are related, rare genetic diseases in which there is abnormal lysosomal enzyme transport in cells of mesenchymal origin. Newly synthesized lysosomal enzymes are secreted into the extracellular medium rather than being targeted correctly to lysosomes. Affected cells show dense storage material-filled inclusions; lysosomal enzymes are present at elevated levels in serum and body fluids of affected patients [27]. Molecular and clinical biology A precise correlation between the numerous genotypes and the phenotypes of these MPS disorders remains elusive. Enzyme activity assays are available for diagnosing most of the MPS disorders, although they are not helpful to definitively identify a carrier. Characterization of the mutation(s) within a family can be helpful. Naturally occurring disease models exist in dogs, cats, rats, mice, and goats; mouse models have been created by targeted gene disruption [26]. Phosphotransferase, deficient in ML, is a low-abundance, membrane-bound enzyme complex of three subunits that are the products of two genes. Biochemical studies suggest that the enzyme contains two domains: catalytic and recognition sites for lysosomal enzymes. In general, ML II and ML III must be distinguished on clinical criteria and on disease progression. Prenatal diagnosis is reliable, and carrier detection is also possible [27]. Clinical description Shared clinical features among the MPS disorders include progressive courses, multiorgan system involvement, organomegaly, dysostosis multiplex, and facial dysmorphia. Hearing, vision, respiratory, and cardiovascular function are often affected. Severe cognitive deficits with developmental delay are observed in Hurler (MPS IH), the severe phenotype of Hunter (MPS IIA), and Sanfilippo syndromes (MPS III) [26]. ML II is characterized by severe psychomotor retardation and shares many clinical and radiologic features with Hurler syndrome. However, clinical differentiation is possible due to earlier onset of signs and symptoms, absence of GAGs in urine, and the more rapidly progressive course, leading to death between 5 and 8 years of age. Neonates with I-cell disease usually show characteristic facial features, craniofacial abnormalities, and restricted joint movement despite generalized hypotonia. Other presenting features may include striking gingival
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hyperplasia, congenital hip dislocation, fractures, hernias, and bilateral talipes equinovarus. The clinical course includes progressive failure to thrive and developmental delay, usually evident by age 6 months; linear growth decelerates during the first year and stops during the second. Joint immobility progresses, with claw hand deformity and kyphoscoliosis ensuing. Hepatomegaly is prominent while splenomegaly is minimal. Respiratory infections are frequent, as is otitis media. Cardiomegaly and murmurs are common, and aortic insufficiency is not infrequent [27]. Nontransplant approaches A wide variety of nontransplant approaches are available for the MPS disorders, including ERT, substrate depletion, and gene transfer. Their applicability, if appropriate, is addressed in Table 77.1. Hematopoietic cell transplantation To date, HCT represents an effective therapy with the longest clinical experience for selected MPS disorders. MPS and ML disorders that can benefit from HCT include Hurler (MPS IH), Maroteaux–Lamy (MPS VI), and Sly (MPS VII) syndromes, as well as I-cell disease (ML II); unfortunately, either limited or lack of effectiveness of HCT in benefiting the CNS and/or the skeletal system has significantly dampened enthusiasm for transplant in patients with Hunter (MPS II), Sanfilippo (MPS III), and Morquio (MPS IV) syndromes (Table 77.1). MPS disorders for which HCT can be beneficial: MPS I, MPS VI, and MPS VII Hurler (MPS IH), Hurler/Scheie (MPS IH/S), and Scheie syndromes (MPS IS). During the last 27 years, since the first allogeneic HCT for MPS IH [12], hundreds of MPS IH patients have been transplanted. Large HCT experiences [28] with both unrelated [17] and related donors [18] have provided guidance for patient selection and timing of HCT, and a realistic assessment of the effects of HCT on various organs and tissues in MPS IH, as well as neurocognitive function (Fig. 77.2). While it is difficult to provide a precise estimate of long-term engrafted survival for Hurler HCT patients, the likelihood is as high as 80–85% using bone marrow or UCB (Fig. 77.3) [22,28]. When performed early in the disease course in conjunction with intensive speech therapy, HCT can preserve intellectual function and prevent manifestations of the classic severe Hurler phenotype [17,18,22,28]. The mechanism by which progressive mental retardation occurs remains unclear; however, increasing atrophy and ventricular size appear to be associated with decreased cognitive ability. Generally, with successful HCT, children with MPS IH can achieve normal neurodevelopment [22] and, in the experience of most HCT centers, do not require ventriculoperitoneal shunting [29,30]. While age and cardiopulmonary and neurodevelopmental status are important determinants of outcome in Hurler patients following HCT, other contributing factors include quality and quantity of developmental services and therapies (e.g. speech and language therapy) as well as the number and severity of specific post-HCT complications such as graft-versushost disease, infections, and organ toxicities [17,18,22,28–36]. On occasions when primary or secondary graft failure has occurred, successful long-term engrafted survival with a favorable quality of life has been achieved following a second HCT [37]. Considerable attention has been paid to issues related to quality of life, including musculoskeletal, motor, and adaptive function in Hurler patients after HCT [38–40], as well as to optimizing patient safety during and after HCT. ERT using laronidase prior to, during, and after HCT has been administered to reduce the total body burden of GAGs, and theoretically to reduce the likelihood of an
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Fig. 77.2 Neurocognitive function as measured by intelligence or developmental quotients (IQ or DQ) in 15 patients with Hurler syndrome before and after haematopoietic cell transplantation. BMT, bone marrow transplantation. (Reproduced from [28], with permission).
Fig. 77.3 Cumulative incidence of neutrophil (a) and platelet (b) engraftment after cord blood transplant, and Kaplan–Meier estimates of the probability of event-free survival (c). In (a), myeloid engraftment was defined as an absolute neutrophil count of at least 500/mm3 on three consecutive days. The one patient who remained aplastic after the first cord blood transplant received a second cord blood transplant on day +63. Myeloid engraftment occurred on day +26 after the second transplant. In (b), platelet engraftment was defined by a platelet count of at least 50,000/mm3 without need for transfusion for at least seven consecutive days. In (c), event-free survival was defined by survival with full (>99%) donor chimerism. Tick marks indicate the most recent follow-up visit for each patient. (Reproduced from [22], with permission.)
MPS-related peritransplant complication [24,25]. Busulfan pharmacokinetics are not altered in children with metabolic storage diseases, including MPS IH, and the rates of donor-derived engraftment are no different in these patients compared with children with other diseases [41]. Discussions continue regarding optimal hematopoietic cell source, preparative regimen, and enzyme activity level following HCT [42,43]. With successful donor-derived engraftment, Hurler patients can experience favorable long-term HCT outcomes. These outcomes include stabilization of the CNS with preservation of neurocognitive function, and motor development within the range of normal and capacity for
independence in activities of daily living. Careful neuropsychologic and neuroradiologic assessments are needed [38–40,44–46]. Hepatosplenomegaly, impaired joint mobility, and upper airway obstruction with sleep apnea [47] resolve within months of HCT; GAG disappears from hepatocytes and Kupffer cells [48]. Corneal clouding stabilizes or slowly resolves in many patients [28], and ocular pressures may normalize; however, for many patients, electroretinogram abnormalities are evident long term [49]. HCT does not reverse the progressive, profound conductive and sensorineural hearing abnormalities observed in many Hurler patients, although nearly 50% of children do
Hematopoietic Cell Transplantation for Storage Diseases
show normalization or improved auditory acuity after HCT [28]. Myocardial muscle function is stabilized or improved, and coronary artery patency has been demonstrated up to 14 years after HCT [50,51]. The long-term outcome of cardiac valvular thickening and insufficiency require continued monitoring [50]. However, some disease features show much poorer responses due to poor penetration of laronidase into the relevant tissue. Principal among these are skeletal abnormalities also known as dysostosis multiplex. Successfully transplanted children often require major orthopedic surgical procedures for genu valgum [52], acetabular hip dysplasia [53], kyphoscoliosis, carpal tunnel syndrome, and trigger digits [54,55] by 6 years of age. Orthopedic aspects of MPS disorders were the subject of a consensus
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conference held in Manchester, United Kingdom, in 2003 (Table 77.6). Interestingly, upper cervical spine stabilization has occurred long term in many donor-engrafted Hurler patients [56]. Nevertheless, severe spinal cord injury has been observed 6 years from successful HCT [57]. The decision of whether to proceed with HCT for patients with Hurler syndrome is a complex one. Factors include the child’s neurocognitive and developmental status with emphasis on the potential for growth, the presence or absence of hydrocephalus with or without a ventriculoperitoneal shunt, and a history of pulmonary and airway complications such as frequent pneumonias, chronic hypoxia, sleep apnea, and cardiac failure. It is imperative that a comprehensive pre-HCT evaluation be performed and that detailed information be provided to families with
Table 77.6 Consensus statement prepared by facilitator Ms Jean Mossman on the orthopaedic management of mucopolysaccharidosis (MPS) patients who have had a hematopoietic cell transplant (HCT). Consensus position statements arising from conference held October 3–4, 2003 in Manchester, UK A. General session Speakers and contributors to this session and their affiliations: Peters, C. currently: The Children’s Mercy Hospital, Kansas City, USA, previously: University of Minnesota, Minneapolis, USA; Wynn, R. Royal Manchester Children’s Hospital, Manchester, UK; Steward, CG. Bone Marrow Transplant Unit, Royal Hospital for Children, Bristol, UK; Wraith, E. Willink Biochemical Genetics Unity, Royal Manchester Children’s Hospital, Manchester, UK; Guffon, N. Centre de Référence des Maladies Héréditaires du Métabolisme, Hôpital Edouard Herriot, Lyon, France. • As a result of HCT and other emerging treatments for MPS diseases, the management of skeletal abnormalities has become increasingly important • HCT has favorably altered the natural history of some MPS diseases and can partially correct the skeletal abnormalities • Multiple lines of accumulating evidence in patients and animals provide compelling evidence that early HCT has a positive impact on skeletal disease, in particular carpal tunnel syndrome and trigger finger • HCT improves the soft tissue abnormalities associated with MPS diseases • HCT is expected to be more effective as prophylaxis than treatment • Skeletal disease varies between types of MPS disease • The etiology of skeletal disease is uncertain: it is not clear how glycosaminoglycan accumulation leads to the skeletal dysplasia • At present, it is not possible accurately to predict which children will develop skeletal abnormalities of sufficient severity to require intervention, and ongoing review of the likely orthopedic problems is essential • The outcome of orthopedic surgery for skeletal and soft tissue problems (genu valgum, hip dislocation, trigger finger, and carpal tunnel syndrome) in the group of patients for whom these symptoms are a problem is encouraging • Most parents of children who have had surgery have been pleased with the results • Assessing the long-term outcome of surgical intervention in children who have received bone marrow transplants is important and requires follow-up into adulthood • An international registry of both treated and untreated patients, including procedures and outcomes, is needed as a matter of urgency • Data fields collected by registries should have input from patients and families to ensure that the data collected are relevant to the decisions future parents will make regarding treatment for their children B. Knee surgery session Speakers and contributors to this session and their affiliations: Ogilvie, JW, currently: Shriners Hospital for Children, Salt Lake City, USA, previously: University of Minnesota, Minneapolis, USA; Meadows, T. Royal Manchester Children’s Hospital, Manchester, UK. • >50% of engrafted patients develop knock knees • Good correction can be achieved with both stapling and osteotomy • Changes with growth can result in failure of both procedures, and more than one procedure may be necessary • Stapling can fail as a result of permanent growth failure of the growth zone on the other side of the knee • Assessment for knee surgery: in children where there is clinical suspicion of genu valgum, a posteroanterior standing X-ray of the entire lower limbs is indicated Stapling • Epiphyseal stapling should be considered when the tibiofemoral angle >~15° and • Epiphysis is ossified enough to hold staple • Age >4–5 years may be optimal and • There is an expectation of a further 1–2 years of growth remaining • The staple should be removed when angle is reduced to 0–5°. Two staples, of a design that provides adequate anchorage but which can be removed, should be used Osteotomy Osteotomy should be considered when: • Degree of deformity >~20° • Stapling has failed to correct the problem • The deformity is not at the knee
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Table 77.6 Continued • The patient is too old and therefore there is not enough growing time left • Acute correction versus graduated correction: Patient preference, in the light of a clear explanation and open discussion of the risks and benefits of both approaches, should contribute to the decision about which approach to use • Patient groups have an important role to play in producing information setting out the risks and benefits Osteotomy
Stapling
More morbidity Several days in hospital Days or weeks to become ambulatory, depending on procedure
Less morbidity Outpatient or short stay if not done alongside other procedures Ambulatory within days
C. Hip surgery session Speakers and contributors to this session and their affiliations: Meadows, T. Royal Manchester Children’s Hospital, Manchester, UK; Ogilvie, JW, currently: Shriners Hospital for Children, Salt Lake City, USA, previously: University of Minnesota, Minneapolis, USA; Garin, C. Hôpital Edouard Herriot, Lyon, France. • The same basic principles for indication for surgery apply as in non-MPS hip dysplasia: • Shallow acetabulum • Shenton’s line broken • Patients for whom longevity is assumed • If the degree of dysplasia is likely to lead to • early or premature arthritis • adverse effect on quality of life is likely The aim of surgery is to: • Reduce the hip • Provide cover • Early rehabilitation Hip surgery should be undertaken before the ability to remodel the acetabulum is lost • Assessment – Anteroposterior X-ray of the pelvis – Consider magnetic resonance imaging Surgery may be to the pelvis, femur or both The operation technique will depend on: • Age • Severity of problem • Surgical preference Both hips may be operated on at the same time, depending on the technique used, the general wellbeing of the patient and the preference of the patient/ family and the surgeon One technique which has been used successfully: 1) Single stage a) bilateral femoral osteotomy b) bilateral iliac osteotomy Femoral varus with or without rotation osteotomy Degas-type iliac osteotomy 2) 6 weeks in a cast a) progressive ambulation b) gentle physical therapy D. Carpal tunnel syndrome session Speakers and contributors to this session and their affiliations: Van Heest, A and Khanna, G. University of Minnesota, Minneapolis, USA. • Screening testing for all children with MPS using nerve conduction velocity is essential. Children do not complain of pain, numbness or weakness until the carpal tunnel syndrome is severe and neurologic damage may be permanent. Nerve conduction velocity testing can diagnose carpal tunnel in its milder form, when median nerve compression is reversible • Nerve conduction velocity with decreased velocity or decreased amplitude or increased latency in median nerve function compared with ulnar nerve function over the same carpal distance is diagnostic of carpal tunnel syndrome • Untreated carpal tunnel syndrome leads to loss of thenar muscle function and loss of sensation in the thumb, index, long, and part of the ring finger • Carpal tunnel syndrome in MPS diseases is due to dysplasia of the carpal bones, leading to a narrower bony carpal tunnel, combined with glycosaminoglycan deposits on the flexor tendons within the carpal tunnel, resulting in increased pressure on the median nerve
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Table 77.6 Continued • Studies have demonstrated that carpal tunnel release surgery improves median nerve function • With improved blood and marrow transplant techniques, treating patient with MPS IH, approximately 50% of children acquire carpal tunnel syndrome. Decreased risk of developing carpal tunnel syndrome exists for patients transplanted at a younger age and in patients with normal or carrier levels of enzyme post transplant E. Trigger finger session Speakers and contributors to this session and their affiliations: Van Heest, A and Khanna, G. University of Minnesota, Minneapolis, USA. • Untreated locked trigger finger can lead to permanent hand joint contractures • Surgical release of the A1 and A3 pulleys with possible resection of half the flexor digitorum superficialis tendon may lead to prevention of contractures if locked digits exist. Stretching exercises for the fingers and night-time splinting do not resolve the underlying cause • Screening for trigger finger includes palpation of the flexor tendons for nodules as well as documentation of active and passive range of motion of each finger joint F. Spinal surgery for kyphosis session Speakers and contributors to this session and their affiliations: Garin, C. Hôpital Edouard Herriot, Lyon, France; Ogilvie, JW, currently: Shriners Hospital for Children, Salt Lake City, USA, previously: University of Minnesota, Minneapolis, USA; Williamson, B. Royal Manchester Children’s Hospital, Manchester, UK. • Purpose of spinal surgery is: • to keep the spine balanced • to protect neural elements • to maximize mobility and function by minimizing the deformity • to retain quality of life • Spinal surgery should only be undertaken after a surgeon with expertise in operating on children with MPS diseases has clinically reviewed the case • Indications for surgery: • these may be different from the indications that would apply to non-MPS children • consistent deterioration on serial X-rays • clinical need as judged by expert opinion • some surgeons operate only when the degree of curvature is in the 60–70° region • There is a role for patient organizations in educating patients and their families on the need to monitor change in the spine and to be alert to neurologic changes that might arise from spinal changes: • Weakness • Pain • Decreasing activity tolerance • Deteriorating bowel or bladder function • Assessment: standing 2 m posteroanterior and lateral X-ray • Surgical approach • Anterior column support • Anterior posterior surgery • The role of braces • There is no evidence that bracing alters the natural history of spinal changes prior to surgery • On this basis, if families can tolerate no bracing, it is an acceptable approach to take • Post-surgical management • A cast should be applied in the operating room at the end of surgery and should remain in place for between 6 weeks and 3 months • Subsequently, the patient should wear a brace until the bones have healed/united/fused • Physiotherapy should only be undertaken with a treatment plan agreed in advance with the surgeon • Normal activity can be resumed at 12 months, although this does not include contact sports and other potentially dangerous activities such as tumbling G. Spinal surgery for scoliosis session Speakers and contributors to this session and their affiliations: Garin, C. Hôpital Edouard Herriot, Lyon, France; Ogilvie, JW, currently: Shriners Hospital for Children, Salt Lake City, USA, previously: University of Minnesota, Minneapolis, USA; Williamson, B. Royal Manchester Children’s Hospital, Manchester, UK. • Purpose of spinal surgery is: • to keep the spine balanced • to protect neural elements • to maximize mobility • to retain quality of life • Indication for surgery: apply same principles as for non-MPS children • Spinal surgery for scoliosis should only be undertaken after a surgeon with expertise in operating on children with MPS diseases has clinically reviewed the case
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particular attention to disease aspects that are typically resistant to the beneficial effects of HCT, such as skeletal abnormalities, some ocular and auditory features, and cardiac aspects. Long-term, coordinated, comprehensive monitoring not only for late effects from HCT (see Chapter 105), but also for disease-specific aspects, should be performed in close collaboration with educators and therapists (e.g. physical, occupational, speech, and language) (Table 77.4). Hurler/Scheie syndrome demonstrates intermediate phenotype between MPS IH and MPS IS, with normal to mildly delayed neurocognitive development, and survival beyond the first decade and often into the third decade [26]. A 10-year-old child with MPS IH/S underwent HCT and demonstrated resolution of hepatosplenomegaly, amelioration of facial features, stabilization of cardiac function, improved joint range of motion, with persistent and progressive skeletal abnormalities, and unchanged corneal haze though visual acuity improved. Neurocognitive function was preserved at a level that was below average [58]. While HCT can successfully treat MPS IH/S or MPS IS as well, such patients are treated typically with α-L-iduronidase ERT [59]. Maroteaux–Lamy syndrome (MPS VI). Maroteaux–Lamy syndrome (MPS VI) is another MPS disorder that can be treated by HCT. The principal clinical features of children with MPS VI are dysostosis multiplex with severe short stature, corneal haze, pulmonary complications related to decreased intrathoracic volume and limited expansion due to severe hepatosplenomegaly, and cardiac valvular abnormalities [26]. HCT has been used successfully to treat MPS VI for nearly 25 years [13,60], with resolution of hepatosplenomegaly, airway obstruction, and sleep apnea, prevention of further cardiopulmonary deterioration, and improved joint mobility. Visual acuity improved in some cases [13,60], although corneal haze does not necessarily resolve. As in other MPS disorders, HCT has not treated effectively skeletal abnormalities (Table 77.6).
shares clinical features with MPS IH, including facial dysmorphia, progressive conductive and sensorineural hearing loss, upper airway obstruction with sleep apnea, cardiopulmonary dysfunction, hepatosplenomegaly, joint stiffness, and short stature [26]. Dysostosis multiplex is less severe and corneal haze is absent in Hunter syndrome. The attenuated form of MPS II is associated with less neurocognitive impairment and, in some cases, normal intelligence; survival can extend into the fifth and sixth decades, in contrast to MPS IIA (severe Hunter syndrome) and its shortened life expectancy of one to two decades [26]. While HCT seems to help patients with MPS IIA and MPS IIB (attenuated Hunter syndrome) with respect to organomegaly, airway obstruction, and cardiac function, boys with MPS IIA still have demonstrated profound neurocognitive disabilities despite early, successful HCT [44,62,64–68]. The failure of HCT to favorably alter the long-term neurocognitive outcome in MPS IIA suggests that iduronate-2-sulfatase is not being effectively delivered to essential components of the CNS. In cases of transplanted MPS IIB patients, there have been somatic benefits, and intellectual function has remained intact as expected based upon the natural history of the disorder. Ethical issues continue to arise in the face of limited somatic benefit and minimal-to-absent neurocognitive gains in patients with MPS IIB undergoing HCT [69]. Sanfilippo syndrome (MPS III). Neurobehavioral manifestations are the hallmarks of all four types of MPS III [26]. Characteristic findings include extreme hyperactivity, aggressive behaviors, attention deficits, and progressive neurocognitive delay often associated with expressive language disabilities. As in MPS II, HCT is able to effectively treat the various somatic aspects of MPS III; however, the uniformly poor neurocognitive and developmental outcomes despite fully donor-engrafted marrow transplants performed early in the disease course (Fig. 77.4)
Sly syndrome (MPS VII). The use of HCT for MPS VII is limited by the rarity of the disorder and its predilection toward hydrops fetalis, although attenuated forms do exist [26]. The neonatal form is one of the few lysosomal storage diseases with clinical manifestations in utero or at birth [26]. MPS VII, in certain circumstances, can be ameliorated by HCT provided the neurodevelopmental and clinical status of the patient is satisfactory at the time of HCT [61]. In this case report, the 12-yearold MPS VII patient experienced major improvements in motor and pulmonary function with diminished upper respiratory tract and middle ear infections, leading to an improved quality of life.
Historical experience with HCT for ML-II (I-cell disease) existed initially only in patients with endstage cardiopulmonary disease [62]. Over the past decade, transplant experience with a small cohort of patients ranging in age from 0.3 to 1.7 years has demonstrated that long-term engrafted survival can be achieved in selected patients who continue to make developmental progress, albeit at slower than normal rates [63]. We have observed that obstructive sleep apnea may require tracheostomy prior to HCT as well as continuous positive airway pressure or bilevel positive airway pressure support. Further, careful long-term monitoring is required as airway complications and pulmonary hypertension can occur. MPS disorders for which HCT is not beneficial: MPS II, MPS III, MPS IV Hunter syndrome (MPS II). There are two distinct clinical phenotypes of MPS II. The more common severe form presents before the age of 3 years with profound neurocognitive and developmental delay, and
Bone marrow transplant
90
80
70
60 DQ or IQ
ML types II and III (I-cell disease and pseudo-Hurler polydystrophy)
100
8
50 7
40
2
4
6
30
20
10 5
1
3
0 0
24
48
72
96
120
144
Age in months
Fig. 77.4 Decline in intellectual function (developmental quotient or intelligence quotient) in eight children with mucopolysaccharidosis III (Sanfilippo syndrome). (Reproduced from [70], with permission.)
168
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The de novo mutation rate in X-ALD is 7.8% of cases, and in 42% of index cases the first recognized mutation occurs in the mother [75]. The ALD gene (located at Xq28) encodes the ALD protein (ALDP, ABCD1), which is a member of the ATP-binding cassette transport protein family (ABC transporters) and is likely involved in transport of fatty acyl coenzyme A substrates or their cofactors into peroxisomes [76]. Impaired ALDP function leads to the accumulation of VLCFAs in body fluids and tissues. However, the contribution to cerebral and adrenal dysfunction is poorly understood, although recent reports reflect significant efforts directed at achieving a better understanding of the underlying pathophysiology [77,78].
resonance imaging [MRI] positive and gadolinium-positive) and the spectrum of clinical manifestations are [80–82]: 1 posterior in approximately 80–85% of cases: graphomotor, spatial perception and visual memory difficulties, and visual agnosia ending in cortical blindness; 2 frontal in 10–15%: “acquired” attention deficit hyperactivity disorderlike presentation, behavioral disinhibition, verbal fluency problems, and memory and new learning difficulties; 3 pyramidal tracts in approximately 5%: corticospinal signs in the extremities. Neurologic deterioration occurs in all boys, with deficits that can include vision/visual processing, hearing/auditory processing, speech, gait, fine motor skills, and activities of daily living. Disability, dementia, and death can occur within months to several years after clinical onset. Adult-onset AMN, typically first manifesting itself in the second or third decades, is characterized by an axonopathy with spinal cord atrophy, initially involving the lower extremities, with progressive long tract signs when progressive to the upper extremities. There is stiffness and clumsiness of the legs which is slowly progressive; mild-to-moderate spastic paraparesis, impaired vibratory sense and graphesthesia of the distal legs; peripheral neuropathy (mild); bowel and bladder control problems; impotence; sparse hair; adrenal insufficiency; and hypogonadism manifested by low testosterone and high luteinizing hormone levels. The MRI of the brain shows characteristic ascending involvement of the pyramidal tracts from the pons to the internal capsules. Of note, men can develop cerebral demyelination that is similar to that seen in childhood cases. A complete evaluation of a boy with cerebral X-ALD includes a thorough neurologic examination; comprehensive neuropsychologic assessment with particular attention to the performance IQ (PIQ), which is a sensitive indicator of deficits in visual perceptual and spatial processing, speed and efficiency of task completion and novel problem solving; and an MRI of the brain. Boys diagnosed with X-ALD due to a family history merit careful, complete serial monitoring beginning at age 3 years and continuing into adolescence, with the objective being to detect cerebral demyelination at a very early stage and to intervene with HCT in a timely and presumably effective manner. To date, MRI scanning of the brain with gadolinium is the most effective clinical tool to perform serial monitoring. It is strongly recommended that scans be performed at least every 6 months, and more often if there is evidence of the earliest stages of dysmyelination. This is particularly relevant for boys under 10 years of age with X-ALD who have the highest risk for developing childhood-onset cerebral demyelination that is rapidly progressive. However, in no circumstances should HCT be performed in the absence of cerebral disease (see below).
Clinical description
Nontransplant approaches
There is no genotype–phenotype correlation in X-ALD; that is, six distinct clinical phenotypes exist independent of VLCFA abnormality or ALDP expression, mutation or family history. Genotypic analysis definitively identifies the 20% of female heterozygous carriers who can have normal levels of fasting plasma VLCFAs. The clinical phenotypes and their relative percentages are as follows [79]: • childhood cerebral (30–40%); • adolescent cerebral (5–10%); • adult cerebral (~5%); • adrenomyeloneuropathy (AMN; 40–50%); • AMN with cerebral involvement (25% of the total AMN cases); • Addison only (variable percentage, dependent on age). Childhood-onset cerebral disease, with its median age of onset of 7 years, encompasses adrenal insufficiency in 90% of affected boys. Locations for the inflammatory demyelinating process (i.e. magnetic
The primary nontransplantation approaches to X-ALD have been Lorenzo’s oil with a special low-fat diet, immunosuppression, and more recently antioxidant agents. There is evidence that, in patients with the biochemical diagnosis of X-ALD who are started on Lorenzo’s oil and the special diet, are compliant, and start at an early age, there is a decreased likelihood of developing childhood-onset cerebral demyelination [83]. However, not all boys are protected, and for those who do develop cerebral manifestations by MRI and/or clinical features, HCT remains the only potentially effective long-term treatment.
[70,71] have been attributed to reduced efficiency of uptake of MPS III-specific hydrolases, leading to diminished clearance of substrate. Morquio syndrome (MPS IV). The two distinct biochemical forms of MPS IV have similar clinical features, including severe dysostosis multiplex, dwarfism, atlantoaxial instability, short trunk, and hyperextensible joints with ligamentous laxity. Corneal haze is mild, hepatosplenomegaly moderate, cardiac abnormalities unusual, and intelligence preserved [26]. HCT is incapable of ameliorating the severe skeletal deformities. Therefore, this shortcoming of HCT and the MPS IV clinical features make HCT a suboptimal intervention for this disease [72].
Leukodystrophies and other white matter diseases In this section, leukodystrophies (X-ALD, GLD, and MLD) will be presented and discussed in detail (see below and Table 77.2); other white matter diseases, such as Alexander disease, Pelizaeus–Merzbacher disease (PMD), vanishing white matter disease (VWMD), and Zellweger syndrome will be presented and discussed in a limited manner (Table 77.2). X-linked adrenoleukodystrophy Epidemiology and etiology Adrenoleukodystrophy is an X-linked peroxisomal disorder of VLCFA metabolism which has a minimum frequency of 1 : 16,800 in the total population and approximately 1 : 20,000 males (Table 77.2) [73,74]. It predominantly affects males, although 40% or more of female heterozygous carriers exhibit mild-to-moderate noncerebral signs of the disease. Molecular and clinical biology
Hematopoietic cell transplantation The first transplant for childhood cerebral X-ALD was performed in 1982 in a boy with advanced-stage disease; despite a successful graft, he died of progressive X-ALD [84]. However, Aubourg and colleagues in Paris successfully transplanted a boy with early stage cerebral X-ALD
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and published their findings in 1990 [14], thus beginning the modern era of successful transplantation for this disorder. Lessons learned and considerations to be given in each case of cerebral X-ALD include: (1) the timing of HCT and disease status; (2) the optimal hematopoietic cell source (marrow, peripheral or UCB, HLA match, cell dose, and carrier status of the donor, if applicable); (3) the conditioning regimen: high dose versus reduced intensity; (4) multispecialty, multidisciplinary evaluation and treatment; and (5) quality of life for the patient and his family. The long-term results (5–10 years) of transplant in 12 boys were reported in 2000 by Shapiro and colleagues [85]. They described the prospects for achieving stability in the extent of MRI demyelination and the changes in neuropsychologic function, with a propensity for deterioration before ultimate stabilization in visual processing as evidenced by PIQ score in boys with a posterior pattern. Interestingly, VLCFA concentrations decreased by 55% but remained elevated in the long term, suggesting that VLCFA levels are not an optimal surrogate marker for this disease. The first extensive report of the international HCT experience for childhood and adolescent cerebral X-ALD was published in 2004 by Peters and colleagues from 43 HCT centers and described 126 subjects, of whom 94 were deemed to have complete data [20]. In addition to providing overall, related, and unrelated donor transplant Kaplan–Meier probabilities of survival, this report described for the first time the extraordinary successes possible in boys transplanted for earlystage cerebral disease (zero or one neurologic deficit and MRI severity score 100 μg/L).
between the degree of crosslinker hypersensitivity and the severity of the phenotype in FA patients or individual patient sensitivity to chemoradiotherapy [22] (Wagner and Auerbach, unpublished data). However, data exist suggesting that genes not in the FA pathway can modify the crosslinker hypersensitivity [23]. Importantly, the clinician should know that the chromosomal breakage test could also be applied to the study of fetal cells obtained by chorionic villous sampling (CVS), amniocentesis, or percutaneous umbilical blood sampling. If the specific mutation is known within the family, testing for the presence of the mutation on limited quantities of uncultured cells is possible and rapid. Alternative diagnostic methods for FA, such as by cell-cycle analysis using flow cytometric methods on
11 breaks
Complementation groups and gene cloning
2 radial figures
Thirteen complementation groups representing distinct genes (FA-A through FA-G, including FA-D1 and FA-D2, FA-I, FA-J, FA-L, FA-M, and FA-N) have been described [27–39]. Complementation groups FAA (~62%), FA-C (~15%), and FA-G (~9%) account for the majority of complement group assignments in FA patients. While the genes responsible for the defect have been identified for each of the 13 FA complementation groups (Table 79.2) [30–47], there are patients for whom all known genes have been excluded, suggesting that additional FA genes exist. Inheritance is autosomal recessive except for FA-B which is Xlinked recessive. The FA genes encode unique proteins, many of which do not exhibit any known functional domains. At least eight FA proteins, FANCA/B/ C/E/F/G/L, and M, form a nuclear multiprotein complex termed the FA core complex, which is required for the monoubiquitination of both FANCD2 and FANCI [39]. Additional FA-associated proteins, including FAAP100 and FAAP24, have also been identified as part of the complex, but mutations in these associated proteins have not yet been detected in FA patients. The genes for FANCD2 and FANCI appear to be ancestrally related, both require ataxia telangiectasia mutated (ATM)/ ATM and Rad3-related (ATR) phosphorylation to be activated, and both require an intact FA core complex for monoubiquitination at specific lysine residues. FANCD2 and FANCI together form a complex, and
1 tri-radial figure
Average number of aberrations/cell (DEB 0.1 mg/mL) Patient
10.78
Control
0.08
Normal range
1181
0.00-0.12
Fig. 79.3 Diepoxybutane (DEB) test. Metaphase cell from a patient with Fanconi’s anemia after treatment with DEB in vitro. Arrows designate chromatid gaps and/or breaks; asterisks designate radial figures. This cell has nine gap/breaks and two multiradial figures. (Reproduced courtesy of Betsy Hirsch, PhD, Director of the Cytogenetics Laboratory of the University of Minnesota Medical Center, Fairview, Minneapolis, MN.) Table 79.2 Fanconi’s anemia (FA) genes: description and frequency
FA group
FA gene
MIM #
Location
Exons
Amino acids
Frequency in the International Fanconi Anemia Registry
A
FANCA
607139
16q24.3
43
1455
62
B C D1 D2 E F G I
FANCB (FAAP95) FANCC FANCD1/BRCA2 FANCD2 FANCE FANCF FANCG/XRCC9 FANCI/KIAA1794
300515 227645 605724 227645 600901 603467 602956 609053
Xp22.31 9q22.3 13q12.3 3p25.3 6p21.3 11p15 9p13 15q26.1
10 14 27 44 10 1 14 38
859 558 3481 1451 536 374 622 1268
2 15 3 3 1 2 9 1
J
FANCJBRIP1/BACH1/
605882
17q22
20
1249
2
L M N
FANCL/PHF9/(FAAP43) FANCM/Hef (FAAP250) FANCN/PALB2
608111 609644 610832
2p16.1 14q21.3 16p12
14 22 13
375 2048 1186
T (p.R185X), c.1916C > T (L554P), and c.1897C > T (p.R548X) [22,50,51]. Like FANCC, FANCA also expresses multiple transcripts, with a major species of 5.5 kilobases (kb), encoding a 4368 nucleotide coding sequence. The predicted FANCA protein is a 1455 amino acid peptide (~163 kDa) that, like FANCC, shares no significant homology with any known protein. Sequence analysis has identified two overlapping nuclear localization signals, as well as a partial leucine zipper consensus domain, but neither of these domains has yet been demonstrated to be functional [41,42]. The FANCA gene contains 43 exons spanning approximately 80 kb, ranging from 34 to 188 base-pairs [52]. FANCA mutation analysis reveals a large number of different mutations, indicating that FANCA is highly polymorphic and may be hypermutable [53]. There are many private or semiprivate mutations, as well as ethnic-specific mutations, making large-scale screening for FANCA mutations difficult [53–59]. The gene for the FA-G complementation group (FANCG) was the third FA gene to be cloned, and was found to be identical with human XRCC9, which maps to 9p13 [43]. The complementary DNA (cDNA) is predicted to encode a polypeptide of 622 amino acids, with no sequence similarities to any other known protein or motifs that could point to a molecular function for FANCG/XRCC9. Four pathogenic mutations in FANCG were originally described; three were in patients of German ancestry, and one was in a consanguineous Lebanese family. FANCG has subsequently been shown to be highly polymorphic, and to exhibit ethnic-specific mutations in Korean/Japanese, Portuguese– Brazilian, French–Acadian, and northern European FA families, as well as many private mutations [60–62]. Notably, only a few FA patients have been described in the literature belonging to groups FA-B, -D1, -D2, -E, -F, -I, -J, -L, -M, and -N. Information regarding FA mutations can be obtained from the Fanconi Anemia Mutation Database at http://www.rockefeller.edu/fanconi/mutate. Proposed functions of FA genes Pathophysiology of FA at a cellular level has yet to be completely defined. Defects in DNA repair, cell-cycle checkpoints, oxygen metabolism, and induction of apoptosis have all been described in FA cells. The published data have been marked by contradictory results, which can be attributed partially to inconsistencies in experimental conditions, including variations in administration and dosage of DNA crosslinking agents administered, the complementation group of the FA cells used, and the use of primary cells versus transformed cell lines. Although the first FA gene was cloned more than 15 years ago, the biochemical function of some FA proteins has only recently being elucidated. The FA genes all encode unique proteins, which typically lack recognizable functional domains. Exceptions are FANCJ/BRIP1, which has a DEAH-box helicase domain, FANCM, which contains a DNA helicase
and endonuclease domain, FANCL, which contains a WD40 repeat, a plant homeo domain finger, and a E3 ubiquitin ligase domain, FANCN, which contains an amino-terminal prefoldin-like domain and two Cterminal WD40-like repeats, and FANCG, which has at least seven tetratricopeptide repeat motifs [33–38]. Recent studies of post-translational modifications of a complex containing FANCD2 and FANCI suggest that FA proteins function in regulating discrete cellular signalling pathways. Formation of the FA nuclear core complex by various FA proteins, and FA-associated proteins, including FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL FANCM, FAAP24, and FAAP100 [39], has been shown to be essential for the monoubiquitination of the FANCD2/FANCI complex in response to DNA damage by crosslinking agents. Activation of the FANCD2/FANCI complex causes it to translocate to nuclear foci and associate with chromatin, suggesting a role for the FANCD2/FANCI complex in DNA repair functions. Recent data suggest that the FANCD1 (BRCA2)/FANCN (PALB2) complex is recruited to these foci, to initiate DNA repair. Phosphorylation of FANCD2 and FANCI by ATM/ATR, is an independent posttranslational modification, which is required for activation of FANCD2 [63,64] and FANCI [31]. The FA core complex possesses a putative helicase and an E3 ubiquitin ligase subunit, as well as subunits with molecular motifs and domains which suggest a general role for the complex as molecular scaffolding during DNA repair, in addition to any other functions for individual subunits [39]. The nature of the interaction of the BRCA1-binding protein FANCJ (BRIP1) in the FA pathway has still not been elucidated. FANCJ exhibits a helicase domain, and a domain required for BRCA1 binding. Mutations in either of these domains results in an FA-like phenotype. Grompe and van de Vrugt [65] have proposed a schematic representation of the FA pathway (Plate 79.3). Monoubiquitination of FANCD2 and FANCI can be detected by immunoblotting with FANCD2 or FANCI antibodies, respectively [32]. Protein extracts from normal cells show both unmodified (short) and the monoubiquitinated (long) forms of these proteins, while extracts from cells from FA complementation groups FA-A, -B, -C, -E, -F, -G, -L, and -M show only the unmodified short FANCD2 and FANCI forms. It is postulated that the entire FA nuclear core complex acts as a multisubunit E3 ubiquitin ligase, which binds to the E2-conjugating enzyme UBE2T, and that this entire complex participates in FANCD2/FANCI monoubiquitination [66]. This is a dynamic and reversible process, with evidence that USP1 is the enzyme that deubiquitinates FANCD2 [67]. It has been suggested that western blotting to detect the presence or absence of the FANCD2 long form be used as a diagnostic screen for FA, and as an assay to detect complementation group after transduction by cDNA-containing retroviral vectors [68]. While this may be a useful adjunct to standard tests of hypersensitivity to crosslinking agents, there are insufficient data at this time to determine its sensitivity, specificity, and reproducibility for routine diagnosis, especially for patients who cannot be complemented by any of the cloned genes and those who exhibit mosaicism. Although the FANCC protein participates in the FA nuclear complex responsible for monobiquitination of FANCD2, abundant levels of endogeneous FANCC are also found in the cytoplasm. There is strong evidence that the cytoplasmic form of FANCC interacts with other cytoplasmic proteins in control of apoptosis [69–72]. Cytoplasmic complexes are also found. There is emerging evidence that FA complexes, often acting in concert with heat shock proteins, function to suppress the activation state of intracellular effectors of apoptosis and facilitate survival signaling pathways, including the Janus kinase (JAK)-dependent signal transducers and activators of transcription (STAT) activation pathway, thus promoting cell survival. The exact composition of the cytoplasmic FA complexes has not been fully defined, but included
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among the cytosolic proteins that bind to FANCC in vitro are the mitotic cyclin-dependent kinase cdc2 [73,74], the molecular chaperone GRP94 [75], NADPH cytochrome p450 reductase [76], and STAT1 [77,78]. FANCC is also suggested to have a role as a redox regulator of glutathione S-transferase P1 (GSTP1) [77,79]. Evidence also indicates that the molecular chaperone heat shock protein 70 (Hsp70) interacts with FANCC to protect hematopoietic cells from cytotoxicity and apoptosis on exposure to interferon-gamma (IFN-γ) and tumor necrosis factor-alfa (TNF-α) [80]. Together these interactions protect cells against a variety of environmental factors, including oxidative stress and radiation, and suppress double-stranded RNA-dependent protein kinase (PKR) activity and caspase 3 activation [81,82]. Subsequent studies have shown that FANCA and FANCG also participate in the complex affecting PKR [83]. More recently, Hsp90, which regulates diverse signaling pathways, has been found to associate with FANCA [84]. In addition, other studies suggest that FANCA may have direct interactions with BRCA1 [85] and brahma-related gene-1 (BRG1), a component of the SWItch/sucrose nonfermentable (SWI/SNF) complex involved in chromatin remodeling [86]. While there has been tremendous progress, the exact role of FA proteins in DNA repair, cell-cycle checkpoints, oxygen metabolism, and induction of apoptosis still remain to be elucidated. Complementation group assignment, mutation analysis, and genotype–phenotype correlations Once a patient is diagnosed as affected with FA, identification of the complementation group assignment should be undertaken, as the risk of developing bone marrow failure or a malignancy at a very young age varies with specific complementation groups and mutations. The complementation group can be determined by correction of crosslink hypersensitivity of the patient’s cells by transduction with retroviral vectors containing the normal FA cDNAs for the various groups. The method of crosslink hypersensitivity correction is similar to that established for testing for crosslink hypersensitivity of lymphoblastoid cell lines, fibroblasts, or peripheral blood-derived patient T cells [87,88]. After the complementation group has been determined by retroviral typing, mutation detection can be targeted to a specific gene, using high-throughput sequencing. Rapid mutation screening methods such as denaturing highperformance liquid chromatography can also provide accurate mutation screening for FA [89–91]. Alternative methods such as multiplex ligation-dependent probe amplification or quantitative polymerase chain reaction are presently used to detect large genomic deletions which comprise at least 40% of mutations in FANCA, as these are not detected by routine genomic DNA sequencing. It is forseen that gene array technology will soon be applied for rapid detecton of mutations, including genomic deletions in most FA genes, with complementation group assignment made directly by finding biallelic mutations in one of the FA genes. The heterogeneous nature of FA makes an understanding of the correlation between genotype and phenotype potentially important in the clinical management of FA patients. IFAR data [10] confirm that specific complementation groups play a significant role in both the timing of bone marrow failure and survival. FA-C patients, for example, have a significantly earlier onset of bone marrow failure (Fig. 79.4) and poorer survival compared with FA-A and FA-G patients. Subdividing the FA-C group based on the region of the gene that is mutated reveals that patients with intron 4 or exon 14 mutations have an earlier onset of bone marrow failure and poorer survival compared with patients with exon 1 mutations, confirming the earlier findings of Gillio et al. and Yamashita et al. [22,92]. As reported by Gillio et al. [22], patients with FANCC genotypes can be divided into three clinical groups: (1) patients
Fig. 79.4 Survival and time to marrow failure was compared for patients with Fanconi’s anemia in complementation groups A, G, and C. Survival and incidence of marrow failure was similar groups A and G. Comparison of the combined groups A/G with group C showed that patients in group C had a significantly shorter survival (a) and higher incidence of bone marrow failure (b). (Reproduced from [10], with permission.)
with the c.711 + 4A > T (IVS4) mutation; (2) patients with at least one exon 14 mutation (p.R548X or p.L554P); and (3) patients with at least one exon 1 mutation (c.322delG or p.Q13X) and no known exon 14 mutation. IVS4 and exon 14 subgroups are associated with a severe phenotype manifested by multiple major congenital malformations, early onset of hematologic disease, and poorer survival compared with exon 1 patients and with the non-FA-C IFAR population (Fig. 79.5) [10].
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1
IVS4 exon 14
exon 1 1
p 15 mg/dL
Diarrhea 500–1000 cm3/d or biopsy-proven upper gastrointesinal involvement Diarrhea 1000–1500 cm3/d Diarrhea 1500–2000 cm3/d Diarrhea >2000 cm3/d or severe abdominal pain with or without ileus
Organ stage
Skin*
Liver
Gut
I II III IV
Stage 1–2 Stage 3 or – Stage 4 or
None Stage 1 or Stage 2–3 or Stage 4
None Stage 1 Stage 2–4 –
* Use the “rule of nines” to determine the body surface area. Adapted with permission from [203].
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Table 86.2 International Bone Marrow Transplant Registry grading of acute graft-versus-host disease Organ stage
Skin*
Liver
Gut
A B C D
Stage 1 Stage 2 Stage 3 Stage 4
None Stage 1 or 2 Stage 3 Stage 4
None Stage 1 or 2 Stage 3 Stage 4
Adapted with permission from [204].
predicted earlier survival better in a comparison of 607 patients. The IBMTR index, however, was easier to use and was associated with less physician bias or error in stage assignment [196]. Reliable incidence figures for acute GVHD are limited by interobserver and intercenter variation. This variability largely derives from uncertainty in establishing grade II GVHD [197,198]. Other factors may influence acute GVHD outcome, independent of severity scores. For example, in a logistic regression training and validation model to assess nonrelapse mortality in patients with chronic myeloid leukemia who had undergone unrelated donor BMT, mortality upon the diagnosis of acute GVHD was influenced not only by bilirubin elevation and the requirement for first- and second-line immunosuppressive therapy, but by protean factors such as caloric intake and performance status [199]. Using this scoring system, algorithms to predict mortality at 200 days from transplantation could be constructed, which can be used in real time by clinicians for prognostic purposes. Similarly, a retrospective analysis demonstrated that up to 20% of patients will develop serious acute (or chronic) GVHD by 1 year from transplantation. In this analysis, serious GVHD was defined by death or major disability related to GVHD or its treatment, three or more major infections in a single year during persistent GVHD, hospitalization for greater than 60 days in a single year because of GVHD or any hospitalization for suicidality because of GVHD or its treatment [200].
Therapy of acute GVHD Primary therapy The mainstay of therapy for acute GVHD is corticosteroids, with the route of administration determined by the severity of acute GVHD and the degree of organ involvement. Corticosteroids are lympholytic and decrease the inflammatory cytokine cascades that propagate acute GVHD. Other agents have been utilized as front-line therapy, but none is superior to corticosteroids [41,201]. There is substantial variability between investigators and centers regarding the stage of acute GVHD that should be treated, and the dose of corticosteroids to be used, particularly in early-stage GVHD. Cutaneous involvement with less than 50% body surface area involvement in the absence of hepatic or gastrointestinal involvement (stage I or II skin disease, or overall grade I acute GVHD) can be treated with the topical administration of medium- or high-potency glucocorticoids alone; however, individuals with multiorgan involvement, as well as some individuals with near-50% body surface cutaneous involvement, require the systemic administration of corticosteroids. The recommended initial dose of corticosteroids for moderate-to-severe (grade II–IV) acute GVHD is 2 mg/kg/day of methylprednisolone, or an equivalent steroid [202,203]. Doses higher than this have not been associated with improvement in response rates. In a prospective trial comparing methylprednisolone at 2 mg/kg/day with 10 mg/kg/day in 94 patients with grade II–IV acute GVHD, response rates, progression to grade III–IV disease,
Fig. 86.1 Nonrelapse mortality following primary therapy for acute graftversus-host disease (GVHD). CR, complete response; MR, mixed response; NC, no change; P, progression; PR, partial response. (Reproduced from [41], with permission.)
nonrelapse mortality, and overall survival were similar in both treatment groups [204]. For steroid-responsive acute GVHD, treatment is generally continued until the complete resolution of the signs and symptoms of acute GVHD and for at least 1–2 weeks thereafter. At this point, a gradual tapering of the corticosteroid dose is initiated, generally at no greater than a 10% reduction per week, but modified by factors such as the durability of the response to steroids and the toxicity of high-dose steroid administration. In a prospective trial, patients with acute GVHD who were responding by day 14 of corticosteroid treatment were randomized to a fast taper of corticosteroids over 86 days (n = 14) or a slow taper over 147 days (n = 16). The median time to resolution of acute GVHD was 42 (range 12–74) days in the short-treatment group, compared with 30 (range 6– 30) days for the long taper schedule (p = 0.01), without significant differences in the number of patients with acute GVHD flares, chronic GVHD, median dose of corticosteroid administered, infection or death, suggesting that lower total cumulative doses of steroids appear equally effective and might minimize steroid-related complications [205]. The response rate to corticosteroid therapy, when analyzed in large retrospective reviews, is approximately 50% [41,206], although, in smaller clinical trials, the response rate has been reported to be higher [207]. It is known that the response to steroids and long-term outcome of acute GVHD is correlated with the initial stage at presentation [193]. Long-term survival varies, and for patients with grade 0–I disease approaches 50%, while for those with grade IV disease, the long-term survival rate has been reported to be as low as 11% [208]. Response to treatment is a key predictor of outcome, as mortality in patients with grade II–IV acute GVHD is lowest in those who achieve a complete response to initial treatment (Fig. 86.1) [41,206,208]. A very early response, as evidenced by the ability to begin a steroid taper on day 5 of therapy, has also been shown to be associated with a favorable prognosis, but this study included patients with grade I disease [204]. It is likely that early responders do better at least in part because the steroid taper begins sooner and reduces the risk of complicating opportunistic infections, which are commonly a cause of death, even among responders. In general, dermal disease responds promptly, but lower gastrointestinal involvement responds poorly to therapy [206]. This may be a
Manifestations and Treatment of Acute Graft-Versus-Host Disease
reflection of the fact that the regeneration of a denuded intestinal mucosa often requires days to weeks to occur, and thus caution must be utilized in establishing failure of therapy in lower gastrointestinal GVHD, since the escalation of immunosuppression to treat refractory or incompletely responding GVHD often can result in fatal infections. Other risks for failure of initial therapy include early onset of GVHD, HLA disparity, and age [41,206]. The complete response rate to corticosteroids is low enough to have prompted numerous investigators to examine the role of additional immunosuppressive agents in the initial therapy of acute GVHD. The most widely studied agent in this setting is antithymocyte globulin (ATG). Dugan et al. reported a response rate of 67% in patients treated with ATG and prednisone as initial therapy of acute GVHD [209], and Graziani et al. reported a response rate of 80% in patients given ATG early in the course of acute GVHD, in conjunction with steroids [210]. Cragg et al. randomly assigned 100 patients to receive prednisolone or prednisolone with equine ATG as initial therapy of grade II–IV acute GVHD, and failed to demonstrate a difference in response rates (76% in both arms) or survival at 1 and 2 years between groups. However, infectious morbidity was higher in the combined immunosuppression group [207]. In order to minimize the systemic effects of long-term high-dose steroids, Hockenbery et al. administered oral beclomethasone and systemic steroids to patients with upper gastrointestinal GVHD, and compared outcomes with patients treated with systemic steroids alone. In patients receiving oral beclomethasone, the relapse rate was significantly lower and mortality was significantly reduced at day 200 when compared with patients receiving steroids alone [211]. A number of other biologic agents have been attempted as part of initial therapy for acute GVHD. Lee et al. randomized 102 patients to receive the monoclonal anti-CD25 (the IL-2 receptor α chain) antibody daclizumab (1 mg/kg) on days 1 and 4 and weekly thereafter or placebo in conjunction with corticosteroids. While response rates were similar in both groups (53% versus 51%), survival at 100 days and 1 year was inferior in the daclizumab group [212]. A previous randomized study evaluating another antibody against the IL-2 receptor (BT563, inolimomab) also did not demonstrate an advantage over placebo when combined with prednisone and cyclosporine [213]. In another randomized trial of 143 patients, methylprednisolone and placebo were found to yield similar clinical results to methylprednisolone when combined with a pan-T-cell ricin-A-chain immunotoxin [214]. Manifestations of acute GVHD responded more quickly to the immunoconjugate, but benefits were not durable. Finally, Uberti et al. reported a 75% response rate to the TNF-α inhibitor etanercept [215]. While none of these experiences has changed the standard of care for the initial therapy of acute
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GVHD, the Bone Marrow Transplant Clinical Trials Network is now evaluating four promising newer agents (denileukin diftitox, etanercept, mycophenolate mofetil, and pentostatin), each in combination with corticosteroids, for the initial therapy of acute GVHD. Secondary therapy Patients in whom initial therapy fails (commonly defined as progression of acute GVHD after 3 days, no change after 7 days or incomplete response after 14 days of methylprednisolone treatment) have been given a variety of salvage regimens. To date, there is no clear standard for salvage treatment, and therapy of steroid-resistant acute GVHD remains unrewarding despite seemingly high response rates to certain second-line therapies, due to the risks of infection during intense immunosuppression. Numerous agents have been tested in this setting. (Table 86.3). The agent used most commonly in the setting of failure of response to corticosteroid therapy is ATG [216,217]. ATG use has been associated with response rates between 30% and 54%, but this translates to long-term survivorship in only 5–20% of treated patients [218–221]. Earlier administration of ATG has been associated with improved survival [221]. The monoclonal murine anti-CD3 antibody OKT3 may be effective as second-line therapy for GVHD [222], but its usefulness may be offset by toxicity and a cytokine release syndrome [223], as well as a higher than anticipated risk of Epstein–Barr virus-associated lymphoproliferative disease [224]. In a trial of OKT3 with high-dose methylprednisolone (10 mg/kg) in comparison with high-dose methylprednisolone alone, the response rate to the combination was higher (53% versus 33%), but this improvement in initial response did not result in superior overall survival at 1 year (45% versus 36%; p = 0.41) [225]. A nonmitogenic anti-CD3 antibody that does not bind the human Fc receptor, visilizumab, has also been tested. Clinical results are similar to OKT3, with a response rate of 32%, and an Epstein–Barr virus reactivation rate of 43% [226]. A pilot study of anti-CD3 and anti-CD7 murine monoclonal antibodies conjugated with the ricin A immunotoxin has also been performed [227]. In an attempt to target activated T cells, antibody therapy directed against the α- subunit of the IL-2 receptor (CD25) has been analyzed. The first antibody (the anti-Tac antibody) was reported in 1981 [228,229]; however, the humanized form (daclizumab), first reported upon in 1989 [230], is used more commonly today. The original trials of anti-Tac therapy as well as therapy with similar anti-IL-2 monoclonal antibodies yielded similar results, with complete responses noted in 10–65% of treated patients [231–234]. The largest of these trials, which evaluated
Table 86.3 Agents used as therapy for steroid-refractory acute graft-versus-host disease (GVHD) Biologic therapy Polycolonal antibodies Monoclonal antibodies Immunotoxin/conjugate Tumor necrosis factor-alpha blockade Chemotherapy Phototherapy Cellular therapy Topical/directed therapy
Antithymocyte globulin (ATGAM, Thymoglobulin) OKT3, visilizumab, ABX-CBL, daclizumab, inolimomab, basiliximab, alemtuzumab, alefacept Denileukin diftitox Infliximab, etanercept Mycophenolate mofetil, pentostatin, calcineurin inhibitors, sirolimus Psoralens + ultraviolet A light, extracorporeal photopheresis Mesenchymal stem cells Oral beclomethasone (as dipropionate) or budesonide for intestinal GVHD, intra-arterial steroid or methotrexate infusion
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85 patients treated with the murine antibody BT563 (inolimomab), demonstrated a complete response in 29% and a partial response in an additional 34%, for an overall response rate of 63% [235]. Three phase II trials of the humanized IL-2 receptor antibody have been published. In the original dose-escalation trial, there was a response rate of 40%, with a predilection for improvement in cutaneous and gastrointestinal disease [236]. Subsequently, a response rate of 50% was shown in a small cohort of patients with grade III–IV acute GVHD [237], and the largest series of patients reported demonstrated a complete response rate of 29% for patients treated with daclizumab weekly for five consecutive weeks, and a complete response rate of 47% for patients treated on a more accelerated schedule (1 mg/kg on days 1, 4, 8, 15, and 22) [238]. A chimeric murine–human monoclonal IL-2 receptor antibody, basiliximab, has also been used in steroid-refractory acute GVHD. In phase II studies using different doses and schedules of basiliximab, the overall response rate was uniformly high (71–82.5%), with the complete response rate varying between 17.5% and 53% [239–241]. Similarly, targeting activated T cells through the IL-2 receptor denileukin diftitox (Ontak) is recombinant fusion protein comprised of amino acids 1 through 133 of IL-2 fused to the membrane translocation and catalytic domains of diphtheria toxin (amino acids 1 through 387). In a phase I study of Ontak in steroid-refractory acute GVHD, 71% of patients had a clinical response to therapy, with 46% of patients achieving a complete response at the maximum tolerated dose level of 9 μg/kg administered on days 1, 3, 5, 15, 17, and 19 [242]. A similar dose-escalation trial that administered 4.5 μg/kg daily for 5 days followed by weekly therapy for 1 month showed a complete response rate of 41% at 36 days [243]. Another approach at targeting activated T cells is with the murine monoclonal antibody ABX-CBL, which targets CD147. CD147, a member of the immunoglobulin superfamily, is found on activated T cells, as well as B cells, monocytes, and dendritic cells [244]. While active, this agent demonstrated inferior survival when compared with ATG in a multicenter, randomized phase III study [245]. In an effort to target T cells as well as antigen-presenting cells, the monoclonal antibody alemtuzumab (Campath) has also been reported to be effective as therapy of steroid-refractory acute GVHD [246–249]. A different approach to therapy of steroid-refractory acute GVHD is to target the inflammatory cytokines that lead to T-cell activation and proliferation, with the soluble factor TNF-α having been targeted the most. Etanercept is a genetically engineered molecule comprising two p75 extracellular domains of the human TNF-α receptor fused with human immunoglobulin G1, which has been used to treat steroidrefractory GVHD. Busca et al. treated 13 patients with steroid-refractory acute GVHD with 25 mg of etanercept administered subcutaneously twice weekly for 4 weeks. Six of the 13 patients responded (46%), with five of six responses being complete [250]. Combination therapy with daclizumab and etanercept has been reported, with a response rate of 66% [251], and there is a report of ATG therapy in combination with etanercept [258,259]. The chimeric antibody infliximab has been used more extensively than etanercept. The antibody both neutralizes TNF-α and lyses cells that produce it. Initial case series and dose-escalation trials documented the potential effectiveness of this agent [253–256], and larger series confirmed that this drug may be effective, with response rates of 59–67% noted, with a predilection for improvement in gastrointestinal GVHD [257,258]. Also noted was a high incidence of fungal infection following therapy [258,259]. The T-cell cytotoxic agent pentostatin has been attempted as therapy in steroid-refractory acute GVHD. The accumulation of 2′deoxyadenosine 5′-triphosphate as a result of pentostatin administration leads to impaired lymphocyte growth and apoptosis of dividing lymphocytes. In a phase I dose-escalation study, the maximum tolerated dose
was determined to be 1.5 mg/m2, and the overall response rate was 77% [269]. Mycophenolate mofetil similarly suppresses T-cell proliferation by reversibly inhibiting purine synthesis. Basara et al. initially reported a 65% response rate in 17 patients with steroid-refractory acute GVHD and updated the series to include 36 patients [261,262]. Similar response rates were noted in other small studies [263–265]. Sirolimus, an inhibitor of the mammalian target of rapamycin, is a T-cell immunosuppressant that acts via a reduction in DNA transcription, DNA translation, protein synthesis, and cell cycling [266]. Sirolimus may also be immunosuppressive through the maintenance of regulatory T-cell populations [267–269] and via inhibition of dendritic cell activity through a reduction in antigen uptake [270,271], cellular maturation [272], intracellular signaling [273], and apoptosis induction [274,275]. Benito et al. reported that 12 of 21 steroid-refractory patients (57%) had an objective response to sirolimus (five complete response and seven partial response), but toxicity was limiting in this series [276]. An alternative approach employs use of ultraviolet A with a psoralen sensitizer to control refractory cutaneous GVHD, where high response rates have been noted [277–279]. More interest has focused on extracorporeal photopheresis (ECP) in management of refractory acute GVHD. Postulated mechanisms of action of ECP, in addition to apoptosis of treated lymphocytes, include a decrease in the production of proinflammatory cytokines and an increase in production of antiinflammatory cytokines, both of which lead to a lower ability to stimulate T-cell responses [280]. In addition, ECP may help eliminate CD8+ T-effector cells while inducing regulatory T cells [280,281]. In the largest reported series of ECP as therapy of acute GVHD, Greinix et al. reported a 60% complete response rate, although there were no responders with gastrointestinal GVHD [282]. Other series have reported similar results [283,284]. The use of cellular therapy to treat resistant acute GVHD has now been explored. Mesenchymal stem cells (MSCs) are immunosuppressive, and transplantation of these cells from haploidentical or third-party donors has been shown to improve GVHD in experimental models [285]. The first report of haploidentical MSC infusion for therapy of resistant acute GVHD was by Le Blanc et al., who reported a single successful outcome [287]. Since then, other case series reporting the successful use of MSCs in resistant visceral acute GVHD have been reported, the largest being a report where six of eight treated patients responded to a single infusion of MSCs [287]. There is even a notion that these cells may help repair the damaged organ, thus speeding up recovery from GVHD even further. There are other experimental approaches to the management of refractory acute GVHD, including intra-arterial steroid [288–290] or methotrexate [291] injection for gastrointestinal and hepatic GVHD, and modulation of T-cell responses using alefacept [292] and IL-1 receptor blocking antibodies [293], but all of these approaches remain experimental. Supportive care In addition to immunosuppressive therapy, supportive care is critical for the favorable outcome of acute GVHD. Organ-specific supportive measures for the skin include the use of topical emollient therapy and meticulous wound care, up to and including care in a burn unit. For the gastrointestinal tract, gut rest and hyperalimentation are required. Antimotility agents such as loperamide are often employed, and there is evidence that topical therapy with nonabsorbable corticosteroids may be of value [211]. Octreotide or other somatostatin analogs may be of benefit in controlling the secretory component of gastrointestinal GVHD [294,295]. Bloody diarrhea often requires frequent transfusion support.
Manifestations and Treatment of Acute Graft-Versus-Host Disease
The use of ursodeoxycholic acid has been advocated as a supportive measure in hepatic acute GVHD, and hepatotoxic agents should be avoided. Systemic supportive measures include the judicious use of antibiotics for proven or suspected infections, the routine use of antiviral prophylaxis against herpesviruses, and routine monitoring for CMV reactivation. Finally, routine antifungal prophylaxis with extended spectrum azoles or echinocandins with activity against Aspergillus species and other molds is recommended.
Special scenarios Hyperacute GVHD Hyperacute GVHD is a very rare complication of allogeneic HCT. Although clinically similar to traditional acute GVHD, hyperacute GVHD occurs earlier, often several days prior to engraftment, as early as 7 days from transplantation, although no standard definition exists. The pathophysiology of hyperacute GVHD remains unclear. It has been postulated to be caused by mature, immunocompetent cells transferred with the stem cell product, including NK-T cells [296]. In one analysis, the incidence of hyperacute GVHD was lower in patients receiving the lowest number of T cells transferred with the hematopoietic cell graft, but this has not been confirmed [297]. Risk factors for hyperacute GVHD include mismatched or unrelated donor transplantation, highdose conditioning, donor–recipient sex mismatching, second transplantation, and the lack of post-transplant immunosuppression [297–300]. Therapy for hyperacute GVHD is similar to that for acute GVHD, although therapy is often less successful when compared with traditional acute GVHD [297], and the long-term outlook for hyperacute GVHD appears no different than for patients with traditional acute GVHD [297,298]. Autologous GVHD Histocompatibility differences between donor and recipient may not always be needed to produce GVHD; instead, inappropriate recognition of self-antigens may produce a GVHD-like syndrome after autologous HCT [301,302]. In a thymus damaged by conditioning therapy after autologous HCT, HLA class II-bearing cells may be absent, and clonal deletion of reactive antiself cells may not occur. Administration of cyclosporine in the context of thymic damage may further prevent the development of immune regulatory cells and permit the development of autoreactive CD8+ and CD4+ T cells that recognize class II determinants [303]. This syndrome has even been purposefully induced with the administration of cyclosporine after autologous transplantation to invoke graft-versus-malignancy effects [304–306]. Clinical involvement of the skin, liver, and gastrointestinal tract have all been reported in autologous GVHD [307–309], although the incidence of each of these complications is low. A recent series of patients conditioned with alemtuzumab prior to transplantation, however, reported a high incidence of autologous GVHD, pointing to the dysregulation of the immune system as the pathophysiology of autologous GVHD [310]. Therapy for autologous GVHD is similar to that for classic acute GVHD, with very favorable results with corticosteroids alone. Transfusion-associated GVHD Risk factors, incidence and etiology Transfusion-associated GVHD (TA-GVHD) is a rare complication of blood component transfusion therapy. Since the original description of
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TA-GVHD over 40 years ago, over 200 patients have been reported worldwide [311]. In most reported cases, patients had received whole blood, packed red cells, platelet or granulocyte transfusions, or fresh plasma [312,313]. No cases have been observed in patients receiving transfusion of cryoprecipitate or fresh frozen plasma [314,315]. The pathobiology of TA-GVHD appears similar to the sequence of GVHD events following allogeneic HCT [316,317]. While normal hosts may be affected by TA-GVHD, several groups are at a distinctly higher risk. Individuals with congenital immune deficiency disorders, cancer patients receiving immunosuppressive chemotherapy, and surgical patients undergoing major surgical procedures or suffering from severe trauma represent the most commonly affected [318]. Among normal individuals, genetic factors appear to be the most important risk factors [319,320]. Particularly in populations with common HLA haplotypes, blood from a donor homozygous for an extended HLA haplotype could be transfused into a recipient who is heterozygous for the same haplotype. In this setting, donor cells would not be rejected as foreign by the recipient and could precipitate TAGVHD [321,322]. As such, in Japan, the incidence is estimated to be approximately 1 in 500 after major cardiothoracic procedures [323]. The incidence of TA-GVHD overall remains elusive, as the disorder is often not recognized and is underreported. Diagnosis and clinical features The clinical features of TA-GVHD are very similar to those of posttransplant GVHD, with an erythematous rash, elevation of liver function tests, and profuse watery diarrhea as the main presenting clinical features. Histologic examination of affected tissues confirms the characteristic lesions of acute GVHD. The onset of TA-GVHD is generally earlier than for traditional GVHD, often occurring 7–10 days after transfusion therapy. The one distinguishing feature of TA-GVHD is the development of pancytopenia, which indicates that host marrow and hematopoietic progenitors are an immunologic target of TA-GVHD. The ensuing pancytopenia and resultant infectious complications of TA-GVHD is the most common cause of death in TA-GVHD, which often occurs 3–4 weeks after the diagnosis is made. Chimerism analysis of circulating peripheral blood cells demonstrates donor origin and represents the sine qua non of TA-GVHD. Prevention The efficacy of blood product irradiation has long been recognized, and initial BMT practice routinely employed irradiation of blood products with 1500 cGy [324]. This dose of gamma irradiation from cobalt-60 or caesium-137 (137Cs) sources effectively prevents lymphocyte proliferation without adverse effects on blood cell morphology or function [325]. Although there has been variation across individual centers, 97% of institutions surveyed by the American Association of Blood Banks employ doses of 1500–3500 cGy [326]. This practice appeared to be highly effective, and only a single case of TA-GVHD has been reported in a patient given blood components irradiated with 2000 cGy from a 137 Cs source [327]. Based upon more sensitive limiting dilution assays of T-cell inactivation, more recent studies have called for higher doses of blood production irradiation, and since July 1993, the Food and Drug Administration has required a dose of 2500 cGy to the internal midplane of the canister with no area receiving less than 15 Gy, with well-defined procedures for quality assurance, dose mapping, and storage times [328– 331]. A Medline search of articles on TA-GVHD published in English since 1960 indicates that most were published between 1985 and 1996, reflecting perhaps more effective current prevention [332]. Newer methods to prevent TA-GVHD are in development. The use of photosensitizing psoralens and ultraviolet A light to inactivate infectious organisms and prevent proliferation of T cells has been explored,
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and in preclinical models this approach appears to be an effective means to prevent TA-GVHD [333]. Another approach that has been attempted is lymphocyte depletion of transfused blood products; however, since the minimum threshold of competent T cells to induce TA-GVHD is yet unknown, this method remains experimental. There are case reports of individuals developing TA-GVHD despite lymphocyte depletion [334,345]. Thus, gamma irradiation remains the standard. Although debate continues as to which patients should receive irradiated blood products, it is universally recommended for transplant recipients. This practice continues for at least 6 months post transplantation, but may be needed for longer periods in patients with active chronic GVHD and severe immunodeficiency. Therapy In the nontransplant setting, therapy with agents used to prevent or treat traditional GVHD after allogeneic transplantation has rarely been successful, since the development of irreversible pancytopenia is often established. This is the main contributor to the near-90% fatality rate associated with TA-GVHD. Despite this, an attempt at immunosuppression using corticosteroids and other immunosuppressive agents is often attempted, and there are some favorable outcomes reported [336,337].
Often, however, allogeneic HCT is required to restore hematopoiesis, since an insufficient number of stem cells are transferred with the blood product that is responsible for the TA-GVHD.
Conclusion Despite a seemingly improved understanding of the biologic mechanisms of acute GVHD, this syndrome is often the first hurdle to challenge the HCT patient. The diagnosis of acute GVHD remains largely a clinical one, with biopsy of affected organs an important adjunct. After decades of research, the standard therapy for this problem remains simple corticosteroids, although novel approaches to the initial management of acute GVHD are being developed. In addition, there remains no standard for secondary therapy of acute GVHD, as numerous agents have modest activity in this setting. Unfortunately, most of these approaches are associated with high mortality related to infectious morbidity. Future endeavors in acute GVHD include novel serologic assays for the diagnosis of acute GVHD, pre-emptive therapy for incipient acute GVHD, and the standardization of therapeutic measures when primary therapy fails.
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275. Woltman AM, van der Kooij SW, Coffer PJ et al. Rapamycin specifically interferes with GM-CSF signaling in human dendritic cells, leading to apoptosis via increased p27KIP1 expression. Blood 2003; 101: 1439–45. 276. Benito AI, Furlong T, Martin PJ et al. Sirolimus (rapamycin) for the treatment of steroid-refractory acute graft-versus-host disease. Transplantation 2001; 72: 1924–9. 277. Wetzig T, Sticherling M, Simon JC et al. Medium dose long-wavelength ultraviolet A (UVA1) phototherapy for the treatment of acute and chronic graft-versus-host disease of the skin. Bone Marrow Transplant 2005; 35: 515–9. 278. Wiesmann A, Weller A, Lischka G et al. Treatment of acute graft-versus-host disease with PUVA (psoralen and ultraviolet irradiation): results of a pilot study. Bone Marrow Transplant 1999; 23: 151–5. 279. Furlong T, Leisenring W, Storb R et al. Psoralen and ultraviolet A irradiation (PUVA) as therapy for steroid-resistant cutaneous acute graft-versushost disease. Biol Blood Marrow Transplant 2002; 8: 206–12. 280. Peritt D. Potential mechanisms of photopheresis in hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2006; 12: 7–12. 281. Biagi E, Di Biaso I, Leoni V et al. Extracorporeal photochemotherapy is accompanied by increasing levels of circulating CD4+CD25+GITR+Foxp3+ CD62 L+ functional regulatory T-cells in patients with graft-versus-host disease. Transplantation 2007; 84: 31–9. 282. Greinix HT, Volc-Platzer B, Kalhs P et al. Extracorporeal photochemotherapy in the treatment of severe steroid-refractory acute graft-versus-host disease: a pilot study. Blood 2000; 96: 2426–31. 283. Richter HI, Stege H, Ruzicka T et al. Extracorporeal photopheresis in the treatment of acute graftversus-host disease. J Am Acad Dermatol 1997; 36: 787–9. 284. Dall’Amico R, Messina C. Extracorporeal photochemotherapy for the treatment of graft-versushost disease. Ther Apher 2002; 6: 296–304. 285. Le Blanc K, Ringden O. Immunobiology of human mesenchymal stem cells and future use in hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2005; 11: 321–34. 286. Le Blanc K, Rasmusson I, Sundberg B et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. The Lancet 2004; 363: 1439–41. 287. Ringden O, Uzunel M, Rasmusson I et al. Mesenchymal stem cells for treatment of therapyresistant graft-versus-host disease. Transplantation 2006; 81: 1390–7. 288. Tfayli A, Selby G, Maqbool F, Bierbaum W, Hamadani M. Role of intra-arterial steroid administration in the management of steroid-refractory acute gastrointestinal graft-versus-host disease. Am J Hematol 2006; 81: 959–62. 289. Sato T, Sakamaki S, Nagaoka Y et al. Intramesenteric artery steroid administration relieved severe refractory gastro-intestinal graft-vs.-host disease in an allogeneic bone marrow transplantation patient. Am J Hematol 1997; 56: 277–80. 290. Shapira MY, Bloom AI, Or R et al. Intra-arterial catheter directed therapy for severe graft-versushost disease. Br J Haematol 2002; 119: 760–4. 291. Bloom AI, Shapira MY, Or R et al. Intrahepatic arterial administration of low-dose methotrexate
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Steven Z. Pavletic & Georgia B. Vogelsang
Chronic Graft-versus-Host Disease: Clinical Manifestations and Therapy
Introduction Chronic graft-versus-host disease (GVHD) is a multisystem chronic alloimmune and autoimmune disorder that occurs later after allogeneic hematopoietic cell transplantation (HCT) [1,2]. It is characterized by immunosuppression, immune dysregulation, decreased organ function, significant morbidity, and impaired survival. Like acute GVHD, there are two opposing aspects: first, chronic GVHD is the main contributor to late post-transplant morbidity, and mortality; and second, in patients with malignant disease, chronic GVHD is associated with important therapeutic graft-versus-tumor (GVT) effects. Overly aggressive immunosuppressive therapy of chronic GVHD may attenuate important GVT reactions and increase the risk of infections, while ineffective (or no) therapy increases chronic GVHD-related transplant morbidity and mortality. Due to insufficient understanding of chronic GVHD biology, our current treatments are based on global immunosuppression and are nonspecific. How to separate beneficial (GVT) effects from harmful effects (GVHD) and use them optimally to the patient’s advantage is a major challenge for HCT research. Depending on a number of factors, chronic GVHD occurs in about half (ranging from 10% to 80%) of 3-month survivors after allogeneic HCT. It is estimated that about 3500 new cases of chronic GVHD are diagnosed each year in North America. Due to its protracted clinical course, an unknown but much larger number of patients live with this diagnosis. There are no contemporary epidemiologic data describing trends in incidence and severity of chronic GVHD. It seems that, due to the changing patterns of utilization of HCT and decreasing early transplant-related mortality, the proportion of patients who develop chronic GVHD is increasing [3]. Reliable incidence estimates are compromised by lack of standardized diagnostic criteria, variability in observer experience, limited expert follow-up, differences in the statistical methods applied, and the sometimes protean nature of chronic GVHD symptoms, which can mimic alternative diagnoses. For many years, chronic GVHD has been a very difficult problem to address. Clinical research in this area lags behind other innovations in HCT. For clinicians, HCT researchers, and patients, chronic GVHD is a major management challenge. The reasons are multiple, and include polymorphic chronic clinical course, disease onset after patients leave transplant centers, poor understanding of disease biology, lack of therapeutic advances, logistical challenges to providing continuous multidis-
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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ciplinary care, and difficulties in obtaining research funding from private or public sources. Consequently, there is no United States Food and Drug Administration-approved medication for chronic GVHD. One of the major obstacles to faster advances in chronic GVHD treatment has been the lack of standardized criteria and definitions for diagnosis and clinical trials. Most recently the international transplant community has focused more on chronic GVHD, as demonstrated by the National Institutes of Health-sponsored chronic GVHD consensus project. A series of consensus documents has been published addressing the areas of diagnosis and staging, histopathology, strategies for the development and validation of biomarkers, response criteria, ancillary therapy and supportive care, and the design of clinical trials (Table 87.1) [4–9]. Although many of these newer criteria still need to be validated and refined, they will also advance the standards and uniformity of chronic GVHD clinical research. This is an opportunity for the transplant community to unite and make a significant impact in our understanding and treatment of chronic GVHD.
Risk factors for onset of chronic GVHD A number of factors have been reported consistently associated with a higher risk of developing chronic GVHD (Table 87.2) [3,10]. Among them, the prior history of acute GVHD seems the most powerful predictor. Therefore, it is not surprising that many risk factors which increase risk of acute GVHD, such as age of the patient or the level of human leukocyte antigen (HLA) and other genetic disparity, also predict chronic GVHD. In interpreting these reports, it is important to remember that all these studies used the traditional definition of chronic GVHD (i.e. any GVHD after day 100). Therefore, the recently described late-onset acute GVHD that occurs after day 100, or persistent acute GVHD after day 100, would have been classified as chronic GVHD. There has been a great deal of discussion on the impact of hematopoietic cell source on the risk of chronic GVHD. A recent individualpatient meta-analysis using data from nine randomized trials enrolling 1111 adult patients convincingly demonstrated the increased risk of overall and extensive chronic GVHD in matched sibling transplant patients who received peripheral blood hematopoietic cells instead of bone marrow. Three-year incidences of extensive and overall chronic GVHD after peripheral blood progenitor cells versus bone marrow were 47% versus 31% (p < 0.000001) and 68% versus 52% (p < 0.000001) respectively [11–13]. An ongoing national randomized clinical trial will address this question in the unrelated donor–recipient setting. The reason for the increased incidence of chronic GVHD after peripheral blood HCT is not yet mechanistically explained. It does not seem to correlate with the number of infused CD3+ cells. The increased chronic GVHD risk may be connected to the infused CD34+ cell fraction or associated
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Table 87.1 Documents published by the National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease (GVHD) Document
Key definitions and recommendations included
Diagnosis and staging [4]
Minimal clinical diagnostic criteria Clinical distinction between acute and chronic GVHD New organ severity scoring system New global scoring system Indications for systemic therapy Strategies for developing better predictors of NRM
Histopathology [5]
Minimal diagnostic criteria for active GVHD Biopsy features suggestive of chronic GVHD Standardized terminology for reporting results List of clinical data that should accompany biopsy Organ-specific criteria for adequate tissue sampling Standardized research forms for histology reporting
Biomarkers [6]
Rationale for developing biomarkers in chronic GVHD Definition of biomarkers and potential applications Proposal for a pathophysiology-based classification Current data in search for biomarkers in chronic GVHD Methodologic considerations in identification and validation of biomarkers in chronic GVHD
Response criteria [7]
Proposal of quantifiable set of chronic GVHD measures suitable for outpatient use by transplant and nontransplant providers and in the adult and pediatric setting Introduction of ancillary objective and patient-reported measures of function, performance, and quality of life Provisional criteria and guidelines for determining partial response and progression Guidelines for use of response assessment in trials
Ancillary and supportive care [8]
Evidence-based rated organ-specific guidelines for ancillary and supportive care including skin, mouth, eyes, vulvar vaginal, gastrointestinal/liver, lungs, hematopoietic system, neurologic system, immunologic system/infection prevention, musculoskeletal system, and psychosocial factors Clinical monitoring guidelines in patients with chronic GVHD Need for multidisciplinary team approach
Design of clinical trials [9]
Approaches and definitions to ensure consistent and interpretable results of clinical studies designed to assess interventions for treatment of chronic GVHD
NRM, nonrelapse mortality
Table 87.2 Factors associated with increased risk for the development of chronic graft-versus-host disease (GVHD) Risk factors consistently demonstrated in published studies as being associated with higher risk of chronic GVHD
Less commonly reported risk factors for the development of chronic GVHD
Prior acute GVHD
Higher infused CD34+ cell dose in peripheral blood stem cell transplantation Lower infused CD34 dose in bone marrow transplantation Faster achievement of complete hematopoietic chimerism Cytomegalovirus seropositivity or reactivation Transplant for chronic myelogenous leukemia or aplastic anemia Corticosteroids in GVHD prophylaxis Lower incidence with umbilical cord transplantations
Increasing recipient age Greater human leukocyte disparity Transplant from a female donor to male recipient Peripheral blood stem cell source Donor leukocyte infusions
cell populations. It does not seem to be related to donor exposure to granulocyte colony-stimulating factor [12]. In some studies, the incidence of extensive chronic GVHD seems to be decreased after umbilical cord blood transplantation [13]. Although a number of studies consistently demonstrated a decreased incidence of acute GVHD with ex vivo T-cell depletion, the effect of these procedures on chronic GVHD is less clear [14].
In spite of increased utilization of reduced-intensity conditioning (RIC) for allogeneic HCT, the impact of RIC on chronic GVHD remains an active question and has been difficult to evaluate systematically. Factors confounding such analyses include: short follow-up of studies; absence of prospective comparative trials due to fundamental differences in the groups receiving RIC versus high-dose conditioning regimens in terms of baseline risk factors for GVHD; use of a variety of
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Chapter 87
Fig. 87.1 Duration of immunosuppressive treatment for chronic graft-versus-host disease (GVHD). (a) Cumulative incidence of discontinued (D/C) immunosuppressive treatment without recurrent malignancy (lower curve and left-hand scale), and the competing risks of death or recurrent malignancy during continued immunosuppressive treatment (upper curve and right-hand scale) among all patients (n = 751). Time to permanent discontinuation of immunosuppression is prolonged among patients who received mobilized peripheral blood (b), among male recipients with female donors (c), and among patients with human leukocyte antigen disparity (d). Other panels show the same results for patients with (– – –) or without (—) the indicated risk factor. In (c), results for female recipients with either male or female donors were similar to those for male recipients with male donors. (Reproduced from [21], with permission.)
RIC regimens; lack of uniform GVHD prophylaxis; and lack of consistent criteria for the diagnosis and staging of chronic GVHD. In some studies, RIC regimens have reduced the incidence of acute GVHD as assessed at day 100 post transplant, but this has not necessarily translated into a demonstrated reduction in the incidence of chronic GVHD [10]. Acute GVHD after RIC occurs often in a delayed fashion, leading to an overlap syndrome of acute and chronic GVHD, which may potentially increase the estimated incidence of chronic GVHD, emphasizing the need for better standardization in future clinical trials. Early achievement of complete donor chimerism, which seems important for the development of chronic GVHD, is frequently delayed after RIC, and may affect the incidence and severity of chronic GVHD [15]. Chronic GVHD after RIC appears to be frequently limited rather than extensive, and patients may be able to be withdrawn from immunosuppressive therapies at an earlier time period [10]. In summary, our ability to predict chronic GVHD remains insufficiently reliable, and there is a need for developing better prognostic criteria, which will likely also include biologic markers [6].
Prognostic factors for outcomes in patients with chronic GVHD Chronic GVHD is major cause of late nonrelapse mortality (NRM) after allogeneic HCT. Across studies, the characteristics most consistently associated with an increased risk of late NRM among patients with chronic GVHD are thrombocytopenia (2 × ULN†; ALT or AST >2 × ULN† Liver biopsy Pulmonary function tests, computed tomography scan with inspiratory and expiratory films, lung biopsy if indicated Physical therapy evaluation to evaluate range of movement, muscle strength, and stiffness, creatine kinase, aldolase, EMG, muscle biopsy, magnetic resonance imaging Complete blood count, differential, quantitative immunoglobulins, Coombs’ test, bone marrow biopsy
Liver
Lung
Bronchiolitis obliterans diagnosed with lung biopsy
Bronchiolitis obliterans diagnosed with pulmonary function tests and radiology‡
Muscles, fascia, joints
Fasciitis Joint stiffness or contractures secondary to sclerosis/fasciitis
Myositis or polymyositis‡
Hematopoietic and immune system
Clinical examination
Edema Muscle cramps Arthralgia or arthritis
Thrombocytopenia Eosinophilia Lymphopenia Hypo- or hypergammaglobulinemia AIHA and ITP
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Table 87.3 Continued
Organ or site
Diagnostic (sufficient to establish the diagnosis of chronic GVHD)
Distinctive (seen in chronic GVHD, but insufficient alone to establish a diagnosis of chronic GVHD)
Other
Other features*
Diagnostic intervention
Pericardial or pleural effusions, Diagnostic ultrasound, computed ascites tomography scans, paracentesis Peripheral Neurology examination, nerve conduction studies and EMG Neuropathy Nephrotic syndrome 24-hour urine collection for urinary protein excretion, protein electrophoresis, kidney biopsy if indicated Myasthenia gravis Neurology examination, EMG, antiacetylcholine receptor antibodies Cardiac conduction Electrocardiogram, Holter monitor, abnormality or creatine kinase, lactate cardiomyopathy dehydrogenase and isoenzymes, troponin
AIHA, autoimmune hemolytic anemia; ALT, alanine aminotransferase; AST, aspartate aminotransferase; EMG, electromyogram; ITP, idiopathic thrombocytopenic purpura; ULN, upper limit of normal. * Can be acknowledged as part of the chronic GVHD symptomatology if the diagnosis is confirmed. † In all cases, infection, drug effects, malignancy, and other causes must be excluded. ‡ Diagnosis of chronic GVHD requires biopsy or radiographic confirmation (or Schirmer test for eyes).
Table 87.4 Categories of acute and chronic graft-versus-host disease (GVHD)
Category
Time of symptoms after hematopoietic cell transplantation or donor lymphocyte infusion
Presence of acute GVHD features*
Presence of chronic GVHD features†
Acute GVHD Classic acute GVHD Persistent, recurrent or late-onset acute GVHD
≤100 days >100 days
Yes Yes
No No
Chronic GVHD Classic chronic GVHD Overlap syndrome
No time limit No time limit
No Yes
Yes Yes
* Maculopapular erythema, gastrointestinal symptoms, and elevated liver function tests. † See Table 87.3.
and loss of body hair. Other characteristics seen with chronic GVHD include premature graying, thinning or brittleness, but these findings are not diagnostic (Plate 87.8).
pain, dry mouth, and sometimes decreased oral range of motion due to fibrosis, which all can interfere with critical oral functioning. Eyes
Mouth Diagnostic features of oral chronic GVHD include: 1 lichen planus-like changes (white lines and lacy-appearing lesions of the buccal mucosa, tongue, palate or lips; Plate 87.9); 2 hyperkeratotic plaques (leukoplakia); 3 decreased oral range of motion in patients with sclerotic features of skin GVHD. Distinctive features of chronic GVHD include xerostomia (dryness), mucoceles, mucosal atrophy, pseudomembranes, and ulcers (infectious pathogens such as yeast or herpesvirus; secondary malignancy must be excluded) (Plates 87.10 and 87.11; see also Plate 27.49). Usual oral symptoms that patients experience include oral sensitivity to food, oral
Distinctive manifestations of chronic GVHD include: 1 new onset of dry, gritty or painful eyes; 2 cicatricial conjunctivitis; 3 keratoconjunctivitis sicca; 4 confluent areas of punctate keratopathy (Plate 87.12). Other features include photophobia, periorbital hyperpigmentation, difficulty in opening the eyes in the morning because of mucoid secretions, and blepharitis (erythema of the eye lids with edema). New ocular sicca documented by low Schirmer test values with a mean value of both eyes 5 mm or less at 5 minutes, or a new onset of keratoconjunctivitis sicca by slit-lamp examination with mean values of 6–10 mm on the Schirmer test, is sufficient for the diagnosis of chronic GVHD if accompanied by
Chronic Graft-versus-Host Disease: Clinical Manifestations and Therapy
Lungs
1.0
P= 0.0005 at 2 years
0.9 0.8 0.7
Chronic GVHD
0.6 0.5 0.4 Late acute GVHD
0.3 0.2 0.1 0.0
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0
5
10
20 15 25 Months post rapamycin
30
35
40
*Unadjusted estimate Fig. 87.3 Survival of patients with late acute graft-versus-host disease (GVHD) versus classic chronic GVHD. The y-axis indicates probability of survival. (Reproduced from [38], with permission.)
distinctive manifestations in at least one other organ. Ocular symptoms include eye dryness, irritation, photophobia, and pain. Genitalia Diagnostic features for the genitalia include lichen planus-like features and vaginal scarring or stenosis (often associated with oral GVHD). Vulvar vaginal symptoms may include itching, pain, dyspareunia, and dysuria (Plate 87.13). Gastrointestinal tract Diagnostic features for the gastrointestinal tract include esophageal web, stricture, or concentric rings documented by endoscopy or barium contrast radiograph. Chronic GVHD may be associated with pancreatic exocrine insufficiency, which may require enzyme supplementation to ameliorate the diarrhea caused by the malabsorption. Manifestations common to both acute and chronic GVHD (as well as other causes, such as drug side-effects, motility disorders, infections or malabsorption) include anorexia, nausea, vomiting, diarrhea, weight loss, and failure to thrive. Wasting syndrome may be a manifestation of chronic GVHD but is often multifactorial (e.g. decreased caloric intake, poor absorption, increased resting energy expenditures, and hypercatabolism). Endoscopic findings of mucosal edema and erythema or focal erosions with histologic changes of apoptotic epithelial cells and crypt cell drop-out may be seen but are not considered diagnostic of chronic GVHD unless the patient also has distinctive features in a non-gastrointestinal system. Patients with unresolved acute GVHD may have more severe intestinal mucosal lesions, including ulcers and mucosal sloughing.
The only diagnostic manifestation of pulmonary chronic GVHD is biopsy-proven bronchiolitis obliterans (BO) (Plate 87.15). BO diagnosed via pulmonary function and radiologic testing requires at least one other distinctive manifestation in a separate organ system to establish the diagnosis of chronic GVHD. BO is characterized by the new onset of an obstructive lung defect. Clinical manifestations may include dyspnea on exertion, cough or wheezing. Some patients may be asymptomatic early in the disease process. Pneumothorax, pneumomediastinum, and subcutaneous emphysema are rare and often represent advanced disease. Restrictive pulmonary function abnormalities secondary to advanced sclerosis of the chest wall are attributable to skin GVHD. BO is clinically diagnosed when all of the following criteria are met: 1 Forced expiratory volume in 1 second (FEV1)/forced vital capacity ratio less than 0.7, and FEV1 less than 75% of predicted. 2 Evidence of air trapping or small airway thickening or bronchiectasis on high-resolution chest computed tomography (with inspiratory and expiratory images), residual volume over 120% or pathologic confirmation of constrictive bronchiolitis. 3 Absence of infection in the respiratory tract, documented with investigations directed by clinical symptoms, such as radiologic studies (radiographs or computed tomographic scans) or microbiologic cultures (sinus aspiration, upper respiratory tract viral screen, sputum culture or bronchoalveolar lavage); 4 BO organizing pneumonia not due to infections may represent a manifestation of either acute or chronic GVHD and is considered a common feature (see also Chapter 96). Musculoskeletal system Diagnostic features include fascial involvement often affecting the forearms or legs and often associated with sclerosis of the overlying skin and subcutaneous tissue (Plate 87.16). Fascial involvement may develop without overlying sclerotic changes of the skin and can result in joint stiffness or contractures when present near joints. Fasciitis is detected on examination by stiffness, a restricted range of motion (e.g. often decreased dorsal wrist flexion or inability to assume a Buddha prayer posture), edema of the extremities with or without erythema (early sign), peau d’orange (edematous skin with prominent pores resembling the surface of an orange) or joint contractures (late complications) (Plate 87.17). Clinical myositis with tender muscles and increased muscle enzymes is a distinctive but nondiagnostic manifestation of chronic GVHD (see Plate 27.54). Myositis may present as proximal myopathy, but this complication is rare and does not explain the frequent complaints of severe cramps. Evaluation of myositis involves electromyography and measurement of creatine phosphokinase or aldolase; imaging by magnetic resonance imaging may also prove useful. Arthralgia and arthritis are uncommon and are occasionally associated with the presence of autoantibodies. Hematopoietic and immune systems
Liver Hepatic acute and chronic GVHD typically presents as cholestasis, with increased bilirubin and/or alkaline phosphatase levels, but it may also present as acute hepatitis [40]. Because of many possible alternative diagnoses, liver biopsy is helpful to confirm GVHD involvement of the liver, but this is often difficult to do because of other medical conditions. Note that because of the histologic similarity between acute and chronic liver GVHD, the diagnosis of chronic GVHD cannot be made on the basis of liver biopsy alone but requires a distinctive manifestation in at least one other organ system (Plate 87.14) (see also Chapter 95).
Although immune deficiency is ubiquitous in chronic GVHD, it is not used to establish the diagnosis. Hematopoietic abnormalities are common in chronic GVHD, but also are not specific and are not used to establish the diagnosis. Cytopenias may result from stromal damage or autoimmune processes. Lymphopenia (≤500/μL), eosinophilia (≥500/μL), hypogammaglobulinemia or hypergammaglobulinemia may be present. Autoantibodies may develop with autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura. Thrombocytopenia (0.5–1.0 mg/kg/day), but this approach remains investigational since the benefits and risks are unknown. All patients receiving immunosuppression should receive Pneumocystis carinii prophylaxis.. It is unknown how long prophylaxis should be continued after stopping immunosuppression, and practices vary widely among centers. Agents used for Pneumocystis prophylaxis include trimethoprim–sulfamethoxazole, pentamidine, dapsone, and atovaquone. Trimethoprim–sulfamethoxazole also provides prophylaxis against Toxoplasma and Nocardia. Some experts use long-term antiviral prophylaxis to prevent recurrent herpes simplex virus and varicella zoster virus (VZV) infection among HCT recipients with severe, longterm immunodeficiency. If VZV-seronegative patients with chronic GVHD are exposed to varicella (either primary or post-vaccination illness), VZV IG should be given within 96 hours. Cytomegalovirus (CMV) disease after day 100 has become more common. Patients with active GVHD, history of CMV reactivation
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Table 87.10 Summary of ancillary therapy and supportive care interventions Organ system
Organ-specific intervention
Skin and appendages
Prevention • Photoprotection. Surveillance for malignancy Treatment • For intact skin – Topical emollients, corticosteroids, antipruritic agents, and others (e.g. PUVA, calcineurin inhibitors) • For erosions/ulcerations – Microbiologic cultures, topical antimicrobials, protective films or other dressings, debridement, hyperbaric oxygen, wound care specialist consultation
Mouth and oral cavity
Prevention • Maintain good oral/dental hygiene. Consider routine dental cleaning and endocarditis prophylaxis. Surveillance for infection and malignancy Treatment • Topical high and ultrahigh potency corticosteroids and analgesics. Therapy for oral dryness
Eyes
Prevention • Photoprotection. Surveillance for infection, cataract formation, and increased intraocular pressure Treatment • Artificial tears, ocular ointments, topical corticosteroids or cyclosporine, punctal occlusion, humidified environment, occlusive eye wear, moisture chamber eyeglasses, cevimeline, pilocarpine, tarsorraphy, gas-permeable scleral contact lens, autologous serum, microbiologic cultures, topical antimicrobials, doxycycline
Vulva and vagina
Prevention • Surveillance for estrogen deficiency, infection (herpes simplex virus, human papilloma virus, yeast, bacteria), and malignancy Treatment • Water-based lubricants, topical estrogens, topical corticosteroids or calcineurin inhibitors, dilators, surgery for extensive synechiae/obliteration, early gynecology consultation
Gastrointestinal tract and liver
Prevention • Surveillance for infection (viral, fungal) Treatment • Eliminate other potential etiologies. Dietary modification, enzyme supplementation for malabsorption, gastroesophageal reflux management, esophageal dilatation, ursodeoxycholic acid
Lungs
Prevention • Surveillance for infection (Pneumocystis carinii, viral, fungal, bacterial) Treatment • Eliminate other potential etiologies (e.g. infection, gastroesophageal reflux). Inhaled corticosteroids, bronchodilators, supplementary oxygen, pulmonary rehabilitation. Consideration of lung transplantation
Hematopoietic
Prevention • Surveillance for infection (cytomegalovirus, parvovirus) Treatment • Eliminate other potential etiologies (e.g. drug toxicity, infection). Hematopoietic growth factors, immunoglobulin for immune cytopenias
Neurologic
Prevention • Calcineurin drug level monitoring. Seizure prophylaxis including blood pressure control, electrolyte replacement, anticonvulsants Treatment • Occupational and physical therapy, treatment of neuropathic syndromes with tricyclic antidepressants, selective serotonin reuptake inhibitors or anticonvulsants
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Table 87.10 Continued Organ system
Organ-specific intervention
Immunologic and infectious diseases
Prevention • Immunizations and prophylaxis against Pneumocystis carinii, varicella zoster virus, and encapsulated bacteria based on Centers for Disease Control guidelines. Consider immunoglobulin replacement based on levels and recurrent infections. No current evidence to support the use of mold-active agents. Surveillance for infection (viral, bacterial, fungal, atypical) Treatment • Organism-specific antimicrobial agents. Empiric parenteral broad-spectrum antibacterial coverage for fever
Musculoskeletal
Prevention • Surveillance for decreased range of motion, bone densitometry, calcium levels and 25-OH vitamin D. Physical therapy, calcium, vitamin D, bisphosphonates Treatment • Physical therapy, bisphosphonates for osteopenia and osteoporosis
PUVA, psoralen and ultraviolet irradiation. Modified with permission from Couriel et al. [8].
during the first 3 months, and lymphopenia are at higher risk of CMV reactivation and death [90]. Some centers continue to monitor for CMV infection after day 100 by pp65 antigenemia or polymerase chain reaction tests, followed by pre-emptive therapy, based on the individual risk as determined by donor and recipient serology. Some investigators advocate early empirical treatment of influenza with neuraminidase inhibitors during influenza outbreaks, using prediction rules based on symptoms and signs, although there is no evidence to support this practice [8].
QOL and other health-related outcomes in chronic GVHD Health related QOL is a multidimensional construct (physical, functional, emotional, social, and spiritual), and in clinical medicine is typically elicited through self-reported assessment that provides a measure of overall satisfaction with life and sense of wellbeing. Chronic GVHD is associated with substantial QOL deficits, particularly in the areas of physical and functional status [3]. Chronic GVHD is also found to be associated with loss of employment in long-term survivors. The need for protracted treatment, particularly with corticosteroids, may result in decreased QOL and functional losses secondary to significant morbidity. It is important to emphasize that QOL questionnaires are measures of the patient’s experiences of the disease, and they do not necessarily accurately represent the severity and activity of the disease. In some studies, it has been found that patients who had chronic GVHD paradoxically reported their QOL as being better than transplant survivors without chronic GVHD [91]. A 30-item survey capturing patient selfreport of chronic GVHD symptoms has been developed and validated for the purpose of use in clinical care and studies in chronic GVHD [92]. It is complementary to the frequently used generic QOL instruments such as SF-36 or FACT-BMT. Prior studies have not thoroughly characterized the impact of chronic GVHD diagnosis on overall health status. Fraser et al. reported recently
a study in 584 individuals who had undergone allogeneic HCT between 1976 and 1999 and survived 2 or more years [93]. These patients completed a 255-item health questionnaire. Global assessment of health status was performed by measurement of six health status domains. In multivariable analyses, subjects with active chronic GVHD were more likely to report adverse general health and mental health, functional impairments, activity limitation, and pain, but not anxiety or fear, when compared with those with no history of chronic GVHD. Importantly, health status did not differ between those with resolved chronic GVHD and those who never had chronic GVHD (Fig. 87.5). This study emphasizes the reversible nature of chronic GVHD, and the need for timely and assertive implementation of effective therapies. A number of valuable web-based resources and organizations exist which can be recommended to patients, including National Bone Marrow Transplant Link (NBMTLink; http://www.nbmtlink.org/), Blood and Marrow Transplant Information Network (BMTInfoNet; http://www.bmtinfonet.org/), and the National Marrow Donor Program (http://www.marrow.org/). Transplantation survivors have an increased long-term risk for new solid cancers. Chronic GVHD and its therapy are independently associated with nearly a threefold higher risk for later developing of squamous cell carcinoma (SCC), particularly of skin and oral mucosa, while there is no additive increased risk for non-SCC cancers [37]. Curtis et al. identified that major risk factors for the development of SCC are long duration of chronic GVHD therapy (p1 HLA mismatch related donor acute GVHD
(Follicular center reaction?)
Resting B cell
Proliferating infected B cells
Resting infected memory B cells
Fig. 93.2 B-cell infection in vivo. Virus infection of resting B cells leads to expression of viral latency genes and proliferation of infected cells. These infected cells may account for several percent of total B cells. Following the development of an adaptive cellular immune response, proliferating infected B cells are eliminated. However, virus persists in resting memory B cells in peripheral blood indefinitely. Viral gene expression is highly restricted in these cells, but viral DNA is present in approximately 1–1000 per million peripheral blood mononuclear cells.
Balanced lymphocyte depletion (Campath, elutration)
Fig. 93.3 Risk factors for Epstein–Barr virus lymphoproliferative disease in the first year after allogeneic marrow transplant. ab, antibody; GVHD, graftversus-host disease; HLA, human leukocyte antigen; TCD, T-cell depletion. (Data from [16].)
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a statistically significant increased relative risk. Smaller studies reinforce these conclusions [15,17,18]. It seems likely that the protective effects of B-cell depletion reflects both decreased numbers of virus-carrying donor lymphocytes and the elimination of the target cell for transformation. Other risk factors identified include the use of unrelated and mismatched donors. In contrast to EBV-LPD in the solid-organ transplant setting, the recipient’s age, EBV seronegativity, and underlying disease are not risk factors for LPD in allogeneic BMT [16]. Cases that occur late after HCT differ in their risk factors, pathology, and EBV association. The only significant risk factor in the IBMTR study was extensive chronic GVHD [16]. These tumors are sometimes of T-cell rather than B-cell origin and are sometimes not EBV associated [19,20]. Late cases of LPD not associated with EBV have also been described in solid-organ transplant recipients [21]. HL occurs at increased frequency following allogeneic BMT. In an IBMTR series, HL occurred about 10-fold less frequently than LPD. This nonetheless indicates a six-fold increased risk of HL in BMT recipients in comparison with the general population, and is similar to the increase in risk in human immunodeficiency virus (HIV)-positive patients [22]. HL is almost always a late event. The median time to developing a tumor is 4.2 years, and it is always more than 2.5 years. It is also virtually always EBV associated, just as it is when it occurs in association with congenital immunodeficiency or HIV infection. Histologically, the mixed-cellularity subtype predominates. As with other late LPDs, T-cell depletion and human leukocyte antigen disparity are not risk factors for HL. The occurrence of severe acute or chronic GVHD was, however, a risk factor. Recipients of allogeneic peripheral blood progenitor cell (PBPC) transplants also are at risk for LPD [23]. The median time to the diagnosis of LPD is similar to that for bone marrow recipients. T-cell depletion of the PBPC product and underlying diagnosis of immune deficiency in the recipient are both identified as risk factors in multivariate analysis. Reduced-intensity transplants have also been associated with EBV-LPD [24,25]. Fludarabine alone may be associated with the development of EBV-LPD [26,27], so the contribution of transplantation to the problem is not yet clear. Since EBV generally does not cross the blood–placenta barrier, cord blood hematopoietic cell (CBHC) transplantation might be anticipated to be associated with a lower incidence of EBV-LPD than transplantation from other donor sources. Alternatively, CBHC transplant recipients might be anticipated to have a higher incidence of lymphoma because they resemble T-cell-depleted bone marrow and blood hematopoietic cell products insofar as they lack EBV-specific cytotoxic T cells. As the incidence of EBV-LPD in CBHC recipients appears not to differ from that in recipients of unmanipulated bone marrow grafts, it may be that these factors balance out [28,29]. An especially high incidence was reported in reduced-intensity unrelated CBHC transplantation recipients that involved antithymocyte globulin in the conditioning regimen [30]. Autologous BMT or PBPC transplantation has also been associated with EBV-LPD, although much less frequently than allogeneic transplantation. T-cell depletion (for the removal of T-cell tumor cells) appears to be the major risk factor, but EBV-LPD has also occurred in association with CD34 cell selection [31–33]. The major determinants of the risk period for LPD are presumed to be immunologic, and several investigators have presented evidence that reconstitution of CD8+ T-cell immunity to EBV generally occurs during the 6-month period following allogeneic BMT [18]. It may occur even more rapidly following autologous PBPC transplantation [34,35]. Hierarchies of cellular responses to classes of EBV antigen (latent versus lytic) and to particular antigens are well recognized [36–38]. Thus different peptide epitopes elicit different magnitudes of T-cell response. Magnitude and patterns of response differ between individuals. In allo-
geneic PBPC transplantation, evidence has been presented that suggests similarities in pattern between patients and their respective donors, suggesting that homeostatic mechanisms in the donor have been recapitulated in the matched sibling recipient [39]. Evidence has been presented suggesting a relationship between EBV CD8+ T-cell frequencies and viral load in peripheral blood mononuclear cells (PBMCs). Whereas EBV reactivation as evidenced by elevated EBV viral loads was not highly predictive of the development of EBV-LPD, impaired EBVspecific T-cell recovery in conjunction with elevated EBV viral load was associated with development of EBV-LPD in five out of five patients [40]. Progressive loss and functional impairment of EBV-specific CD4+ and CD8+ T cells has also been associated with high-risk for developing EBV-associated lymphomas in acquired immune deficiency syndrome patients [41,42]. However, the relative importance of CD8+ and CD4+ responses, responses to latent versus lytic viral antigens expressed on tumor cells, or responses to specific individual viral antigens remains to be determined. Several investigators have presented evidence for a critical role of CD4+ T cells. These may exert direct cytotoxic effects or suppress the outgrowth of EBV-transformed B-cell lines [24,43,44].
Clinical and pathologic aspects LPD most commonly presents early after transplantation and as disseminated disease [16]. Fever, generalized lymphadenopathy, respiratory compromise, and rising liver transaminase levels are typical and have usually been associated with a rapidly progressive multiorgan failure and death. Lesions are nodal and extranodal, frequently involving Waldeyer’s ring, the gastrointestinal tract, the liver, and the central nervous system. Tumors that arise later after transplantation (>1 year) are more commonly localized and often have an indolent course. The definitive diagnosis of EBV-LPD requires biopsy with in situ hybridization or immunohistochemistry to define the viral association and immunohistochemistry or flow cytometry [2]. EBV-encoded RNA in situ hybridization is the most sensitive tool for detecting virus in tumor insofar as these small viral RNAs which do not code for protein are expressed in all EBV-associated tumors. LMP-1 staining is also available in most pathology laboratories but misses the presence of virus in the subset of tumors that do not express this viral antigen. Immunohistochemistry for EBNA-1 would in theory be broadly applicable, but the antibodies available are technically less satisfactory. A variety of morphologic classifications have been suggested [45–49]. Four categories of post-transplant LPD are recognized in the World Health Organization classification: early lesions, polymorphic lesions, monomorphic lesions, and HL (Table 93.1). Monoclonal, oligoclonal, and polyclonal lesions as determined by immunoglobulin gene rearrangements are all recognized. There is no consensus on the prognostic predictive value of either morphology or clonality. However, it is clear that, on occasions, patients with monoclonal disease will have spontaneous remissions and that patients with polyclonal disease may progress despite aggressive therapy including donor lymphocyte infusion (DLI) [23]. Whereas in solid-organ transplant recipients, EBV-LPD most commonly arises in host B cells, in allogeneic HCT recipients EBV-LPD usually arises in donor B lymphocytes (although exceptions associated with autologous recovery are well documented). Lesions are typically CD19+ and CD20+, and often show restricted light chain expression [47,50]. EBV is usually associated with B-cell lymphoproliferative disease in the post-transplant setting, with the exception of lesions arising several years after transplantation. T-cell lymphoma in the post-transplant setting is often EBV associated. EBV detection requires in situ hybridization for the EBV-encoded RNA transcripts or the detection of viral antigens [49]. Because of variability of
Epstein–Barr Virus Infection
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Table 93.1 Epstein–Barr virus lymphoproliferative disorder Classification
Description
Early lesions
Plasmacytic hyperplasia which resembles infectious mononucleosis. Architecture is preserved. Little or no cytologic atypia.
Polymorphic lesions
Polymorphic lesions show loss of architecture. These lesions are heterogeneous in their cell population. Cells may or may not show plasmacytic differentiation. There is a spectrum of cytologic atypia.
Monomorphic lesions
The monomorphic category includes diffuse large B cell lymphoma, Burkitt or Burkitt-like lymphoma, plasma cell myeloma, plasmacytoma and some peripheral T-cell lymphoma. The histologic features are those seen in immunocompetent individuals.
Data from [49].
antigen expression in EBV-LPD, the inability to detect particular viral antigens, such as LMP-1 or EBNA-2, should not be regarded as evidence that the lesion is not virus associated. Some EBV-associated tumors, such as Burkitt’s lymphoma, and a subset of EBV-LPD will not express any viral antigens other than EBNA-1, and reagents to detect EBNA-1 are not widely used because of lack of commercial availability and issues related to cross-reactivity. With the availability of effective interventions for the prevention or treatment of LPD in BMT recipients, early diagnosis has become clinically more important. Whereas serology is useful in the diagnosis of primary EBV infection in the general population, in the post-transplant setting it is difficult to interpret because blood products passively transfer antibodies to the viral capsid antigen and EBNA. Thus, most patients who are seronegative prior to transplant and have seronegative hematopoietic cell donors show antibodies to viral capsid antigen and EBNA within a few days after transplantation if they have been transfused (since most blood products are from EBV-seropositive donors). In addition, the typical serologic changes of IM, i.e. a rise in the immunoglobulin M anti-viral capsid antigen antibody or the appearance of heterophile antibodies, do not generally occur in the BMT setting.
Virologic aspects High-dose therapy destroys the viral B-cell reservoir and, at least in some cases, seems to eliminate the host virus [51]. EBV-seropositive BMT recipients receiving allografts from seronegative donors may become seronegative and, in EBV-seropositive BMT recipients receiving allografts from seropositive donors, the host strain of virus may be replaced by the donor strain. These observations are consistent with the observation that B cells are required for long-term maintenance of infection, as exemplified by the absence of viral infection in patients with X-linked agammaglobulinemia [52]. Viral strain or “biotype” has been studied. The A strain, which is also known as the type 1 virus, is most common in LPD in the transplant setting [53,54]. In vitro and in murine xenograft studies, this strain is more efficient in immortalization and transformation than the less common B strain. Many variations in viral genes have been described, including variants in EBNA-1 and LMP-1. However, there is little evidence for a high-risk donor strain. Expression of the full spectrum of latency viral antigens is often detected in LPD following BMT as it is in LPD following organ transplantation [55]. T-cell products expanded using LCL stimulators are generally expected to target LPD insofar as the viral antigens expressed parallel those expressed in LCLs. Antigen escape by mutation in EBVLPD following transplantation has been documented in some instances [54,56].
Viral copy number and monitoring Polymerase chain reaction quantitation of viral DNA shows promise in facilitating diagnosis and guiding early interventions to prevent or treat EBV-LPD. However, there are, as of yet, no simple universally accepted standards with regards to what should be measured, in whom the measurements should be made, and how the results should be interpreted. Aspects of monitoring are discussed below and in Fig. 93.4. EBV DNA in PBMC Results are variously reported as EBV copies (or genome equivalents) per million PBMCs or per microgram PBMC DNA. There is striking variability in the absolute values in healthy seropositive individuals across studies but general agreement that the number of EBV genomes in PBMCs is increased in association with EBV-LPD [57–62]. Measurement of EBV DNA in donors is not a standard part of donor evaluation. There is no evidence to suggest that a healthy donor with high copy number is at greater risk than a healthy donor with an undetectable copy number. Transplant recipients who have normal or undetectable EBV copy number in PBMCs almost never develop EBV-LPD. High copy number following solid organ or HCT identifies a group of patients at risk for EBV LPD with high sensitivity but limited specificity [63]. It should be noted that copy number will generally be low or undetectable in patients treated with rituximab in the months prior to assay (reflecting depletion of B cells). Copy number is high in HIV patients independent of the presence of EBV-associated or other tumor, independent of CD4 count, and independent of HIV RNA load. EBV DNA in plasma This is reported as EBV copies (or genome equivalents) per millilitre of plasma. EBV DNA is only occasionally detectable in healthy EBVseropositive individuals. It is detected in plasma from patients with nasopharyngeal carcinoma, EBV-associated HL, EBV-associated NK/ T-cell lymphoma, EBV-associated gastric carcinoma, IM, HIV, and chronic active EBV. It is also detected in patients with EBV-LPD (outside the central nervous system) [64–66]. In very high-risk HCT recipients, weekly monitoring has been used to trigger “pre-emptive” therapy with rituximab. Monitoring and pre-emptive therapy were credited with reducing the incidence of EBV-LPD from 49% to 18%. PBMC versus plasma versus whole blood It might be presumed that changes in PBMC parallel changes in cell-free blood (plasma or serum). However, this supposition is clearly wrong with regard to nasopharyngeal carcinoma patients. Monitoring EBV copy number in PBMCs does not distinguish patients from healthy
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T est EBV DNA in PBMC
Comments
viral episomes in lymphocytes EBV DNA in plasma
Values >10,000,000 copies/million PBMC are useful in prompting investigation of a diagnosis of EBV LPD in highrisk settings In patients treated with rituximab in previous several months low or undetectable values should be regarded as of no diagnostic or prognostic value Routine monitoring is only advised in conjunction with HCT procedures with a risk of EBV LPD >5% When PBMC and plasma monitoring are both available, elevated PBMC values without elevated plasma values should be interpreted with great caution Values >1000 copies/mL are useful in prompting investigation of a diagnosis of EBV LPD in high-risk settings
virion DNA
free DNA
Other sources of EBV DNA in plasma include EBVassociated cancers, primary EBV infection (IM), immunocompromise (including HIV) Monitoring may be valuable even in rituximab-treated patients Routine monitoring is only advised in conjunction with HCT procedures with a risk of EBV LPD >5%
controls, whereas monitoring EBV copy number in cell-free blood not only distinguishes patients from healthy controls, but is also arguably more useful than anatomic staging for prognosis [67–70]. The reason for the difference lies in what is being measured. Measurements of EBV in PBMCs generally reflect the presence of EBV in resting B cells. Increased numbers of B cells harboring virus or an increase in the tendency of infected cells to produce virus both manifest as increased copy number in PBMCs. In contrast, measurements in plasma or serum indicate either the presence of virion DNA or DNA released from cells. In immunocompetent patients, except in the setting of a primary EBV infection, free virion DNA is rarely detected. In nasopharyngeal carcinoma patients, the EBV DNA detected in plasma or serum is not virion DNA but is derived from tumor cells that have undergone apoptosis, releasing tumor DNA including viral DNA into the circulation. Thus, local radiation therapy or surgical excision of nasopharyngeal tumors results in no change in DNA in PBMCs (a reservoir of EBV-infected cells unrelated to the tumor) but a very rapid fall in cell-free EBV DNA (EBV tumor DNA). In HCT recipients, EBV DNA in cell-free blood likely reflects the presence of virions and the presence of viral DNA released by dying cells. Measurements in whole blood ignore these distinctions but are often convenient [71,72].
Suggested guidelines The authors’ suggested guidelines are presented in Fig. 93.4. At the authors’ institution (Johns Hopkins Hospital), there is no routine monitoring of EBV copy number in PBMCs or plasma in the absence of clinical signs of EBV LPD. High viral copy number in PBMCs or plasma increases suspicion of EBV LPD but never triggers preemptive treatment.
Treatment A variety of treatments for LPD have been explored [73]. Discontinuation of immunosuppression is sometimes associated with regression of LPD in solid-organ transplant recipients, but it is very rarely effective in BMT recipients. The use of antivirals, such as aciclovir, ganciclovir,
Fig. 93.4 Viral copy number measurements and guidelines for interpretation. Commonly used assays measure Epstein–Barr virus (EBV) DNA in mononuclear cells (top) or in plasma (bottom). In plasma, viral DNA may be packaged as a virion or free (released from cells, commonly tumor cells). Virion and free DNA are not distinguished in routine clinical testing. HCT, hematopoietic cell transplantation; HIV, human immunodeficiency virus; IM, infectious mononucleosis; LPD, lymphoproliferative disorder; PBMC, peripheral blood mononuclear cells.
and their congeners, has been advocated, but there is little evidence to suggest therapeutic efficacy against EBV in any setting. These drugs do not inhibit the proliferation of lymphocytes latently infected by EBV either in vitro or in vivo. Furthermore, high rates of EBV-LPD have been noted in recipients of T-cell-depleted grafts despite the use of these drugs [23,74]. Interferon-alpha has also been used with some success [75]. Cytotoxic chemotherapy has been curative in organ transplant recipients with LPD, but there was no similar track record of success in a series of BMT recipients [76]. In contrast, therapies with antibodies, DLI, or adoptive T-cell transfer of EBV-specific cytotoxic T cells have all yielded good results. Experience with DLI to treat EBV-LPD was first reported from the Memorial Sloan Kettering Cancer Center, where patients with EBVLPD following T-cell depletion, allogeneic marrow transplantation were treated with donor leukocytes (1 × 106 CD3+ cells/kg) [77]. Durable remissions were rapidly achieved (14–30 days after infusion) with only modest GVHD. Since the LPD was of donor origin, the effectiveness of the therapy is presumed to reflect EBV-specific reactivity rather than alloreactivity. DLI has also been used in other settings, but not always successfully. In recipients of mismatched-related transplants, DLI given to prevent primary disease did not prevent late LPD (>15 months) [78]. Similarly, in the allogeneic peripheral blood HCT setting, DLI has been used with mixed results [23]. This use of DLI in the treatment of EBV-LPD after BMT was followed by the demonstration that infusion of EBV-specific T cells can also induce tumor regression or prevent the development of tumors [79]. Expanded EBV-specific donor T-cell lines were prepared by stimulating donor lymphocytes with irradiated donor-derived LCLs. The resultant cell lines were mixes of CD8+ and CD4+ T cells as well as NK cells. Patients responded to 4-weekly T-cell infusions with a return of viral copy numbers to normal values and resolution of fever, lymphadenopathy, and pulmonary infiltrates. High-risk patients were treated prophylactically. These infusions prevented the development of EBV-LPD, restored EBV cellular immune responses, and established populations of cytotoxic T-cell precursors that could respond to in vivo or ex vivo challenge with the virus for as long as 18 months [79,80]. Anti-B-cell antibodies have also been used to treat EBV-LPD [57,81]. These include antibodies with specificity for CD21, CD24, and CD20
Epstein–Barr Virus Infection Table 93.2 Treatment for Epstein–Barr virus (EBV) lymphoproliferative disorder (LPD) and Hodgkin’s lymphoma (HL) Early LPD Recommended Rituximab Donor lymphocyte infusion or EBV T cells from donor if available Other sources of EBV-specific T cells Standard lymphoma chemotherapy Not recommended Ganciclovir
HL
Standard HL chemotherapy
Ganciclovir
(rituximab). Each of the monoclonals has met with substantial success and been associated with very modest or no toxicity. They are effective in many instances, particularly when used early. The complete response rate in solid organ transplant recipients was 44% in a multicenter trial [81]. Rituximab has also proven effective in HCT patients with EBVLPD [82,83], although the number of patients studied remains small. Recommended treatment approaches are summarized in Table 93.2. The ready availability of rituximab and its lack of toxicity suggest that it should be considered appropriate as first-line therapy, or as a part of first-line therapy, in most instances of EBV B-cell LPD. It may also have a role as pre-emptive treatment in high-risk patients, although no consensus has yet emerged in this regard [63,66,84]. EBV-specific T cells, when available, are an attractive alternative or adjunct. Similarly, DLI from allogeneic HCT donors is often effective, even at lymphocyte doses not usually associated with GVHD. For monomorphic lymphoma occurring late after transplant, conventional chemotherapy may be effective. EBV-associated adoptive targeted immunotherapy, when available, is appropriate. For post-transplant HL, the response to conventional chemotherapy is excellent, and most patients in the CIBMTR report were alive and well after therapy [85].
T-cell therapy for EBV-associated tumors The successes in the treatment of EBV-LPD in BMT recipients have stimulated interest in the treatment of other EBV-associated lymphoid tumors. Two issues need to be considered in regard to such immunotherapeutic approaches. The first relates to the ability of tumor cells to present antigens and to be killed by cognate T cells. The second relates to the pattern of viral antigen expression associated with the tumor. Defects in antigen processing, downregulation of major histocompatibility complex (MHC) class I molecules, and downregulation of adhesion molecules are well recognized in Burkitt’s lymphoma. At least some HL tumors are not likely to be susceptible to CD8+-mediated killing because Reed–Sternberg cells often fail to express MHC class I antigens [86]. However, downregulation of MHC class I antigens is almost exclusively a phenomenon of HL not associated with EBV. EBVassociated HL very consistently expresses MHC class I and high levels of transporter-associated proteins (TAP-1 and -2), irrespective of the presence of latent EBV infection. Studies of the HL cell lines support susceptibility to lysis by antigen-specific cytotoxic T-lymphocytes in a class I-restricted manner [87]. In HL, cytokines and chemokines elaborated by tumor cells may lead to dysregulation of cellular immune responses. Much of the T-cell infiltrate in HL consists of regulatory cells [88–90]. These cells suppress interferon-γ4 production by lymphocytes including the CD8+ cells specific for EBV antigens expressed in HL.
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Note that the presence of T-regulatory cells is characteristic of HL and not specifically associated with EBV(+) HL. The spectrum of viral antigen expression is also an obstacle. Tumor types have characteristic patterns of viral gene expression. Several patterns of expression are recognized. Burkitt’s lymphoma expresses only a single viral antigen (EBNA-1) in most tumor cells. HL expresses EBNA-1 and the latency membrane antigens (LMP-1 and -2). Nasopharyngeal carcinoma less consistently expresses the membrane antigens. EBV-LPD in organ transplant recipients sometimes expresses the complete spectrum of latency antigens. The patterns of expression are of key importance because the viral latency antigens (EBNA-3A, -3B, and -3C) most commonly targeted by EBV-specific T cells expanded in vitro with LCLs are not expressed in HL, nasopharyngeal carcinoma, and many other EBV-associated malignancies. Thus, adoptive T-cell therapy using LCL stimulators for T-cell expansion has been explored in a number of small studies of patients with LPD following organ transplantation, nasopharyngeal carcinoma, and HL [91]. In LPD, the tumors are generally sensitive to T-cell killing, and there is likely to be a match between antigens expressed by tumor cells and antigens targeted by expanded T cells. EBV-specific T cells are readily expanded in vitro, an increase in EBV-specific T-cell frequency is detected in vivo, and tumor regression often results [91]. However, the approach is limited by the time required (several weeks) to expand autologous EBV-specific T-cell products. An alternative approach has been to use partially matched products expanded from allogeneic donors [92]. In a phase II study, 21 of 33 patients showed a clinical response as measured by tumor regression (12 complete responders) despite evidence that infused T cells survived for only a few days or weeks. In HL and nasopharyngeal carcinoma, the results with LCL-expanded T-cell products are intriguing but less certain [93–95]. In patients with EBV-associated HL, expansion of LMP-2-specific T cells in vivo and trafficking to tumor sites were seen in some patients, although measurable tumor responses were seen in only three of 11 patients with measurable disease [93]. Similar results were seen in small numbers of patients with metastatic nasopharyngeal carcinoma [94,95]. Promising new approaches to expanding antigen-specific T cells, which will more specifically target viral antigens expressed in a given tumor type, are emerging. These include improved methods in expanding T cells directed toward LMP-1 or LMP-2 [96] and in vitro modification of T cells via transduction with recombinant T-cell receptors [97,98]. Expansion of T cells from allogeneic HCT donors may in some instances allow expansion of CD8 T cells with specificities for EBV-antigens expressed in HL that are absent from expansions of autologous products [96].
Summary EBV infection is ubiquitous. Following primary infection, latency is established in B cells and infection is spread throughout the B-cell compartment by virus-“driven” proliferation of infected B cells. The cellular immune response helps to limit the proliferation of infected cells. A reservoir of resting infected B cells with very limited viral antigen expression eludes immune surveillance. With high-dose conditioning regimens and marrow transplantation, the host reservoir of latently infected B cells may be replaced by infected donor B cells. In the 6-month period following HCT, patients are at risk for developing EBV-LPD. This tumor usually arises in the donor B cells. The major risk factors are T-cell depletion and HCT from unrelated or mismatched donors. Treatment with adoptive cellular immunotherapy, either DLI or EBV-specific T-cell infusion, has been associated with impressive clinical results in a small number of patients, as has treatment with monoclonal antibodies. Adoptive immunotherapy in the setting of HCT may also have a role to play in the management of EBV-associated malignancies such as HL.
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virus+ Hodgkin’s disease. J Exp Med 2004; 200: 1623–33. 94. Comoli P, Pedrazzoli P, Maccario R et al. Cell therapy of stage IV nasopharyngeal carcinoma with autologous Epstein–Barr virus-targeted cytotoxic T lymphocytes. J Clin Oncol 2005; 23: 8942– 9. 95. Straathof KC, Bollard CM, Popat U et al. Treatment of nasopharyngeal carcinoma with Epstein– Barr virus–specific T lymphocytes. Blood 2005; 105: 1898–904. 96. Bollard CM, Gottschalk S, Huls MH et al. In vivo expansion of LMP 1- and 2-specific T-cells in a patient who received donor-derived EBV-specific
T-cells after allogeneic stem cell transplantation. Leuk Lymphoma 2006; 47: 837–42. 97. Savoldo B, Rooney CM, Di Stasi A et al. Epstein Barr virus specific cytotoxic T lymphocytes expressing the anti-CD30zeta artificial chimeric Tcell receptor for immunotherapy of Hodgkin disease. Blood 2007; 110: 2620–30. 98. Jurgens LA, Khanna R, Weber J, Orentas RJ. Transduction of primary lymphocytes with EpsteinBarr virus (EBV) latent membrane protein-specific T-cell receptor induces lysis of virus-infected cells: a novel strategy for the treatment of Hodgkin’s disease and nasopharyngeal carcinoma. J Clin Immunol 2006; 26: 22–32.
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Michael Boeckh
Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation
Introduction Viral infections are major cause of morbidity and mortality after hematopoietic cell transplantation (HCT). There has been significant progress in the management of herpes simplex virus, varicella zoster virus, and cytomegalovirus (CMV) after transplantation, but, at the same time, other viruses have gained importance. Herpesviruses 1–5 (herpes simplex virus-1 and -2, varicella zoster virus, CMV, and EBV) are reviewed in Chapters 90–93. This chapter will review the significance of adenoviruses, herpesviruses 6–8, community respiratory viruses, papovaviruses (papillomavirus and polyomaviruses), parvovirus B19, enteroviruses, rotavirus, and Norovirus in the HCT setting. The term “infection” generally refers to asymptomatic shedding or viremia, while “disease” refers to end-organ symptomatic manifestations of the infection, which require detection of the virus in the particular organ. Adenovirus Virology Adenovirus is a nonenveloped DNA virus that replicates and assembles in the nucleus. Virions are released when the cell is lysed. Adenovirus causes both lytic and latent infection [1]. Infection may persist from days to years, and may occur in host tissue (e.g. adenoidal) without causing apparent symptoms in immunocompetent hosts. All adenoviruses share a common group-specific complementfixing antigen. Adenoviruses are categorized in subgroups A to F, and there are presently 51 human serotypes known, although only approximately half have been implicated in human disease. Serotypes from all six subgroups can cause disease in both immunocompetent and immunocompromised subjects, although the disease spectrum associated with strains may differ [2,3]. After primary infection, adenovirus establishes latency in adenoidal tissues [3], with lifelong persistence of specific antibodies. Pathogenesis and immunity Transmission of adenovirus occurs by either respiratory droplet or the oral–fecal route. Adenovirus enters the mucosa and infects epithelial cells, resulting in inflammation and necrosis. Subsequent viremia may lead to infection of kidney, bladder, liver, and lungs in HCT recipients;
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
central nervous system (CNS) disease is rare. After HCT, reactivation from latency is probably more important than exogenous infection [2]. Epidemiology and clinical manifestations Primary infection is usually established in early life with either asymptomatic or symptomatic infection. By age 5, most people have been infected by one or more serotypes of adenovirus. Adenovirus infections are common after HCT [2,4,5], and some recent reports suggest that they may be increasing. This increase may be related to transplantation practices (e.g. T-cell depletion) [6]. However, the incidence of end-organ adenovirus disease is dependent on the degree of immunosuppression and seems to be increasing only in recipients of T-cell-depleted grafts [6]. Clinical manifestations include pneumonia, hepatitis, gastrointestinal disease, nephritis, cystitis, and eye infections [6,7]. Risk factors for adenovirus infection after HCT include graft-versushost diseases, HCT from unrelated donors, use of total body irradiation, T-cell depletion, and younger age [4,6–8]. The degree of T-cell depletion and post-transplant immunosuppression directed at T-cell function seems to be important. For example, transplant protocols employed with haploidentical transplantation are associated with a particularly high incidence of adenovirus infection and invasive disease [9]. Almost all studies report a higher incidence in children [10–12]. In recipients of non-T-cell-depleted grafts, adenovirus disease is rare and seems to be restricted to allogeneic transplant recipients [2]. However, cases of disease in reduced-intensity regimens that include T-cell depletion have been reported [8]. Risk factors for adenovirus disease include younger age [12], viremia and shedding from more than two sites [12–14], total body irradiation [15], GVHD [6], and transplantation from an unrelated donor [13]. High-level plasma viremia (>1000 copies/mL) is a marker for invasive infection in recipients of non-T-celldepleted grafts [16]. Adenovirus disease is associated with a fatality rate of 30–50% [2,6]. Response to treatment seems to be particularly poor in patients with pneumonia or disseminated disease [17]. A recent multivariable analysis in recipients of T-cell-replete grafts indicated that adenovirus infection is independently associated with mortality [2]. Diagnostic techniques Direct detection methods are necessary to establish the diagnosis of adenovirus infection and disease in HCT. As with other viral infections in HCT recipients, serology is not recommended. Adenovirus can be grown in cell culture. Shell vial assays and direct antigen-detection methods are used routinely in clinical practice. For detection of adeno-
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virus in stool, enzyme immunoassays or the polymerase chain reaction (PCR) is required because enteropathogenic serotypes 40 and 41 usually do not grow in culture. Quantitative PCR assays are now routinely used to diagnose adenovirus viremia [14,18–20]. For diagnosis of adenovirus disease, detection of the virus in bronchoalveolar lavage (BAL) or tissue samples is required, using rapid culture techniques or direct fluorescent antibody testing on BAL, and immunohistochemistry or in situ hybridization techniques for tissue samples. The role of quantitative PCR for BAL and tissue is currently not defined. Prevention and treatment There are no controlled treatment studies for adenovirus infection in the immunocompromised host. Intravenous ribavirin has been used, but results are conflicting [21–24]. Recently, cidofovir has been shown to have in vitro activity, and several uncontrolled case series have shown promising results [19,23,25–30]. Ganciclovir has in vitro activity against adenovirus, and has a moderate effect in prevention of adenovirus infection in non-T-cell-depleted patients [10]. However, for treatment of adenovirus disease, ganciclovir is not recommended. The antiretroviral drugs zalcitabine (DDC), alovudine, and stavudine have activity in vitro [31,32]. Differences in the results of treatment studies in terms of in vitro susceptibility and outcome may be due to strain and serotype differences, which often have not been reported in these studies. Specific and non-specific T-cell therapy have been reported in small series [33–35]. In high-risk settings such as haploidentical transplantation or cord blood transplantation [18,36], weekly PCR surveillance for adenovirus viremia and pre-emptive treatment with cidofovir is now used at several centers [9,29,30]. However, this strategy does not completely eliminate adenovirus disease in the highest-risk patients [29]. It has also been suggested that adenovirus DNA in stool may be predictive for disease in highest-risk patients [37], which might represent an earlier opportunity to intervene. Adenovirus-specific T cells therapy is an area of active research [35]. Prospective trials are needed to evaluate prevention strategies.
Respiratory viruses Community-acquired respiratory virus infections are an important cause of morbidity and mortality after HCT [38,39]. The best studied viruses include respiratory syncytial virus (RSV), parainfluenza viruses, and influenza viruses. Less information is available on rhinoviruses and coronaviruses, human metapneumovirus (HMPV), and the recently described human bocavirus (HBoV). The infection epidemiology in HCT recipients usually parallels that observed in the community. This
phenomenon is due to the fact that these viruses circulate in immunocompetent individuals (including health-care personnel and family members). A seasonal distribution with a peak during the winter and spring months is well described for RSV, influenza viruses, and MPV; the other respiratory viruses may show different patterns of seasonality. The most significant impact on morbidity and mortality after HCT has been reported from RSV, parainfluenza viruses, and influenza viruses [39–42]. The significance of the emerging respiratory viruses has not been fully examined. Molecular detection techniques are rapidly replacing traditional methods [43–45]. One feature of respiratory virus infections is that the clinical signs and symptoms at presentation are not specific. This syndromic nature of illness requires multiplex testing platforms [46–48]. Specimen handling is important for maximal diagnostic yield. Nasal wash or swab specimens should be placed on ice or in the refrigerator immediately and transported to the laboratory without delay [49]. For nonmolecular methods, specimen set-up in the laboratory should occur within 2–4 hours. Nonmolecular methods available for testing include standard viral cultures (results available in several days), shell vial centrifugation cultures using specific monoclonal antibodies (results after 1–3 days), direct fluorescent antibody tests (2 hours), and enzyme immunoassays (2 hours). On tissue sections from lung biopsy or autopsy specimens, virus-specific monoclonal antibody staining, viral cultures, or PCR can be used. Respiratory syncytial virus Clinical significance RSV is an RNA virus (paramyxovirus) that causes a wide spectrum of respiratory diseases. These diseases range from life-threatening bronchiolitis in infants and potentially fatal pneumonia in transplant recipients to a mild upper respiratory infection (URI) in immunocompetent adults and older children. The virus is increasingly implicated in a number of respiratory illnesses in immunocompetent or mildly immunosuppressed individuals, such as otitis media, exacerbation of chronic obstructive lung disease, and community-acquired pneumonia. After HCT, and also in severely immunosuppressed nontransplant patients with hematologic malignancies [50], RSV causes upper respiratory tract infection which may progress to fatal pneumonia [51]. Recently, RSV has been linked to late airflow obstruction, a debilitating condition of accelerated loss of lung function after HCT [52]. During the respiratory virus season, the incidence may be as high as 10%, and both allogeneic and autologous transplant recipients can be infected (Table 94.1 and Fig. 94.1) [39,53]. Most of the infections occur during the first 2 months after HCT, but late cases have also been reported [54].
Table 94.1 Respiratory virus infections after hematopoietic cell transplantation: comparison of respiratory syncytial virus, parainfluenza virus-3, and influenza viruses
Virus
Incidence of infection
Progression from URI to pneumonia
Time from URI to pneumonia (median)
Proportion of pneumonia without URI
Pulmonary copathogens in cases with pneumonia
Overall mortality at 1 month after diagnosis of pneumonia
Respiratory syncytial virus Parainfluenza virus 3 Influenza viruses A and B
1.8–6%* 4–7% 1.3–2.6%†
40% 18–44% 18%
7 days 7 days 11 days
20–50% 31% 18%
2.5–33% 53% 50%
45% 35–37% 25–28%
URI, upper respiratory infection. * During the winter season, the incidence may be as high as 10%. † May be significantly higher during outbreaks [217]. Data from [38,39,53,75,76,84].
Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation
Risk factors In a large study, winter season, male gender, and use of bone marrow as stem cell source were identified for the acquisition of RSV in HCT recipients [39]. URI precedes pneumonia in 80% of patients, and approximately 30–50% of patients with RSV URI progress to pneumonia after a median of 7 days. The strongest risk factors for progression to pneumonia are older age and lymphopenia [39]. Patients receiving nonmyeloablative conditioning regimens seem to be largely protected
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from progression to lower respiratory tract disease during the first 3 months after HCT [55]. Following reduced-intensity conditioning regimens, progression to lower tract disease has been described, but fatal disease may be less common [56]; however, larger studies are needed to better define the risk of progression and outcome of disease. One study in pediatric HCT recipients indicates that asymptomatic shedding of RSV can occur [57]. Two studies in adult HCT recipients, one of which used weekly PCR-based surveillance, did not detect asymptomatic shedding [45,58]. Treatment and prevention
Fig. 94.1 Time to first respiratory virus infection after hematopoietic cell transplantation. MPV, metapneumonia virus; PIV, parainfluenza virus; RSV, respiratory syncytial virus. (Reproduced from [45], with permission.)
Without treatment, RSV pneumonia is almost uniformly fatal in highly immunosuppressed HCT recipients [51]. However, recovery from RSV lower respiratory tract disease without specific treatment has been reported [59]. These differences are likely due to less intense immunosuppressive regimens. Pulmonary co-pathogens are detected in up to one-third of the patients with RSV pneumonia and require aggressive treatment. No adequately powered controlled trials exist for the treatment of RSV infection and pneumonia in the HCT setting. Available evidence comes from small uncontrolled cohort studies and one small randomized trial [60]. The data suggest that treatment of early pneumonia (i.e. prior to mechanical ventilation) is associated with improved outcome (Table 94.2). Intermittent short duration (2 g over 2 hours three times per day) or continuous aerosolized ribavirin is considered the treatment of choice for RSV pneumonia. With this regimen, the 30-day all-cause mortality is approximately 40% (Table 94.2). In a large multivariable analysis, RSV associated pneumonia (but not URI) was independently associated with mortality [39]. Systemic ribavirin alone does not seem to be effective for the treatment of pneumonia [61,62]; better response rates are reported for treatment of RSV URI, but small sample sizes limit the
Table 94.2 Outcome of radiographically and virologically documented respiratory syncytial virus (RSV) pneumonia with antiviral therapy (only series with five or more patients considered)
Strategy, authors
Reference
Number of patients
Aerosolized ribavirin alone Harrington et al., 1992 Ljungman, 2001 Nichols et al., 2001
[51] [42] [39]
13 9 5
Survival
Respiratory failure at start of treatment
22% 66% 60%
NR NR NR
Aerosolized ribavirin + pooled intravenous immunoglobulin [218] 12 58% Whimbey et al., 1995 [219] 6 66% Ghosh et al., 2000 Ljungman, 2001 [42] 5 60% [39] 19 68% Nichols et al., 2002
Survival 78% if therapy was started before respiratory failure; 0% if after respiratory failure Survival 80% if therapy was started before respiratory failure; 0% if after respiratory failure NR NR
Aerosolized ribavirin + palivizumab [64] Boeckh et al., 2001
None of the patients had respiratory failure at the start of treatment
12
83%
Aerosolized ribavarin + RSV-specific immunoglobulin 7* 86% De Vincenzo et al., 2000 [65] [53] 11 91% Small et al., 2002
None of the patients had respiratory failure at the start of treatment Including two patients with respiratory failure at the start of treatment; both survived†
Intravenous ribavirin alone Lewinsohn et al., 1996
[61]
10
20%
Survival 40% if therapy was started before respiratory failure; 0% if after respiratory failure
Intravenous ribavirin + aerosolized ribavirin Ljungman, 2001 [42] 5
40%
NR
NR, not reported. * Only patients with radiologic signs of lower tract involvement considered. † T. Small, personal communication, 2003.
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strength of the data [63]. It has not been studied whether combined oral and aerosolized ribavirin is more effective than using aerosolized ribavirin alone. The role of concomitant intravenous immunoglobulin or RSV-specific immunoglobulin or palivizumab, an RSV-specific monoclonal antibody directed at the F protein of RSV, remains poorly defined. Uncontrolled data suggest that high-titer antibody preparations or palivizumab may be required if such adjunctive therapy is given [53,64,65]. However, this issue has not been studied in a controlled fashion. Outcome results of recent series are depicted in Table 94.2. There are several factors that may account for differences in outcome in the available cohort studies. Perhaps the most important factor seems to be the timing of initiation of therapy. Several studies suggest that treatment that is started after respiratory failure has occurred is almost uniformly unsuccessful (Table 94.2). Other factors that may explain the differences in outcome between studies include the presence of lymphopenia at the time of diagnosis, the presence of co-pathogens (e.g. invasive molds and CMV), and the use of immunosuppressive agents. Recent data suggest that lymphocyte, rather than neutrophil, engraftment is an important risk factor for progression and outcome in HCT recipients [38,42,66]. Available studies are too small to control for these parameters, making the interpretation of results of pharmacologic interventions difficult. Most centers now agree that aerosolized ribavirin should be given to HCT patients with RSV lower respiratory tract disease. There is no consensus on the role of antibody preparations. Due to the high mortality of RSV pneumonia, much interest has focused on prevention. Possible strategies are similar to those employed for prevention of CMV. While it is well established that RSV-associated URI precedes pneumonia in the majority of cases, limited information is available on the extent of asymptomatic shedding of respiratory viruses in HCT recipients [57,58]. A recent small controlled study of pre-emptive antiviral therapy based on RSV-associated URI showed a decline in viral load but no significant difference in RSV pneumonia; the study was stopped prematurely due to slow accrual [60]. A retrospective analysis of this strategy carried out at the M.D. Anderson Cancer Center, suggests a reduced risk of progression to lower tract disease [38]. Whether aerosolized ribavirin is indicated for RSV-associated URI remains controversial. Some centers administer it in the presence of lymphopenia (less than 300 per mm3) when the risk of progression is particularly high [38,66]. A nonrandomized study suggests that palivizumab given pre-emptively to patients with RSV-associated URI does not prevent progression to pneumonia [67]. Prophylactic measures recommended throughout the respiratory virus season include isolation of infected patients, hand washing prior to every patient contact, educational efforts targeted at health-care personnel and family members, restricting patient contact with health-care personnel and family members with URI symptoms [66], as well as influenza vaccination of health-care personnel and family members [68,69]. Whether pharmacologic prophylaxis (e.g. palivizumab or RSV immunoglobulin) throughout the respiratory virus season is effective in preventing infection and disease in HCT recipients has not been studied. For pretransplant infections with RSV, parainfluenza virus or influenza virus, most transplant centers postpone transplantation until resolution and cessation of viral shedding occurs, especially when a high-dose allogeneic HCT is planned. Indeed, Centers for Disease Control (CDC) guidelines suggest postponing transplantation in all patients with URI symptoms. This approach is supported by a recent study of pretransplant RSV infection, which showed rapid progression to RSV pneumonia in patients where the transplant procedure was not postponed [70]. However, there is some evidence that low-risk autologous transplant patients may be transplanted without adverse outcome [71]. In addition to this, the outcome of respiratory virus infections appears to be less
severe with nonmyeloablative conditioning regimens [55]. Thus, delaying the transplant procedures based on URI symptoms is recommended if patients are to undergo transplants after high-dose conditioning, but less is known in the setting of reduced-intensity conditioning. Use of prophylaxis (e.g. RSV immunoglobulin or palivizumab) during transplantation in patients with pretransplant respiratory virus infections has not been studied. Parainfluenza viruses Clinical significance Parainfluenza, an enveloped paramyxovirus containing single-stranded RNA, is classified into four serotypes. Of the four types of parainfluenza virus, parainfluenza virus-3 is most common (approximately 90%), followed by serotypes 1 and 2. The incubation time is 1–4 days. Parainfluenza virus infections remain detectable throughout the summer season (Fig. 94.2). The incidence of 7% in two studies (using nonmolecular detection methods) is higher than that reported for RSV (4%) [72,73].
60 RSV 50 40 30 20 10 0 60 Influenzavirus A and B 50 Number of cases
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40 30 20 10 0 60 Parainfluenzavirus 3 50 40 30 20 10 0 J
F
M
A
M
J
J
A
S
O
N
D
Month
Fig. 94.2 Monthly distribution of respiratory syncytial virus (RSV), parainfluenza virus-3, and influenza infections after hematopoietic stem cell transplant in the 1990s at the Fred Hutchinson Cancer Research Center. (Data from [39].)
Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation
Risk factors Only HCT from an unrelated donor has been identified as a risk factor for the acquisition of the parainfluenza virus [73]. In Tcell-depleted patients, CD4 lymphopenia has been reported to increase the risk of all respiratory virus infections, including parainfluenza virus infections [56]. Similar to RSV, URI is the predominant clinical presentation. Progression to pneumonia seems to be less common than with RSV (Table 94.1) [73]. Late airflow obstruction has been strongly linked to parainfluenza virus pneumonia (adjusted odds ratio 18), and to a lesser degree to URI [52]. The most important risk factors for the progression from URI to pneumonia are use of systemic corticosteroids and lymphopenia [56,73]. Recipients of nonmyeloablative conditioning regimens (defined as lowdose fludarabine and low-dose total body irradiation) appear to have a small risk of progression during the first 3 months after HCT [55]; progression may still occur with reduced-intensity regimens containing anti-T-cell antibodies, but larger studies are needed to confirm this [56]. Although the overall progression rate to pneumonia is only 18%, in allograft recipients receiving more than 1 mg/kg of prednisone, the risk is 40%, and 65% with 2 mg/kg [73]. Parainfluenza virus 3 pneumonia may also occur after autologous HCT, but it mainly arises in the setting of CD34 selection or use of high-dose steroids [73]. Parainfluenza virus 3 pneumonia is often associated with serious pulmonary co-pathogens (53%), such as Aspergillus fumigatus. Factors associated with poor outcome after pneumonia include the presence of co-pathogens and mechanical ventilation [73]. Thus, aggressive diagnostic intervention (i.e. BAL) and therapy are indicated in patients with suspected parainfluenza virus pneumonia. In a large retrospective analysis, both URI and pneumonia due to parainfluenza virus 3 were associated with overall mortality in multivariable models [73]. Treatment and prevention The mortality of pneumonia is approximately 35% in recipients of allografts following high-dose conditioning [40,74,75]. Outcome may be improved in reduced-intensity conditioning regimens [56]. In a retrospective analysis, neither aerosolized ribavirin nor intravenous immunoglobulin led to an improved outcome of pneumonia or a reduction in viral shedding following pneumonia [74]. Systemic ribavirin has only been reported in case reports [76]. Randomized treatment studies have not been performed. Whether earlier antiviral treatment (i.e. pre-emptive treatment for URI) is effective in the prevention of pneumonia or late airflow obstruction is unknown [76]. The association of high-dose steroid treatment with progression to disease might suggest that a reduction of immunosuppression may be useful [73]. However, such an approach has not been tested. The role of immunoglobulin for treatment is also poorly defined. One retrospective analysis did not find a benefit of pooled immunoglobulin given for parainfluenza virus pneumonia [73]. However, the antibody content of pooled preparations is highly variable. The highest titers can be obtained from RSV-specific immunoglobulin, which also contains a high titer of antibodies directed against parainfluenza virus [77]; RSVspecific immunoglobulin is not available in the United States. Infection control is the mainstay of prevention strategies. Unfortunately, current infection control practices seem to be far from perfect in keeping parainfluenza virus out of HCT units, as indicated by the high incidence figures (Table 94.2). HCT units have been prone to persistent parainfluenza virus outbreaks [44,78–80]. Possible explanations for the difficulty in preventing parainfluenza virus from entering HCT units include lack of a vaccine for health-care workers and close contacts, very mild or a lack of symptoms in immunocompetent individuals,
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prolonged asymptomatic shedding in infected patients, and persistence of the virus on environmental surfaces [45]. Influenza viruses Clinical significance Influenza viruses belong to the family of orthomyxoviruses and are enveloped, single-stranded, pleomorphic RNA viruses. Influenza is classified into three major types, of which type A is most common, followed by type B [41,81,82]. Influenza type C is very uncommon, even in the immunocompetent population; there are no reports of influenza C in HCT recipients. Influenza virus infections seem to be less common than RSV and parainfluenza virus infections. Both subtypes can cause infection, although type A appears to be more common. Clinical characteristics are listed in Table 94.1. Risk factors Acquisition of influenza is increased in patients with high-risk underlying disease [83,84]. Progression to severe pneumonia can occur similar to RSV and parainfluenza virus [82], but progression appears to be less common and risk factors for progression differ (Table 94.2). In contrast to parainfluenza virus infection, where corticosteroids are an important risk factor for the development of pneumonia, with influenza virus, lower tract infection appears to be less common in patients treated with corticosteroids [83,84,85]. Also, recipients of low-dose conditioning appear to be less likely to develop pneumonia during the first 3 months after HCT [55]. Interestingly, the clinical presentation in HCT often lacks myalgia and high fever, which is commonly seen in immunocompetent individuals (Fig. 94.3) [45]. Treatment and prevention Effective prevention is available for influenza, which may explain the lower incidence. Health-care personnel, family members, and visitors are advised to be vaccinated against influenza early in the season. Antiviral therapy is available for influenza virus infection; however, available agents have not been studied prospectively in HCT recipients. Due to the recent emergence of resistance against the M2-inhibitors amantadine and rimantidine, with a more favorable side-effect profile, these agents should not be used to treat influenza A [86]. Neuraminidase inhibitors (i.e. zanamavir and oseltamivir) are now available [87,88] and are active against most strains of influenza A and B. Uncontrolled studies suggest that pre-emptive therapy with neuraminidase inhibitors (i.e. oseltamivir and zanamivir) may be effective in preventing progression to lower tract disease [38,83,84,89]. Widespread chemoprophylaxis for susceptible immunosuppressed patients in outbreak situations has been recommended [90], and a case-control study of oseltamivir suggests that this approach is safe [91].
Other community respiratory viruses Human rhinovirus Human rhinovirus (HRhV) is classified as a picornavirus characterized by small, naked, single-stranded RNA. There are approximately 100 serotypes, which usually cause mild upper respiratory tract symptoms in immunocompetent hosts. Using culture techniques, rhinovirus is infrequently reported as a cause of respiratory infection in HCT recipients. However, several reports found the organism in patients with pneumonia. Ghosh et al. reported lower tract disease in seven of 22 patients with HRhV infection [92], while Bowden documented lower tract disease in only one of 29 infected patients [82]. HRhV appears to occasionally cause lower respiratory tract disease [93], although a clear
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RSV
PIV
MPV
Influenza
Negative samples
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Runny nose
Cough
Watery eyes
Sore throat
Shortness Sputum of breath
Wheezing
Sinus Stuffy Sinus congestion headache nose
Muscle aches
Fever >38ºC
Fig. 94.3 Symptoms associated with respiratory virus infection after hematopoietic cell transplantation. The y-axis shows the fraction of patients showing each symptom. MPV, metapneumonia virus; PIV, parainfluenza virus; RSV, respiratory syncytial virus. (Reproduced from [45], with permission.)
causative role is sometimes difficult to ascertain because of the presence of co-pathogens [82,92,94]. Surveillance studies using reverse transcriptase PCR are ongoing to more precisely define the role of HRhV in HCT recipients. There are presently no drugs or vaccines available for the treatment or prevention of rhinovirus infections. Pleconaril, a capsid-binding compound, was the first anti-HRhV antiviral to be submitted to the United States Food and Drug Administration for regulatory approval, but was not approved for licensure for the treatment of colds in adults, primarily based on drug interactions and modest treatment effects. Other antiviral agents, including protease inhibitors, are now being studied. Human coronaviruses Human coronaviruses (HCoVs) are RNA viruses that can cause URI, croup, wheezing, and pneumonia in immunocompetent individuals. Recently, several new strains have been described, including NL63 (identified in 2004) and HKU1 (identified in 2005), in addition to the previously described OC43 and 229E strains [95]. In a recent study, 5.7% of 823 patients admitted to the hospital had HCoV detected. HCoV infection occurred in 8.8% of immunocompromised patients compared with 4.5% of immunocompetent patients. Fourteen (30%) of the 47 HCoV-infected patients, including three HCT recipients, were also infected with other pathogens (RSV and Aspergillus; influenza A; RSV and HRhV, respectively), and these patients had more severe diseases [96]. The four HCoVs have also been detected in solid organ transplant recipients, patients with underlying malignancies, and HIV-infected individuals [97]. HCoV infections have also been associated with longterm decline in forced expiratory volume in 1 second in lung transplant recipients [97]. Surveillance studies are ongoing to better define the incidence and pathogenesis of HCoV in HCT recipients. Currently, there is no specific treatment. Measles virus Measles virus (rubeola), a paramyxovirus, is a recognized cause of giant cell pneumonia, developing with and without rash, and severe CNS disease in immunocompromised hosts, including HCT recipients [98,99]. The incidence of measles is extremely low at most centers due to the widespread use of measles immunization in the general population. However, recently an outbreak among HCT recipients has been reported [100]. During that outbreak, among 122 susceptible patients, clinically
severe disease occurred in one patient, and seven had mild symptoms. Exanthema was present in all of these patients, although it was often atypical. Koplik spots were observed in five patients. All but one patient had fever and nonproductive cough [100]. The use of ribavirin has been reported for measles in two critically ill immunocompromised patients, but no systematic assessment of this intervention can be made [101]. Human metapneumovirus HMPV is a newly discovered negative-sense nonsegmented RNA paramyxovirus [102]. By age 5, virtually all children are seropositive. The virus can cause upper and lower tract infection during the winter season [103]. Serious lower respiratory tract disease associated with HMPV has been reported in immunocompromised patients. HMPV infection occurs in up to 5% of HCT recipients [45]. Whether asymptomatic shedding occurs is controversial [45,104]. The progression rate to lower respiratory tract disease is presently unknown. Among HCT recipients undergoing BAL for clinical and radiographic pneumonia, 3–4% had HMPV detected [105]. Most of these patients were clinically classified as having idiopathic pneumonia syndrome [105]. HMPV disease in HCT recipients immediately post transplant typically presents with upper respiratory symptoms, including fever, nasal congestion, and cough. Once pneumonia develops, rapidly progressive pulmonary infiltrates can occur. This is frequently accompanied by hypotension, septic shock or both [105]. Radiographic findings range from diffuse bilateral alveolar and interstitial infiltrates to emphysema without infiltrate. Histologic assessment has shown that diffuse alveolar damage with hyaline membrane formation, foci of bronchiolitis obliterans and organizing pneumonia, and diffuse alveolar hemorrhage are common [105]. There is no proven treatment for HMPV disease, which may be fatal in HCT recipients [105]. Although intravenous immunoglobulin has been shown to effectively neutralize HMPV in vitro, its effect in vivo is unknown [106]. The role of steroids is also not defined. Ribavirin has been shown to have in vitro antiviral activity against HMPV [106], but there are no reports in regards to treatment with ribavirin. Human bocavirus HBoV is a newly identified human parvovirus originally identified by random PCR amplification/cloning technique from hospitalized children
Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation
in Sweden [107]. HBoV has been detected worldwide [108–110], and appears to be common in young children. Symptoms associated with HBoV infections include rhinorrhea, cough, fever, wheezing, hypoxia, and diarrhea. Viremia is common [111]. To date there is only limited information on the clinical significance of HBoV infection in immunocompromised patients [112]. Infection control practices Recommendations have been summarized by the CDC/American Society for Blood and Marrow Transplantation [90]. Presently, the guidelines are undergoing revision. Hand washing is the single most effective way of preventing the spread of respiratory viruses and should be performed after each patient contact by all health-care personnel and visitors. Respiratory isolation is used for HCT recipients with respiratory virus infections (masks, gowns, gloves, and eye protection) [113]. The use of masks among health-care workers, family members, and asymptomatic patients remains controversial. One nonrandomized study suggests a benefit of masks [68]. However, questions remain on the type of mask, the frequency of changing the mask, and who should wear masks. Not all transplant centers have a universal mask policy. Recent CDC guidelines recommend using masks during patient transport [90]. Another approach that is likely to reduce transmission is to restrict health-care workers and family members from patient contact if they have a URI and systemic symptoms such as rhinorrhea, watery eyes, sneezing, fever, and myalgia [39,70]. While this is not uniformly done in adult patients, most centers restrict small children from direct patient contact during the respiratory virus season due to their predisposition to URIs and prolonged high-titer shedding [90]. A strategy that restricts access of personnel and other close contacts will only be effective for infections that present with significant drainage and symptoms, such as RSV, rhinovirus, HMPV, and influenza infections. Infections which may present with only mild symptoms (e.g. parainfluenza virus infections) may be missed by this approach [44,45].
Human herpesviruses-6, -7, and -8 Human herpesvirus-6
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Whether monitoring for HHV-6 viremia is useful in predicting these clinical syndromes is not known. In solid organ transplant recipients, there seems to be an interaction between HHV-6 and CMV [132–137]. One study showed a trend toward more CMV infection among patients with over 100 copies of HHV-6 DNA/5 × 104 lymphocytes in peripheral blood lymphocytes [117], and another study suggests interaction of HHV-6 and CMV in BAL fluid of HCT recipients [127]. Data in HCT recipients are limited and results are inconclusive. HHV-6 reactivation typically occurs 2–4 weeks after HCT [116,122–124], preceding CMV reactivation, which occurs a median of 40–42 days following transplant [138]. An interaction between HHV-6 and CMV may be explained by a generalized immunosuppressive effect of HHV-6 [139–142]. Early HHV-6 reactivation leads to a delay in CMV-specific T helper immune responses in HCT recipients [143], but the exact mechanism of the interaction remains unclear. Treatment and prevention Currently, there is no widely accepted clinical approach to HHV-6 screening or prevention following HCT. In vitro studies suggest that HHV-6 is susceptible to foscarnet, ganciclovir, and cidofovir, and moderately susceptible to acyclovir [144–146]. Foscarnet and ganciclovir have been used to treat HHV-6-associated disease, and a decline in viral load has been documented with therapy [147,148]. The high risk of HHV-6 disease in cord blood transplant recipients may justify PCR surveillance and pre-emptive therapy [131]; however, no randomized trials or systematic evaluations have been reported for prophylaxis or pre-emptive therapy strategies. Human herpesvirus-7 HHV-7 is a β herpesvirus (similar to HHV-6 and CMV) and can be detected by PCR [149]. One study found both HHV-6 and HHV-7 in peripheral blood and marrow of healthy subjects [150]. The impact of HHV-7 after HCT has been examined in longitudinal studies [114,125]. So far, it has not been possible to assign a clinical syndrome to HHV-7 – with the exception of one recent report of a CNS infection with myelitis in an unrelated donor transplant recipient [151].
Clinical significance Human herpesvirus-6 (HHV-6) is a β herpesvirus with similarities to CMV and HHV-7. Several longitudinal studies have examined the role of HHV-6 after HCT [114–117]. Similar to other HHVs, it is ubiquitous, infecting most people by 2 years of age [118]. After primary infection, the virus persists in lymphocytes and salivary glands, with 95% of adults having detectable viral DNA in their peripheral blood mononuclear cells and saliva [119]. During times of immunosuppression, the virus may reactivate and cause disease. Type B is far more common than type A. Rarely, HHV-6 DNA is integrated into the human DNA [120]. This may lead to continued detection of the virus in the blood after HCT [120,121]. It is estimated that this occurs in approximately 1% of patients [120]; detection of integrated DNA does not require treatment. Active HHV-6 infection has been documented in 38–46% of patients following HCT [116,122–125]. Clinical signs and symptoms that have been associated with HHV-6 include rash, a CNS syndrome consisting of encephalitis and impaired memory [126], interstitial lung disease [119,127], and delayed platelet engraftment [117]. Secondary graft failure has been documented in case reports [128]. Early nonrelapse mortality has also been associated with HHV-6 in one small series [129]. The best evidence exists for encephalitis. HHV-6 encephalitis appears to be particularly common in cord blood transplant recipients [130,131]. HHV-6-associated encephalitis is diagnosed by detection of HHV-6 DNA in the cerebrospinal fluid and compatible clinical symptoms [126].
Human herpesvirus-8 HHV-8 is a γ herpesvirus (similar to EBV). HHV-8 is associated with Kaposi’s sarcoma in HIV-infected individuals. Two recent case series from areas with high seroprevalence suggest that it can cause febrile syndrome and marrow failure in HCT recipients [152,153]. HHV-8 can be detected by PCR [146]. While transmission from donor to recipient has been documented in the kidney transplant setting [154], there have been no reports of transmission via stem cells or cord blood to date [155]. A case of HHV-8-associated Kaposi’s sarcoma has been reported in an HCT recipient [156].
Popovaviruses BK virus BK and JC virus are human polyomaviruses, and both can cause disease in HCT recipients [157]. BK virus is a nonenveloped DNA virus which multiplies in the nucleus. Seroprevalence in the population is between 60% and 80%. Primary infection usually occurs during childhood, possibly by the respiratory route. The virus can be detected by PCR [158,159]. BK virus is associated with postengraftment hemorrhagic cystitis after HCT [160,161]. BK-associated hemorrhagic cystitis appears to be more
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common after high-dose conditioning than after reduced-intensity conditioning [162]; unrelated donor status is an additional risk factor [162]. The statistical and epidemiologic evidence that links BK virus to hemorrhagic cystitis is quite strong. However, tissue-invasive BK hemorrhagic cystitis has only recently been demonstrated in a few cases [163,164]. Whether BK virus causes hemorrhagic cystitis through lytic viral infection remains controversial. An alternative hypothesis is that BK virusassociated hemorrhagic cystitis in HCT recipients is an immune reconstitution phenomenon [165]. According to this theory, hemorrhagic cystitis is the result of an inflammatory process that occurs when antigenspecific T cells recognize BK virus in the bladder. Immune reconstitution disease has been described for JC virus infection in HIV infected individuals treated with antiretroviral combination therapy. The immune reconstitution model is probably not sufficient to explain BK virusassociated hemorrhagic cystitis in the HCT setting since BK virus has been shown in bladder biopsies, and patients can develop postengraftment hemorrhagic cystitis during profound lymphocytopenia and in the presence of high-dose steroids [163,166]. Several cases of BK virus-associated pneumonia have been reported [167]. After kidney transplantation, BK virus is an important cause of late nephritis [168]. No comprehensive data exist on BK nephropathy in HCT recipients, although individual cases have been reported [169]. Although BK virus is often detected in urine by PCR, an association with clinical symptoms may be difficult to prove [170]. High viral load in urine was associated with a higher risk of BK-associated cystitis, but no threshold was defined [158]. BK plasma viremia is associated with high risk of nephropathy in renal transplant recipients [159,168]. Three recent studies indicate that BK viremia is also a marker of BK virusassociated hemorrhagic cystitis in HCT recipients [161,163,166]. Erard et al. showed that BK viremia occurs in about one-third of HCT recipients at a median of 41 days after transplantation, and is associated with post-engraftment hemorrhagic cystitis [163]. In a case-control study of hemorrhagic cystitis, BK plasma levels greater than 10,000 copies/mL were highly associated with post-engraftment BK-associated hemorrhagic cystitis [166]. However, detection in blood was not associated with hemorrhagic cystitis in two earlier studies [158,171]. In a study of pediatric HCT recipients, BK virus-associated hemorrhagic cystitis occurred between day 24 and 50 after transplant in nine of 117 patients. Infection was characterized by a long duration, correlated with use of busulfan, and resulted in bladder tamponade in two of nine patients [172]. There is no established treatment other than supportive care. Cidofovir has activity in vitro against murine polyomavirus, but there are only anecdotal treatment results in humans to date [173]. Pharmacokinetic studies have demonstrated that cidofovir accumulates in renal tissue and urine [165]. Treatment of infections localized to the urinary tract, with doses lower than those used in systemic CMV infection, might thus be effective. Initial data from renal transplant patients with BK nephropathy suggest that lower doses of cidofovir may be sufficient to treat BK disease [174]. Studies are ongoing to define the optimal dose of cidofovir for treatment of BK virus-associated disease. Leflunomide, an immunosuppressive agent used for treatment of rheumatoid arthritis, also appears to have antiviral activity and has been used in uncontrolled case series [175]. Gyrase inhibitors such as fluoroquinolones appear to have moderate preventative activity [157]. Prospective randomized trials are needed to assess the treatment options.
HCT, and in patients with hematologic malignancies [176–180]. Overall, this complication seems to be rare. PCR detection of JC virus in the cerebrospinal fluid is used for diagnosis [181]; however, its sensitivity is not 100%. Therefore, brain biopsies may be required. Additionally, plasma PCR may also be useful [180]. The optimal treatment has not been defined. Cidofovir has activity in vitro, but little is known about its in vivo efficacy in HCT recipients [182]. A study in HIV-infected individuals failed to show a therapeutic effect of cidofovir. Other treatment options include interleukin-2, cytarabine, chlorpromazine or the antipsychotic drugs ziprasidone, risperidone, mirtazipine and olanzapine [180,183]. However, only individual patients have been treated with these drugs, which makes an assessment of efficacy difficult [184]. Human papillomavirus Human papillomavirus (HPV) is a nonenveloped DNA virus. HPV infections occur rarely after transplantation [185,186]. HPV causes cutaneous infections (warts), and an association with cervical carcinoma has been identified. In one series, three of 238 allogeneic HCT recipients developed anogenital condylomata associated with HPV [185]. Rapid progression of pre-existing warts has been reported [187]. One retrospective study found a higher rate of cervical cytologic abnormalities, both before and after marrow transplantation, when compared with the general population [188]. Severe HPV disease has been reported in HCT recipients with severe combined immunodeficiency caused by common cytokine receptor subunit of janus kinase-3 (JAK-3) deficiency [189]. The clinical significance of these findings is unknown. There are no recommendations about the appropriate use of the recently licensed HPV vaccine in HCT recipients.
Parvovirus B19 Parvoviruses are made of small, naked, single-stranded DNA. Parvovirus infects a variety of animals, including cats and dogs. In 1975, human parvovirus B19 was isolated from asymptomatic blood donors. Seroepidemiologic studies showed evidence of past infection in approximately 60% of young adults. It is most likely spread by respiratory transmission or by blood. Parvovirus B19 can infect erythroid progenitor cells. Parvovirus B19 commonly is associated with aplastic crisis in patients with hemolytic anemia. It is also the cause of erythema infectiosum, a self-limited disease of childhood characterized by fever, fatigue, myalgias, a lace-like rash, and a “slapped cheek” appearance. Parvovirus B19 also is associated with some cases of rheumatoid arthritis. Immunity to parvovirus B19 is thought to be primarily humoral; neutralizing antibodies to capsid protein have a major role in host defense. IgM antibody detection may be useful in the diagnosis of acute infection in immunocompetent persons. The reliability of serologic testing in HCT recipients is questionable. In addition to transient marrow failure, parvovirus B19 can cause chronic anemia, and rarely, pancytopenia in HCT recipients [190–192]. Blood PCR is useful as a diagnostic tool [191]. Intravenous immunoglobulin is effective in treating parvovirus B19 symptomatic infection [193]. Prophylactic intravenous immunoglobulin seems to have a protective effect against parvovirus B19, although this has not been established in a randomized study [194].
JC virus JC virus is a nonenveloped DNA virus classified as a polyomavirus. JC virus is the cause of progressive multifocal leukoencephalopathy in immunosuppressed patients. Several cases have been reported after
Enteroviruses Enteroviruses belong to the picornavirus family. They include polioviruses, Coxsackie viruses, echoviruses, and other serotypes simply
Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation
referred to as enteroviruses. They are naked virions containing singlestranded RNA that replicate in the cellular cytoplasm. Both tissue culture and molecular methods are used for diagnosis. The virus can be detected in throat washes, stool, cerebrospinal fluid, and other body fluids. Asymptomatic infection is common in normal hosts, and these viruses are most prevalent during the summer and fall months. Infection is usually by the fecal–oral route; shedding of the virus from the oropharynx may persist for 1–4 weeks, and from the gastrointestinal tract, for up to 18 weeks. Viremic spread in the host is common and can affect a variety of organs. Infection by enteroviruses in HCT recipients has been reported [11,195,196]. One outbreak of Coxsackie A1-associated diarrhea affected patients in a 13-bed unit over a 3-week period, resulting in death in six of seven infected patients [197]. Coxsackie virus was also identified in stools of four of 78 patients with gastrointestinal infections [198]. All four patients with Coxsackie virus died; two had co-infections with adenovirus and rotavirus [198]. Additional cases of disseminated and life-threatening infection have been reported [199–203]. Severe cases of echovirus infections have been reported, including a disseminated infection with pneumonitis and pericarditis [204,205]. Treatment of enterovirus infections is supportive. The antiviral drug pleconaril is active against enteroviruses, but the drug is not licensed by the Food and Drug Administration.
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Rotaviruses Rotaviruses consist of double-stranded RNA and have a double-shelled capsid. They infect the duodenum and the jejunum, causing vomiting and diarrhea in normal hosts, occasionally accompanied by low-grade fever. Infection usually occurs in the winter, and transmission is by the fecal–oral route. During one outbreak, contaminated toys in a cancer center play room were implicated as the mode of transmission [206]; hospital personnel can transmit the virus as well [207]. Diagnosis is by electron microscopy, enzyme-linked immunoabsorbent assay or PCR. Repeated testing does not appear to increase the diagnostic yield [208]. Rotavirus is a common cause of diarrhea in HCT recipients presenting with gastroenteritis [195,198,209–212], and fatalities have been reported [195]. There is no proven specific treatment other than supportive care. Oral immunoglobulin has been used in individual cases [213], but this approach has not been studied in larger series or randomized trials.
Caliciviruses Caliciviruses (Norovirus [prototype Norwalk virus] and Sapovirus [prototype Sapporo virus]) are made of small, nonenveloped, single-stranded RNA-containing virions that have been associated with outbreaks of
Table 94.3 Overview of antibody preparations and antiviral agents with in vitro activity against other viral infections after hematopoietic cell transplantation (HCT). For details about indications, doses and limitations, see text Virus
Drugs
Clinical use in HCT recipients*
Comment
Adenovirus
Ribavirin (intravenous) Cidofovir
Adenovirus disease Adenovirus disease, pre-emptive therapy
RSV
Ribavirin
RSV pneumonia, pre-emptive therapy for upper respiratory infection Combination therapy for RSV pneumonia Combination therapy for RSV pneumonia
High rate of failures, possibly due to selection bias Response to therapy reported, no direct comparison with ribavirin reported Moderate-to-strong evidence for pneumonia,
Human metapneumonia virus Parainfluenza viruses
RSV-specific monoclonal antibodies (palivizumab) Pooled immunoglobulin Ribavirin
Combination with immunoglobulin preparations appear to be more effective when RSV-specific preparations are used (Table 94.2)
No data
Ribavirin Pooled immunoglobulin
Parainfluenza virus pneumonia Combination therapy for parainfluenza pneumonia
Influenza viruses
M2-Inhibitors (amantidine, rimantidine) Neuraminidase inhibitors (oseltemavir, zanamivir)
Influenza infection and disease, prophylaxis; combination therapy Influenza infection and disease, prophylaxis; combination therapy
Rhinovirus HHV-6
Pleconaril Ganciclovir, valganciclovir Foscarnet Cidofovir Foscarnet Cidofovir Ganciclovir, valganciclovir Cidofovir Leflunomide JC virus Cidofovir, risperidone, Mirtazipine interleukin-2, cytarabine
No data HHV-6 CNS disease HHV-6 CNS disease No data No data No data No data Hemorrhagic cystitis
Small case series
Multifocal leukoencephalopathy
Case studies only
HHV-7 HHV-8 BK virus
CNS, central nervous system; HHV, human herpes virus; RSV, respiratory syncytial virus. * All clinical results in HCT recipients are from non-randomized studies.
No apparent effect for pneumonia of either ribavirin or the combination with immunoglobulin; pre-emptive therapy not systematically tested; reduction of immunosuppression may be effective (see text) M2-inhibitors only effective against influenza A, most isolates now resistant; both groups of drugs extensively studied in immunocompetent subjects; neuraminidase inhibitors appear to have improved toxicity profile, although experience in HCT is limited Drug is not Food and Drug Administration approved Both foscarnet and ganciclovir have been used for the treatment of CNS disease; anti-HHV-6 activity does not appear to be HHV-6 variant-dependent [144]
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diarrhea [214]. There have been no reports of calicivirus outbreaks in HCT recipients, but the potential is high, especially for Noroviruses. Diagnosis is by PCR or electron microscopy. Treatment is supportive.
Conclusion and future perspective Major progress has been made in the diagnosis and prevention of viral infections after HCT over the last decade. Perhaps the most impressive examples are the prevention of CMV disease by ganciclovir and the recognition of the host defense mechanisms that control CMV. During the same time period, the significance and our knowledge of other viral infections has increased, especially that of respiratory viruses, HHV-6, BK, and adenoviruses. The increasing availability of sensitive and specific multiplex diagnostic platforms that detect a large number of viruses will soon lead to better understanding of the infection epidemiology, improved infection control, and the development of more therapeutic options [215]. A systematic and comprehensive evaluation of the disease burden of viral infections is needed. Historically, direct organ involvement, such as pneumonia or gastrointestinal disease, and death were reported as endpoints; however, data on airflow obstruction (for respiratory viruses), health-care utilization, including duration of hospitalization, intensive care, and mechanical ventilation are important as well. For most of the infections discussed in this chapter, very little information is available on disease pathogenesis. Although several studies point towards lymphopenia as an important risk factor for severe manifestations of disease, the exact host defense mechanisms that lead to the often devastating consequences of viral infections in HCT recipients are
poorly understood. Studies of the importance of virus load, T-cell and humoral immunity, as well as cytokine and chemokine expression and genetics, are needed. Genetic studies may be useful to better understand the risk of virus acquisition and progression to end-organ and fatal disease. Most epidemiologic and outcome studies to date have focused on the early period after transplantation. More information is needed on the risk of progression and outcome late after HCT (i.e. after the first 3 months), when lymphopenia is rare. For instance, there may be bidirectional relationships between respiratory viruses and lung function late after transplantation: viral infections may lead to prolonged airflow obstruction, and pre-existing airflow obstruction may predispose individuals to progression from upper to lower respiratory tract disease and a poor outcome. Major progress is needed with regards to prevention and treatment options for the viral infections discussed in this chapter. With the exception of treatment for influenza virus, there is a very limited armamentarium of potent and easy to administer drug treatments (Table 94.3) [215]. Drug development, as well as research of the host defense mechanisms, may ultimately lead to novel drug or immune-based interventions. Immune augmentation strategies could consist of vaccination, T-cell or antibody therapy, or nonspecific immune reconstitution strategies (e.g. keratinocyte growth factor). Finally, the explosion of technical capabilities has led to the discovery of several new viruses in recent years, with novel coronaviruses, HBoV, polyomaviruses, and arenaviruses [216] being the latest additions. It is likely that additional viruses will be discovered. Well-designed disease association studies will be needed in order to determine the significance of these viruses in HCT recipients.
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stem-cell transplantation in patients with severe combined immune deficiency caused by common gamma cytokine receptor subunit or JAK-3 deficiency. Lancet 2004; 363: 2051–4. Azzi A, Fanci R, Ciappi S, Zakrzewska K, Bosi A. Human parvovirus B19 infection in bone marrow transplantation patients. Am J Hematol 1993; 44: 207–9. Schleuning M, Jager G, Holler E et al. Human parvovirus B19-associated disease in bone marrow transplantation. Infection 1999; 27: 114–17. Florea AV, Ionescu DN, Melhem MF. Parvovirus B19 infection in the immunocompromised host. Arch Pathol Lab Med 2007; 131: 799–804. Kurtzman G, Frickhofen N, Kimball J, Jenkins DW, Nienhuis AW, Young NS. Pure red-cell aplasia of 10 years’ duration due to persistent parvovirus B19 infection and its cure with immunoglobulin therapy [see comments]. N Engl J Med 1989; 321: 519–23. Frickhofen N, Arnold R, Hertenstein B, Wiesneth M, Young NS. Parvovirus B19 infection and bone marrow transplantation. Ann Hematol 1992; 64(Suppl): A121–4. Yolken RH, Bishop CA, Townsend TR et al. Infectious gastroenteritis in bone-marrowtransplant recipients. N Engl J Med 1982; 306: 1010–12. Chakrabarti S, Collingham KE, Stevens RH, Pillay D, Fegan CD, Milligan DW. Isolation of viruses from stools in stem cell transplant recipients: a prospective surveillance study. Bone Marrow Transplant 2000; 25: 277–82. Townsend TR, Bolyard EA, Yolken RH et al. Outbreak of Coxsackie A1 gastroenteritis: a complication of bone-marrow transplantation. Lancet 1982; 1: 820–3. Cox GJ, Matsui SM, Lo RS et al. Etiology and outcome of diarrhea after marrow transplantation: a prospective study. Gastroenterology 1994; 107: 1398–407. Aquino VM, Farah RA, Lee MC, Sandler ES. Disseminated coxsackie A9 infection complicating bone marrow transplantation. Pediatr Infect Dis J 1996; 15: 1053–4. Galama JM, de Leeuw N, Wittebol S, Peters H, Melchers WJ. Prolonged enteroviral infection in a patient who developed pericarditis and heart failure after bone marrow transplantation. Clin Infect Dis 1996; 22: 1004–8. Gonzalez Y, Martino R, Badell I et al. Pulmonary enterovirus infections in stem cell transplant recipients. Bone Marrow Transplant 1999; 23: 511–13. Fischmeister G, Wiesbauer P, Holzmann HM, Peters C, Eibl M, Gadner H. Enteroviral meningoencephalitis in immunocompromised children after matched unrelated donor-bone marrow transplantation. Pediatr Hematol Oncol 2000; 17: 393–9. Tan PL, Verneris MR, Charnas LR, Reck SJ, van Burik JA, Blazar BR. Outcome of CNS and pulmonary enteroviral infections after hematopoietic cell transplantation. Pediatr Blood Cancer 2005; 45: 74–5. Biggs DD, Toorkey BC, Carrigan DR, Hanson GA, Ash RC. Disseminated echovirus infection complicating bone marrow transplantation. Am J Med 1990; 88: 421–5. Schwarer AP, Opat SS, Watson AM, Spelman D, Firkin F, Lee N. Disseminated echovirus infection
Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation
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after allogeneic bone marrow transplantation. Pathology 1997; 29: 424–5. Rogers M, Weinstock DM, Eagan J, Kiehn T, Armstrong D, Sepkowitz KA. Rotavirus outbreak on a pediatric oncology floor: possible association with toys. Am J Infect Control 2000; 28: 378– 80. Kruger W, Stockschlader M, Zander AR. Transmission of rotavirus diarrhea in a bone marrow transplantation unit by a hospital worker. Bone Marrow Transplant 1991; 8: 507–8. Kamboj M, Mihu CN, Sepkowitz K, Kernan NA, Papanicolaou GA. Work-up for infectious diarrhea after allogeneic hematopoietic stem cell transplantation: single specimen testing results in cost savings without compromising diagnostic yield. Transpl Infect Dis 2007; 9: 265–9. van Kraaij MG, Dekker AW, Verdonck LF et al. Infectious gastro-enteritis: an uncommon cause of diarrhoea in adult allogeneic and autologous stem cell transplant recipients. [In process citation.] Bone Marrow Transplant 2000; 26: 299–303.
210. Troussard X, Bauduer F, Gallet E et al. Virus recovery from stools of patients undergoing bone marrow transplantation. Bone Marrow Transplant 1993; 12: 573–6. 211. Yeager AM, Kanof ME, Kramer SS et al. Pneumatosis intestinalis in children after allogeneic bone marrow transplantation. Pediatr Radiol 1987; 17: 18–22. 212. Liakopoulou E, Mutton K, Carrington D et al. Rotavirus as a significant cause of prolonged diarrhoeal illness and morbidity following allogeneic bone marrow transplantation. Bone Marrow Transplant 2005; 36: 691–4. 213. Kanfer EJ, Abrahamson G, Taylor J, Coleman JC, Samson DM. Severe rotavirus-associated diarrhoea following bone marrow transplantation: treatment with oral immunoglobulin. Bone Marrow Transplant 1994; 14: 651–2. 214. Green KY. The role of human caliciviruses in epidemic gastroenteritis. Arch Virol Suppl 1997; 13: 153–65. 215. Nichols WG, Peck-Campbell AJ, Boeckh M. Respiratory viruses other than influenza virus:
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impact and therapeutic advances. Clin Microbiol Rev 2008; 21: 274–90. Palacios G, Druce J, Du L et al. A new arenavirus in a cluster of fatal transplantassociated diseases. N Engl J Med 2008; 358: 991–8. Martino R, Ramila E, Rabella N et al. Respiratory virus infections in adults with hematologic malignancies: a prospective study. Clin Infect Dis 2003; 36: 1–8. Whimbey E, Champlin RE, Englund JA et al. Combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients. Bone Marrow Transplant 1995; 16: 393–9. Ghosh S, Champlin RE, Englund J et al. Respiratory syncytial virus upper respiratory tract illnesses in adult blood and marrow transplant recipients: combination therapy with aerosolized ribavirin and intravenous immunoglobulin. Bone Marrow Transplant 2000; 25: 751–5.
95
Simone I. Strasser & George B. McDonald
Gastrointestinal and Hepatic Complications
Introduction The frequency and severity of intestinal and liver complications of hematopoietic cell transplantation (HCT) have changed in the last few years. Fewer patients have severe gut and liver damage from acute graftversus-host disease (GVHD), as the development of more effective strategies to prevent GVHD has had a marked effect on its clinical presentation. Protracted jaundice is now far less common, as the frequency of sinusoidal liver injury caused by myeloablative regimens has fallen, and prophylaxis with ursodiol has been effective in mitigating cholestatic liver injury. Antiviral agents have almost completely eliminated intestinal and hepatic infections caused by herpes simplex virus (HSV) and cytomegalovirus (CMV), but sporadic cases of adenovirus, rotavirus, astrovirus, hepatitis B virus (HBV), and norovirus infection may be seen. Fungal infections of the intestine and liver have become uncommon since the advent of more effective prophylaxis. Clinicians have become adept at recognizing unusual presentations of infectious diseases, for example abdominal pain as a manifestation of varicella zoster virus (VZV) infection, and diarrhea with multiorgan failure with adenovirus infection. New challenges, however, have come from the greater use of human leukocyte antigen (HLA)-mismatched and unrelated donors, the increase in certain diseases as indications for HCT (e.g. sickle cell disease, renal carcinoma, myeloma, and autoimmune diseases), the older age of patients being considered for HCT, and the growth of reduced-intensity allogeneic HCT. This chapter is organized by the problems a clinician might encounter in caring for an HCT recipient, in the chronologic order in which these problems are likely to appear. Differential diagnoses of specific problems provide a framework for addressing clinical issues.
Evaluation of intestinal and liver problems before transplant Ulcers, tumors, and infections in the gastrointestinal tract Mucosal ulcerations may bleed profusely when platelet counts drop after HCT, and in immunocompromised patients, ulcers may have an infectious etiology (e.g. CMV, HSV or fungal infection) that requires specific antimicrobial treatment. Thus, symptoms of esophageal pain, heartburn, dysphagia, abdominal pain, nausea, and vomiting should be investigated
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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with endoscopy before HCT, and all intestinal ulcerations healed before the start of conditioning therapy. The goal of pre-HCT therapy in patients with ulcerative colitis or Crohn’s disease is to minimize the extent of gross intestinal ulceration to lower the risk of bleeding after HCT. One must also rule out infections as a cause of colonic ulceration. CMV, Entamoeba histolytica, and clostridial infections are causes of colonic ulceration that may mimic inflammatory bowel disease. CMV disease before transplant is associated with a high risk for early CMV disease and death after HCT [1]. Selected patients with ulcerative colitis and Crohn’s disease have undergone both allogeneic and autologous HCT without complications of bleeding, perforation or dissemination of microorganisms [2,3]. Long-term resolution of Crohn’s disease has been observed following myeloablative allogeneic HCT, and autologous HCT has resulted in improvement as well [2,3]. Patients who are to undergo HCT for lymphoma, leukemia in relapse, myeloma or metastatic breast cancer may have intestinal involvement with malignant cells. Cytoreductive therapy results in necrosis of these cells. Both large intestinal ulcers and perforation due to tumor lysis have been seen, but the low frequency of this complication (estimated at less than one case in 1000 HCTs) does not warrant screening in asymptomatic patients. Intra-abdominal abscesses and fistulae should be managed surgically before HCT. Patients over the age of 50 are at risk for the development of colorectal polyps and cancer. These are seldom considerations in younger patients, but the presence of occult blood in stool specimens from patients over 50 years should prompt endoscopic evaluation, including colonoscopy. Diarrhea Patients with diarrhea should be investigated for organisms that may cause morbidity during the period of immunosuppression after HCT (E. histolytica, Strongyloides, Giardia lamblia, Cryptosporidium, clostridial infections, CMV, rotavirus, and adenovirus) [1,4–6]. Persistent eosinophilia in a patient from an area where parasites are endemic is also an indication for screening for parasitic disease, including coccidial and microsporidial organisms if the patient is immunosuppressed. Specific therapy is available for amebiasis, strongyloidiasis, giardiasis, other helminths, and most coccidia and microsporidia. Cryptosporidiosis may be resistant to therapy in an immunosuppressed patient [5], but restoration of normal immunity after allogeneic HCT can result in clearance of cryptosporidia [7]. Similarly, protracted diarrhea related to immune dysregulation can be treated with allogeneic HCT, for example by restoration of T regulatory cells in children with IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome [8].
Gastrointestinal and Hepatic Complications
Other treatable causes of diarrhea in patients with hematologic malignancies include clostridial infections (C. difficile, C. perfringens, and C. septicum), CMV enterocolitis, adenovirus infections, and overgrowth with Candida albicans. The patient who has had previous typhlitis during neutropenia can present problems if there is persistent fever or tenderness over the cecum. Typhlitis is a syndrome of cecal edema, mucosal friability and ulceration, with fever often associated with polymicrobial sepsis; its cause is usually intestinal clostridial infection, particularly C. septicum [9]. After treatment, the risk of post-HCT typhlitis is no different than that of other patients. Pericolic phlegmons, abscesses, and persistent colitis caused by clostridial infections must be treated before the start of conditioning therapy. Perianal pain Pain near the anal canal in a granulocytopenic patient is due to bacterial infection until proven otherwise. Most anorectal infections begin in the anal glands deep to the crypts at the dentate line. Inflammation of anal crypts may cause pain in the absence of an abscess. Abscesses tend to originate in the intersphincteric space and may extend into the perianal, ischiorectal, or supralevator space [10]. Extensive supralevator and intersphincteric abscesses may be present without being apparent on external examination. Perianal infections must be dealt with before HCT, as extensive tissue necrosis and septicemia may result from uncontrolled infection [11,12]. Antibiotic treatment should cover anaerobic as well as aerobic bacteria [13]. Perineal HSV infection and rarely fungal infections may also lead to painful ulcerations. Fungal liver infections A search for hepatic fungal infection should be undertaken in patients with tender hepatomegaly, fevers, and abnormal liver enzymes. Diagnosis may be established by sensitive liver imaging (high-resolution computed tomography [CT] or magnetic resonance imaging [MRI]) [14] in conjunction with fungal biomarkers such as serology for fungal antigens (galactomannan and glucan assays) [15,16], the polymerase chain reaction (PCR) or culture of liver biopsy material. Fungal liver infection should be controlled with systemic antifungal agents, with newer agents offering reduced toxicity and a broader range of coverage [17,18]. Therapy should be continued through HCT until engraftment is established, which can effect resolution of intractable fungal liver abscesses [19]. Viral hepatitis in allogeneic HCT donors Hepatitis B and hepatitis C will be transmitted from viremic donors to transplant recipients [20]. When equally HLA-matched donors are available, a donor who is not infected by hepatitis viruses should be selected. If the most suitable donor has chronic hepatitis B or C, however, preventing passage of virus and mitigating the effects of infection in the recipient become priorities. Oral nucleos(t)ide analogs, such as lamivudine, adefovir, entecavir, telbivudine, and tenofovir result in rapid HBV suppression and, where possible, should be used to reduce viral load prior to stem cell collection [21,22]. However, HBV may persist in donor peripheral blood stem cells despite clearance from serum [23]. If donor hematopoietic cells are rendered negative for HBV DNA before harvest, passage can be prevented [22]. Regardless of whether a donor has been treated with antiviral therapy, a recipient transplanted from a hepatitis B surface antigen (HBsAg)-positive donor should receive prophylaxis with oral antiviral drugs. In addition, hepatitis B vaccination administered to anti-HBs-negative recipients prior to the start of chemotherapy may reduce the development of post-HCT hepa-
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titis B. Antiviral treatment after HCT should be continued for at least 1 year. HBsAg negative-, anti-hepatitis B core antigen (HBc)-positive donors are viremic in fewer than 5% of cases and can be used as donors if their serum and peripheral blood stem cells are HBV DNA negative using a sensitive quantitative PCR assay. A donor who is naturally antiHBs positive may be the preferred donor if the recipient is HBsAg positive or anti-HBc positive, as adoptive transfer of immunity can effect clearance of virus [24]. If a preferred donor is hepatitis C virus (HCV) antibody and RNA positive, HCV will usually be transmitted, and the rate of spontaneous viral clearance is likely to be low. If time permits, treatment of the donor with pegylated interferon plus ribavirin prior to harvest of donor cells may render them nonviremic and much less likely to transmit infection [25,26]. New small-molecule antiviral drugs in current development may offer clinical benefit in rendering donors nonviremic. If virus is transmitted, the acute phase of HCV infection may cause elevated liver enzymes at 2–3 months post HCT, after recovery of T-cell function; however, severe hepatitis is rare, and the outcome in 10 years of followup is no different than in transplant recipients without hepatitis C infection [27]. In the long-term, antiviral treatment with pegylated interferon plus ribavirin should be offered to the recipient who remains with active hepatitis, as such a patient is at risk for development of cirrhosis and hepatocellular carcinoma [28]. Chronic liver disease in candidates for HCT Patients with active liver disease, particularly those with severe hepatic fibrosis or cirrhosis, are at increased risk for fatal sinusoidal obstructive syndrome (SOS; formerly known as veno-occlusive disease of the liver) following hepatotoxic myeloablative regimens, and the presence of cirrhosis may provide a contraindication to any high-dose conditioning regimen. Patients with cirrhosis are at risk for fatal hepatic decompensation after HCT even if given a reduced-intensity conditioning regimen [29]. Liver biopsy should be considered if there is a clinical suspicion of cirrhosis or extensive fibrosis resulting from chronic viral infection, ethanol or nonalcoholic steatohepatitis. This caution also extends to patients with myelofibrosis, in whom extramedullary hematopoiesis can lead to extensive sinusoidal fibrosis. A reduced-intensity conditioning regimen may allow congenitally immunodeficient children with liver disease to be successfully transplanted without SOS, with subsequent normalization of liver abnormalities [30]. Hepatitis B-infected HCT recipients are at risk for severe hepatitis flares and fulminant liver failure. In the absence of antiviral prophylaxis, HBV reactivation is common, and fatal fulminant hepatitis develops in approximately 15% [20]. Prophylaxis with oral nucleoside therapy starting prior to chemotherapy is therefore recommended for all HBsAgpositive recipients. Even in patients with isolated anti-HBc antibodies, there is a 35% risk of post-HCT HBV reactivation, usually during treatment for acute GVHD; the pathogenesis of infection in these cases is related to a reservoir of virus within the hepatocytes [31]. Severe hepatitis B reactivation has also been seen in anti-HBc/anti-HBs-positive patients and in a patient with occult hepatitis B [32]. Patients from areas where hepatitis B is endemic need close scrutiny after HCT. Recent liver dysfunction in candidates for HCT Patients who come to HCT following recent chemotherapy or radiation therapy may be at risk for unexpected toxicities following myeloablative conditioning regimens, particularly if these therapies have resulted in liver dysfunction. Many chemotherapy drugs cause liver dysfunction either directly (cholestatic injury, hepatocyte necrosis or sinusoidal damage) or indirectly (cholestasis caused by sepsis or endotoxemia, i.e.
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cholangitis lenta) (reviewed in [33,34]). Although recent exposure to standard chemotherapy has not been associated with an increased risk of fatal SOS after cyclophosphamide (CY)-based regimens [35], patients with persistent jaundice and elevations of aminotransferase enzymes are likely at risk. Two newer drugs, Imatinib (Gleevec) and gemtuzumab ozogamicin (Mylotarg) deserve special mention in this context. Imatinib mesylate may cause acute hepatocellular necrosis and multiacinar collapse, with eventual healing by focal fibrosis [36,37]; we have successfully given CY-based myeloablative conditioning to patients who recovered from the acute injury but who had patchy fibrosis on biopsy. Gemtuzumab ozogamicin causes sinusoidal liver injury in 3–15% of patients [38]; if a patient is transplanted following a hepatotoxic myeloablative conditioning regimen within 3 months of exposure to high doses of gemtuzumab ozogamicin, SOS develops in 15–40% of cases [38,39].
Table 95.1 Differential diagnosis of anorexia, nausea and vomiting after transplant
Gall bladder and bile duct stones
Infectious causes in bold are now so rare that they should be considered only when patients are at risk.
HCT candidates with asymptomatic gallstones (incidentally discovered during a CT scan or ultrasound) do not require operative intervention [40]. Patients with symptomatic gall bladder or common duct stones are at significant risk of sepsis after HCT, and consideration should be given to pretransplant cholecystectomy or an endoscopic biliary procedure. Iron overload HCT candidates with diseases such as thalassemia, aplastic anemia, and chronic leukemia or lymphoma may come to HCT with marked hepatic iron overload, best documented by either measurement of iron in liver biopsy tissue or iron-specific MRI imaging (FerriScan) [41]. Excess iron may interfere with Kupffer cell function and predispose to mold infections [42–44]. In patients with extreme iron overload, effective pre-HCT chelation therapy improves post-HCT survival [45]. While some studies suggest an association between excess tissue iron stores and regimenrelated toxicity, others have failed to demonstrate this. Severe iron overload has been associated with nonspecific liver dysfunction post HCT, which may respond to erythropoietin-assisted phlebotomy or chelation [46,47]. In most patients, the quantitation of tissue iron stores and consideration of iron removal can be deferred until after recovery from HCT.
Problems from transplant through day 200 Nausea, vomiting, and anorexia (Table 95.1) Myeloablative conditioning therapy makes most patients nauseated and anorexic, leading to delayed gastric emptying [48] and poor oral intake, with a nadir at day 10–12 post transplant but often extending to day 20 [49]. Serotonin-antagonist drugs are very effective in relieving symptoms during conditioning therapy but have little effect afterwards. Protracted anorexia may be related to circulating [interleukin(IL)-1, IL-6,] and tumor necrosis factor-alpha, cytokines known to affect appetite centers [49]. Mucositis caused by conditioning therapy leads to oral mucosal swelling, pain, and, in severe cases, sloughing of pharyngeal and esophageal epithelium, intense gagging, an inability to swallow, vomiting, retrosternal pain, and airway obstruction. Opioid therapy is effective in relieving pain but can lead to gastric stasis and intestinal pseudo-obstruction. Some regimens, notably those that contain cytarabine, etoposide, high-dose melphalan or multiple alkylating agents, may cause unusually severe intestinal mucosal necrosis and further delay the return of eating.
Infectious causes
Noninfectious causes
Cytomegalovirus Herpes simplex virus Varicella zoster virus Helicobacter pylori Giardia lamblia Cryptosporidia Rotavirus Oropharyngeal bacteria (phlegmonous gastritis)
Residual effects of conditioning therapy Acute graft-versus-host disease Medication intolerance Intestinal obstruction or pseudo-obstruction Gastroparesis Uremia/dialysis Acute hepatitis Acute pancreatitis Cholecystitis Increased intracranial pressure (central nervous system lesions)
An early sign of acute GVHD is loss of appetite, followed by nausea and vomiting. When the onset of GVHD is before day 15, following a peripheral blood allograft for example, these symptoms are indistinguishable from those resulting from conditioning therapy. Some patients never experience improvement in appetite before the onset of GVHD, but rather have a seamless persistence of anorexia and nausea. After day 20, over 80% of patients with intractable anorexia, nausea or vomiting will have gastric and duodenal GVHD as the sole explanation [50,51]. Endoscopy shows edema of the gastric antral and duodenal mucosa, patchy erythema, and bilious gastric fluid (Plates 27.26, 27.27, and 27.28), and histology may show epithelial cell apoptosis and drop-out, often with localized lymphocytic infiltrates (Fig. 27.5, and Plates 27.20, 27.27, 27.28, and 27.29) [52,53]. However, the predictive value of negative histology for GVHD is not high, as what the endoscopist sees as mucosal edema (Plate 27.26) is not what the pathologist relies upon to make a diagnosis of GVHD (apoptosis of crypt cells; Plates 27.20, 27.27, 27.28, and 27.29). Thus, the gamut of clinical presentation, endoscopic appearance, absence of infection, and histology needs to be integrated to arrive at a diagnosis when histology is equivocal. Immunosuppressive therapy using a 10-day induction course of prednisone 1 mg/kg/day plus oral beclomethasone dipropionate 8 mg/day is an effective therapy that avoids prolonged prednisone exposure [54]. Recipients of autologous grafts may also develop a syndrome of anorexia, nausea, and vomiting that is associated with diffuse gastric edema and erythema [55]. Gastric histology shows typical GVHD (Plate 27.37), and symptoms respond to a 10-day course of prednisone 1 mg/kg/day. Endoscopy also serves to rule out intestinal infection with herpesviruses, bacteria, and fungi. CMV infection of the esophagus and upper intestine accounted for a third of patients with unexplained nausea and vomiting during the pre-ganciclovir era, but CMV infection is now an uncommon cause of these symptoms. CMV infections in the gastrointestinal tract (Plates 27.35 and 27.36) are usually diagnosed between 50 and 150 days after HCT [56], but often appear earlier if patients have CMV infection before HCT [1]. CMV infections can be detected in the gastrointestinal tract in the absence of CMV antigen or DNA in peripheral blood. HSV esophagitis may present similarly in patients not receiving prophylactic acyclovir. Fungal esophagitis may cause anorexia but not the incessant vomiting often seen with GVHD or CMV infection. Studies of gastric emptying and myeloelectric activity in HCT patients have shown that symptoms of nausea and vomiting are frequently accompanied by retention of radionuclide meals and disordered
Gastrointestinal and Hepatic Complications
electrical activity [57]. Promotility agents such as metoaclopramide, domperidone, and low-dose erythromycin are occasionally useful, but in patients with persistent symptoms, endoscopic diagnosis of the underlying cause of altered motility should come before empiric promotility therapy. Anorexia and vomiting may also be manifestations of increased intracranial pressure; other neurologic signs and symptoms usually dominate the clinical picture with these disorders. Oral nonabsorbable antibiotics (particularly nystatin), cyclosporine, mycophenolate mofetil, trimethoprim–sulfamethoxazole, voriconazole, posaconazole, itraconazole, intravenous amphotericin, and high-dose opioids also cause nausea and occasionally vomiting that can be confused with symptoms of GVHD [17,18]. Anorexia and nausea are also common, sometimes intolerable, side-effects of treatment with maribavir. Parenteral infusions of fat, glucose, and amino acids reduce food intake, slow gastric emptying, and cause nausea. Even after total parenteral nutrition has been stopped, appetite suppression may linger for 1–3 weeks [58].
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lary hematopoiesis) [27,35]. At our center, the overall incidence of SOS among patients with hematologic malignancy conditioned with CY 120 mg/kg plus TBI 12–13.2 Gy is 38% (7% severe), and among patients with myelodysplastic syndrome conditioned with targeted busulfan (BU)/CY 120 mg/kg it is 12% (2% severe) [64,66]. Overall, the frequency and severity of SOS have fallen dramatically over the last few years, for several reasons: (1) the strategy of dose escalation of conditioning regimens has been abandoned; (2) fludarabine is replacing CY in many centers; and (3) patients at risk for SOS (with underlying fibroinflammatory liver disease) are being directed to regimens that do not contain liver toxins. Evidence has been presented that prophylaxis with ursodiol prevents SOS [67], but the largest randomized trial of ursodiol that specifically tracked SOS as an endpoint found no such effect [60]. The effect of ursodiol is primarily on cholestatic liver disease, and thus it seems likely that many past patients diagnosed as having SOS on clinical grounds had instead a combination of sinusoidal and cholestatic liver injury. Clinical presentation and diagnosis
Jaundice, hepatomegaly, and abnormal liver tests Development of jaundice following HCT is an ominous prognostic sign, with increased nonrelapse mortality in patients whose total serum bilirubin exceeds 4 mg/dL [59]. Because treatment of severe liver dysfunction is often futile, transplant physicians must recognize risk factors for hepatobiliary problems before the transplant process starts, implement measures to prevent liver damage, and identify post-transplant liver disorders that have specific treatments. Over the last decade, this approach has resulted in a marked reduction in the incidence of liver infections and clinical jaundice, resulting from prevention of hepatic sinusoidal injury, fungal and viral prophylaxis, and the introduction of prophylactic ursodiol for all patients undergoing transplant [17,20,60,61]. Nonetheless, there remain multiple hepatic causes of jaundice and elevations of serum alanine aminotransferase (ALT) and alkaline phosphatase (Table 95.2). Additional processes can contribute to jaundice following HCT while not usually being important causes of jaundice in isolation, for example hemolysis and renal insufficiency. It is imperative to search for causes of progressive jaundice as early intervention to prevent severe hyperbilirubinemia may reduce the risk of death, although this is unproven. Sinusoidal obstructive syndrome Definition SOS is a clinical syndrome of tender hepatomegaly, fluid retention and weight gain, and elevated serum bilirubin that follows high-dose myeloablative therapy [62]. This syndrome is sometimes called veno-occlusive disease, but this is an inaccurate descriptor of the underlying pathobiology. This liver injury is initiated by changes in hepatic sinusoids, and occlusion of hepatic venules is not essential to the development of clinical signs and symptoms [63]. Incidence As SOS is caused by toxins in certain conditioning regimens, and not by the transplant process per se, the reported incidence varies with the composition and intensity of the conditioning regimen, from zero after most reduced-intensity regimens [29] to 50% after CY plus total body irradiation (TBI) of more than 14 Gy [35]. Other important contributors to variability in the incidence of SOS are large variations in the metabolism of CY from patient to patient [64,65] and underlying fibroinflammatory liver diseases (for example, chronic viral hepatitis, nonalcoholic steatohepatitis, cirrhosis, and sinusoidal fibrosis related to extramedul-
The diagnosis of SOS rests on the findings of tender hepatomegaly, weight gain, and jaundice following conditioning therapy, in the absence of other explanations for these signs and symptoms (Table 95.3) [35]. Cholestatic liver injury related to cytokine effects on bilirubin transport by hepatocytes may occur during episodes of neutropenic fever before engraftment, and thus be confused with SOS. The most common combinations of illnesses that mimic SOS are (1) sepsis syndrome requiring large volumes of crystalloid, followed by renal insufficiency and sepsisrelated cholestasis; (2) cholestatic liver disease, hemolysis, and weight gain related to total parenteral nutrition; and (3) hyperacute GVHD and sepsis syndrome. SOS may also coexist with these disease processes. The onset of SOS is heralded by an increase in liver size, right upper quadrant tenderness, renal sodium retention, and weight gain, occurring 10–20 days after the start of CY-based cytoreductive therapy [35] and later after other myeloablative regimens [68–70]. Patients then develop hyperbilirubinemia, usually before day 20 [35]. Other clinical manifestations of portal hypertension may strongly suggest SOS (Table 95.3). Some authors have described a syndrome of “late veno-occlusive disease” following conditioning with BU-containing regimens, where signs of liver disease are first recognized after day 30 after transplantation [70]. After HCT, treatment of relapsed acute myeloid leukemia (AML) with gemtuzumab ozogamicin may also result in SOS [38,71]. Measurement of total serum bilirubin is a sensitive test for SOS but not a specific one, as there are many causes of jaundice after HCT (Table 95.2). Elevations of serum aspartate aminotransferase (AST) and ALT can occur late in the course of SOS, reflecting ischemic hepatocyte necrosis from sinusoidal fibrosis (Plate 27.12), as peak AST/ALT levels (sometimes over 1500 U/L) are seen weeks after toxin exposure. Several plasma proteins have been reported to be abnormally high in patients with SOS, including endothelial cell markers (hyaluronic acid, von Willebrand factor, plasminogen activator inhibitor-1, and tissue plasminogen activator), thrombopoietin, proinflammatory cytokines, vascular endothelial growth factor, and procollagen peptides; some laboratory tests are abnormally low in patients with SOS, including the anticoagulant proteins protein C and antithrombin III and platelet counts (reviewed in [62]). Maximum levels of plasminogen activator inhibitor-1 less than 120 ng/mL have a strong negative predictive value for the diagnosis of SOS and may be useful in excluding this diagnosis [72]. It is not clear whether any other laboratory tests have diagnostic or prognostic utility beyond the clinical criteria of weight gain, jaundice, and hepatomegaly. Imaging studies of the liver are useful for demonstrating hepatomegaly, ascites, periportal edema, attenuated hepatic venous flow, and gall bladder wall edema consistent with SOS [73–75], as well as
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Table 95.2 Liver diseases after hematopoietic cell transplant Disease
Frequency
Timing
Diagnosis
Sinusoidal obstructive syndrome
10–35% (regimen dependent)
Onset before day 20
Cholestasis of sepsis (cholangitis lenta)
Common in neutropenic patients
Acute GVHD
10–20% of allograft recipients Rarely after autograft Uncommon when prophylaxis is used against herpes viruses or hepatitis B
Following sepsis or neutropenic fever (usually before day 30) Day 15–50
Typical clinical features plus exclusion of other causes of jaundice and weight gain Imaging (Doppler ultrasound or CT) Transvenous measurement of wedged hepatic venous pressure gradient and liver biopsy Note atypical presentations (acute hepatitis, anasarca) Exclude other causes of cholestasis Inferential diagnosis in a patient with cholestatic jaundice Confirm GVHD in skin and gut Exclude other causes of cholestasis Liver biopsy Pretransplant blood tests (antigen, antibodies, PCR results) Isolation of virus from other sites (stool and urine for adenovirus) PCR of serum for specific viruses Liver biopsy, histology/PCR/immunostatins Hepatic pain, fever Liver imaging (MRI > CT) Serum fungal antigen(s) Hepatic pain, fever Liver imaging Liver biopsy, culture Clinical evidence linking elevations of serum ALT or alkaline phosphatase to drugs known to cause liver injury Clinical evidence linking shock to subsequent rises in serum ALT
Acute viral hepatitis
HSV, day 20–50 Adenovirus, day 30–80 VZV, day 80–250 HBV and HCV, during immune reconstitution
Fungal abscess
Rare when prophylaxis is used
Day 10–60
Bacterial infection
Rare (pyogenic abscess, mycobacterial infection) Common
Day 10–80
Confined to patients with septic or hemorrhagic shock Transient biliary sludge, common Stones or chloromas, rare Rare
Day 0–30
Formerly common
After day 80
Iron overload
Very common
Pre transplant Long-term follow-up after transplant
Chronic GVHD
Common after allografts
After day 80
Nodular regenerative hyperplasia
Rare complication of toxic liver injury
Years after transplant
Drug liver injury
Ischemic liver disease Biliary obstruction
Idiopathic hyperammonemia Chronic hepatitis C
Day 0–100
Day 15–60
History, examination Biliary ultrasound ERCP > magnetic resonance cholangiography
Day 10–50
Unexplained confusion, coma Venous blood ammonia HCV RNA in serum Elevation of serum ALT after immune reconstitution Transferrin saturation Marrow iron quantitation Liver iron quantitation (FerriScan MRI, liver biopsy quantitation) Prior acute GVHD history Chronic GVHD in other organs Consistent elevations of serum ALT and alkaline phosphatase Liver biopsy Signs of portal hypertension but preserved liver function Liver biopsy histology (reticulin stain), laparoscopic appearance of the liver
ERCP, endoscopic retrograde cholangiopancreatography. See text for other abbreviations.
Gastrointestinal and Hepatic Complications Table 95.3 Clinical, laboratory, and imaging manifestations of SOS Usually present
May be present
Hepatomegaly (change from baseline) Weight gain (usually abrupt) Jaundice
Low urine sodium concentration or fractional excretion of sodium Peripheral edema, ascites, anasarca Elevation of serum alanine aminotransferase Gall bladder wall edema on ultrasound, pain over the gall bladder fossa Isolated weight gain Isolated jaundice Thrombocytopenia Appearance of esophageal varices Pleural effusions, pulmonary vascular congestion, hypoxia Acute renal failure
excluding biliary dilation or infiltrative lesions in the liver and hepatic veins that might also explain hepatomegaly and jaundice. Other abnormal findings may include an enlarged portal vein diameter, slow or reversed flow in the portal vein or its segmental branches [76], high congestion index, portal vein thrombosis, and increased resistive index to hepatic artery flow. Unfortunately, ultrasound findings very early in the course of SOS do not appear to add to the information provided by clinical criteria [77]; however, Doppler evaluation of flow in segmental branches of the portal vein can be useful [76]. Later in the course of SOS, especially in patients with severe disease, ultrasound evidence of altered liver blood flow, particularly reversal of portal flow and portal vein thrombosis, is more common [77]. There may be value in following vascular parameters as indices of improvement in sinusoidal blood flow. Liver biopsy and measurement of the hepatic venous pressure gradient In cases where the cause of liver dysfunction is unclear, a transvenous approach that allows both biopsy and hepatic venous pressure measurements is the most accurate diagnostic test [78,79]. Laparoscopic needle biopsy is an alternative method of obtaining liver tissue [80]. Percutaneous biopsy poses a high risk of bleeding in the thrombocytopenic patient, whereas transvenous biopsy methods can be done safely with platelet counts as low as 30,000/mm3. In HCT patients, a hepatic venous pressure gradient (use of an occlusive balloon technique [81] is essential here) above 10 mmHg is highly specific for SOS [78,79]. Initial histologic changes of SOS are dilation of sinusoids, extravasation of red cells through the space of Disse, necrosis of perivenular hepatocytes, and widening of the subendothelial zone in central veins (Plates 27.11, 27.13, 27.14, and 27.15) [62,63]. A finding of “hemorrhage” in zone 3 of the liver acinus is the result of destruction of sinusoidal endothelium (Plate 27.14). In severe SOS, fragmented hepatocyte cords can be seen, with dislodgement of clusters of hepatocytes (Plate 27.15) into portal veins through disrupted pores into lumina of damaged central veins. The later stages of SOS are characterized by extensive collagenization of sinusoids with a variable degree of obstruction of venular lumens by collagenized walls, leading to an obliteration of sinusoidal blood flow (Plates 27.10, 27.12, and 27.16). In severe SOS – if patients survive beyond day 50 post transplant – a pattern of reverse cirrhosis may develop, with extensive linkage between obliterated central venules by fibrous bridges, collapse, and acinar extinction.
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Clinical course and prognosis The severity of SOS has been classified as mild (SOS that is clinically obvious, requires no treatment, and resolves completely), moderate (SOS that causes signs and symptoms requiring treatment such as diuretics or pain medications, but that resolves completely), or severe (SOS that requires treatment but that does not resolve before death or day 100). There is a range of clinical and laboratory findings that correspond to these operational definitions of disease severity [35]. Some patients have subclinical liver damage, evinced by histologic signs of liver toxicity in the absence of clinical signs and symptoms. Published case fatality rates for SOS vary widely, depending on whether all cases of jaundice or only biopsy-proven cases are classified as SOS [82]. Recovery from SOS was seen in over 70% of patients whose SOS followed CY-containing regimens, and in 84% when SOS was caused by other alkylating agents [35,69]. Despite deep jaundice, patients with severe SOS seldom die of liver failure, but rather from renal and cardiopulmonary failure [35,83,84]. A clinically useful model has been developed that predicts the outcome of SOS after CY-based regimens, derived from rates of increase of both bilirubin and weight in the first 2 weeks following HCT [85]. In some patients, there is a bimodal presentation of SOS; that is, clinical signs of SOS appear in the first 2 weeks post HCT, then wane, and then reappear later, probably related to stellate cell proliferation and collagenization of sinusoids. This clinical pattern is associated with a worse prognosis. In some cases, signs of SOS resolve, but ascites later recurs following development of inflammatory liver disease (e.g. GVHD). A poor prognosis correlates with overt signs of portal hypertension, hepatocyte necrosis, and renal and pulmonary failure. Pathogenesis of SOS: insights from animal models The use of animal and in vitro models has clarified the cellular mechanisms of toxic sinusoidal injury (reviewed in [62]). Sinusoidal endothelial cells are more susceptible than hepatocytes to drugs that cause SOS in patients, notably CY. In animals exposed to toxin, the first morphologic change is loss of sinusoidal endothelial cell fenestration, appearance of gaps in the sinusoidal endothelial cell barrier, rounding up of sinusoidal endothelial cells, and penetration of red cells into the space of Disse [86]. Sloughed sinusoidal lining cells, that is, Kupffer cells, sinusoidal endothelial cells, and stellate cells, embolize downstream and obstruct sinusoidal flow. By the time hepatocyte necrosis is observed, there is extensive denudation of the sinusoidal lining. Drugs and toxins that cause SOS profoundly deplete sinusoidal endothelial cell glutathione (GSH) prior to cell death, and support of sinusoidal endothelial cell GSH prevents cell death [87]. One possible explanation for the rounding up of sinusoidal endothelial cells may be increased activity of matrix metalloproteinases (MMPs), as inhibition of MMP activity completely prevents SOS in animal models [88]. Although SOS is defined as a nonthrombotic obstruction of blood flow, the issue of thrombosis has been a recurring topic of research interest, based largely on plasma studies in patients with SOS and an immunohistologic study [89]. Sequential observations during the development of SOS in the animal model have not demonstrated any evidence of thrombosis, perhaps because of obliteration of sinusoidal endothelial cells. Pathogenesis of SOS: clinical studies The proximate cause of SOS is the conditioning regimen, specifically chemotherapy drugs or irradiation that damage sinusoidal endothelium. Two observations about SOS in patients provide clues as to the mechanism of disease. First, unlike other intrinsic liver diseases, the signs and symptoms of portal hypertension precede evidence of parenchymal damage. In SOS, disruption of the liver circulation (Plate 27.14) is the cause and not the consequence of the parenchymal disease. Second,
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involvement of the hepatic veins is not essential to the development of the clinical picture: 45% of patients with mild or moderate disease and 25% of patients with severe SOS did not have occluded hepatic venules at autopsy [63]. Occlusion of central veins of the liver lobule (Plates 27.10 and 27.12) is associated with more severe disease and the development of ascites. This finding suggests that occlusive lesions involving the central veins, a later development, may exacerbate the acute circulatory impairment that occurs at the level of the sinusoid. Chemotherapy drugs CY is common to the conditioning regimens with the highest incidence of fatal SOS: CY/TBI, BU/CY, and BCV (BCNU, CY, and etoposide). The metabolism of CY is highly variable; patients who generate a greater quantity of toxic metabolites are more likely to develop severe SOS following conditioning with CY and TBI [64]. The liver toxin generated by CY metabolism is acrolein (a metabolite formed simultaneously along with the desired metabolite, phosphoramide mustard), via mechanisms dependent upon GSH. Exposure to one CY metabolite (carboxyethyl phosphoramide mustard [CEPM]) was significantly related to SOS, bilirubin elevation, nonrelapse mortality, and survival in patients receiving conditioning with CY/TBI, but there was no relationship to either engraftment or tumor relapse [64]. These data suggest that a strategy that targets the dose of CY on the basis of a patient’s metabolism will substantially reduce the risk of fatal SOS, without jeopardizing engraftment. Accurate methods to target the dose of CY to a metabolic endpoint, thereby eliminating variable exposure to liver toxins, have been developed [65,90]. BU is another component of regimens with a high frequency of SOS, but BU itself is not hepatotoxic [91,92]. A relationship between BU exposure (measured by area under the curve or average steady-state – concentration, C ss, Bu) following oral dosing in the BU/CY regimen has been reported; however, in adults with chronic myeloid leukemia in chronic phase and children with acute leukemia, there is no correlation between BU exposure and SOS [93,94]. BU may contribute to liver injury by inducing oxidative stress, reducing GSH levels in hepatocytes and sinusoidal endothelial cells [91] and altering CY metabolism [66]. Total body irradiation The doses of TBI given in the setting of HCT are in the range of 10–16 Gy, far less than the dose that causes radiationinduced liver disease. In combination with CY, however, there is a clear relationship between the total dose of TBI and the frequency of severe SOS [64]. The frequency of severe SOS is approximately 1% after CY/ TBI 10 Gy [95], 4–7% after CY/TBI 12–14 Gy [64,96], and 20% after CY/TBI greater than 14 Gy [35]. The synergism between CY and TBI in causing sinusoidal injury may be due to the fact that both agents cause depletion of hepatic reduced GSH as well as sublethal damage to sinusoidal endothelial cells. The order of administration (CY/TBI versus TBI/ CY) does not appear to affect the frequency of liver injury. Gemtuzumab ozogamicin Gemtuzumab ozogamicin specifically targets leukemia cells expressing the CD33 receptor by means of a humanized antibody conjugated to a modified cytotoxic agent, calicheamicin. Gemtuzumab ozogamicin may cause sinusoidal liver injury when used to treat patients with AML [38]. Pretransplant sinusoidal liver injury is a risk factor for SOS; thus, the risk of SOS is 15–40% when high-dose gemtuzumab ozogamicin is given in proximity to a CY-containing myeloablative conditioning regimen [38,97]. Lower doses of gemtuzumab ozogamicin appear to eliminate this risk. The drug may also cause SOS when given after transplant for relapsed AML [38,71]. The clinical presentation is one of acute portal hypertension, ascites, weight gain, elevations of serum AST/ALT, and moderate jaundice, with histology showing intense sinusoidal fibrosis and centrilobular hepatocyte necrosis (Plate 27.1) [70].
Intrahepatic coagulation Some see SOS as a disease of disordered coagulation, in which damage to endothelium in the sinusoids and central veins leads to exposure of tissue factor and initiation of the coagulation cascade. However, sinusoidal endothelial cells embolize downstream in SOS, heparin and antithrombin III infusions are ineffective in preventing fatal SOS, and thrombolytic therapy effects improvement in only a minority of patients. Genetic disorders predisposing to coagulation (factor V Leiden and prothrombin gene 20210 G-A) have no associations with SOS after HCT. Current evidence suggests that disordered coagulation in SOS is an epiphenomenon secondary to widespread centrilobular damage (Plate 27.11), and not a cause of sinusoidal damage. However, thrombosis in the portal vein may result from a hypercoagulable state in patients with severe SOS [98]. Stellate cells and sinusoidal fibrosis Several series have documented the early appearance of a procollagen peptide in serum of patients who develop more severe SOS [99], along with inhibitors of fibrolysis, consistent with the intense fibrosis in centrilobular sinusoids and venular walls that is common in fatal SOS [63]. Immunohistology of liver specimens from patients with SOS for alpha-actin (a marker for stellate cell activation) shows intense staining in sinusoids (Plate 27.16) [62]. The stimuli for stellate cell activation and proliferation in the HCT setting have not been identified; candidates include centrilobular hypoxia, endotoxemia, Kupffer cell damage, loss of sinusoidal endothelial cells, and a melange of circulating growth factors and cytokines [100]. Genetic factors Genetically determined differences in drug metabolism or susceptibility to toxic injury might explain some of the variability in the frequency of SOS. Case-control studies using single-nucleotide polymorphisms have reported associations between SOS and carbamyl phosphate synthetase-1 c.4340C>A (CPS1), factor V c.1691G>A (FV Leiden), HFE C282Y, and glutathione S-transferase (GSTM1 and GSTT1) genes [101–103]. These associations could not be confirmed in a cohort of Seattle patients receiving a uniform conditioning regimen (CY/TBI). Patients with Gilbert’s syndrome, a common polygenic disorder that leads to unconjugated hyperbilirubinemia, have defective hepatic glucuronidation; drugs that are normally excreted in bile as glucuronides may cause increased toxicity in patients with Gilbert’s syndrome [104]. Recently, the genetic cause of a naturally occurring form of hepatic vascular damage that is indistinguishable from SOS at the histologic level has been described in kindreds with SOS with immunodeficiency syndrome that results from truncating mutations in SP110 [105]. When patients conditioned with CY/TBI were studied, six singlenucleotide polymorphisms, as well as one haplotype within the SP110 locus, reached nominal significance, but none was significant after correction for multiple testing. Detection of genetic polymorphisms that lead to SOS will require more highly powered studies. Prevention of SOS in patients receiving myeloablative therapy Prevention of severe sinusoidal liver injury begins with an assessment of the risk of hepatotoxicity from a given myeloablative conditioning regimen. Table 95.4 lists underlying liver disorders that increase the risk of severe sinusoidal liver injury following CY-based conditioning regimens. Patients at risk have several options: (1) conventional therapy that does not involve HCT; (2) a reduced-intensity conditioning regimen [29]; (3) a myeloablative regimen that does not contain CY, for example targeted BU and fludarabine for allogeneic [106,107] or BCNU, etoposide, cytosine arabinoside, and melphan (BEAM) [108] for autologous HCT; (4) modification of CY-based regimens [65,90]; and (5) use of pharmacologic approaches to prevent sinusoidal liver injury. The transplant oncologist must achieve a balance between the optimal
Gastrointestinal and Hepatic Complications Table 95.4 Liver disorders that increase the risk of severe sinusoidal liver injury following cyclophosphamide-based regimens Necroinflammatory disorders Chronic hepatitis B or C Nonalcoholic steatohepatitis Alcoholic hepatitis Fibrotic disorders Cirrhosis Lobular fibrosis Extramedullary hematopoiesis with sinusoidal fibrosis Recent exposure to gemtuzumab ozogamicin Prior liver irradiation Cholestatic disorders Jaundice caused by intrahepatic cholestasis Dubin-Johnson syndrome
conditioning regimen for a given malignancy and its potential for fatal liver toxicity in a given patient. Patients with cirrhosis, for example, may decompensate even after a reduced-intensity allograft [29]. If a CY/TBI regimen must be used for a patient at risk for fatal SOS, modifications should be considered for both CY and TBI dosing, with the understanding that clinical trials to prove efficacy and safety have not been done. The total dose of CY should be in the range 75–100 mg/ kg [65], and TBI doses should not exceed 12 Gy [64,68]. Shielding the liver during TBI will lessen liver injury but leads to relapse of underlying hematologic disease [109]. Accurate methods are available to target CY doses to a metabolic endpoint, based on exposure to the CY metabolites 4-HCY and CEPM [65,90]. If a BU/CY regimen must be used for a patient at risk for fatal SOS, liver toxicity may be less frequent if CY is given before targeted BU [110] (this requires intravenous BU, as oral BU is poorly tolerated after CY infusions), or if dosing of CY is delayed for 1–2 days after completion of BU [111]. BU (and the use of phenytoin to prevent seizures) has marked effects on CY metabolism when CY is given second in order; that is, there is increased exposure to 4-HCY, phosphoramide mustard, and acrolein compared with giving CY first in order [66]. BU itself is not a significant sinusoidal liver toxin [91,92], but appears to contribute to SOS by causing depletion of reduced GSH in the liver, thus making sinusoidal endothelial cells more susceptible to injury from metabolites of CY or TBI [87,112]. It is not clear whether intravenous BU offers any advantage over targeted oral BU with regard to liver toxicity from a BU/CY regimen. A lower incidence of SOS has been reported following intravenous BU/CY, compared with oral BU/CY, when neither BU formulation was adjusted for metabolism [113]. There are no studies that examine the frequency of severe SOS following intravenous versus oral BU/CY when both are dosed to the same steady-state concentration (e.g. 900 ng/mL) [93]. The metabolism of intravenous BU is variable, with a several-fold range in area under the curve for BU, a problem that can be addressed only with therapeutic drug monitoring [114]. Liver toxicity has remained a complication of conditioning with both targeted oral BU/CY [66,115,116] and weight-based dosing of intravenous BU/CY [113,117,118]. Lower hepatic exposure to BU may indeed reduce the frequency of fatal SOS following BU/CY regimens by causing less extreme reductions in hepatic GSH, and not because BU is a liver toxin. In patients with no identifiable risk factors for severe SOS who receive a CY-based myeloablative regimen, fatal SOS develops in around 7% of cases following CY/TBI 12–14 Gy [64] and in less than 5% of cases
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following targeted oral BU/CY [66]. The unpredictability of liver toxicity after CY-based regimens is related to aberrant CY metabolism [64], in turn likely related to polymorphisms in hepatic enzymes or transporters. The only certain way to prevent fatal SOS is to avoid giving conditioning therapy that damages hepatic sinusoidal cells, that is, CY and TBI. Substitution of fludarabine for CY (e.g. a BU plus fludarabine regimen) appears to avoid sinusoidal liver damage [106,107]. Use of drugs to prevent SOS has been achieved in animal models of liver injury (reviewed in [62]), but these strategies have not been proved in the clinical setting. There may be value in prophylaxis of SOS with repletion of intracellular GSH [87], inhibition of MMP enzymes [88] or infusion of defibrotide [119,120]. Adequately powered clinical trials of these modalities with proper stratification for risk of fatal outcomes have not been reported. Prospective studies have shown no benefit from use of heparin [121] or antithrombin III [122] for prevention of fatal SOS. A meta-analysis suggests that ursodiol may prevent SOS [67], but SOS was not differentiated from cholestatic liver disease in these studies [82], and the largest randomized trial showed no effect of ursodiol on the frequency of SOS [60]. Treatment of patients with SOS For the 70–85% of patients with SOS who will recover spontaneously, treatment involves management of sodium and water balance with diuretics, preservation of renal blood flow, and repeated paracenteses for ascites that is associated with discomfort or pulmonary compromise. Patients with a poor prognosis can be recognized soon after disease onset by steep rises in total serum bilirubin and body weight, serum ALT values greater than 750 U/L, portal pressures over 20 mmHg, development of portal vein thrombosis, and multiorgan failure requiring dialysis or mechanical ventilation [59,83–85]. There are no satisfactory therapies for severe SOS; the best current results (45% response) are with intravenous defibrotide (25 mg/kg/day) [123]. Defibrotide, a polydisperse mixture of single-stranded phosphodiester oligodeoxyribonucleotides obtained from controlled depolymerization of porcine intestinal mucosal genomic DNA, induces antithrombotic and profibrinolytic effects in vitro and in vivo. However, its mechanism of action in the treatment of SOS is not known [124]. Clinical responses to defibrotide infusions may take weeks to be seen. Although randomized, placebo-controlled trials of defibrotide for therapy of severe SOS have not been carried out, the complete recovery of some patients with severe SOS and multiorgan failure suggests that the drug has biologic effects in man [123,125]. Numerous other approaches to treatment of severe SOS have been reported over the last 20 years, but none can be currently recommended. Thrombolytic therapy with tissue plasminogen activator was ineffective in patients with SOS and organ failure, and resulted in fatal bleeding. Other reported therapies have included intravenous N-acetylcysteine, human antithrombin III concentrate, activated protein C, prostaglandin E1, prednisone, topical nitrate, vitamin E plus glutamine, and use of a liver-assist device. Transhepatic shunts have been placed in patients with SOS to reduce portal pressure and mobilize ascites, but neither serum bilirubin levels nor patient outcomes were improved [126]. In one case, a transjugular intrahepatic portosytemic shunt led to acute respiratory distress syndrome that was fatal [127]. Patients with SOS have undergone successful portosystemic shunts for persistent ascites, but liver dysfunction had resolved long before these shunts were placed. Peritoneovenous shunts for intractable ascites have been unsuccessful. Successful liver transplants for severe SOS have been reported [128]. When severe SOS develops in a patient with a benign condition (a rare event) or in a patient with a favorable outcome post HCT (e.g. chronic myelogenous leukemia in chronic phase), liver transplantation should be considered. Prevention of sinusoidal injury is likely to be a more effective strategy for improving transplant outcomes than treatment.
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Cholestatic disorders Cyclosporine inhibits canalicular bile transport and commonly causes mild increases in serum bilirubin without an effect on serum ALT or alkaline phosphatase [129]. Tacrolimus less commonly causes cholestasis, except in the setting of toxic blood levels. Sepsis-associated cholestasis is an important contributor to hyperbilirubinemia in the weeks after HCT, mediated by endotoxins, IL6, and tumor necrosis factor-alpha [130,131]. Jaundice may be progressive and marked in patients with ongoing sepsis, particularly when other cholestatic disorders are present. Many drugs used after HCT have been associated with liver dysfunction (e.g. trimethoprim–sulfamethoxazole, itraconazole, voriconazole, fluconazole, posaconazole, and ribavirin), although drugs are usually not responsible for severe liver injury in this setting. Prolonged parenteral nutrition may also contribute to cholestasis. Acute GVHD is the most common cause of severe cholestatic injury, as alloreactive T-cells recognize foreign major and minor histocompatability antigens as well as adhesion molecules expressed on biliary epithelial cells. Hepatic GVHD usually follows cutaneous and/or intestinal GVHD, and is heralded by a gradual rise in serum bilirubin, alkaline phosphatase, and aminotransferase enzymes. However, a blinded histologic study could identify no features characteristic of GVHD when biopsies were done within several weeks of the onset of GVHD [132], suggesting that jaundice occurring early after the onset of GHVD is related to cholestasis caused by exposure to cytokines such as IL-6 [49]. In allograft recipients on minimal immunosuppression or after donor lymphocyte infusion, GVHD may also present as an acute hepatitis (Plate 27.39) with marked elevation of serum ALT [133–135]. A cholestatic condition identical to GVHD occurs rarely in autologous HCT recipients. Characteristic liver biopsy findings in GVHD include lymphocytic infiltration of small bile ducts with nuclear pleomorphism and epithelial cell drop-out (Fig. 27.6 and Plate 27.38). Because these patients are frequently receiving immunosuppressive therapy, inflammatory infiltrates may be minimal, and bile duct epithelial changes may be the only abnormality. Severe, ongoing hepatic GVHD and worsening jaundice are associated with progressive destruction of small bile ducts (Fig. 27.7 and Plate 27.41). Only 30% of patients with liver GVHD have resolution of liver abnormalities after initial immunosuppressive treatment. Prophylactic ursodeoxycholic acid reduces the frequency of cholestasis in general and GVHD-related cholestasis specifically, compared with placebo, and it is recommended that all allograft recipients be routinely treated through day 80 post transplantation [60]. Over 50% of patients with acute liver GVHD will develop chronic GVHD.
Acute hepatitis In most cases, a sudden rise in the serum aminotransferase enzymes (AST and ALT) following HCT is due to a noninfective cause such as zone 3 hepatocyte necrosis in SOS, ischemic hepatopathy (septic shock), acute biliary obstruction (choledocholithiasis), drug-induced liver injury, or the acute hepatitic presentation of GVHD [133–135]. If a likely cause is not apparent, acute viral hepatitis should be suspected, as early diagnosis and treatment may prevent a fatal outcome. Serum ALT levels in patients with severe SOS peak at day 23 on average, with extreme elevations indicating a poor prognosis. Acute hepatitis caused by HSV, VZV (Plate 27.58), adenovirus (Plate 27.59), and HBV can lead to fatal fulminant hepatic failure after HCT [20,136–140], whereas hepatic infections caused by CMV and HCV are seldom severe [27]. With routine use of prophylactic acyclovir/valacyclovir, acute hepatitis due to HSV and VZV is now rare; however human herpesvirus-6 (HHV-6) and HHV-8 reactivation causing hepatitis despite
prophylaxis has been reported [141]. When there is uncertainty about the cause of rising serum ALT and AST levels, DNA blood tests for herpesviruses, adenovirus, and HBV should be performed. Transvenous measurement of the wedged hepatic venous pressure gradient can exclude acute portal hypertension due to SOS, and transvenous liver biopsy (via the transjugular or via femoral vein) may demonstrate a specific diagnosis (Plates 27.58 and 27.59). If acyclovir is not being given, it should be started empirically, particularly if the patient presents with abdominal complaints typical of VZV infection [142]. Adenovirus hepatitis should be suspected if the patient has concomitant pulmonary, renal, bladder or intestinal symptoms; the most effective treatment is cidofovir when given early in the course of infection [137,143,144]. Fulminant hepatitis B may develop during immune reconstitution in patients at risk, but can be prevented with prophylactic antiviral agents [20,145]. If severe hepatitis B reactivation does occur, usually because a diagnosis of HBV was not made prior to HCT [32], antiviral therapy with the most potent antiviral drug available (lamivudine, entecavir or telbivudine or tenofovir) should be initiated immediately; however progression to fatal liver failure is not uncommon [146]. Fulminant hepatitis B has also been reported following discontinuation of prophylactic antiviral therapy, and all patients, particularly those with high pretransplant HBV DNA levels, should be monitored following antiviral drug withdrawal [147,148]. Chronic hepatitis C in HCT recipients usually results in asymptomatic elevation of ALT from days 60 to 120, coinciding with the tapering of immunosuppressive drugs [27]. Severe hepatitis due to HCV has only rarely been reported, and specific antiviral therapy for HCV is not indicated early after HCT. Therapy directed at chronic HCV infection should be considered once the patient has ceased all immunosuppressive drugs and has no evidence of active GVHD [149]. Fungal and bacterial infections Antifungal prophylaxis with fluconazole, itraconazole, and more recently posaconazole has had a significant impact on the incidence of invasive fungal disease in HCT recipients, particularly in those requiring ongoing immunosuppression for treatment of GVHD [17,150–152]. If invasive fungal disease does occur, infection with resistant Candida species or molds may involve the liver [150]. Hepatic infection should be suspected in the presence of fever, tender hepatomegaly, and increased serum alkaline phosphatase levels. High-resolution CT scan or MRI may demonstrate multiple fungal abscesses, and serologic tests for fungal antigens may be useful for diagnosis. Treatment courses may need to be protracted in immunosuppressed patients with visceral fungal infection, but return of neutrophil function after HCT can effect resolution of previously treatment-refractory mold infection [19]. Bacterial liver abscesses are rare in HCT recipients, probably because of the high use of systemic antibiotics; however, latent mycobacterial infection may reactivate within the liver with prolonged immunosuppressive therapy. Disseminated Bacille Calmette-Guerin infection with liver involvement has been reported. Disseminated clostridial infection and gall bladder infection with gas-producing organisms may lead to air in the liver and biliary system. Gall bladder and biliary disease Biliary sludge (calcium bilirubinate and crystals of calcineurin inhibitors) is a common finding in HCT recipients, being identified by ultrasound in approximately 70% and at autopsy in 100% of patients [153]. Biliary sludge is usually asymptomatic; however, passage down the bile duct may cause epigastric pain, nausea, and abnormal serum liver enzymes. Biliary sludge may be a cause of acute “acalculous” cholecystitis, acute
Gastrointestinal and Hepatic Complications
pancreatitis, and bacterial cholangitis [154–156]. Acute cholecystitis is uncommonly seen in HCT recipients and is frequently acalculous [157]. Cholecystitis in this setting may also be due to leukemic relapse with gall bladder involvement or infection by CMV or fungi. Diagnosis is difficult because of the high frequency of gall bladder abnormalities on ultrasound following HCT. Pericholecystic fluid, gall bladder wall necrosis or localized tenderness suggest cholecystitis. A radionuclide bile excretion study, with morphine infusion to enhance gall bladder filling, can be useful: nonvisualization of the gall bladder suggests cholecystitis. Biliary obstruction is a rare event, caused by a variety of disorders (e.g. common bile duct calculi or inspissated biliary sludge, GVHD of the ampullary mucosa; lymphoblastic infiltration of the common bile duct, gall bladder, and ampulla of Vater in Epstein–Barr virus (EBV) lymphoproliferative disease (Plate 27.3); CMV-related biliary disease; dissecting duodenal hematoma complicating endoscopic biopsy; and leukemic relapse [chloroma] in the head of the pancreas) [155,156]. Noninvasive imaging (ultrasound, magnetic resonance cholangiopancreatography or CT cholangiography) is usually adequate to demonstrate biliary pathology, and therapeutic endoscopic retrograde cholangiopancreatography should be required only in patients with clinical evidence of cholangitis following HCT and radiologic evidence of extrahepatic biliary obstruction [156]. Malignant disorders EBV lymphoproliferative disease is now an infrequent complication of HCT, largely because of EBV DNA surveillance and pre-emptive treatment. The highest incidence is in recipients of HLA-mismatched, T-celldepleted grafts and those receiving potent anti-T-cell therapies for GVHD. Symptoms include fever, sweats, generalized malaise, enlarged tonsils, and cervical lymphadenopathy with liver involvement (Plate 27.4) occurring in over 50%, manifest by abnormal serum alkaline phosphatase and massive hepatosplenomegaly. Recurrent cancer in patients transplanted for hematologic malignancy or solid tumors may present with abnormal liver enzymes, hepatomegaly or abnormal imaging studies within the first year after HCT. In the absence of disease elsewhere, fine-needle aspiration or needle biopsy will usually be required for definitive diagnosis. Idiopathic hyperammonemia and coma A syndrome of hyperammonemia and coma has been described in patients who received high-dose chemotherapy, including conditioning for HCT [158]. Patients present with progressive lethargy, confusion, weakness, incoordination, vomiting, and hyperventilation with respiratory alkalosis. The diagnosis is confirmed when the plasma ammonia exceeds 200 μmol/L and there is no evidence of liver failure. This syndrome is rare, but is associated with a high mortality. The pathogenesis of idiopathic hyperammonemia likely involves the unmasking of a latent genetic disorder similar to ornithine transcarbamylase deficiency in children [159,160]. Gastrointestinal bleeding Bleeding that does not require transfusion is very common, particularly when platelet counts are low. Causes include retching trauma to esophageal or gastric mucosa, mucosal injury from conditioning therapy, peptic esophagitis, C. difficile colitis, anal fissures, hemorrhoids, and acute GVHD. The incidence of severe gastrointestinal bleeding after HCT is 1–2% – lower than in the past because of effective prophylaxis against viruses, fungi, and acute GVHD [161]. Mortality from severe intestinal bleeding, however, remains at 40% [161,162].
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Table 95.5 Differential diagnosis of gastrointestinal bleeding after transplant Infectious causes
Noninfectious causes
Cytomegalovirus ulcers Fungal infection (usually molds) Adenovirus infection Clostridial infection (C. difficile, C. septicum) Varicella zoster virus ulcers (esophagus, stomach) Helicobacter pylori-related ulcers Epstein–Barr virus lymphoproliferative disease Herpes simplex virus esophagitis
Mucosal necrosis from conditioning therapy Retching trauma (Mallory–Weiss tear) Acute/chronic graft-versus-host disease Lysis of intramucosal tumor Peptic esophagitis Gastric antral vascular ectasia (may also involve other sites) Mucosal biopsy sites Mycophenolate mofetil ulcerations Amyloidosis (mucosal ischemia)
Infectious causes in bold are now so rare that they should be considered only when patients are at risk.
The most common cause of severe bleeding is refractory acute GVHD, with bleeding from extensive ulceration in the small intestine and cecum (Table 95.5 and Plate 27.32). In some patients with GVHD, bleeding may appear to be coming from specific areas of the mucosa, but when such patients are operated on or come to autopsy, diffuse rather than focal mucosal ulceration is the rule [163,164]. Ulcers in the stomach or duodenum that develop after HCT are usually caused by acute GVHD or CMV infection, but with ganciclovir prophylaxis, bleeding CMV ulcers have become rare [56,161]. Gastric ulcerations may also be caused by infection by VZV, bacteria (phlegmonous gastritis) or EBV (lymphoproliferative disease; see Plate 27.3). Gastric antral vascular ectasia is also a cause of severe upper intestinal bleeding in HCT recipients, particularly those who have received oral BU [165]. Diffuse areas of hemorrhage are seen in the gastric antrum and proximal duodenum, but the underlying mucosa is intact. Histology is diagnostic, revealing abnormal dilated capillaries, thromboses, and fibromuscular hyperplasia in the lamina propria. Patients who are transplanted for advanced systemic sclerosis may bleed from similar vascular lesions. Endoscopic laser therapy is the treatment of choice to control bleeding from vascular ectasia, but multiple laser treatments may be required to obliterate ectatic lesions [165]. Similar vascular ectasias can rarely be found in the small intestine and colon [166]. Other rare causes of bleeding post HCT include ulcers caused by molds [167,168], Dieulafoy lesions, Curling’s (stress) ulcers, duodenal biopsy sites, adenovirus colitis, and C. septicum infection (typhlitis) [161]. Patients with severe amyloidosis who undergo autologous HCT may bleed from multiple ischemic ulcers throughout the intestine. There is no effective therapy for mucosa that is diffusely oozing blood other than raising the platelet count and treating the underlying condition. Continuous infusion of octreotide at 25 μg/hour has resulted in cessation of bleeding from GVHD, but recurrent bleeding developed when infusion was decreased [169]. In GVHD, re-epithelialization of ulcerated intestinal mucosa is very slow. Focal lesions, especially mucosal infection, can be treated with endoscopic cautery, heater probe or epinephrine injection provided platelet counts are adequate, but these therapies are futile in mucosa that is oozing blood diffusely. Unless the underlying disease process is eliminated, endoscopic methods will not cure the bleeding problem. Attempts to resect large segments of diffusely bleeding intestine involved with GVHD have not been successful [163].
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Table 95.6 Differential diagnosis of dysphagia, painful swallowing, and esophageal pain after transplant Infectious causes
Noninfectious causes
Cytomegalovirus Candida albicans Other fungal species (e.g. Candida glabrata, molds) Oropharyngeal bacteria (bacterial esophagitis) Herpes simplex virus Mycobacterium tuberculosis Varicella zoster virus
Acid–peptic reflux Intramural hematoma Pill esophagitis Postinflammatory esophageal stricture Acute GVHD of the esophagus Esophageal perforation Chronic GVHD of the esophagus (see section on problems in long-term transplant survivors)
Infectious causes in bold are now so rare that they should be considered only when patients are at risk. GVHD, graft-versus-host disease.
Dysphagia, painful swallowing, and esophageal pain Mucositis, acid–peptic esophagitis, and pill esophagitis are currently the leading causes of dysphagia and esophageal pain (Table 95.6). Infections of the esophagus (Plates 27.35 and 27.36) have largely disappeared because of antiviral and antifungal prophylaxis; when fungal esophagitis is discovered in a patient receiving fluconazole, the organism is likely to be a resistant candidal species or a mold. Rarely, fungal esophagitis can lead to perforation [170]. Mucositis caused by conditioning therapy may lead to pain on initiating a swallow and inability to move a bolus past the cricopharyngeus, symptoms that can also be seen with oropharyngeal GVHD and HSV infection. Rarely, nonhealing esophageal ulcerations, strictures, and dysphagia result from conditioning therapy [171]. In patients with gastric stasis related to acute GVHD, painful esophagitis is common, caused by reflux of both gastric acid and bilious fluid. The use of proton pump inhibitor therapy, while effective in treating acid–peptic reflux, may lead to colonization of the aerodigestive system with bacteria and fungi [172]. Symptom relief can be obtained by treating the cause of gastric stasis and placing the patient in the reverse Trendelenburg position when in bed. The abrupt onset of severe retrosternal pain, hematemesis, and painful swallowing suggests a hematoma in the wall of the esophagus, a result of retching when platelet counts are very low [173]. Endoscopy is relatively contraindicated, as many intramural hematomas represent contained perforations. The course of intramural hematomas is one of slow resolution over 1–2 weeks. In patients with severe GVHD, esophageal edema, erythema, and a peeling epithelium lead to ulcerations [174]. Pill esophagitis occurs after ingestion of some medications that might be used after HCT, for example phenytoin (dilantin), foscarnet, captopril, oral bisphosphonates, ascorbic acid, ciprofloxacin, clindamycin, and oral potassium chloride. Diarrhea Diarrhea caused by mucosal damage from conditioning therapy is seldom severe, usually resolving by day 12–15 (Plates 27.8 and 27.9). Cytarabine-containing regimens, high-dose melphalan (200 mg/m2), and regimens containing multiple alkylating agents may cause more severe, protracted diarrhea. Intravenous infusion of octreotide and oral loperamide (at 4 mg orally every 6 hours) may be effective for severe diarrhea associated with conditioning therapy [175]. There is a correlation between the amount of diarrhea in the week following completion of conditioning therapy and the risk of subsequent GVHD [176].
Table 95.7 Differential diagnosis of diarrhea after transplant Infectious causes
Noninfectious causes
Clostridium difficile Cytomegalovirus Adenovirus Rotavirus Clostridium septicum and other clostridial species Astrovirus, norovirus, other small round viruses Mycobacterial infection Fungal infection Giardia lamblia Cryptosporidia Microsporidia Strongyloides stercoralis Epstein–Barr virus lymphoproliferative disease Herpes simplex virus Bacterial enteric pathogens (e.g. Salmonella, Shigella, Campylobacter spp., Yersinia)
Residual effects of conditioning therapy Acute graft-versus-host disease Antibiotic-associated diarrhea Mycophenolate mofetil toxicity Promotility drugs Magnesium salts Carbohydrate malabsorption (e.g. genetic or acquired lactase, sucrase/isomaltase deficiency) Medication side-effect Pancreatic insufficiency Intestinal thrombotic microangiography
Infectious causes in bold are now so rare that they should be considered only when patients are at risk.
Acute GVHD is the most common cause of diarrhea after day 15 [162,177]. The onset of diarrhea can be sudden, with volumes in excess of 2 L daily in severe cases. GVHD in patients who were grafted after reduced-intensity conditioning regimens may have its presentation delayed, occurring after day 100 in many patients [178]. The diarrheal fluid of GVHD is watery, green in color, with ropy strands of mucoid material that reflect transmucosal protein loss [179]. In an allografted patient with skin and liver abnormalities typical of acute GVHD, this diarrheal syndrome is almost diagnostic of intestinal GVHD, particularly when there is falling serum albumin and negative stool studies for infection. In GVHD, abdominal imaging with CT or ultrasound may reveal intestinal edema, particularly in the ileum and right colon [75,180,181], but does not differentiate between CMV infection and acute GVHD. Pneumatosis intestinalis, which may be associated with GVHD or CMV enteritis, may be seen by plain X-ray, CT or MRI. A definitive diagnosis of GVHD in problematic cases requires mucosal biopsy. In mild cases, gastroduodenal and rectosigmoid mucosa are grossly normal, but moderately severe GVHD causes diffusely edematous and erythematous mucosa throughout the gastrointestinal tract [52]. Severe GVHD may lead to ulcerations and large areas of mucosal sloughing in the stomach, small intestine and colon [52,163]. Even when the appearance is normal, biopsies from the gastric antrum or rectosigmoid colon often reveal intestinal crypt cell necrosis and apoptotic bodies diagnostic of acute GVHD (Figs 27.2–27.4 and Plates 27.20 and 27.27–27.30) [117,182]. The diagnostic yield of mucosal biopsy is best when biopsies are obtained either from the stomach and distal colon or from the colon and ileum [182]. Reports by endoscopist and pathologist can be discordant, as mucosal edema and erythema are not criteria that are used for the histologic diagnosis of GVHD; visual inspection of the mucosa and histology should be viewed as complementary [52]. Other histologic findings that support the diagnosis of GVHD include pericapillary hemorrhage [183], infiltrating neutrophils [184] or eosinophils [185]. The use of capsule endoscopy for diagnosis of GVHD has been
Gastrointestinal and Hepatic Complications
described; this method does not allow biopsy, but can provide visual inspection of the small intestine that cannot be seen with routine endoscopy [186]. The predictive value of a negative capsule endoscopy examination of the small intestine appears to be high, and thus useful for excluding more severe acute GVHD [186]. In severe cases of GVHD, whole crypts are destroyed, then adjacent crypts, and finally whole segments of intestinal mucosa (Figs 27.3 and 27.4, and Plates 27.31 and 27.32). Bleeding often accompanies diarrhea in patients with mucosal ulceration [161]. Successful treatment of acute GVHD with immunosuppressive therapy results in a dramatic reduction in stool volume, with resolution of accompanying symptoms of abdominal pain, nausea, and vomiting. The management of patients whose diarrhea and other symptoms of intestinal GVHD persist after 7–14 days of immunosuppressive therapy is unsatisfactory, as the rate of failure of secondary therapy is high [187]. In allograft recipients, infectious causes of diarrhea are far less common than GVHD, accounting for only 10–15% of diarrheal episodes [162,177]. In countries where intestinal parasitism and bacterial contamination of water are endemic, the spectrum of infections may be wider [4]. Clostridium difficile colitis is usually a relatively mild, treatable disease when diagnosed at the onset of diarrhea [177]. The recent emergence of more virulent strains of C. difficile has changed the natural history of this infection, and thus prevention of nosocomial C. difficile infection in both hospital and outpatient settings has become even more important (Plate 27.34). One often overlooked factor is the inappropriate use of proton-pump inhibitors for marginal indications [172], which increases the risk of C. difficile colitis twofold [188]. The most common cause of infectious diarrhea in one prospective study was astrovirus (a small round virus similar to norovirus); diagnosis can be made by PCR of stool specimens or by typical histologic findings in small intestinal biopsies [177,189]. Some serotypes of adenovirus cause necrotizing enteritis and rapidly fatal multiorgan failure involving the gut, liver, lungs, and kidneys [138–140]. There should be a sense of urgency in identifying adenovirus as a cause of enteritis, as early treatment with cidofovir appears to be effective [143,144]. CMV is the only common infectious cause of enteritis after HCT that requires an intestinal biopsy for diagnosis [177]. CMV can be found in mucosal biopsies in patients whose blood is negative for CMV antigen or DNA [1,190]. Otherwise the predictive value of a negative stool examination for other viruses, bacteria, fungi, and parasites is high,
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particularly if molecular methods are used [5,177,191]. After HCT, watery diarrhea secondary to intestinal parasite infection (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica) is rare among patients to come to transplant without diarrhea, but sporadic cases are seen that can be confused with GVHD [4,6]. Strongyloides infection and hyperinfection syndrome have been described after HCT; patients from endemic areas should be screened before HCT. There are several disorders that can closely mimic intestinal GVHD or, more commonly, co-exist with GVHD, including infection with nonculturable enteric viruses, mycobacteria, fungi, and parasites (Table 95.8). Brush border disaccharidase deficiency, bile salt malabsorption, pancreatic insufficiency, mucosal toxicity from mycophenolate mofetil (MMF), and intestinal thrombotic microangiopathy may contribute to diarrhea that might otherwise be attributed to GVHD. That is, persistent diarrhea in an allograft recipient may not be caused directly by the necroinflammatory process of GVHD, but by related disorders. For example, intestinal inflammation often results in downregulation of brush border disaccharidases such as lactase and sucrase/isomaltase, leading to diarrhea if lactose or sucrose is ingested. Failure of bile salt absorption in the small intestine leads to inefficient water transport in the colon. Transient pancreatic insufficiency has been described as a consequence of mucosal edema at the ampulla of Vater [197]. Japanese investigators have described intestinal microangiopathy related to calcineurin inhibitors as a disorder that mimics GVHD [194]. MMF causes intestinal ulcerations and apoptotic crypt cells indistinguishable from the histology of acute GVHD [192,193]. In a patient with intestinal symptoms whose immunosuppressive drug regimen includes MMF, it may be difficult to make a diagnosis of GVHD until MMF has been discontinued. Diarrhea may also result from carbohydrate malabsorption (particularly in patients on antibiotics that affect the colon flora’s ability to salvage carbohydrate), oral magnesium salts, tacrolimus (a motilin agonist), and metoclopramide. The multiple causes of diarrhea not directly caused by GVHD in an allograft recipient may explain why diarrhea is not a component of the acute GVHD Activity Index that predicts day 200 nonrelapse mortality [198]. Abdominal pain It is extremely important to distinguish abdominal pain as an indicator of a rapidly progressive, fatal illness from illnesses with a benign natural
Table 95.8 Difficult-to-diagnose disorders that can mimic or complicate gastrointestinal acute GVHD Disorders
Comments
References
Mycophenolate mofetil toxicity
A wide range of gastrointestinal pathology has been described in mycophenolate mofetil-treated organ transplant patients. Very difficult to differentiate from GVHD in the setting of hematopoietic cell transplantation Diagnosis may be difficult when cytomegalovirus in present only in gastrointestinal mucosa (blood is virus free) Rare and treatable only by withdrawing immunosuppression. Molecular diagnosis is far more accurate than microscopic methods of diagnosis The most common infection(s) in a prospective study, but seldom diagnosed because of lack of commercial diagnostic tests (polymerase chain reaction) The hypothesis is that tacrolimus-related vascular injury leads to ischemic damage to mucosa. Withdrawal of tacrolimus has been followed by improvement in some cases Granulomatous mucosal involvement is part of the spectrum of mycobacterial infections in transplant patients Case report of a mold leading to granulomatous enteritis
[192,193]
Cytomegalovirus infection Cryptosporidial infection Nonculturable virus infections (astrovirus, norovirus) Tacrolimus-related thrombotic microangiography Nontuberculous mycobacterial infections Diffuse fungal infection
[1,56] [6,7] [177,189] [194]
[191,195] [196]
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Table 95.9 Differential diagnosis of abdominal pain after transplant Infectious causes
Noninfectious causes
Cytomegalovirus enteritis Clostridial colitis (C. difficile, C. septicum) Intestinal perforation (abscess/ peritonitis) Varicella zoster infection Adenovirus enteritis Acute cholecystitis Cystitis (JC/BK virus, adenovirus) Viral pancreatitis Aspergillus vasculitis/intestinal infarction Epstein–Barr virus lymphoproliferative disease Phlegmonous gastritis Helicobacter pylori-related ulcers
Colonic pseudo-obstruction Narcotic bowel syndrome Intestinal mucosal necrosis from myeloablative conditioning Sinusoidal obstructive syndrome Acute pancreatitis/pseudocyst Hemorrhagic cystitis (cyclophosphamide) Intramural hematoma of intestine/rectus sheath Acute graft-versus-host disease Biliary sludge syndrome
Infectious causes in bold are now so rare that they should be considered only when patients are at risk.
history that require only conservative management. The causes of abdominal pain after HCT are listed in Table 95.9. Dilation of the bowel in the absence of a mechanical obstruction (i.e. intestinal pseudo-obstruction) is the most common cause of moderateto-severe abdominal pain. The illnesses that may progress rapidly include intestinal perforation, some infections (e.g. typhlitis caused by C. septicum, adenovirus enteritis, and visceral VZV infection), gall bladder necrosis, and bacterial liver abscess. Fortunately, these disorders are far less common than intestinal pseudo-obstruction, liver pain related to SOS, acute GVHD, and hemorrhagic cystitis – causes of severe pain to be sure, but not imminent death. A systematic approach in evaluating abdominal pain in these difficult patients is important in excluding rapidly fatal diseases.
Does the patient need urgent surgery? Surgery is indicated for intestinal perforation, acute cholecystitis, drainage of abscesses, appendicitis, and in some patients with intestinal or biliary obstruction, typhlitis, and dissecting hematomas [199]. The majority of patients with abdominal pain do not require surgery [199], and in patients who have SOS, general anesthesia may jeopardize liver blood flow and lead to progressive liver failure. SOS presenting as severe abdominal pain can usually be recognized by its timing post HCT, by the finding of liver tenderness in either the epigastrium or the right lobe, away from the gall bladder fossa, and by consistent findings on Doppler ultrasound, which also serves to examine the right upper quadrant for abscess or gall bladder disease. Intestinal perforation may develop in the setting of lysis of a transmural lymphoma or metastatic carcinoma shortly after conditioning therapy, or later from CMV ulcers or diverticular perforation. Perforation may present with only mild-tomoderate abdominal pain and pneumoperitoneum on abdominal CT. Pneumoperitoneum on upright X-ray or CT scan was present in all Seattle patients with perforation, but this finding can be a manifestation of pneumatosis intestinalis, a more benign process than free perforation [200]. Recognition of acute cholecystitis is more difficult, as right upper quadrant pain (usually from SOS) and fever are common in the early post-HCT period, and imaging studies frequently show gall bladder wall
thickening and luminal sludge in completely asymptomatic patients [201]. A radionuclide study with morphine that shows filling of the gall bladder suggests that surgical cholecystitis is not present, but falsepositive results (i.e. lack of gall bladder filling) do occur in severely ill patients on total parenteral nutrition [202]. Dilation of the bowel in the absence of a mechanical obstruction is the most common cause of moderate-to-severe abdominal pain. Most patients with pseudo-obstruction have an underlying intestinal disease, such as enteritis from conditioning therapy, GVHD or infection, but frequently the acute presentation is related to increasing use of μ-opioid and anticholinergic medications. Pseudo-obstruction is more frequent among patients transplanted for lymphoma or myeloma, as a result of intestinal neuropathy from repeated use of vincristine. In visceral VZV infection, abdominal distention, severe pain, fever, and rising serum ALT levels may precede cutaneous manifestations by up to 10 days [136,142]. In rare instances, a skin rash never develops. Acyclovir should be started on clinical suspicion while serum is analyzed by PCR for VZV DNA. Pancreatitis is an uncommon cause of abdominal pain in HCT patients, but in a study of autopsied patients, the prevalence of acute pancreatitis was 28% [154]. Symptoms of pancreatitis were absent in many patients with florid pancreatitis at autopsy, suggesting that symptoms were masked by immunosuppressive drugs. Patients with low platelet counts or prolongation of blood clotting may rarely bleed into the retroperitoneum, abdominal wall or intra-abdominal viscera, particularly after duodenal biopsy, causing significant pain. Intestinal infections presenting with significant pain are listed in Table 95.9. Clostridium difficile colitis is generally mild, but severe colitis can be seen with more virulent strains (Plate 27.34). Typhlitis occurs in granulocytopenic patients infected with C. septicum but is not common after HCT. Symptoms include fever, right lower quadrant pain, nausea and vomiting, diarrhea, occult blood in the stool, and shock [9]. Diagnosis of typhlitis is usually made clinically by imaging studies [203]; laparotomy is rarely necessary provided that imipenem and oral vancomycin therapy for C. septicum are started along with systemic coverage for luminal bacteria and fungi in febrile patients [204]. Modern imaging tests (ultrasound and CT scan) and careful examination should allow the surgeon to be highly selective in choosing candidates for operation, as there is little to be gained by diagnosis of GVHD at operation [163,199]. Clinical judgment may lead to surgical exploration or laparoscopy in problematic cases where there is pneumoperitoneum of unknown cause or possible gall bladder necrosis.
Is the pain a manifestation of acute GVHD? Acute gastrointestinal GVHD usually presents with nausea, vomiting, anorexia, abdominal pain, and diarrhea [179]. The sudden onset of intestinal edema can cause a rigid abdomen with rebound tenderness preceding the development of a skin rash or diarrhea [163]; pain is usually crampy and periumbilical, but can be localized to the epigastrium. The decision to treat a patient empirically when definitive evidence of GVHD is not at hand is difficult, but when the pretest probability of GVHD is high and that of perforation or infection low, prednisone therapy at 2 mg/kg/day should be started [187]. CT abdominal imaging shows intestinal wall thickening in more severe acute GVHD, a nonspecific but highly suggestive finding, particularly when CMV infection is unlikely [75,180]. CT is preferred to ultrasound because of better sensitivity for detecting perforation or intra-abdominal abscess. Although one might withhold prednisone therapy until a definitive biopsy diagnosis has been made, delays in therapy may lead to more extensive mucosal necrosis and morbidity. If the pain later proves to be due to causes other than GVHD, prednisone can be discontinued.
Gastrointestinal and Hepatic Complications
Is visceral VZV infection a possibility? Disseminated VZV may present with difficult-to-explain abdominal pain 2–6 months after HCT. This presentation is most likely in immunosuppressed patients who had positive pretransplant VZV serology and who are not currently receiving acyclovir or ganciclovir therapy. Importantly, the abdominal pain may precede typical VZV skin lesions by several days [142,205,206]. Marked hyponatremia due to the syndrome of inappropriate antidiuretic hormone secretion has also been observed in this setting [207]. Patients should be started on intravenous acyclovir while a definitive diagnosis is sought (VZV DNA in serum, gastric mucosal or liver biopsy; Plate 27.58) [206,208]. Pain from visceral VZV infection may be poorly localized, and the patient may not look particularly unwell at presentation, but if not treated promptly, the disease can follow a rapidly fatal course. Is the pain due to a treatable infection? Recent advances in antiviral and antifungal prophylaxis [17,152,209] have made intestinal and liver infections unusual causes of abdominal pain. Nonetheless, infections of the liver and intestinal tract must be considered in patients with pain, particularly infection caused by CMV, adenovirus, C. difficile, C. septicum (typhlitis) or mold (either blood borne or mucosally invasive) [9,56,138–140,168,210,211]. Is the pain caused by intestinal pseudo-obstruction? In patients with intestinal pseudo-obstruction and gaseous distention, medical management is almost always effective and perforation rare. While there are some infections (VZV [212] and CMV [213]) that lead to pseudo-obstruction, opioid and anticholinergic medications are the usual cause. There are only two sources of intestinal gas: swallowed air (or, in the case of mechanical ventilation, leakage around the endotracheal tube) and fermentation of carbohydrate by colon bacteria. A nasoesophageal sump tube connected to suction will prevent additional swallowed air from accumulating in the gut. Switching from a μ-opioid to a κ-opioid agonist (e.g. butorphanol) may allow pain relief while allowing gut motility to recover. Peripheral μ-opioid blockade with methylnaltrexone can also be effective [214]. Neostigmine (2 mg intravenously) has been successfully used in patients with acute colonic pseudo-obstruction after HCT [215]. If distention is caused by fluid, as in severe acute GVHD, or by true obstruction, a rare event, the approach is to place a nasogastric or nasoenteric sump tube to prevent further accumulation, and then address the underlying cause. Identification of other causes of abdominal pain Once the diseases that need urgent surgical or medical care have been ruled out, one can perform directed diagnostic tests to identify specific causes of pain. Hemorrhagic cystitis is usually seen after CY exposure but also occurs with adenovirus or polyomavirus infection; it is always accompanied by hematuria. Hematomas of the abdominal wall or intestine should be suspected in patients with a rapidly expanding anterior abdominal wall mass or obstructive symptoms after intestinal biopsy in the setting of profound thrombocytopenia. Perianal pain Perianal pain after HCT can be caused by an anal fissure, a thrombosed external hemorrhoid, cellulitis related to tissue maceration, and infections. In patients with granulocytopenia, infections in the perineum or perianal spaces are usually polymicrobial, arising either from anal glands or from tears in the anal canal. After HCT, these infections can be difficult to recognize because they may not produce abscesses but rather a spreading cellulitis. Extensive supralevator and intersphincteric abscesses
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may be present without being apparent on external examination [10,12]. CT or MRI scans or endoscopic ultrasound can give a clear view of the anatomy involved, particularly if there is pus present [216]. When antibiotics covering both anaerobic and aerobic bacteria are given to patients with “cryptitis” (incipient perianal infection), far fewer patients require surgical drainage than in the past [11]. Rarely, surgical drainage will be needed for patients with abscesses and rapidly progressive tissue necrosis.
Problems in long-term transplant survivors Liver disorders Chronic GVHD Patients with chronic hepatic GVHD usually have features of chronic GVHD elsewhere in the body. Evidence of cholestasis (elevated alkaline phosphatase and gamma glutamyl transpeptidase, usually without pruritus) is present in approximately 80% of patients with extensive chronic GVHD. Patients with isolated elevations of alkaline phosphatase in the absence of jaundice should be followed closely, but may not require extended doses of high-dose immunosuppressive therapy. Jaundice is a late feature of the disease process, and by the time that jaundice develops, liver biopsy shows extensive damage to, and loss of, small bile ducts (Plates 27.40 and 27.41) [132,133]. In patients receiving no, or tapering doses of, immunosuppression, chronic liver GVHD may present acutely with abrupt elevations of aminotransferase levels to over 2000 U/ L [133,135]. Serologic and nucleic acid testing and liver biopsy are essential to exclude acute viral hepatitis due to a herpesvirus (HSV or VZV) or a hepatitis virus (hepatitis A–E), and to make a definitive diagnosis of chronic GVHD (Plate 27.39) [217]. A serum autoantibody test for CYP1A2 may prove diagnostically useful in the diagnosis of hepatitic GVHD, as this enzyme appears to be a target antigen in GVHD [218]. Acyclovir should be started pending results of viral tests, and, if negative, treatment with a calcineurin inhibitor and prednisone 1–2 mg/ kg/day should be begun. Immunosuppressive drug treatment of chronic GVHD is successful in 50–80% of patients with extensive multiorgan disease. The use of long-term ursodeoxycholic acid (15 mg/kg/day) is safe and well tolerated, and may result in normalization or improvement of liver enzymes [219]. In longstanding chronic GVHD of the liver, small bile ducts may be absent (Plate 27.41), analogous to ductopenic rejection in liver allografts. Ductopenic GVHD is potentially reversible if ongoing immunologic destruction of epithelium ceases, but this process may take months before resolution of jaundice [133]. Liver transplantation, including living-donor transplantation from the original stem cell donor [220], has been performed for patients with liver failure due to chronic hepatic GVHD, although frequently there are contraindications to this approach [221]. Chronic viral hepatitis and cirrhosis HCV infection in HCT survivors almost always results in chronic hepatitis [27,28]. In the first 10 years of HCV infection after HCT, there is little liver-related morbidity. However, cirrhosis of the liver related to chronic HCV infection is rising in frequency among patients transplanted before the 1990s [28,222]. The rate of progression of HCV is higher in HCT recipients than in matched controls, with reports of cirrhosis in 24% by 20 years follow-up [28]. The reasons for more rapid fibrosis progression after HCT are unclear, but may be related to concomitant liver involvement with GVHD, iron overload [223], and immunosuppression. Clues to the presence of cirrhosis include a switch in the normal ratio of serum aminotransferases so that AST is higher than ALT, thrombocytopenia, and hepatic nodularity and splenomegaly on imaging,
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although thrombocytopenia may be unrelated to liver disease in HCT survivors. It is important to establish a diagnosis of cirrhosis in patients with chronic hepatitis C, so that monitoring for complications can be undertaken. Hepatocellular carcinoma develops in 2–8% per annum of patients with HCV cirrhosis, and screening (usually with 6-monthly ultrasound and serum alfafetoprotein levels) is recommended [224]. Chronic hepatitis C may also be a risk factor for development of lymphoma [225] and other lymphoproliferative disorders [226] after transplant. All patients with chronic HCV, including those with compensated cirrhosis, should be considered for therapy with combination pegylated interferon-α plus ribavirin, unless contraindications exist [227,228]. Pegylated interferon is currently the standard of care for treatment of chronic hepatitis C; however, the long half-life may be associated with rapid falls in platelet and granulocyte counts. Patients with renal impairment may experience increased hemolysis and anemia with ribavirin therapy. Growth factors (e.g. filgastrim and erythropoietin) may be needed to support patients through therapy. New direct antivirals for hepatitis C are under development and may offer enhanced viral responses in the future [229]. Interferon-based therapy may activate chronic GVHD, and patients should be closely monitored. Liver transplantation should be considered in any HCT survivor with hepatic decompensation or early hepatocellular carcinoma. Living-donor transplantation from the original stem cell donor is an attractive consideration in this situation, as minimal immunosuppression would likely be required in the long term [230]. The prevalence of chronic HBV infection among HCT survivors varies widely depending on the country. The serologic pattern of HBV infection may be atypical in HCT survivors, probably as a consequence of immunosuppression. Clearance of surface antigenemia may be observed, and is particularly likely if the donor was anti-HBs positive because of prior HBV infection [20]. Patients who remain HBsAg positive after HCT are at risk of flares of hepatitis activity, particularly at times of reduction of immunosuppression, such as during taper or cessation of treatment for chronic GVHD. All long-term survivors with chronic hepatitis B should be regularly monitored to assess virologic and disease status, and the need for antiviral therapy. Hepatitis B e antigen and antibody status should be determined in all patients, and liver enzymes monitored every 6–12 months. The HBV DNA level should be determined in those with abnormal ALT levels, and liver biopsy considered to assess the severity of hepatic inflammation and fibrosis. The need for antiviral treatment with an oral nucleoside or nucleotide analogue (e.g. entecavir, telbivudine adefovir, or tenofovir) is based upon the ALT and HBV DNA levels, and the severity of hepatic fibrosis [231], and may change over time, hence the need to continually reassess patients who are not on treatment. Long-term survivors with chronic hepatitis B do not seem to have an increased rate of progression to cirrhosis compared with non-HCT patients. As HCT survivors are at increased risk of second malignancy or recurrent malignancy, care should be taken to reassess HBV viral status prior to reintroduction of chemotherapy. Newer agents, such as rituximab used in the treatment of B-cell malignancy, have a particularly high risk of reactivation of latent hepatitis B (HBsAg negative and anti-HBc positive), and careful monitoring of HBV DNA levels on therapy and early introduction of an antiviral drug (lamivudine, entecavir adefovir, or tenofovir) is strongly recommended [232]. As in the peritransplant period, patients with positive HBsAg should receive an antiviral agent whenever they receive immunosuppressive or cytotoxic therapy [233]. Fungal abscess Fungal abscesses can recur after apparently successful antifungal therapy, when high-dose immunosuppressive drugs are started for GVHD. Non-
sterile herbal remedies contaminated by molds may lead to liver abscesses in immunosuppressed HCT survivors [234]. Acute hepatocellular injury The differential diagnosis of extreme elevations of serum ALT in an HCT survivor includes VZV infection, a hepatitic presentation of chronic GVHD [133,134], flares of chronic hepatitis B or C, and drug-induced liver injury. Drug-induced liver injury may be related to antihypertensive drugs, lipid-lowering agents, hypoglycemic agents, nonsteroidal antiinflammatory drugs, antidepressants, antibiotics or herbal preparations. Some drug reactions may result in chronic liver disease [235]. Iron overload Iron overload may be an important cofactor in liver disease in long-term survivors of HCT, and should be part of a screening panel [236,237]. Iron overload is particularly severe in thalassemic patients who have undergone HCT [238]. Iron overload is caused by a combination of multiple red cell transfusions and dyserythropoiesis leading to aberrant hepcidin regulation and increased iron transport by the intestine. In patients where a marked degree of iron overload was unexpected, HFE gene testing (on buccal mucosa swab or stored DNA sample) should be performed. After HCT, iron accumulation stops and body iron stores fall slowly over time [239]. Clinically significant iron overload is usually present only when serum ferritin levels are over 1000 μg/dL [240]. An elevated serum ferritin level, particularly in patients with chronic GVHD, chronic viral hepatitis or other causes of liver disease, may not reflect tissue iron stores, and it may be important to document the degree of tissue iron overload. In the past, liver biopsy with liver iron determination was required; however, increasingly noninvasive methods (e.g. MRI and FerriScan [41]) are being utilized to provide assessments of liver iron concentration and distribution. The consequences of extreme iron overload in HCT survivors are primarily those of cardiac, pituitary, and pancreatic endocrine dysfunction. Patients with a liver iron content greater than 15,000 μg/g dry weight (or equivalent) should be treated aggressively with both phlebotomy and chelation; when the liver iron content is 7000–15,000 μg/g dry weight, phlebotomy is indicated; but when the liver iron content is under 7000 μg/g dry weight, treatment is indicated only if there is evidence of liver disease [241]. Mobilization of iron from heavily overloaded patients improves cardiac function, normalizes serum ALT levels, and results in improved liver histology [46,241,242]. Iron overload may also be a cause of persistent hepatic dysfunction after HCT that responds to iron mobilization [46,47]. Hepatocellular carcinoma Compared with the general population, patients who survive over 10 years post HCT have an eightfold risk of developing a new solid malignancy. Because of the increased rate of chronic viral hepatitis, particularly hepatitis C, the risk of hepatocellular carcinoma is particularly elevated [243]. Transplant survivors with risk factors for hepatocellular carcinoma (HCV or HBV infection, obesity, diabetes or low platelet count) should be screened at yearly intervals [244]. Nodular regenerative hyperplasia Rarely, patients who have experienced toxic liver injury (SOS) from either chemotherapy or a myeloablative conditioning regimen will develop hepatic nodularity caused by atrophy of zone 3 and hypertrophy of zone 1 hepatocytes, without fibrosis [245]. This process is usually clinically silent unless portal hypertension develops, manifest by variceal bleeding, ascites, splenomegaly, and thrombocytopenia, but with preserved liver function.
Gastrointestinal and Hepatic Complications
Gall bladder and biliary tract disease There appears to be a higher-than-expected incidence of gallstones and stone-related biliary problems after HCT than in an age-matched population, probably related to earlier formation of nucleating microliths (biliary sludge). Chronic cyclosporine or tacrolimus dosing may also lead to gallstones, biliary symptoms, and acute pancreatitis [246–248]. Esophageal diseases Chronic GVHD Some patients with extensive chronic GVHD have esophageal desquamation, webs, submucosal fibrous rings, bullae, and long, narrow strictures in the upper and mid-esophagus [249–251]. Development of severe esophageal GVHD is most frequent in patients whose chronic GVHD has been either neglected or inadequately treated. The most common symptom is dysphagia; some patients present with insidious weight loss, retrosternal pain, and aspiration of gastric contents, leading to pulmonary disease that can be mistaken for bronchiolitis obliterans. In children, a common presentation is weight loss and failure to thrive. The diagnosis is made by barium contrast X-ray and endoscopy, which should be done with caution, as perforations have been reported [249]. Tight strictures are difficult to dilate safely, but failure to dilate strictures may lead to progressive esophageal narrowing. Although strictures that have developed recently can be successfully dilated under cover of immunosuppression, chronic strictures that involve most of the upper esophagus may prove intractable. Esophageal involvement can be prevented by prompt treatment of chronic GVHD at its early stages. Therapy with proton pump inhibitors should be considered because patients with esophageal chronic GVHD usually have uncontrolled acid reflux as a result of poor salivary bicarbonate secretion and lack of esophageal peristalsis. Myasthenia gravis may also complicate chronic GVHD, with dysphagia as its presenting complaint [252].
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usually in patients with concomitant chronic GVHD of the oropharynx [253]. Gastrointestinal symptoms: diarrhea, anorexia, nausea, and weight loss The incidence of diarrhea falls sharply after day 100 except in some patients who have developed acute GVHD after reduced-intensity conditioning regimens [178] and in patients whose severe acute GVHD has never resolved. Some long-term survivors have intestinal symptoms for years following HCT, caused by protracted acute GVHD whose symptoms wax and wane with the intensity of immunosuppressive therapy, with each exacerbation similar to the presenting signs of acute GVHD (satiety, poor appetite, nausea, episodic diarrhea, and weight loss) [254,255]. The endoscopic and histologic appearance of the intestinal mucosa is identical to that seen in acute GVHD. This form of GVHD is not considered typical of chronic GVHD, but frequently occurs in patients with chronic GVHD in other sites [256]. Use of oral beclomethasone dipropionate can be effective in treating patients with protracted acute GVHD involving the gastrointestinal tract [257]. Before the introduction of more effective immunosuppressive drugs, chronic GVHD resulted in extensive collagen deposition in submucosal and subserosal areas of the intestinal tract (Plate 27.33), resulting in refractory malabsorption [258]; this process has not been seen in recent years. There are sporadic cases of C. difficile, CMV [56], and rarely Giardia and Cryptosporidium infection in long-term survivors. Rarely, chronic intestinal viral infection can be seen in patients who remain on immunosuppressive drugs, for example rotavirus, norovirus, and adenovirus. Rare cases of “transmission” of intestinal diseases have been reported, such as inflammatory bowel disease and celiac sprue [259]. Pancreatic insufficiency may also cause diarrhea after transplant [197,260,261]. Pancreatic disease
Strictures Esophageal strictures may also be sequelae of earlier herpesvirus infection, severe mucositis involving the esophagus or chronic reflux of gastric contents. Sporadic cases of fungal and rarely viral esophagitis may occur in patients with chronic GVHD on immunosuppressive and antibiotic therapy. Esophageal cancer In series of secondary cancers developing late after transplant, squamous cell carcinoma of the esophagus has been reported,
Sporadic cases of acute pancreatitis are seen in transplant survivors, usually related to passage of gallstones or sludge; cyclosporine and tacrolimus have also been implicated [246–248]. Diarrhea, steatorrhea, and weight loss secondary to pancreatic insufficiency have developed in some long-term HCT survivors [260,261]. The most likely cause is pancreatic acinar atrophy from previous pancreatic necrosis or prolonged corticosteroid exposure [154]. Transient pancreatic insufficiency has been noted in patients with gut and liver GVHD [197]. Chronic GVHD and extreme iron overload may also contribute to pancreatic damage.
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Kenneth R. Cooke & Gregory A. Yanik
Lung Injury Following Hematopoietic Cell Transplantation
Introduction Allogeneic hematopoietic cell transplantation (HCT) is the only curative therapy for a number of malignant and nonmalignant conditions, but successful outcomes are limited by several side-effects, including pulmonary toxicity. Diffuse lung injury remains a significant problem following allogeneic HCT both in the immediate post-transplant period and in the months to years that follow. Lung injury occurs in 25–55% of HCT recipients and accounts for approximately 50% of transplantrelated mortality [1–6]. Historically, approximately one-half of all pneumonias seen after HCT have been secondary to infection, but the judicious use of broad-spectrum antimicrobial agents has tipped the balance toward noninfectious causes [7]. Despite advances in preventing and treating opportunistic organisms, infectious lung injury remains a significant problem, particularly in patients with acute or chronic graftversus-host disease (GVHD) or in individuals who have poor or delayed immune reconstitution. Noninfectious lung injury can be either acute (idiopathic pneumonia syndrome [IPS]) or chronic, depending upon the onset after HCT and the tempo of disease progression. Chronic lung injury is further subdivided into two types: obstructive and restrictive [8–14]. Although noninfectious lung injury occasionally occurs following autologous transplants, the allogeneic setting significantly exacerbates the severity of disease in both the acute and chronic timeframes; in each scenario, pulmonary toxicity is associated with significant morbidity and mortality, and responds poorly to standard therapy. This chapter will review the definitions, risk factors, and pathogeneses of lung injury occurring both early and late after allogeneic HCT.
Infectious interstitial pneumonias Infections contribute to interstitial pulmonary infiltrates in a significant number of HCT recipients. The most common pathogens include community acquired respiratory viruses (e.g. parainfluenza, respiratory syncytial virus [RSV], influenza, and metapneumonia), Mycoplasma, and opportunistic pathogens such as Pneumocystis jiroveci (previously Pneumocystis carinii) (Table 96.1). Fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) or surgical lung biopsy is required to distinguish infectious from noninfectious causes (Table 96.2). Besides bacterial, fungal, and cytologic stains, quantitative cultures should be performed on BAL fluid for diagnostic purposes. In addition, direct fluorescent antibody stains, centrifugation cultures (shell vial), and
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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polymerase chain reaction (PCR) assays may be very useful in isolating and/or identifying various viral pathogens. Pneumocystis jiroveci pneumonia may be identified through a number of techniques, including cytologic studies, special stains, and PCR-based assays. Cytomegalovirus (CMV) pneumonitis remains a significant cause of morbidity and mortality following allogeneic HCT. In the absence of a CMV prevention strategy (e.g. pre-emptive monitoring of CMV by plasma PCR or antigenemia, or universal prophylaxis), CMV pneumonitis may develop during the first 100 days following HCT with a peak incidence at approximately 8 weeks. In the current era, most CMV infections occur after the monitoring or prophylaxis period ends. CMV pneumonitis in patients with chronic GVHD has also been well documented [15,16]. With the availability of improved antiviral therapy, the mortality rate associated with CMV pneumonitis has declined significantly in recent years [17]. Risk factors for the development of CMV disease include older patient age, the presence of acute GVHD, recipient CMV seropositivity, transplantation for a hematologic malignancy, and the use of antithymocyte globulin during the transplant process [16,18]. Radiologic manifestations of CMV range from diffuse reticular opacities to widespread air space consolidation [19]. Histopathology remains the gold standard for identification of CMV pulmonary disease. Although molecular techniques have an excellent sensitivity for detection of infection, they may be less specific in regard to identifying CMV pneumonitis. RSV is a single-stranded, enveloped RNA virus that presents as a self-limiting upper respiratory tract infection in immunocompetent individuals, or a potentially fatal pneumonitis in immunocompromised patients [20]. Outbreaks in patients undergoing allogeneic HCT have been associated with mortality rates as high as 78% [21,22]. In the United States, the onset of RSV infections typically begins in November and continues for approximately 24 weeks. The organism is highly contagious, with transmission occurring primarily through surfaces contaminated with virus-laden nasal or oral secretions. Even in immunocompetent patients, native memory responses are incomplete, allowing for repeated infections. The overall virulence of this agent places the immunocompromised patient at particular risk for fatal lower respiratory tract infections during the seasonal period [20]. Radiographically, patchy alveolar or diffuse interstitial infiltrates may be seen. Clinically, affected patients may exhibit a profound dyspnea and hypoxemia, with or without concurrent upper respiratory tract symptoms. Less than 50% of patients with lower tract involvement have preceding or concurrent nasopharyngeal symptoms [20,22]. The use of aerosolized ribavirin (6 g/day) using small-particle generators has resulted in a decrease in RSV shedding, but clinical efficacy has not been proven [20,23]. The clinical impact of recently described viruses, including human metapneumovirus, and non-severe acute respiratory syndrome (SARS)
Lung Injury Following Hematopoietic Cell Transplantation
human coronaviruses is not yet clear. Human metapneumovirus is a paramyxovirus recently recognized as the first human pathogen within the genus Metapneumovirus [24]. The virus was first identified in the Netherlands in 2001, and was recently reported as a potential pathogen in allogeneic HCT recipients [25]. Infected patients typically present within the first 50 days post transplant, and exhibit clinical and radiographic findings similar in appearance to those of IPS. Mortality rates as high as 80% have been reported. Rapid progression of respiratory symptoms may occur, with a median of 4 days between initiation of oxygen support and death [25]. The clinical spectrum of human metapneumovirus as a cause of interstitial pneumonia post transplant remains ill-defined, with issues such as the frequency of asymptomatic shedding, improved detection methods, and treatment strategies all under investigation. The reactivation of latent viruses, especially herpes family and adenovirus, may also clinically mimic IPS (discussed below) [26]. However,
Table 96.1 Common infectious pathogens of diffuse interstitial pneumonia Viral
Cytomegalovirus Adenovirus Human herpes virus-6 Varicella virus Respiratory syncytial virus Parainfluenza – types 1 and 2 Influenza A, influenza B Rhinovirus Metapneumovirus
Protozoan
Pneumocystis jiroveci pneumonia Toxoplasma gondii Mycoplasma Chlamydia
Mycobacterial infections
Miliary tuberculosis
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in the vast majority of cases, clinical symptoms related to systemic involvement are manifested by signs of disease in other organs, including elevation of serum hepatic transaminases with CMV, varicella zoster virus or adenovirus, oral mucosal involvement with herpes simplex viruses, or cutaneous involvement secondary to varicella. Abdominal pain with diarrhea may represent concurrent viral enteritis, as commonly seen in association with CMV, herpes simplex or adenovirus.
Noninfectious, acute lung injury: IPS Definition, clinical course, and spectrum of disease In 1993, IPS was defined by a panel of experts as widespread alveolar injury following hematopoietic HCT that occurs in the absence of an active lower respiratory tract infection and cardiogenic causes [27]. As shown in Table 96.2, diagnostic criteria of IPS include signs and symptoms of pneumonia, nonlobar radiographic infiltrates, abnormal pulmonary function, and the absence of infectious organisms as determined by BAL or lung biopsy [2,27]. A variety of histopathologic findings have been associated with IPS, including diffuse alveolar damage with hyaline membranes, lymphocytic bronchitis, bronchiolitis obliterans organizing pneumonia (BOOP) [28], and interstitial pneumonitis (IP). The term IP has historically been used interchangeably with IPS, and it is the most frequently reported histologic pattern associated with this syndrome [3]. IP is seen in association with diffuse alveolar damage and hemorrhage early after HCT and is accompanied by bronchiolar inflammation and epithelial damage at later time points [28]. The incidence of IPS in the first 120 days after allogeneic HCT following high-dose conditioning ranges from 3% to 15% [5–7,27]. The median time of onset for IPS was initially reported to be 6–7 weeks (range 14–90 days) after HCT [27]. Mortality rates ranged from 50% to 80% overall, and to greater than 95% for patients requiring mechanical ventilation [1,3,5–7,27]. A retrospective study from Seattle showed a lower incidence and earlier onset of IPS than previously reported, but the typical clinical course involving the rapid onset of respiratory failure leading to death remained unchanged [6]. In a more recent publication, the frequency of IPS after allogeneic HCT ranged from 5% to 25%
Table 96.2 Definition of idiopathic pneumonia syndrome I: Evidence of widespread alveolar injury: a. Multilobar infiltrates on routine chest radiographs or computed tomography b. Symptoms and signs of pneumonia (cough, dyspnea, tachypnea, rales) c. Evidence of abnormal pulmonary physiology 1. Increased alveolar to arterial oxygen difference 2. New or increased restrictive pulmonary function test abnormality II: Absence of active lower respiratory tract infection based upon: a. Bronchoalveolar lavage negative for significant bacterial pathogens including: acid-fast bacilli, Nocardia and Legionella species b. Bronchoalveolar lavage negative for pathogenic nonbacterial microorganisms: 1. Routine culture for viruses and fungi 2. Shell vial culture for CMV and RSV 3. Cytology for CMV inclusions, fungi and Pneumocystis jiroveci 4. Direct fluorescence staining with antibodies against CMV, RSV, HSV, VZV, influenza virus, parainfluenza virus, adenovirus, and other organisms c. Other organisms/tests to also consider: 1. PCR for HMP, rhinovirus, coronavirus, and HHV-6 2. PCR for Chlamydia, Mycoplasma, and Aspergillus species 3. Serum galactomannan ELISA for Aspergillus species d. Transbronchial biopsy if the patient’s condition permits III: Absence of cardiac dysfunction, acute renal failure or iatrogenic fluid overload as etiology for pulmonary symptomatology CMV, cytomegalovirus; ELISA, immune-linked immunosorbent assay; HHV, human herpes virus; HMP, human metapneumovirus; HSV, herpes simplex virus; PRC, polymerase chain reaction; RSV, respiratory syncytial virus; VZV, varicella zoster virus.
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Chemoradiation toxicity
TRALI
IP CLS Infection
DAH
Cardiogenic edema
IPS PERDS
ARDS
RLD: BOOP / IP
OLD: BrOb/BOS
Pulmonary fibrosis
depending upon donor source and the degree of antigenic mismatch between donor and recipient [29]. Day 100 mortality was 80%, and the median time from diagnosis to death was 13 days despite aggressive treatment with high-dose steroids and broad-spectrum antimicrobial therapy. The clinical spectrum of IPS encompasses several forms of pulmonary toxicity (Fig. 96.1 and Table 96.3). In one small subset of patients with IPS, acute pulmonary hemorrhage or hemorrhagic alveolitis occurs. Diffuse alveolar hemorrhage (DAH) generally develops in the immediate post-HCT period, and is characterized by progressive shortness of breath, cough, and hypoxemia with or without fever [7,30–32]. Classically, DAH is defined by the demonstration of progressively bloodier aliquots of BAL fluid, but frank hemoptysis is rare [30]. Mortality from DAH is as high as 75% despite aggressive treatment with high dose (2 mg/kg to 1 g/m2) steroids, and death usually occurs within weeks of diagnosis [31]. Some patients with DAH can have microorganisms isolated from blood, BAL fluid or tracheal aspirate within 1 week of alveolar hemorrhage. Majhail and colleagues recently compared patients with DAH and infection-associated alveolar hemorrhage who presented with similar clinical and radiographic findings in the setting of progressively bloodier BAL fluid following allogeneic HCT. Alveolar hemorrhage from infectious and noninfectious causes was found to be related but distinct entities with extremely poor outcomes following therapy with conventional agents, including steroids [33]. Peri-engraftment respiratory distress syndrome (PERDS) also falls within the definition of IPS [7]. PERDS is characterized by fever, dyspnea, and hypoxemia that, by definition, occur within 5–7 days of neutrophil engraftment [34–36]. Although PERDS after autologous HCT appears similar to IPS after allogeneic HCT with respect to clinical presentation and time of onset, the two entities differ sharply with respect to overall outcome: inflammation in the autologous setting responds promptly to corticosteroids and is associated with a favorable
Fig. 96.1. Clinical spectrum of idiopathic pneumonia syndrome (IPS) after hematopoietic cell transplantation. The clinical spectrum of IPS includes a variety of descriptive forms of lung injury that may share clinical features with toxicity incurred by chemoradiotherapy, cardiogenic edema, pulmonary fibrosis, infection, and transfusionassociated lung injury (TRALI). ARDS, acute respiratory distress syndrome; BOOP, bronchiolitis obliterans organizing pneumonia; BOS, bronchiolitis obliterans syndrome; BrOb, bronchiolitis obliterans; CLS, capillary leak syndrome; DAH, diffuse alveolar hemorrhage; IP, interstitial pneumonitis; OLD, obstructive lung disease; PERDS, peri-engraftment respiratory distress syndrome; RLD, restrictive lung disease.
prognosis [34], whereas PERDS occurring in an allogeneic environment responds poorly to standard therapy and commonly results in rapid respiratory failure and death in the majority of patients [6,29,37]. Lung inflammation following the administration of 1,3-bis(2choloroethyl)-1-nitrosurea (BCNU; carmustine) and transfusionassociated lung injury (TRALI) are two forms of noninfectious pulmonary toxicity that can be mistaken for IPS but have distinct etiologies. Initially described in the 1970s, chemotherapy-associated pulmonary toxicity has been reported in association with multiple chemotherapeutic agents, including BCNU, busulfan, and melphalan. In particular, BCNU-related lung injury is acute in onset and develops within the first 3 months following HCT in 10–40% of patients receiving this therapy [38]. Clinically, BCNU-related lung injury is typically associated with a nonproductive cough with increasing dyspnea in the context rapidly progressing, bilateral, interstitial infiltrates on both chest radiograph and computed tomography. Pulmonary function tests (PFTs) reveal a restrictive pattern of lung injury, with diminished forced vital capacity (FVC) and total lung capacity (TLC) noted. Risk factors for BCNU pneumonitis include prior pulmonary irradiation, cigarette smoking, and a dosage greater than 450 mg/m2 [39]. The pathogenesis of BCNU-related lung injury has been ill-defined, although increased production of fibrogenic factors such as platelet-derived growth factor-β, insulin-like growth factor I and transforming growth factor-β1 have been implicated [40]. Treatment with pulsed doses of corticosteroids early in the clinical course significantly decreases the morbidity and mortality associated with this condition and is key for successful outcomes; if left untreated, or recognized late in the clinical course, severe pulmonary fibrosis may develop [40]. TRALI is one of the leading causes of mortality following infusions of plasma containing blood products, estimated to occur in 1 in 1000 to 1 in 5000 transfusions [41,42]. Symptoms present acutely, with the onset of dyspnea and respiratory distress typically 6–8 hours following
Lung Injury Following Hematopoietic Cell Transplantation
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Table 96.3 The spectrum of noncardiogenic, pulmonary toxicity defined by idiopathic pneumonia syndrome Interstitial pneumonitis: • Clinical symptoms: fever, cough, dyspnea, hypoxemia • Onset: within first 100 days post transplant • Etiology: infectious (i.e. CMV, Pneumocystis jiroveci pneumonia) or noninfectious factors (chemotoxicity: BCNU, bleomycin, busulfan, methotrexate) • Radiographic findings: bilateral interstitial infiltrates Diffuse alveolar hemorrhage: • Clinical symptoms: progressive dyspnea, cough, rare hemoptysis • Key finding: progressively bloodier aliquots of lavage fluid • Onset: early, within first 100 days post transplant. • Radiographic findings: diffuse infiltrates, central appearance initially noted • Histology: diffuse alveolar damage with alveolar hemorrhage Peri-engraftment respiratory distress syndrome: • Clinical symptoms: fever, dyspnea, hypoxemia • Onset: very early, within 5–7 days of engraftment, classically after autologous stem cell transplantation • Radiographic findings: bilateral interstitial infiltrates Noncardiogenic capillary leak syndrome: • Clinical symptoms: dyspnea, cough, weight gain, edema • Onset: early, within first 30 days post transplant • Radiographic findings: bilateral perihilar infiltrates, pulmonary edema, pleural effusions Bronchiolitis obliterans organizing pneumonia: • Clinical symptoms: fever, dry cough, dyspnea • Onset: 2–12 months post transplant • Radiographic findings: patchy airspace disease, ground glass appearance, nodular opacities • Histology: peribronchiolar infiltration and fibrosis and the presence of intraluminal granulation tissue Bronchiolitis obliterans syndrome: • Clinical symptoms: cough, dyspnea, wheezing, lack of fever • Pulmonary function testing: obstructive findings (diminished FEV1 or FEV1:FVC) • Onset: 3–24 months post transplant • Radiographic findings: hyperinflation. Otherwise routinely normal • Computed tomography: bronchiectasis, centrilobular nodules, septal lines, ground glass appearance • Histology: lymphocytic bronchitis; bronchiolar inflammation with luminal obliteration CMV, cytomegalovirus; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity.
transfusion. Chest radiographs reveal diffuse pulmonary infiltrates reflecting edema from increased pulmonary vascular permeability. Treatment is generally supportive. Discontinuation of the blood product, corticosteroid administration, forced diuresis, and respiratory support results in recovery within 3–4 days in the majority of patients. In over 70% of cases, antibodies directed against human leukocyte antigen class I or II epitopes on recipient hematopoietic cells have been identified as the primary cause of the TRALI event, but in rare cases, the antibody may be present in the recipient’s plasma, and may be directed against transfused donor leukocytes [43,44].
Table 96.4 Risk factors for idiopathic pneumonia syndrome Graft-versus-host disease prophylaxis (methotrexate) Acute graft-versus-host disease Increasing recipient age Total body irradiation (≥1200 cGy) Myeloablative conditioning Decreased pretransplant performance status Longer duration from diagnosis to transplant Transplantation for malignancy other than leukemia Human leukocyte antigen disparity (donor: recipient)
Risk factors for IPS As shown in Table 96.4, risk factors for IPS include conditioning with high-dose, total body irradiation, acute GVHD, older recipient age, initial diagnosis of malignancy other than leukemia, and the use of methotrexate for GVHD prophylaxis [5,45]. Although recipient age and the use of methotrexate are not always risk factors, total body irradiation and the development of acute GVHD have been identified as factors in multiple reports [2,5,6,46,47]. Recently, the cumulative incidence of IPS within 120 days of HCT was found to be significantly lower after reduced-intensity conditioning than observed following conventional
conditioning, despite greater patient age and a similar incidence of acute GVHD in the reduced-intensity group [48]. Once established, however, pulmonary toxicity was severe in each group and resulted in respiratory failure in the majority of patients. These findings suggest that the intensity of HCT conditioning plays an important role in the development of IPS, and are consistent with data generated from mouse HCT models showing that the lung is sensitive to the combined effects of radiation and alloreactive T cells [49,50].
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Etiology of IPS Potential etiologies for IPS include direct toxic effects of HCT conditioning regimens, occult pulmonary infections, and the release of inflammatory cytokines that have been implicated in other forms of pulmonary injury. The association between IPS and severe GVHD reported in several large series [2,3,5–7] suggests that immunologic factors may also be operative in the development of lung injury. Acute GVHD often precedes IPS, suggesting a possible causal relationship between the two disorders [5,51,52]. Although IPS can also occur when signs and symptoms of GVHD are limited or absent, the consistent association between lung injury and GVHD in experimental models also supports such an etiology [2,3,5,6,50,53–56]. Despite the aforementioned clinical association, the lung has not been traditionally recognized as a classic GVHD target organ, and the specific role of alloreactive donor T lymphocytes in the pathogenesis of IPS remains a topic of considerable debate. Epithelial apoptosis is usually attributed to T-cell-mediated injury and is considered pathognomonic for acute GVHD. Although identified in the lungs of some patients with IPS [28,52], epithelial apoptosis has not been consistently observed in allogeneic HCT recipients with pulmonary dysfunction. A histologic spectrum of pulmonary GVHD has recently been described that ranges from diffuse alveolar injury early after HCT to cicatrical bronchiolitis obliterans, a late and irreversible form of lung injury [28]. Bronchitis/ bronchiolitis with IP was the most common finding and included a lymphocytic infiltration around bronchial structures along with a mononuclear inflammation in the perivascular zones and alveolar septa. The heterogeneity of pulmonary histopathology after allogeneic HCT is complicated further by the nonspecific changes that occur after mechanical ventilation and by the limited quality and quantity of lung biopsy tissue. Animal models of human disease The relationship between alloreactivity and IPS has been explored by several laboratories using a variety of murine models (Table 96.5). Rodent HCT models have consistently shown that animals with systemic GVHD develop lung injury, whereas syngeneic, non-GVHD controls do not [50,53,55,57]. Several patterns of lung injury have been identified including acute alveolitis, late-onset IP, and lymphocytic bronchiolitis [53]. In models where the graft-versus-host reaction is induced to (1) minor histocompatibility antigens, (2) class I or class II major histocompatibility complex (MHC) antigens only or (3) both major and minor histocompatibility antigens, two major abnormalities are apparent after allogeneic HCT: a dense mononuclear cell infiltrate around both pulmonary vessels and bronchioles, and an acute pneumonitis involving the
interstitium and alveolar spaces [55,58,59]. These patterns of inflammation closely resemble those reported in allogeneic HCT recipients [27,28,52,60]. Significant alterations in pulmonary function are associated with lung histopathology, demonstrating that the observed lung inflammation is physiologically relevant [56,57]. Furthermore, lung injury correlates with the presence but not the severity of GVHD, consistent with clinical reports of IPS in allogeneic HCT recipients whose signs and symptoms of GVHD were mild [8,9,29,61–63]. Thus, mouse models of IPS reproduce many of the histologic and functional changes observed during human disease. The pathogenesis of IPS Soluble inflammatory effectors: tumor necrosis factor-alpha and lipopolysaccharide The mixed inflammatory alveolar infiltrates found in mice with IPS are accompanied by significant increases in the total number of lymphocytes, macrophages, and neutrophils in the bronchoalveolar space [55], and by increased tumor necrosis factor-alpha (TNF-α) levels in both lung tissue and BAL fluid [50,54,55,64,65]. The presence of neutrophils and TNF-α in the absence of infection suggests that endogenous endotoxin/lipopolysaccharide (LPS) may contribute to the pathophysiology of IPS. LPS levels are elevated in the BAL fluid of mice with IPS, and the intravenous administration of LPS to mice with advanced GVHD significantly amplifies lung injury [55]. The enhanced inflammation is associated with large increases in TNF-α and LPS in the BAL fluid, and the development of alveolar hemorrhage [55,65]. Further, direct antagonism of LPS early in the time course of HCT reduces systemic levels of TNF-α and significantly decreases the severity of GVHD and IPS compared with control treated animals [66]. A causal role for TNF-α in the development of IPS has been established using strategies that either neutralize its effects [65,67] or use TNF-α-deficient mice as HCT donors [68,69]. Administration of recombinant human TNF-α receptor fusion protein (rhTNFR:Fc), a soluble, dimeric, TNF-binding protein, at the time of LPS challenge effectively prevents enhanced pulmonary inflammation, confirming the linkage between LPS and TNF-α in this setting [65]. Neutralization of TNF-α during the development of IPS also reduces the severity of lung injury during the natural course of disease [65]. Recent studies using genetically altered mice have shown that IPS is dependent upon donor-, rather than host-, derived TNF-α. While TNF-α from both donor accessory cells (macrophage/monocytes) and T cells significantly contributes to lung injury, the T-cell component predominates [69]. The actions of TNF-α are mediated by two receptors: a 55–60 kDa type I receptor (TNFRI; p55/60; CD120a) and a 75–80 kDa type II
Table 96.5 Animal models of idiopathic pneumonia syndrome Hematopoietic cell transplant donors
Stem cell transplant recipients
Mismatch
Conditioning
Reference
B10.BR C57BL/6 C57BL/6 C57BL/6 B6C3F1 hybrid C57BL/6 C57BL/6 LP/J
CBA B10.BR B6.C-H2bm1/By B6.C-H2bm12/KhEg B6C3F1 hybrid B6D2F1 hybrid B6.C-H2bm1/By C57BL/6
Multiple minor antigens Complete mismatch MHC class I MHC class II None (syngeneic) Haploidentical MHC class I Multiple minor antigens
TBI: 1100 cGy TBI: 750 cGy ± cyclophosphamide TBI: 675 cGy TBI: 675 cGy Cytoxan, cisplatin, BCNU TBI: 1100–1300 cGy TBI: 1100 cGy TBI: 1300 cGy
[55,56,65,92] [57,74,97,101] [100] [100] [79] [69,85,96] [99,72] [59,72]
MHC, major histocompatability complex; TBI, total body irradiation.
Lung Injury Following Hematopoietic Cell Transplantation
receptor (TNFRII; p75/80; CD120b) [70]. TNFRI is constitutively expressed, while the expression of TNFRII is strongly modulated by various cytokines and other inflammatory stimuli, including LPS. In an animal model, the absence of TNFRI was associated with decreased pulmonary edema and improved lung compliance on day 7 of allogeneic HCT [71]. However, cellular infiltration into the lung and BAL fluid levels of proinflammatory cytokines were actually higher in TNFRI−/− mice than controls. These findings are consistent with a recent report showing that compared with allogeneic controls, TNFRII-deficient HCT recipients develop significantly less severe IPS, which is associated with reductions in expression of pulmonary intercellular adhesion molecule-1 (ICAM-1) and in leukocyte infiltration into the lungs. This protective effect is comparable to that observed when wild-type mice are transplanted with allogeneic TNF-α-deficient donor cells, thereby strengthening the view of the role of TNF-α–TNFRII interactions in the pathophysiology of IPS [72]. TNF-α likely contributes to the development of IPS through both direct and indirect mechanisms. In addition to being directly cytotoxic, TNF-α increases expression of inflammatory chemokines [69] and MHC antigens, modulates leukocyte migration, and facilitates cell-mediated cytotoxicity. TNF-α may also contribute to lung injury by increasing the severity of GVHD in other target organs such as the gut and liver, thus promoting the release of other inflammatory mediators and their ultimate passage to the pulmonary vascular bed. From this perspective, the structural and functional integrity of the liver is critical; the liver is pivotally located between the intestinal reservoir of Gram-negative bacteria and their toxic byproducts, and the rich capillary network in the lung. In the setting of acute GVHD, an endotoxin surge into the systemic blood stream can arise from increased leakage of LPS across damaged intestinal mucosa, and underlying damage to the liver could decrease its capacity for LPS uptake and clearance. Consistent with this scenario, animals with mild or no GVHD can effectively detoxify endotoxin and protect their lungs from further damage, whereas mice with severe GVHD are unable to do so, and develop severe extensive lung injury including alveolar hemorrhage [55]. These data suggest that the inflammatory mediators TNF-α and LPS both contribute to experimental IPS and may do so along a “gut– liver–lung” axis of inflammation (Fig. 96.2). A clinical linkage of hepatic dysfunction to lung injury after HCT is also suggested by associations between sinusoidal obstructive syndrome and IPS, and between hepatic failure and death from IPS [2,29]. Further, evidence for cytokine activation and LPS amplification has been demonstrated in patients with IPS after allogeneic HCT; increased pulmonary vascular permeability and increases in BAL fluid levels of several cytokines, including LPSbinding protein and soluble CD14, were also observed in these patients [1]. Other soluble mediators Strategies that neutralize TNF-α in experimental models do not completely abrogate lung injury [53,65,67,69,73], suggesting that other inflammatory and cellular mechanisms may also contribute to the development of IPS. For example, IL-1β, nitric oxide, and reactive oxygen species have also been implicated in the development of lung injury after HCT, particularly when cyclophosphamide is included in the conditioning regimen [57,74,75]. From a clinical perspective, a recent analysis of plasma and BAL fluid protein profiles in patients with IPS showed that, in addition to increases in the levels of TNF-α and its soluble receptors, significant elevations in other molecules that participate in the cellular activating effects of LPS (soluble CD14, LPS-binding protein), along with three inflammatory chemokines (interleukin-8 [IL-8], monocyte chemoattractant protein-1 [MCP-1], and monokine induced by interferon-gamma [IFN-γ] [MIG]) that regulate leukocyte recruitment to sites of inflammation, were also evident [76].
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The proinflammatory environment observed following allogeneic HCT is frequently associated with the generation of oxidative stress and increased production of reactive oxygen species [77,78]. Exposure to radiochemotherapy further increases oxidant stress by depleting antioxidants, including reduced glutathione, the major antioxidant in the epithelial lining fluid of the lung, findings which have also been observed in murine IPS models after both allogeneic and autologous HCT [79,80]. While direct evidence that oxidative stress plays a distinct role in the development of IPS injury in human is lacking, experimental models demonstrate that the administration of n-acetylcysteine to HCT recipients substantially reduces lung injury compared with mice receiving high-dose chemotherapy without n-acetylcysteine [79]. Taken together, these data suggest that amelioration of oxidative stress may be an effective strategy to reduce the severity of pulmonary toxicity during IPS.
Cellular effectors and the development of IPS Donor-derived T-cell effectors The role of alloreactive donor T cells in the pathogenesis of IPS remains a topic of considerable debate. The importance of lymphocytes to lung injury after experimental HCT has also been shown by several groups [54–57,69]. Donor T cells are critical to the early proinflammatory events associated with lung injury that develops within the first week of HCT across MHC antigens, whereas in minor histocompatibility antigen-mismatch systems, donor lymphocytes continue to contribute to physiologically significant lung injury at later time points [56,57]. Donor T-cell clones that recognize CD45 polymorphisms result in a rapidly progressive pulmonary vasculitis within 3 days of their injection into nonirradiated recipients [54]. The origin and functional capacity of T cells infiltrating the lung have been examined by using differences in the T-cell Vβ repertoire between donor and recipient [56]. Flow cytometry demonstrated that the TCRαβ+ T cells found in the lung 6 weeks after allogeneic HCT were of donor origin, and when these T cells were recultured with irradiated host antigen-presenting cells, they proliferated vigorously and produced significant amounts of IFN-γ [56]. Donor cytotoxic lymphocytes can contribute to lung injury via three primary cytolytic mechanisms: the perforin–granzyme, Fas/Fas ligand (FasL), and TNF-α pathways. Each pathway has been shown to contribute to lung injury in non-HCT models [81,82]. Further, cytolytic T cells expressing granzyme B are present in the lungs of mice after allogeneic HCT, and they colocalize with macrophages expressing the costimulatory molecules B7.1/CD80 and B7.2/CD86 [57]. Alloantigenspecific killing by donor T cells using both perforin and Fas/FasL pathways has also been identified in the lung after HCT. Pulmonary cytotoxic lymphocyte activity is present as early as week 2 after transplant, and cytotoxicity mediated by Fas/FasL contributes to the progression of lung inflammation [58]. Despite these compelling data supporting a role for alloreactive donor lymphocytes in the development of noninfectious lung injury after HCT, IPS has been reported in patients in whom systemic GVHD is mild or absent, making a causal relationship between the two entities difficult to establish. The relationship between lung injury and GVHD severity has been examined in a HCT model across minor histocompatibility antigens. T-cell depletion at the time of allogeneic HCT reduced the number of T cells by greater than 99% and eliminated evidence of clinical or histologic GVHD. Nevertheless, significant lung injury was noted after allogeneic T-cell-depleted HCT, and donor lymphocytes reactive to host antigens were present in the BAL fluid, but not the spleens, of these animals [56]. The precise mechanisms and locations in which T cells interact with host antigens and ultimately cause injury remain unresolved, but this process is likely to involve interactions with dendritic cells (DCs) either
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BMT Conditioning HOST TISSUE INFa
GUT
IL-1 IL-6
LPS LIVER
Host APC
Stage two
Donor T Cell
Mf
IL-2 CTL Stage one
LPS
IFNg
NK
TNFa
Stage three PMNs
LUNG
Apoptosis Lymphocyte axis
Cytokine axis
Fig. 96.2. Pathophysiology of idiopathic pneumonia syndrome (IPS) after allogeneic hematopoietic cell transplantation (HCT). Data generated using murine HCT models have been incorporated into a working hypothesis of IPS physiology. This schema postulates that the lung is susceptible to two distinct but interrelated pathways of immune-mediated injury that occur along a T-lymphocyte activation axis and a “gut–liver–lung” axis of inflammation. Chemoradiotherapy of HCT conditioning causes cytokine release that enhances the ability of host antigen-presenting cells (APCs) to present alloantigens to mature donor T cells and upregulates chemokine expression in the lung. Once engaged, donor T cells become activated and secrete IFNγ and interleukin-2 (IL-2). Interferon-gamma (IFNγ) primes donor macrophages (Mφ) and monocytes, whereas IL-2 facilitates T-cell activation and the generation of CXCR3expressing type 1 T helper lymphocyte effectors that migrate to the lung early after HCT (stage one) in response to inflammatory chemokine gradients, and contribute to pulmonary toxicity via Fas–FasL-mediated cell killing. The inflammatory axis focuses on the relationship between the cellular activating effects of lipopolysaccharide (LPS) and the downstream production of tumor necrosis factor-alpha (TNFα) as it occurs along a gut–liver–lung axis of inflammation. LPS enters the systemic circulation through gaps in the intestinal mucosa. The ability of systemic endotoxin to reach the alveolar space is related to the consequences of graft-versus-host disease in other target organs, particularly the liver, which is pivotally located immediately downstream (via the splanchnic circulation) of the intestinal reservoir of Gram-negative bacteria and their toxic byproducts. CCR2-expressing donor macrophages primed by IFNγ are recruited to the lung (stage two), where they are triggered by LPS to secrete inflammatory cytokines like TNFα, resulting in enhanced chemokine expression, the recruitment of neutrophils to the lung, and increased tissue damage (stage three). NK, natural killer; PMN, polymorphonucleocyte.
resident in the respiratory system or in peripheral, secondary lymphoid organs. Pulmonary DCs are located in the interstitium and in the bronchial epithelium and submucosa, where DC tissue density diminishes with decreasing airway diameter. During steady-state conditions, pulmonary DCs constitute the sole source of MHC class II expression within the epithelial lining of the airway, and they have been shown to play a critical role in the initiation of both acute and chronic rejection of lung allografts [83]. It is possible that radioresistant host DCs persist longer in the lung than in other organs and allow for sustained presentation of host antigens [84]. Activated donor T cells might therefore remain within the pulmonary microvascular circulation because hosttype DCs function as a persistent site of alloantigen presentation. This scenario could account for the apparent “sanctuary” status of the lung with respect to alloreactive donor T cells, and may have important implications with regard to the evaluation and treatment of IPS after allogeneic HCT even when clinical GVHD is absent. Donor accessory cells Experimental data suggest that synergistic interactions between cells from the lymphoid and myeloid lineage are critical to the development of IPS. Specifically, the production of IFN-γ from activated donor T cells primes mononuclear cells and macrophages to secrete inflammatory cytokines when stimulated with LPS. The contribution of donor accessory cells (monocytes/macrophages/neutrophils)
to IPS has been investigated using several models. Kinetic studies of macrophage recruitment to the lung after allogeneic HCT show that the percentage of donor macrophages in the BAL fluid increases from approximately 40% at week 1 to over 90% by week 4 [85]. Additional experiments have shown that these donor-derived macrophages are a significant, albeit not the primary, source of TNF-α after HCT [69]. Studies completed using HCT donors that differ in their response to LPS (by virtue of a genetic mutation in the Toll-like receptor-4 [Tlr4] gene or the absence of CD14, a key cell surface receptor for the LPS– LPS-binding protein complex) have shown that recipients of LPS-resistant donor cells develop significantly less lung injury compared with recipients of wild-type, LPS-sensitive donors [68,86]. The results obtained using CD14-deficient donors are consistent with the report that monocytes recruited to an inflamed lung upregulate CD14 expression and show enhanced sensitivity to LPS stimulation [87], and with the clinical observations made in the BAL fluid of patients with IPS [1]. Collectively, these data demonstrate that donor macrophages/monocytes cells are recruited to the lungs after allogeneic HCT, and their ability to secrete TNF-α in response to LPS stimulation directly correlates with IPS severity. Polymorphonuclear cells are a major component of the BAL fluid of animals with IPS [55]. In mouse IPS models, the BAL fluid neutrophilia is prominent between weeks 4 and 6 after HCT, and is associated with increases in BAL fluid levels of TNF-α and LPS [55,65]. Neutralization
Lung Injury Following Hematopoietic Cell Transplantation
of TNF-α with rhTNFR:Fc during this time interval prevents the influx of neutrophils and reduces the progression of lung injury and dysfunction [65]. Administration of rhTNFR:Fc following LPS challenge completely abrogates the recruitment of neutrophils into the lungs and prevents further damage (including hemorrhage), underscoring the relationship between neutrophils, TNF-α, and LPS in this setting. Neutrophil products such as elastase, myeloperoxidase, metalloproteinases, and oxidants are abundant in the BAL fluid of patients with acute respiratory distress syndrome, and are believed to contribute to the endothelial and epithelial damage that occurs in this setting [88,89]. Neutrophils are likely to play a role in patients with IPS as well; their appearance in the blood stream is often temporarily associated with the onset of lung inflammation [29,34]. Mechanisms of leukocyte recruitment to the lung during IPS Cellular effectors play a significant role in the development of IPS, but the molecular mechanisms by which leukocytes traffic to the lung and cause damage have yet to be fully elucidated. White blood cell migration to sites of inflammation involves interactions between leukocytes and endothelial cells that are mediated by adhesion molecules, chemokines, and their receptors [90,91]. The recruitment of leukocytes from the vascular space and into target tissue can be divided into four steps: (1) weak adhesion of leukocytes to the vascular endothelial cells; (2) firm adhesion to endothelial cells; (3) transmigration of leukocytes through the vascular wall; and (4) migration of cells through the extracellular matrix along a chemotactic gradient. The role of adhesion molecules in the development of IPS has been examined using mouse models. Various adhesion molecules are upregulated by proinflammatory cytokines such as TNF-α and the messenger RNA (mRNA) expression of ICAM-1, vascular cell adhesion molecule-1, and E-selectin is increased in the lungs of mice with IPS after allogeneic HCT [92]. Further, the severity of IPS is dramatically reduced when ICAM-1-deficient (ICAM-1−/−) mice are used as HCT recipients of either MHC matched or mismatched allogeneic donor cells [59,93]. Surprisingly, in each scenario, ICAM-1 deficiency selectively protects the lung from injury even though ICAM-1 expression is elevated in the liver, colon, and spleen of animals with acute GVHD [94]. Chemokine receptor–ligand interactions facilitate the recruitment of leukocytes to the lung in a variety of inflammatory states [95], and investigators have recently begun to explore their role in IPS. The mixed pulmonary infiltrate observed after experimental allogeneic HCT therefore suggests that those chemokines responsible for the recruitment of lymphocytes, monocytes, and neutrophils will be upregulated during the development of IPS. The pulmonary expression of inflammatory chemokine receptors for T-cell effectors (CCR1 and CXCR3), monocytes and macrophages (CCR2), and neutrophils (CXCR2), and their respective ligands, have been analyzed in mouse IPS models. Compared with syngeneic controls, mRNA expression of each chemokine receptor and ligand is increased in allogeneic HCT recipients [96,97]. Elevations in CCR1 and CXCR3 expression peak early after allogeneic HCT, whereas expression of CCR2 and CXCR2 continue to rise over time. Importantly, the kinetics of chemokine ligand expression correlated with the corresponding receptors; increases in RANTES, macrophage inflammatory protein-1 (MIP-1α; CCR1), and IFN-γ-inducible protein-10 (IP-10; CXCR3) peaked at week 1 and then tapered off, whereas increases in MCP-1 (CCR2) and MIP-2 (CXCR2) peaked at weeks 2 and 4, respectively. These data are consistent with prominent increases in the expression of IFNγ-inducible chemokines observed when lung injury is induced by alloreactive type 1 T helper (Th1) cells [98]. Significant increases in chemokine ligand expression in the lung therefore precede the development of IPS and herald the influx of leukocyte subsets bearing the corresponding chemokine receptors. Enhanced chemokine expression and leukocyte infiltration during IPS can be conceptu-
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alized in three distinct stages. In stage 1 of the process, HCT conditioning and the initial allogeneic donor T-cell response result in systemic inflammation during the first week after transplantation. This proinflammatory environment is characterized by the release of IFN-γ, TNF-α, and LPS, and causes increased chemokine expression in the lung. Production of RANTES, MIP-1α, and IP-10 recruits donor-derived, Th1/type 1 cytotoxic lymphocyte (Tc1) lymphocyte effectors (CXCR3+) within the first 2 weeks of HCT [96–99]. In stage 2, T cells cause local tissue injury and activate endothelial and epithelial cells to secrete additional chemokines, including MCP-1. Enhanced expression of MCP-1 in turn recruits CCR2+ donor monocytes, macrophages, and additional T cells [96,97]. In stage 3, recruited macrophages secrete TNF-α in response to LPS stimulation, which results in additional tissue injury, the upregulation of the chemokine ligands cytokine-induced neutrophil chemoattractant (KC) and MIP-2, and the recruitment of donor, CXCR2+ neutrophils. These neutrophils amplify progressive lung injury and dysfunction. This hypothesis stipulates that the migration of each leukocyte subset controls the recruitment of the next wave of effectors. Thus, the recruitment of Th1/Tc1 effectors initiates the cascade, and interruption of this first stage should have profound effects on the development of IPS. Data in support of this hypothesis have recently been generated by using congenic HCT donors that express allelic differences of CD45 on all leukocytes. Using this system, greater than 95% of CD4+ and CD8+ lymphocytes in the bronchoalveolar space are of donor origin by the first week, and turnover is complete by week 2 after HCT [69]. These findings were complemented by a subsequent study using an irradiated murine MHC class I mismatched model wherein IPS and GVHD are mediated by CD8+ T cells. Elevated levels of MIG (CXCL9) and IP-10 (CXCL10) correlated with the recruitment of CXCR3-expressing CD8+ T cells to the lung as early as day 7. In vivo neutralization of CXCL9 or CXCL10, or HCT using CXCR3−/− donor leukocytes, resulted in a near-complete abrogation of infiltrating CD8+ T cells and IPS severity [99]. Donor T cells initially recruited to the lung may play an active role in regulating the inflammation engendered during the evolution of IPS. For example, experiments using genetically altered mice have shown that TNF-α secreted by donor lymphocytes regulates the chemokine milieu in the lung within the first 2 weeks after HCT, which directly contributes to the subsequent recruitment of monocytes and macrophages as lung injury progresses [69]. Similarly, enhanced pulmonary expression of CCL5 (RANTES) correlates with the initial influx of donor T cells into the lungs after allogeneic HCT, and the autocrine production of CCL5 by these infiltrating cells significantly contributes to the subsequent recruitment of additional T cells and accessory cells over time [85]. By contrast, the role of donor-derived CCL3 (MIP-1α), a chemokine that shares common receptors with CCL5, has been less clear. CCL3 contributes to the recruitment of leukocytes during IPS following nonmyeloablative conditions [100]. However using Mip-1 α−/− donor mice in a myeloablative model exacerbated rather than reduced early lung injury [101], providing further support that receptor–ligand interactions of CCL5, likely via its co-receptor CCR1, may be dominant in the evolution of IPS [102]. Targets of inflammation and injury during IPS Pulmonary endothelial and epithelial cells can express MHC class I, MHC class II, and minor histocompatibility antigens, and the expression of these molecules is enhanced by TNF-α and IFN-γ. It is therefore conceivable that pulmonary parenchymal cells can serve as targets for direct cell-mediated damage. Endothelial cell injury has been observed after allogeneic HCT, and has been implicated as a direct contributor to the development of several complications, including GVHD, sinusoidal obstructive syndrome, and thrombotic microangiopathy [103]. Clinical and experimental IPS is associated with evidence for endothelial cell
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injury and leak, as demonstrated by pulmonary edema, enhanced total protein levels in BAL fluid, and increased wet-to-dry lung weight ratios [57,99]. Moreover, leukocyte infiltration during IPS is accompanied by significant apoptosis of the pulmonary vascular endothelium [92]. Endothelial cell apoptosis coincides with the onset of pulmonary pathology, is associated with elevations in BAL fluid TNF-α levels, and is accompanied by evidence for endothelial cell activation as measured by enhanced mRNA expression of adhesion molecules [92]. The administration of a soluble TNF-α-binding protein (rhTNFR:Fc) from week 4 to week 6 after allogeneic HCT significantly reduces the endothelial cell apoptosis and lung histopathology observed in mice [92]. In this light, TNF-α may therefore function as both an effector and a facilitator of lung injury by contributing directly to endothelial cell injury and death, and regulating the chemokine milieu in the lung during the early stages of IPS. By contrast, epithelial apoptosis, generally ascribed to T-cell-mediated injury and considered pathognomonic for acute GVHD in other target tissues, has not been consistently observed in allogeneic HCT recipients with lung injury. The unique aspects of epithelial anatomy in the lung may help explain this discrepancy. Since there is no stratification or layering of pulmonary epithelial cells as in the skin or intestine, identification of epithelial cell apoptosis by histologic criteria can be more challenging. Experimental studies have, however, provided evidence for epithelial injury during IPS. Panoskaltsis-Mortari and coworkers demonstrated that IPS was associated with injured alveolar type II cells and increased frequencies of cytotoxic T lymphocytes in a model of early-onset IPS [57]. The same group later showed that keratinocyte growth factor, a mediator of epithelial cell proliferation and a growth factor for type II pneumocytes, diminished IPS injury by dampening the immune response to chemoradiotherapy and by accelerating repair of the damaged tissue, specifically alveolar type II epithelial cells [104]. The approach to HCT patients with acute respiratory dysfunction The approach to HCT patients with acute pulmonary dysfunction is complex and requires a variety of diagnostic tests and possible consultation with experts in the fields of pulmonology, cardiology, nephrology, radiology, and critical care medicine. Because symptoms of respiratory distress can progress rapidly once established, the timely coordination of care is essential to optimizing outcomes. In addition, there are several diagnostic challenges to address in this setting, as both pulmonary and nonpulmonary causes of respiratory compromise are possible. Determination of the severity of respiratory dysfunction, including an assessment of the need for supplemental oxygen support, overall fluid balance, renal function, and cardiac output should be followed by radiographic imaging. In general, an initial chest X-ray or computed tomography scan will identify the presence of lobar, multilobar or diffuse pulmonary infiltrates. While such findings may impact the decision-making process (Fig. 96.3), they are nondiagnostic in and of themselves. In the absence of obvious left-sided heart failure or iatrogenic fluid overload, bronchoscopy with BAL should be strongly considered when multilobar or diffuse infiltrates are present. As outlined in Table 96.2, BAL samples should be sent for a variety of diagnostic tests to determine the presence of community-acquired, hospitalacquired, and otherwise opportunistic infections. While accepted at many large transplant centers, the need to complete a BAL in HCT recipients with significant respiratory compromise remains a matter of debate, particularly in those patients who are critically ill. Recently, Yanik and colleagues reported on the results of 444 bronchoscopy procedures completed on 300 (20% of all patients) who received HCT at the University of Michigan from 2001 to 2007 [105]. Thirteen percent of BAL specimens collected in the first 30 days of HCT were positive for infection, and this number increased to 33% between
days 31 and 100. Hence, while the majority of HCT patients requiring BAL within the first 100 days may have IPS, a significant number of individuals will have evidence for infection. To this end, BAL data resulted in changes in medical management in approximately 60% of cases, and modifications in antimicrobial therapy in just under half. Since the medical management of IPS (immunosuppression) and infection (antimicrobial therapy) are rather divergent, making the appropriate diagnosis has significant merit. As shown in Table 96.6, current standard treatment regimens for IPS include supportive care measures in conjunction with broad-spectrum antimicrobial agents and intravenous corticosteroids [6,29]. Unfortunately, responses to standard therapy are limited, and the mortality of patients diagnosed with IPS remains unacceptably high [7]. Moreover, high-dose corticosteroid therapy (>2 mg/kg/day of methylprednisolone equivalent) has not been shown to improve outcome when compared with lower doses of corticosteroids (≤2 mg/kg/day) [48]. Advances in supportive care including the early institution of continuous veno-venous hemofiltration may help to improve survival in some patients, but prospective studies addressing the treatment of IPS, including the specific use of steroids, are lacking in the literature. Translational research studies suggest that etanercept may be a useful therapeutic option for IPS [29]. Etanercept was administered in combination with systemic steroids and empiric antimicrobial therapy to a total of 18 patients with IPS [106]. Etanercept was given subcutaneously at a dose of 0.4 mg/kg twice weekly for a maximum of eight doses. Therapy was well tolerated overall. Thirteen of 18 patients were able to completely withdraw from supplemental oxygen support within 28 days of therapy. Survival at day 28 and day 56 (from the first etanercept dose) was 73% and 60%, respectively. Based upon these encouraging results, larger phase II (pediatric) and phase III (adult) trials are ongoing within the Bone Marrow Transplant Clinical Trials Network (phase III) and the Children’s Oncology Group (phase II). Summary IPS remains a frequent and severe complication of allogeneic HCT, and mortality rates remain unacceptably high with standard therapy. Preclinical and clinical data suggest that both inflammatory and cellular effectors participate in the development of IPS after allogeneic HCT. TNF-α and LPS are significant, albeit not exclusive, contributors to IPS, and cells of both lymphoid and myeloid origin play a direct role in lung injury that occurs in this setting. The contribution of donor, nonlymphoid, accessory cells may be linked to cellular activation by LPS and the ultimate secretion of TNF-α within a “gut–liver–lung” axis of inflammation, whereas donor T-cell effectors can home to and damage the lung even when systemic GVHD is mild or absent. Hence, significant research efforts have resulted in a paradigm shift away from identifying lung injury after HCT solely as an idiopathic clinical syndrome and toward understanding IPS as a process involving two distinct, but interrelated, pathways of immune-mediated injury involving aspects of both the adaptive and the innate immune response. Importantly, new laboratory insights are currently being translated to the clinic and will likely prove important to the development of future strategies to prevent or treat this frequently fatal complication.
Chronic pulmonary dysfunction after HCT: obstructive and restrictive lung disease Definition, risk factors, and clinical course Decline in lung function has long been identified as a significant complication in the months to years that follow allogeneic HCT. Two forms
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S/S of respiratory distress Cough, rales Supplemental oxygen requirement
Obtain CXR or chest CT scan Close observation Assess fluid status
Normal Abnormal
Diffuse infiltrates
Lobar infiltrate
Yes Yes
S/S of heart failure or fluid overload? Empiric antibiotic treatment
Yes
Treat
Yes
Treat
No
S/S of sepsis? Response
No
No response
Bronchoscopy/BAL Continue antibiotics
Bronchoscopy
Normal
Abnormal
Treat identified infection
Diagnosis: IPS
Modify antimicrobial regimen
Corticosteroids 2 mg/kg/day Consider
Investigational trials
TNF inhibitors
CVVH
Other
Fig. 96.3. Approach to hematopoietic cell transplantation (HCT) patients with acute respiratory dysfunction. The approach to HCT patients with acute pulmonary dysfunction is complex. The timely determination of the severity of respiratory dysfunction, including an assessment of the need for supplemental oxygen support, overall fluid balance, renal function, and cardiac output should be followed by radiographic imaging and consideration for bronchoscopy and bronchoalveolar lavage (BAL). CT, computed tomography; CVVH, continuous veno-venous hemofiltration; CXR, chest X-ray; IPS, idiopathic pneumonia syndrome; S/S, signs and symptoms; TNF, tumor necrosis factor. Table 96.6 Treatment options for idiopathic pneumonia syndrome Supportive therapy
Immunosuppressive therapy
Supplemental oxygen, mechanical ventilation Empiric broad-spectrum antimicrobial agents pending culture results Management of iatrogenic fluid overload Continuous veno-venous hemofiltration Corticosteroids (2 mg/kg/day) Investigational: cytokine inhibitors, including anti-tumor necrosis factor agents
of chronic pulmonary dysfunction are common in patients surviving longer than 100 days after allogeneic HCT: obstructive lung disease (OLD) and restrictive lung disease (RLD). The incidence of both patterns of lung toxicity ranges from 20% to 50% depending upon donor
source and the time interval after HCT [8–14,63]. In each scenario, collagen deposition and the development of fibrosis either in the interstitial (RLD) or peribronchiolar space (OLD) are believed to contribute to the patterns of lung dysfunction displayed on pulmonary function testing. While both forms of pulmonary dysfunction exist as late-onset, noninfectious lung complication following allogeneic HCT, RLD and OLD can be distinguished by a number of clinical parameters as described below (Table 96.7). Restrictive lung disease RLD is defined by reductions in forced vital capacity (FVC), total lung capacity (TLC), and diffusion capacity of the lung for carbon monoxide (DLCO) as measured by standard PFTs. In restrictive disease, the ratio of the forced expiratory volume in 1 second (FEV1) to FVC (FEV1/FVC) is maintained near 100% [7,10,13,62]. RLD is common after HCT; significant decreases in FVC or TLC have been reported in as many as 25–45%
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Table 96.7 Clinical factors present in obstructive versus restrictive lung disease Clinical factor
Obstructive lung disease
Restrictive lung disease
Onset Symptoms Physical examination Pulmonary function tests FEV1:FVC Total lung capacity DLCO Computed tomography findings
Late (3–12 months) Dyspnea, nonproductive cough Wheezing Obstructive physiology Decreased Normal Decreased Air trapping Bronchial wall thickening Centrilobular nodules Strong association
Early (within 3 months) Dyspnea, nonproductive cough Rales Restrictive physiology Normal Decreased Decreased Patchy consolidation: BOOP “Ground glass” opacities
Chronic graft-versus-host disease
Variable, association with BOOP
BOOP, bronchiolitis obliterans organizing pneumonia; DLCO, diffusing capacity of the lung for carbon dioxide; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity.
of allogeneic HCT recipients at day 100 [10,13,14]. A decline in TLC or FVC at 100 days and 1 year after HCT compared to pretransplant values, even if the absolute values for each measurement remained within the normal range, has been associated with an increase in nonrelapse mortality [10,13]. Total body irradiation-containing conditioning regimens and the presence of acute GVHD have been associated with RLD [10,13,14], but the impact of age on the development of RLD is less clear. Early reports suggested that the incidence of RLD is lower in children compared with adults, and that the incidence increases with advancing recipient age [14], but more recent studies have revealed significant RLD in children receiving HCT [107]. In contrast to OLD, RLD after HCT has not been clearly associated with the presence of chronic GVHD. Histologic features of RLD after HCT are rarely described in the clinical literature, although varying degrees of interstitial and alveolar inflammation and fibrosis, as seen in patients with other forms of interstitial pulmonary fibrosis, would be expected. One exception is BOOP. Although reported in less than 2% of HCT recipients, BOOP is associated with restrictive (rather than obstructive) changes on PFTs [7,108]. Clinical features resemble idiopathic BOOP and include dry cough, shortness of breath, and fever, and radiographic findings show diffuse, peripheral, fluffy infiltrates consistent with air space consolidation. In the context of allogeneic HCT, the development of BOOP is strongly associated with prior acute and chronic GVHD [109]. The diagnosis of BOOP requires histologic evidence on lung biopsy of several signature features: patchy fibrosis, granulation tissue within alveolar spaces, alveolar ducts, and respiratory bronchioles, and the absence of infectious organisms. The term BOOP should not be used interchangeably with bronchiolitis obliterans to describe a patient with chronic lung dysfunction after HCT, although such usage is unfortunately widespread. The two disorders differ with respect to histopathology, pulmonary function characteristics, and, most importantly, response to therapy: BOOP after HCT is quite responsive to corticosteroids and in other settings may resolve spontaneously; bronchiolitis obliterans is not [7,108].
Obstructive lung disease OLD was first recognized as a complication of allogeneic HCT in the mid 1980s and is now a well-documented cause of morbidity and mortality [8,62,110–113]. OLD is characterized by enhanced resistance to airflow on expiration and reflects conditions in the smaller airways and bronchioles. The diagnosis of OLD is based primarily on spirometric
measurements as demonstrated by decreases in FEV1 and FEV1:FVC [110]. Obstructive defects as defined by an FEV1:FVC ratio of less than 70% have been observed in approximately 15–25% of allogeneic HCT recipients by day 100 and can persist for years [10,62,63]. OLD results from extensive narrowing and/or destruction of the small airways. Lung biopsies from patients with OLD have shown lymphocytic bronchitis, acute and chronic IP, and bronchiolar inflammation, including bronchiolitis obliterans [8,63,111,114]. This variation in histopathology is complicated further by the methods used to procure lung tissue: transbronchial biopsies rarely include an adequate sampling of distal bronchial structures, and therefore such specimens can reveal cellular infiltrates involving larger airways and the interstitium but may not detect bronchiolar inflammation. Bronchiolitis obliterans depicts small airway inflammation with fibrinous obliteration of the bronchiolar lumen, and remains the most common form of histopathology associated with OLD, and has been used historically to describe “chronic GVHD of the lung” [8,63,111,114]. Airflow obstruction may, however, occasionally exist without bronchiolitis obliterans and vice versa [115]. Moreover, in the vast majority of cases, OLD is diagnosed by PFT findings without histopathologic confirmation, and in this context two phrases that identify affected patients are found in the literature. “Airflow obstruction” has recently been defined as a more than 5% per year decline in percent predicted FEV1 with the lowest post-transplant FEV1:FVC ratio less than 0.8 [112], and “bronchiolitis obliterans syndrome” describes the deterioration of graft function that accompanies chronic lung allograft rejection [116]. The lack of consistent terminology and the variability in diagnostic criteria have contributed to the wide variation in the reported incidence of OLD after HCT. Afessa and colleagues found that OLD was reported in 8.3% of over 2000 allogeneic HCT patients in nine studies and was identified in 6–20% of long-term survivors with chronic GVHD [7]. By contrast, using the definition described above for airflow obstruction, Chien and coworkers found that new OLD following HCT with highdose conditioning was more frequent (26% overall and 32% among patients with chronic GVHD) than in previous estimates [112]. The onset of OLD is later than IPS (ranging from 3 to 18 months after HCT), and more insidious [110]. Respiratory symptoms include cough, dyspnea, and wheezing, but many patients remain asymptomatic despite showing signs of moderate-to-severe airway obstruction on PFTs [8,62]. Chest radiographs are most often normal except for signs of hyperinflation, but patchy, diffuse infiltrates can be present [8,61,63]. Likewise, chest
Lung Injury Following Hematopoietic Cell Transplantation
computed tomography findings range from normal early in the course of disease to extensive peribronchial inflammation and bronchiectasis with significant air trapping and diffuse parenchymal hypoattenuation at later time points [9,117]. The clinical course of OLD also varies from mild to severe with necrotizing bronchiolitis, a rapid decline in FEV1, and a mortality rate of 25–50%, and, unfortunately, clear predictors of outcome have not been identified [8,9,11,12,61,110]. Since airflow obstruction tends to be fixed rather than reversible, response to bronchodilator therapy is usually marginal. Similarly, responses to immunosuppressive therapy (including various combinations of steroids, calcineurin inhibitors, and azathioprine) are limited, and are typically associated with preservation of remaining lung function rather than in significant improvement. Since enhanced immunosuppression significantly increases the risk of infection, the utility of such therapy is questionable when a clinical response is not seen within the first several months of treatment or when pulmonary dysfunction is longstanding. The partial response to immunsuppressive therapy suggests that early detection of disease may be important [8,9,12,110]. In this light, two reports have suggested that a decrease of maximum mid-expiratory flow rates may be an earlier indicator of OLD than changes in FEV1 [12,61]. Further, faster rates of airflow decline between day 100 and 1 year after HCT correlate with higher mortality risk [113]. More importantly, investigators have found that pretransplantation risk scores based solely or in part on pre-HCT lung function tests can be used to predict early respiratory failure and death in patients undergoing allogeneic HCT [118,119]. Risk factors for OLD are several and include lower pretransplant FEV1:FVC, concomitant infections, chronic aspiration, acute and chronic GVHD, older recipient age, and the use of mismatched donors and high-dose (versus reduced-intensity) conditioning regimens [8,9,62,110,112,120]. Recently, the impact of age and intensity of HCT conditioning was examined by Chien and colleagues, who found that the risk for experiencing a yearly decline in FEV1 in excess of 20% was significantly lower for recipients of reduced-intensity conditioning compared with patients receiving high-dose therapy who were greater than 50 years of age [120]. The CMV status of donor and recipient prior to HCT does not correlate with the development of OLD, but previous RSV and adenoviral infections appear to be possible risk factors for the higher incidence of OLD in the pediatric population [9]. The development of OLD is strongly associated with chronic GVHD [121], particularly in patients with low serum IgG levels [8,110] and with chronic hepatic GVHD [9]. Pathogenesis of chronic pulmonary toxicity The pathophysiology of chronic lung injury after HCT is poorly defined, in part because of the paucity of correlative clinical data and the lack of suitable animal models for either OLD or RLD. The development of chronic pulmonary toxicity likely involves an initial insult to the lung parenchyma, followed by an ongoing inflammatory process involving the interplay between recruited immune effector cells and the resident cells of the pulmonary vascular endothelium and interstitium. Most of what is known about the pathogenesis of OLD is based upon observations made in lung allograft recipients and from data generated in murine heterotopic tracheal transplant models. The absence of an initial inflammatory response from HCT conditioning regimens and the presence of a “host-versus-graft” rather than a “graft-versus-host” reaction are just two of the issues that limit the extrapolation of such data to OLD after HCT. Similarly, our understanding of the mechanisms responsible for RLD is inferred from patients with various forms of interstitial fibrosis and from animal models of these diseases. Despite these limitations, lung allograft rejection and pulmonary fibrosis are characterized by
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epithelial cell injury in the terminal bronchioles or alveoli, respectively, and by a profound defect in re-epithelialization and normal repair. Each response also involves T-cell activation, leukocyte recruitment, and enhanced expression of inflammatory mediators, all of which are likely operative in chronic lung injury after HCT. Murine models have begun to elucidate the mechanisms that contribute to leukocyte migration and rejection of lung allograft epithelium, and may shed light on mechanisms contributing to OLD after HCT. Boehler and colleagues found that rat heterotopic lung allografts undergoing rejection showed a strong Th1 immune response even after fibrosis and airway obliteration was complete [122]. This response was also associated with upregulation of MCP-1/CCL2 and RANTES/CCL5. Subsequent studies by several groups have shown enhanced expression of TNF-α, IL-8, transforming growth factor-beta and IL-1β during clinical allograft rejection [123–126], and ultimately revealed critical roles for both RANTES and MCP-1 in the development of experimental bronchiolitis obliterans [127,128]. Although direct cytotoxicity by cellular or cytokine effectors may be responsible for damage and loss of airway epithelium in the transplanted lung, the inability to regenerate and heal injured epithelium is believed to be an equally important factor in the development of bronchiolitis obliterans. In this context, interactions between activated epithelial cells and lung fibroblasts may be critical [129,130]. The impact that innate immune dysregulation may have on these interactions has been recently highlighted by two studies demonstrating that genetic variations in bactericidal/permeabilityincreasing (BPI) protein and oligomerization domain containing-2/ caspase recruitment domain family member 15 (NOD2/CARD15) influence the risk of airflow obstruction and bronchiolitis obliterans after allogeneic HCT [131,132]. Such genetic approaches, along with the development of a new animal model for bronchiolitis obliterans [133], may ultimately prove useful for deciphering mechanisms of disease that are more relevant to OLD that occurs in the context of HCT. The mechanisms responsible for RLD after HCT are also poorly characterized. The prevailing hypothesis holds that most forms of interstitial fibrosis are initiated by an acute inflammatory event that injures the lung, results in the secretion of inflammatory mediators, and subsequently modulates pulmonary fibrogenesis. The etiology of the initial parenchymal insult may; (1) include direct toxicity from drugs, toxins or ionizing irradiation, (2) represent a dysregulated immune response to an aberrantly expressed environmental or self-antigen, and (3) may or may not involve a role for activated T cells. Like OLD, several proteins with profibrotic activity, including TNF-α, IL-1β, IL-6, transforming growth factor-beta, IL-8, and MCP-1/CCL2 have been identified in the BAL fluid of patients and animals with interstitial fibrosis. A growing body of scientific evidence suggests that the cytokine profile present during an evolving immune response determines its pathologic outcome. In this context, a Th1/Th2 explanation has emerged to help predict whether a specific pulmonary response will ultimately resolve or progress toward end-stage fibrosis; IFN-γ has profound regulatory activity on collagen synthesis and possesses potent antifibrotic effects, whereas Th2 cytokines like IL-4 and IL-13 activate fibroblasts and stimulate the production of extracellular matrix proteins [129,130]. This working hypothesis has been recently supported in dramatic fashion by Burman and colleagues, who showed that complete abrogation of IFN-γ signaling using IFN-γ-deficient donors and IFN-γ receptor-deficient recipients resulted in robust pulmonary inflammation and death early after HCT [134]. The evolution of chronic lung injury after HCT A triphasic model of chronic, noninfectious lung injury after HCT is proposed wherein alloantigen recognition represents the inciting stimulus of the immune response (Plate 96.1). Phase one of the disease is
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Chapter 96
characterized by the development of a mixed leukocytic infiltrate and acute interstitial and peribronchial inflammation similar to that observed in IPS (Fig. 96.1(a)). This injury is initiated early after allogeneic HCT by a systemic proinflammatory environment that leads to chemokine upregulation, the promotion of leukocyte recruitment, and the secretion of inflammatory cytokines in the lung. In phase two, the expression of MHC antigens on the pulmonary epithelial and vascular endothelial cells results in a sustained inflammatory signal and a dysregulated reparative response that promotes the transition from acute to chronic lung injury. This transition is accompanied by a change in the character of the leukocytic infiltrate (to one that is predominantly lymphocytic in nature) along with a shift to a profibrotic environment and a proactive role of the lung fibroblast. If the inciting injurious stimuli involve the bronchiolar epithelium, phase two is associated with a progressive, concentric lymphocytic infiltration, collagen deposition, and early fibrosis in the peribronchial areas, resulting in a chronic bronchiolitis (Plate 96.1(b)). Activated lymphocytes then migrate through the basement membrane of the respiratory epithelium and into the airway mucosa and result in epithelial cell apoptosis and necrosis. Continued epithelial injury leads to areas of denudation and ulceration. As chronic inflammation proceeds into phase three, lung fibroblasts increase dramatically in number and contribute to (1) the proliferation of endothelial cells, (2) enhanced collagen deposition, and (3) the development of intraluminal granulation tissue and dense, concentric, periluminal fibrous bands (Plate 96.1(c)). Ultimately, this process results in complete obliteration of the small airways (cicatricial bronchiolitis obliterans) and significant OLD. By contrast, if epithelial cells in the alveolar septae are the principal targets of injury, persistent antigenic stimulation results in recruitment of lymphocytes and monocytes into the interstitial space, eventually resulting in RLD (Plate 96.1(d)). The resultant inflammation is associated with apoptosis and loss of septal epithelial cells, exudation of proteinaceous material, the recruitment and proliferation of fibroblasts, and the deposition of intraseptal granulation tissue. If this extracellular matrix is not resorbed, chronic inflammation progresses to phase three, wherein collagen is deposited within the alveolar septae, and the chronic leukocytic infiltrates are less evident. This results in interstitial thickening, septal fibrosis, and loss of alveolar architecture leading to dilated cystic air spaces or “honeycombing” (Plate 96.1(e)). Such end-stage histopathology is associated with the significant volume reduction and severely impaired gas exchange that is characteristic of severe RLD. Precisely what determines the anatomic specificity (peribronchiolar versus interstitial) of chronic lung injury remains unclear, and the development of either pattern of chronic lung injury (OLD or RLD) does not necessarily exclude the other [135]. Further, the role of acute inflammation and specifically of alloreactive effector cells in the initial damage to the alveolar or bronchiolar epithelium and the subsequent progression to chronic pulmonary injury are not well established. Moreover, an early, robust inflammatory phase may not be a prerequisite for subsequent fibrosis; persistent epithelial damage and subsequent aberrant “cross talk” between epithelial cells and fibroblasts may be sufficient to cause an exuberant reparative response characterized by proliferation, excess matrix deposition, and the development of fibrotic lung disease [136,137]. This mechanism could explain why some patients with chronic lung dysfunction do not have a clear antecedent history of acute lung inflammation. In the setting of imbalanced immune regulation, a subclinical injury, such as an allogeneic response to lung epithelial cells, could initiate a dysregulated reparative response resulting in scarring of either the terminal airways (OLD) or the interstitial space (RLD). In this context, pulmonary fibroblasts may represent an important link in the development of both RLD and OLD after allogeneic HCT.
TNF-α may be a central factor in the triphasic model proposed above. TNF-α is an important mediator of acute lung injury after HCT, it directly contributes to the development of interstitial fibrosis in several non-HCT models [129,138,139], and the abrogation of TNF-α signaling, either by antibody neutralization or by using mutant mice deficient in both TNF-α receptors, significantly reduces lung fibrosis [140,141]. Strong evidence for a role of TNF-α in the transition from acute to chronic lung injury comes from studies using transgenic rodents with targeted overexpression of TNF-α in the lungs [138,139]. Early lung histopathology includes a lymphocytic infiltrate similar to that seen in experimental IPS models, whereas the histologic changes associated with more prolonged exposure to TNF-α closely resemble those seen at later time points after HCT. The linkage between TNF-α and OLD after HCT is more indirect: increased levels of TNF-α have been shown to increase the expression of several proteins that may contribute to this process. For example, increased levels of TNF-α are associated with enhanced MCP-1 expression in a murine lung allograft rejection model and correlate with upregulation of CCR2 and the recruitment of mononuclear phagocytes into the allograft [127]. Interruption of MCP-1/CCR2 signaling using CCR2deficient recipients results in a reduction of macrophage infiltration and less rejection [127], and modulates the induction of profibrotic cytokine cascades following the intratracheal administration of bleomycin and other irritants such as fluorescein isothiocyanate [142]. Finally, clinical studies have shown that enhanced MCP-1 is associated with the progression from acute to chronic allograft rejection and with the evolution of pulmonary fibrosis [127,143]. Treatment of chronic lung injury after HCT The association of OLD and chronic GVHD has resulted in a general consensus that this form of lung damage is immunologically mediated. Thus, “standard” therapy of OLD combines enhanced immunosuppression in conjunction with supportive care including supplemental oxygen therapy and broad-spectrum antimicrobial prophylaxis. Unfortunately, the response to multiple agents including steroids, cyclosporine, tacrolimus, and azathioprine is limited and tends to occur only early in the course of treatment [8,12,61,63,110]. Patients with more severe disease at the start of treatment have poor prognoses and high mortality rates, suggesting that early recognition of OLD may be important [12,61,110]. The poor response to standard therapy and the unacceptable morbidity and mortality associated with chronic lung injury after HCT are underscored by the need for lung transplant in some HCT recipients with severe OLD [144]. The published literature contains a paucity of therapeutic trials for chronic lung injury after HCT, and no agent or combination of agents has been shown to be particularly efficacious. Whereas a clinical study using inhaled steroids in addition to standard systemic immunosuppression to prevent bronchiolitis obliterans syndrome after lung allografting showed no benefit compared with controls [145], a recent randomized, placebo controlled trial of 58 patients demonstrated that while inhaled cyclosporine did not improve the rate of acute rejection, it did improve survival and extend periods of chronic rejection-free survival in lung allograft recipients [146]. Three recently published case series have exploited the anti-inflammatory effects of the antibiotic azithromycin to treat OLD in both allogeneic HCT and lung allograft recipients. Each study suggested a beneficial effect of this drug on pulmonary function when administered for 12 or more weeks [147–149]. The potential role for TNF-α in the pathogenesis of both OLD and RLD suggests that agents such as etanercept, which neutralize this protein, may hold promise, and several studies have demonstrated a potential benefit of this drug in some HCT patients with chronic lung
Lung Injury Following Hematopoietic Cell Transplantation
injury [150,151]. A phase I–II clinical trial using etanercept in this context has recently been completed at the University of Michigan. Preliminary results demonstrate that etanercept can be safely administered in this patient population, and six of 15 patients showed at least a 10% improvement from baseline in either FEV1, FVC or DLCO within the first 2 months of finishing therapy [151]. Based upon these findings, a larger phase II trial has been developed and is currently accruing patients.
Conclusion Diffuse lung injury remains a significant problem following allogeneic HCT both in the immediate post-transplant period and in the months to years that follow. Although lung injury occasionally occurs following autologous transplantation, the allogeneic setting significantly exacerbates toxicity in both the acute and chronic conditions. Historically, much of this injury was assumed to be due to occult and unidentifiable infections, but a large preponderance of experimental data now demonstrates that IPS has a major immunologic component. Despite these findings, establishing the lung as a true target of GVHD remains a hotly debated topic. Like the gut and skin, the lung is a critical immunologic interface between the sterile body sanctuary and the outside environment. As such, the lung is a rich source of histocompatibility antigens and professional antigen-presenting cells, and is the site of complex immunologic
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networks that involve cytokine production and lymphocyte activation. Inflammatory mediators such as TNF-α and LPS, along with donorderived effector cells, which contribute to GVHD, are associated with acute lung injury in the experimental and clinical settings. Clinically, evidence supporting the concept that the lung is a target organ of acute GVHD is limited, and the major obstacle has been the lack of apoptotic epithelial injury. However, other GVHD target organs such as the thymus do not express this particular form of injury, and recent experimental data demonstrate that direct recognition of alloantigen on host epithelium by cytotoxic effectors may not be required for GVHD induction or target organ injury. In the chronic setting, the lung is widely accepted as a target of GVHD. The striking similarities between the histopathologic features of bronchiolitis obliterans seen in association with OLD after allogeneic HCT and those observed during lung allograft rejection, along with reports of improvement in lung function with immunosuppressive agents, strongly support this concept. The case for immunologic mechanisms contributing to RLD is less strong, and the data from experimental allogeneic models are still very scant. Nonetheless, TNF-α may be viewed as a common thread between acute IPS and chronic lung disease of either the obstructive or restrictive type. It is hoped that as animal models of lung injury after HCT yield further insights, our understanding of these disease processes will improve and ultimately lead to new therapeutic strategies to diagnose, treat, and prevent pulmonary toxicity in our allogeneic HCT recipients.
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following allogeneic murine bone marrow transplantation. Blood 2000; 96: 834–9. Dixon AE, Mandac JB, Madtes DK, Martin PJ, Clark JG. Chemokine expression in Th1 cellinduced lung injury: prominence of IFN-gammainducible chemokines. Am J Physiol Lung Cell Mol Physiol 2000; 279: L592–9. Hildebrandt GC, Corrion LA, Olkiewicz KM et al. Blockade of CXCR3 receptor:ligand interactions reduces leukocyte recruitment to the lung and the severity of experimental idiopathic pneumonia syndrome. J Immunol 2004; 173: 2050–9. Serody JS, Burkett SE, Panoskaltsis-Mortari A et al. T-lymphocyte production of macrophage inflammatory protein-1alpha is critical to the recruitment of CD8(+) T cells to the liver, lung, and spleen during graft-versus-host disease. Blood 2000; 96: 2973–80. Panoskaltsis-Mortari A, Hermanson JR, Taras E, Wangensteen OD, Serody JS, Blazar BR. Acceleration of idiopathic pneumonia syndrome (IPS) in the absence of donor MIP-1 alpha (CCL3) after allogeneic BMT in mice. Blood 2003; 101: 3714– 21. Choi SW, Hildebrandt GC, Olkiewicz KM et al. CCR1/CCL5 (RANTES) receptor–ligand interactions modulate allogeneic T-cell responses and graft-versus-host disease following stem-cell transplantation. Blood 2007; 110: 3447–55. Cooke KR, Jannin A, Ho V. The contribution of endothelial activation and injury to end-organ toxicity following allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2008; 14: 23–32. Panoskaltsis-Mortari A, Ingbar DH, Jung P et al. KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT. Am J Physiol Lung Cell Mol Physiol 2000; 278: L988–99. Yanik G et al. Impact of broncho-alveolar lavage on the diagnosis and management of pulmonary complications post transplant. Biol Blood Marrow Transplant 2008; 14: 89. Yanik GA, Levine JE, Ferrara JLM et al. Survival following etanercept therapy for the treatment of idiopathic pneumonia syndrome following allogeneic stem cell transplantation. Blood 2004; 104: 104a. Cerveri I, Zoia MC, Fulgoni P et al. Late pulmonary sequelae after childhood bone marrow transplantation. Thorax 1999; 54: 131–5. Yoshihara S, Yanik G, Cooke KR, Mineishi S. Bronchiolitis obliterans syndrome (BOS), bronchiolitis obliterans organizing pneumonia (BOOP), and other late-onset noninfectious pulmonary complications following allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2007; 13: 749–59. Freudenberger TD, Madtes DK, Curtis JR, Cummings P, Storer BE, Hackman RC. Association between acute and chronic graft-versus-host disease and bronchiolitis obliterans organizing pneumonia in recipients of hematopoietic stem cell transplants. Blood 2003; 102: 3822–8. Clark JG, Crawford SW, Madtes DK, Sullivan KM. Obstructive lung disease after allogeneic marrow transplantation. Clinical presentation and course. Ann Intern Med 1989; 111: 368–76. Ralph DD, Springmeyer SC, Sullivan KM, Hackman RC, Storb R, Thomas ED. Rapidly progressive air-flow obstruction in marrow transplant
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recipients. Possible association between obliterative bronchiolitis and chronic graft-versus-host disease. Am Rev Respir Dis 1984; 129: 641– 4. Chien JW, Martin PJ, Gooley TA et al. Airflow obstruction after myeloablative allogeneic hematopoietic stem cell transplantation. Am J Respir Crit Care Med 2003; 168: 208–14. Chien JW, Martin PJ, Flowers ME, Nichols WG, Clark JG. Implications of early airflow decline after myeloablative allogeneic stem cell transplantation. Bone Marrow Transplant 2004; 33: 759–64. Wyatt SE, Nunn P, Hows JM et al. Airways obstruction associated with graft-versus-host disease after bone marrow transplantation. Thorax 1984; 39: 887–94. Crawford SW, Clark JG. Bronchiolitis associated with bone marrow transplantation. Clin Chest Med 1993; 14: 741–9. Cooper JD, Billingham M, Egan T et al. A working formulation for the standardization of nomenclature and for clinical staging of chronic dysfunction in lung allografts. International Society for Heart and Lung Transplantation. J Heart Lung Transplant 1993; 12: 713–16. Ooi GC, Peh WC, Ip M. High-resolution computed tomography of bronchiolitis obliterans syndrome after bone marrow transplantation. Respiration 1998; 65: 187–91. Parimon T, Madtes DK, Au DH, Clark JG, Chien JW. Pretransplant lung function, respiratory failure, and mortality after stem cell transplantation. Am J Respir Crit Care Med 2005; 172: 384–90. Parimon T, Au DH, Martin PJ, Chien JW. A risk score for mortality after allogeneic hematopoietic cell transplantation. Ann Intern Med 2006; 144: 407–14. Chien JW, Maris MB, Sandmaier BM, Maloney DG, Storb RF, Clark JG. Comparison of lung function after myeloablative and 2 Gy of total body irradiation-based regimens for hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2005; 11: 288–96. Watkins TR, Chien JW, Crawford SW. Graft versus host-associated pulmonary disease and other idiopathic pulmonary complications after hematopoietic stem cell transplant. Semin Respir Crit Care Med 2005; 26: 482–9. Boehler A, Bai XH, Liu M et al. Upregulation of T-helper 1 cytokines and chemokine expression in post-transplant airway obliteration. Am J Respir Crit Care Med 1999; 159: 1910–17. Belperio JA, DiGiovine B, Keane MP et al. Interleukin-1 receptor antagonist as a biomarker for bronchiolitis obliterans syndrome in lung transplant recipients. Transplantation 2002; 73: 591– 9. Elssner A, Jaumann F, Dobmann S et al. Elevated levels of interleukin-8 and transforming growth factor-beta in bronchoalveolar lavage fluid from patients with bronchiolitis obliterans syndrome:
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proinflammatory role of bronchial epithelial cells. Munich Lung Transplant Group. Transplantation 2000; 70: 362–7. Fattal-German M, Le Roy Ladurie F, Cerrina J, Lecerf F, Berrih-Aknin S. Expression and modulation of ICAM-1, TNF-alpha and RANTES in human alveolar macrophages from lungtransplant recipients in vitro. Transpl Immunol 1998; 6: 183–92. El-Gamel A, Sim E, Hasleton P et al. Transforming growth factor beta (TGF-beta) and obliterative bronchiolitis following pulmonary transplantation. J Heart Lung Transplant 1999; 18: 828–37. Belperio JA, Keane MP, Burdick MD et al. Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome. J Clin Invest 2001; 108: 547–56. Belperio JA, Burdick MD, Keane MP et al. The role of the CC chemokine, RANTES, in acute lung allograft rejection. J Immunol 2000; 165: 461–72. Coker RK, Laurent GJ. Pulmonary fibrosis: cytokines in the balance. Eur Respir J 1998; 11: 1218–21. Hogaboam CM, Smith RE, Kunkel SL. Dynamic interactions between lung fibroblasts and leukocytes: implications for fibrotic lung disease. Proc Assoc Am Physicians 1998; 110: 313–20. Chien JW, Zhao LP, Hansen JA, Fan WH, Parimon T, Clark JG. Genetic variation in bactericidal/permeability-increasing protein influences the risk of developing rapid airflow decline after hematopoietic cell transplantation. Blood 2006; 107: 2200–7. Hildebrandt GC, Granell M, Urbano-Ispizua A et al. Recipient NOD2/CARD15 variants: a novel independent risk factor for the development of bronchiolitis obliterans after allogeneic stem cell transplantation. Biol Blood Marrow Transplant 2008; 14: 67–74. Panoskaltsis-Mortari A, Tram KV, Price AP, Wendt CH, Blazar BR. A new murine model for bronchiolitis obliterans post-bone marrow transplant. Am J Respir Crit Care Med 2007; 176: 713–23. Burman AC, Banovic T, Kuns RD et al. IFNgamma differentially controls the development of idiopathic pneumonia syndrome and GVHD of the gastrointestinal tract. Blood 2007; 110: 1064– 72. Trisolini R, Bandini G, Stanzani M et al. Morphologic changes leading to bronchiolitis obliterans in a patient with delayed non-infectious lung disease after allogeneic bone marrow transplantation. Bone Marrow Transplant 2001; 28: 1167– 70. Pardo A, Selman M. Idiopathic pulmonary fibrosis: new insights in its pathogenesis. Int J Biochem Cell Biol 2002; 34: 1534–8. Selman M, King TE, Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 2001; 134: 136–51.
138. Sime PJ, Marr RA, Gauldie D et al. Transfer of tumor necrosis factor-a to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming growth factor-b1 and myofibroblasts. Am J Pathol 1998; 153: 825–32. 139. Miyazaki Y, Araki K, Vesin C et al. Expression of a tumor necrosis factor-a transgene in murine lung causes lymphocytic and fibrosing alveolitis. J Clin Invest 1995; 96: 250–9. 140. Ortiz LA, Lasky J, Lungarella G et al. Upregulation of the p75 but not the p55 TNFa receptor mRNA after silica and bleomycin exposure and protection from lung injury in double receptor knockout mice. Am J Respir Cell Mol Biol 1999; 20: 825–33. 141. Piguet PF, Vesin C. Treatment by human recombinant soluble TNF receptor of pulmonary fibrosis induced by bleomycin or silica in mice. Eur Respir J 1994; 7: 515–18. 142. Moore BB, Paine R 3rd, Christensen PJ et al. Protection from pulmonary fibrosis in the absence of CCR2 signaling. J Immunol 2001; 167: 4368– 77. 143. Antoniades HN, Neville-Golden J, Galanopoulos T, Kradin RL, Valente AJ, Graves DT. Expression of monocyte chemoattractant protein 1 mRNA in human idiopathic pulmonary fibrosis. Proc Natl Acad Sci U S A 1992; 89: 5371–5. 144. Rabitsch W, Deviatko E, Keil F et al. Successful lung transplantation for bronchiolitis obliterans after allogeneic marrow transplantation. Transplantation 2001; 71: 1341–3. 145. Whitford H, Walters EH, Levvey B et al. Addition of inhaled corticosteroids to systemic immunosuppression after lung transplantation: a double-blind, placebo-controlled trial. Transplantation 2002; 73: 1793–9. 146. Iacono AT, Johnson BA, Grgurich WF et al. A randomized trial of inhaled cyclosporine in lung-transplant recipients. N Engl J Med 2006; 354: 141–50. 147. Verleden GM, Dupont LJ. Azithromycin therapy for patients with bronchiolitis obliterans syndrome after lung transplantation. Transplantation 2004; 77: 1465–7. 148. Gerhardt SG, McDyer JF, Girgis RE, Conte JV, Yang SC, Orens JB. Maintenance azithromycin therapy for bronchiolitis obliterans syndrome: results of a pilot study. Am J Respir Crit Care Med 2003; 168: 121–5. 149. Khalid M, Al Saghir A, Saleemi S et al. Azithromycin in bronchiolitis obliterans complicating bone marrow transplantation: a preliminary study. Eur Respir J 2005; 25: 490–3. 150. Chiang KY, Abhyankar S, Bridges K, Godder K, Henslee-Downey JP. Recombinant human tumor necrosis factor receptor fusion protein as complementary treatment for chronic graft-versus-host disease. Transplantation 2002; 73: 665–7. 151. Yanik G, Uberti J, Ferrara JLM et al. Etanercept for sub-acute lung injury following allogeneic stem cell transplantation. Blood 2003; 102: 471a.
97
Sangeeta Hingorani
Kidney and Bladder Complications of Hematopoietic Cell Transplantation
Introduction Renal dysfunction is a common complication of hematopoietic cell transplantation (HCT) and a major cause of morbidity and mortality. There are many definitions used to describe both the acute kidney injury (AKI) and chronic kidney disease (CKD) that occur after HCT (Table 97.1). The heterogeneous nature of the definitions, types of HCT, and diverse patient populations often make it difficult to draw comparisons across studies. The bladder is also commonly involved after HCT, and processes that affect the bladder can also lead to renal injury. This chapter will focus on the epidemiology, pathogenesis, and treatment of renal and urologic problems. The time course of renal injury varies from days after transplant to months later. The difference between acute and chronic is arbitrarily defined by timing as CKD usually refers to manifestations of renal disease that occur after day 100, and “acute” covers the early injury up to day 100. Similarly, bladder injury, most commonly hemorrhagic cystitis (HC), is divided into early onset and late onset, and the distinction between the two is also one of timing, lending itself to different etiologic factors. In this chapter, the earlyonset or acute injuries will be discussed first, followed by the later or chronic injuries. The disease processes can overlap: AKI that persists is later termed CKD. Although this chapter will discuss the injury that occurs after HCT, baseline or pretransplant renal function can impact outcome [1]. Therefore, baseline assessments not only of serum creatinine, but also urinalyses and more formal estimations of glomerular filtration rate (GFR) are warranted. Accurate assessment of baseline renal function can help guide later medication dosing.
Acute kidney injury Epidemiology AKI is defined as a doubling of baseline serum creatinine within the first 60–100 days after HCT. The incidence of AKI varies from a low of 6.5% in autologous transplants to as high as 79% in patients admitted to the intensive care unit (ICU) after transplant. The incidence also varies based on the type of HCT and the conditioning regimen. AKI is more common in allogeneic transplant recipients compared with autologous transplant recipients, and high-dose regimens compared with reduced-intensity conditioning (RIC). The less severe, or grade 2, AKI is defined as less than a doubling of baseline serum creatinine with a
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
greater than 25% decrease in GFR. Grade 2 is a doubling of baseline serum creatinine without the need for dialysis, and grade 3 is dialysisrequiring AKF (Table 97.1). Grade 3 AKF carries with it a higher mortality. Table 97.1 Definition of terms Term
Definition
Acute kidney injury
Doubling of baseline serum creatinine or fall in GFR to >50% Tripling of baseline serum creatinine, fall in GFR to 75% or creatinine >4.0 mg/dL GFR 0.7 mg/dL pre transplant Nephrotoxic drugs, SOS, older age Liver and lung toxicity, sepsis
28 days
[7]
329 180 57
100% allogeneic 100% allogeneic 26% autologous, 73% allogeneic
76% 80% 60%/79%
Grades 2 and 3 Grades 2 and 3 Grades 2 and 3
Within 56 days Within 100 days 27 days
[8] [9] [10]
269
100% allogeneic
60%
Grades 2 and 3
22 days
[11]
147 88
100% allogeneic 100% allogeneic
36% 69%
Grade 2 Grades 2 and 3
33 days 16 days
[3] [12]
47
53% autologous, 47% allogeneic 100% allogeneic, nonmyeloablative
32%/68%
Grades 2 and 3
44 days
[4]
40%
Grades 2 and 3
60 days
[13]
232
253
GVHD, graft-versus host disease; SOS, sinusoidal obstruction syndrome.
Liver failure, low serum albumin, APACHE II score Myeloablative conditioning, female gender, high-risk malignancy, comorbidity SOS, amphotericin use SOS, sepsis, lung and liver toxicity SOS, sepsis, cyclosporine Ventilator use, GVHD, product source (marrow versus peripheral blood)
Kidney and Bladder Complications of Hematopoietic Cell Transplantation
intrarenal injury [15]. In addition to the effects of sepsis, the medications used to treat the infections are often nephrotoxic. Several reports have identified amphotericin, in the conventional or liposomal formulation, as a risk factor for AKI. Hepatic SOS There is a well-known association between sinusoidal liver injury and renal insufficiency. It has been postulated that portal hypertension resulting from hepatic sinusoidal injury leads to both decreased renal perfusion and tubular injury, the former probably being the more important in the genesis of AKI. Clinically, SOS is associated with marked sodium avidity, low urinary sodium resulting in volume overload, edema, and weight gain, as seen in hepatorenal syndromes [16]. Weight gain likely serves as a marker of impending renal failure rather than being a result of the renal injury; in addition, the association between weight gain and the development of AKI described in the literature is conflicting [2,3,17]. Although some have advocated keeping weight gain per se to a minimum (3.0 mg/dL) compared with those patients on conventional amphotericin [22,23]. In a subset analysis of the allogeneic HCT recipients, Cagnoni et al. found that patients receiving liposomal amphotericin were less likely to develop nephrotoxicity compared with those who received conventional amphotericin: 32% versus 66%, respectively. Fewer patients in the liposomal group required dialysis. In a prospective, randomized controlled trial comparing conventional amphotericin with liposomal amphotericin in ICU patients with candidal infections, Sorkine et al. found that 66.7% of patients treated with conventional amphotericin experienced a significant increase in their serum creatinine compared with 4% of patients in the group treated with liposomal amphotericin [24]. Use of amphotericin for fever of unclear etiology carries significantly increased risks of AKI in the transplant population. Given the marked increase in risk of AKI in patients who have received either conventional or liposomal amphotericin, we recommend that patients be treated with amphotericin only if clear indications exist, such as documented fungal infection or prolonged use of prophylactic triazoles. Fortunately, newer antifungal drugs such as fluconazole, itraconazole, voriconazole, and caspofungin are now available for indications formerly covered by amphotericin. Depending on the endpoints chosen, each of these medications has been shown to be equally efficacious to amphotericin but with better safety profiles [25]. The new definition proposed by nephrologists for acute kidney failure (AKF) is based on the so-called “RIFLE – Risk, Injury, Failure, Loss,
1475
and End-stage – criteria. As one progresses through the stages, the criteria are based on changes in GFR and serum creatinine and/or decreases in urine output. Risk is an increase in serum creatinine of 1.5× baseline or a decrease in GFR of over 25%. Injury is a doubling of baseline serum creatinine or a 50% decrease in GFR. Failure is either a tripling of baseline serum creatinine, a serum creatinine of over 4 mg/dL or a 75% decrease in GFR. Loss and end-stage refer to duration of renal failure of 1 and 3 months, respectively [26]. These criteria were established because the degree of renal failure affects outcome in adult patients admitted to ICU. They are also applicable to the HCT population and should be used in future studies of AKI after HCT. Management of AKI In the majority of cases, the management of AKI is supportive. Studies comparing intermittent hemodialysis with continuous renal replacement therapy in patients admitted to ICU are equivocal (reviewed in [15]). The choice of dialysis modality often depends on hemodynamic stability of the patient, degree of volume overload, and blood pressure, which makes it difficult to interpret outcomes as sicker patients are more likely to be placed on continuous renal replacement therapy. What is apparent is that early intervention, regardless of modality choice, can improve outcome.
Chronic kidney disease Epidemiology The cumulative incidence of CKD varies from 13–60% in adult studies [27–29] to as high as 62% in children [30]. CKD usually becomes apparent 6–12 months after HCT, although it has been described as early as 2 months and as late as 10 years post transplant. As in AKI, there are numerous definitions used to define CKD in these patients. Most definitions are based on serum creatinine or an estimated or calculated measurement of GFR based on the Modification in Diet and Renal Disease or the Schwartz formula in children. The National Kidney Foundation has divided GFR into stages 1–5 based on an estimated GFR (eGFR) to help guide clinical treatment plans. The range of GFR is ≥90 mL/min/ 1.73 m2 with some renal damage for stage 1, to a GFR of 20-fold ↑ lovastatin acid AUC 20-fold and Cmax 13-fold ↑ simvastatin AUC and Cmax 10-fold ↑ simvastatin acid AUC 19-fold and Cmax 17-fold ↓ itraconazole [ ] ↓ itraconazole AUC >90%, Cmax 80% Itraconazole ↑ phenytoin AUC 10% ↓ itraconazole [ ] ↓ itraconazole AUC ~ 88% in healthy volunteers and 64% in AIDS patients ↑ sirolimus [ ] >8-fold
↑ unmetabolized terfenadine [ ] → QTc prolongation, torsades de pointes ↑ INR >8 → bruising/bleeding
Avoid concomitant use. Use alternative antifungal agent until busulfan elimination is completed ↓ calcineurin inhibitor dose by ~50% when starting itraconazole. Monitor [ ] and adjust dose accordingly Avoid combination therapy if possible, otherwise monitor itraconazole [ ] to prevent therapeutic failure Avoid concomitant use. Use alternative antifungal agent until cyclophosphamide elimination is completed ↓ digoxin dose 50% when starting itraconazole. Monitor digoxin [ ] and signs/symptoms of toxicity Monitor heart rate and blood pressure. ↓ dose of felodipine if needed Avoid concomitant use
Avoid combination therapy if possible, otherwise monitor itraconazole [ ] to prevent therapeutic failure Avoid concomitant use, as it may lead to therapeutic failure Avoid concomitant use, as it may lead to therapeutic failure Avoid concomitant use, as it may lead to therapeutic failure Monitor sirolimus [ ] and signs of toxicity. Adjust dose as needed. Refer to text for empiric dose adjustment for azole Use is contraindicated Monitor INR and for signs of bleeding. Dosage adjustment may be needed
(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; →, leading to; CYP3A4, cytochrome P450 3A4 isoenzyme; CYP2C9, cytochrome P450 2C9 isoenzyme; AUC, area under the curve; AUCPO, area under the curve (oral); Cl, clearance; CmaxPO, maximum concentration (oral); Cmax, maximum concentration; INR, international normalized ratio; P-gp, P-glycoprotein.
Voriconazole Voriconazole is also extensively metabolized by the CYP enzyme system. It is a substrate for and an inhibitor of CYP2C9, CYP2C19, and CYP3A4. Voriconazole has the highest affinity for inhibiting CYP2C9 and the lowest for CYP3A4, which explains why it is a less potent inhibitor of CYP3A4 when compared with itraconazole and ketoconazole. In contrast, voriconazole-N-oxide, one of three major metabolites, has higher inhibition affinity for CYP3A4 and CYP2C19 than CYP2C9 [30]. Since voriconazole is dependent on three CYP enzyme pathways, it is reasonable to expect that it could potentially affect the metabolism of many
drugs. Likewise, its metabolism may also be affected by other agents. The use of voriconazole in combination with sirolimus, terfenadine, astemizole, cisapride, pimozide, quinidine, and ergot alkaloids is contraindicated by the manufacturer because of the high risk for fatal adverse effects, such as significant elevation of drug levels, QTc interval prolongation, and ergotism [31]. Voriconazole also decreases the metabolism of drugs such as calcineurin inhibitors, HMG-CoA reductase inhibitors, omeprazole, phenytoin, and warfarin [30]. On the other hand, it has minor to negligible effects on prednisolone, digoxin, and mycophenolate. The use of voriconazole with potent inducers such as rifampin and rifabutin is contraindicated because they significantly reduce serum con-
Common Potential Drug Interactions Following Hematopoietic Cell Transplantation
centrations of voriconazole to undetectable levels. Increasing the voriconazole dose is incapable of overcoming the induction effects of rifampin; however, the effects of rifabutin can be overcome, although this is not advised. Voriconazole levels are also decreased by phenytoin and human immunodeficiency virus antivirals, whereas drugs such as cimetidine, ranitidine, erythromycin, azithromycin, and indinavir have negligible effects on voriconazole metabolism [32]. Additionally, concomitant administration with carbamazepine and long-acting barbiturates is also contraindicated. Pharmacokinetic studies of voriconazole’s effect on chemotherapy have also been conducted. In these studies, CYP3A4 inhibition by voriconazole resulted in an increase in the concentration of irinotecan, alltrans-retinoic acid, and imatinib, leading to a risk for increased toxicity [31]. Other drugs not tested but likely to interact with voriconazole
1527
include cyclophosphamide (a substrate of CYP2C9 and CYP3A) and dasatinib (a substrate of CYP3A). Alternatives to voriconazole should be employed during the co-administration of these chemotherapy agents if antifungal therapy is necessary. If temporary cessation of voriconazole is possible, it should be discontinued at least 30 hours prior to the start of chemotherapy and resumed when five half-lives of the antineoplastic agent have elapsed [1]. Table 99.4 further describes the clinically relevant drug interactions with voriconazole [1,31]. Posaconazole The primary metabolism of posaconazole is through glucuronidation by UGT enzymes [33]. When the effect of posaconazole was tested on CYP
Table 99.4 Selected drug interactions with voriconazole Drug or class
Mechanism
Effect
Management
(−) CYP3A4
Use is contraindicated
(+) CYP metabolism (−) CYP3A4
↑ antihistamine [ ] → QTc prolongation, torsade de pointes ↓ voriconazole [ ] → ↓ efficacy and therapeutic failure ↑ benzodiazepine [ ] → ↑ sedation
Calcineurin inhibitors Cyclosporine Tacrolimus
(−) CYP3A4
See Tables 99.6 and 99.7
See Tables 99.6 and 99.7
Calcium channel blockers [31] Diltiazem Verapamil
(−) CYP3A4
↑ calcium channel blocker [ ] → hypotension
Carbamazepine [31]
(+) CYP3A4
Cisapride [31]
(−) CYP3A4
Ergot alkaloids [31]
(−) CYP3A4
Ethinyl estradiol/norethindrone [31]
(−) CYP2C19
HMG-CoA reductase inhibitors [31]
(−) CYP3A4
↓ voriconazole [ ] → ↓ efficacy and therapeutic failure ↑ cisapride [ ] → QTc prolongation, torsade de pointes ↑ ergot alkaloid [ ] → ↑ risk of ergotism ↑ Cmax and AUC of voriconazole. ↑ Cmax and AUC of ethinyl estradiol/ norethindrone ↑ statin [ ] → ↑ risk for rhabdomyolysis
Monitor blood pressure and heart rate. Dosage reduction in calcium channel blocker may be necessary (if symptomatic) Use is contraindicated
Imatinib [1]
(−) CYP3A4
↑ imatinib [ ] → ↑ dose-dependent toxicities
Macrolide antibiotics [31] Clarithromycin
(−) CYP3A4
↑ clarithromycin [ ] → ↑ risk of QTc prolongation ↑ erythromycin [ ] → ↑ risk of QTc prolongation
Monitor cardiac function and symptoms of bradycardia. Consider the use of another macrolide antibiotic (i.e., azithromycin)
(−) CYP2C19, CYP2C9, and CYP3A4
↑ Cmax and AUC of active (R) methadone by 31% and 47%
Monitor for signs/symptoms of methadone toxicity including QTc prolongation. Methadone dose adjustment may be needed
Antihistamines [31] Astemizole Terfenadine Barbiturates (long acting) [31] Benzodiazepines [1]
Erythromycin
Methadone [31]
Use is contraindicated Monitor for ↑ benzodiazepine toxicity (sedation). Dose reduction in benzodiazepine may be necessary
Use is contraindicated Use is contraindicated Monitor for signs of voriconazole toxicity and for signs of abnormal menstruation Monitor for rhabdomyolysis, muscle pain and/or weakness. Consider dosage adjustment of statin and use of a non-CYP3A4 metabolized statin (pravastatin) Monitor blood counts, nausea/vomiting, fluid retention. Dosage reduction of imatinib may be necessary
1528
Chapter 99
Table 99.4 Continued Drug or class
Mechanism
Effect
Management
Phenytoin [31]
↓ Cmax of voriconazole by 50% and AUC by 70% → ↓ voriconazole efficacy and/or therapeutic failure ↑ Cmax and AUC of phenytoin by at least twofold → phenytoin toxicity
↑ voriconazole maintenance to 5 mg/kg intravenously twice a day (from 4 mg/kg) or to 400 mg orally twice a day (from 200 mg) Frequent monitoring of plasma phenytoin [ ] and phenytoin-related adverse effects
↑ [ ] of proton pump inhibitors
If omeprazole dose is >40 mg daily, ↓ omeprazole dose by 50%
Rifabutin [31]
Phenytoin (+) CYP metabolism of voriconazole, Voriconazole (−) CYP2C9 metabolism of phenytoin (−) CYP2C19, CYP2C9, and CYP3A4 (+) CYP3A4
Use is contraindicated
Rifampin [31] Sirolimus Sulfonylureas [31]
(−) CYP3A4 (−) CYP2C9
↓ Cmax of voriconazole by 93% → ↓ efficacy ↓ voriconazole [ ] → ↓ efficacy See Table 99.9 ↑ sulfonylurea [ ] → hypoglycemia
Proton pump inhibitors [31]
Tretinoin (all-trans retinoic acid; ATRA) [1]
(−) CYP3A4
↑ tretinoin [ ] → ↑ risk of hypercalcemia
Vinca alkaloids [1]
(−) CYP3A4
↑ vinca [ ]
Warfarin [31]
(−) CYP2C9
↑ PT, INR → ↑ bleeding risk
Use is contraindicated See Table 99.9 Frequent monitoring of blood glucose. Dosage reduction of sulfonylureas may be necessary Limit concurrent use. Otherwise, monitor for hypercalcemia. Discontinue tretinoin and/or add a bisphosphonate Monitor for vinca-associated neurotoxicity. Avoid concomitant administration. Dosage reduction of the vinca alkaloid may be necessary Monitor closely for signs/symptoms of bleeding, PT and INR. ↓ warfarin dose as needed
(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; →, leading to; CYP, cytochrome P450 isoenzyme; CYP3A4, cytochrome P450 3A4 isoenzyme; CYP2C19, cytochrome P450 2C19 isoenzyme; AUC, area under the curve; Cmax, maximum concentration; PT, prothrombin time; INR, international normalized ratio.
Table 99.5 Selected drug interactions with posaconazole Drug
Mechanism
Effect
Management
Cimetidine [33]
Alteration of gastric pH
Avoid concomitant use unless benefit outweighs risk
CSP [33]
(−) CYP3A4
↓ posaconazole AUC and Cmax 39% ↑ CSP [ ]
Midazolam [33] Phenytoin [33]
(−) CYP3A4 (+) UGT (−) CYP3A4
Rifabutin [33]
(+) UGT (−) CYP3A4
Tacrolimus [33]
(−) CYP3A4
↑ midazolam AUC 83% ↓ posaconazole AUC 50% and Cmax 41% ↑ phenytoin AUC and Cmax 16% ↓ posaconazole AUC 49% and Cmax 43% ↑ rifabutin AUC 72% and Cmax 31% ↑ tacrolimus AUC 358% and Cmax 121%
↓ CSP dose by 25% when starting posaconazole. Monitor CSP [ ] and adjust dose accordingly Monitor for adverse effects of benzodiazepines metabolized through this route Avoid concomitant use unless benefit outweighs risk. If used, monitor phenytoin [ ] and adjust dose accordingly
Avoid concomitant use unless benefit outweighs risk. If used, monitor for adverse effects of rifabutin
↓ tacrolimus dose by 66% when starting posaconazole. Monitor tacrolimus [ ] and adjust dose accordingly
(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; →, leading to; CYP3A4, cytochrome P450 3A4 isoenzyme; AUC, area under the curve; Cmax, maximum concentration; CSP, cyclosporine; UGT = uridine 5′-diphosphate glucuronosyltransferase.
enzymes, it was shown to inhibit hepatic CYP3A4, but not CYP1A2, CYP2C8/9, CYP2D6, or CYP2E1 [34]. Terfenadine, astemizole, cisapride, pimozide, and quinidine are contraindicated with posaconazole administration because of the potential development of QTc prolongation and torsades de pointes. Ergot alka-
loids are also contraindicated because they can result in ergotism. Similar to itraconazole and voriconazole, posaconazole has inhibitory effects on the calcineurin inhibitors, requiring initial dosage adjustments and frequent monitoring of their levels. Sirolimus, vinca alkaloids, calcium channel antagonists, and HMG-CoA reductase inhibitors are
Common Potential Drug Interactions Following Hematopoietic Cell Transplantation
examples of drugs metabolized through CYP3A4 that have not yet been tested but are likely to have an interaction with posaconazole. Other interactions occurring with posaconazole are summarized in Table 99.5 [33,35].
1529
clinically significant increases in CSP levels. Patients at highest risk for this interaction are those with the highest initial oral clearance of CSP. In these patients, careful monitoring of CSP levels and for signs of CSP toxicity is recommended with micafungin initiation or discontinuation. Adjustment in CSP levels may also be warranted in this patient population.
Echinocandins Caspofungin
Anidulafungin
Caspofungin is a poor substrate for CYP, and is neither an inhibitor nor an inducer of CYP3A4 [36]. To date, drug interaction data for caspofungin are minimal. The one of most relevance to clinicians in HCT is the interaction involving CSP. In two small studies, the area under the curve (AUC) for caspofungin was increased approximately 35% by CSP [36,37]. In addition, co-administration resulted in elevated alanine transaminase and aspartate transaminase levels. Three of four subjects developed transient alanine transaminase elevations two to three times the upper limit of normal. When lower doses of caspofungin were used, slight elevations were seen in 25% of the subjects. Increases in aspartate transaminase were also experienced, but were smaller in the two study groups. This prompted the manufacturer to caution against combination therapy unless the potential benefits outweigh the risks. If these drugs are used together, liver function tests should be monitored. The same interaction between caspofungin and CSP did not occur when tacrolimus, another calcineurin inhibitor, was given concomitantly with caspofungin. In fact, caspofungin decreased the tacrolimus AUC by 20%, maximum concentration (Cmax) by 16%, and 12-hour blood levels by 26% [36,37]. Therefore, routine monitoring of tacrolimus concentrations is advised with appropriate dosage adjustments. Another drug interaction study was performed involving rifampin and caspofungin. When both drugs were given together, trough concentrations of caspofungin were reduced by 30% at steady state [38]. To avoid possible therapeutic failure, it is recommended that the daily dose of caspofungin be increased to 70 mg. The higher dose of caspofungin should also be considered when used concomitantly with other agents known to be inducers of drug clearance, such as carbamazepine and phenytoin.
Unlike the other echinocandins, anidulafungin undergoes a slow chemical degradation (>90%) upon intravenous administration. It is not dependent on the CYP isoenzymes, nor does it appear to induce or inhibit them [42]. Consequently, the likelihood of drug interactions between anidulafungin and other agents administered to HCT patients is rare. No differences in the Cmax and AUC of anidulafungin were observed when comparing anidulafungin monotherapy, voriconazole monotherapy or the combination in a randomized, blinded, crossover pharmacokinetic study in healthy volunteers [42]. Similar results were described in a pharmacokinetic study of healthy volunteers where anidulafungin was combined with tacrolimus [43]. In another study with CSP, a statistically significant difference in the AUC was observed with combination therapy, but it was not considered clinically significant [44].
Micafungin Micafungin is primarily metabolized in the liver by arylsulfatase and catechol-O-methyltransferase [39]. In vitro data showed that micafungin was a substrate and a weak inhibitor of CYP3A4; however, in vivo data suggested that the CYP system played a negligible role in the drug’s metabolism. Additional published drug interaction studies involving micafungin are limited. Pharmacokinetic studies between micafungin 150 mg and voriconazole, and micafungin 100 mg and both calcineurin inhibitors, have been performed in healthy volunteers. The pharmacokinetics of micafungin was unaffected by the co-administration of these agents [39–41]. Micafungin had no effects on the pharmacokinetics of voriconazole or tacrolimus, although the authors of the tacrolimus study warned that an interaction remains possible if a dose of micafungin of over 100 mg/day is used with tacrolimus [39–40]. It appeared to be a mild inhibitor of CSP metabolism in the majority of the healthy volunteers [41]. This however, did not result in a significant change in CSP concentrations in 80% of the studied patients. In the remaining 20% of patients, the inhibition of CSP metabolism led to
Immunosuppressants Calcineurin inhibitors The metabolism of both CSP and tacrolimus is mediated predominantly through the CYP3A4 pathway. They are substrates for that isoenzyme as well as the P-gp transporter system. Therefore, they have similar drug interaction profiles. These are depicted in Tables 99.6 [12,33,36,45–52] and 99.7 [12,33,36,45,53–59]. Knowing which agents may affect the concentrations of these medications is crucial because the calcineurin inhibitors have a narrow therapeutic window. Too much of either agent will result in toxicity (nephrotoxicity, neurotoxicity, etc.), while too little will cause inadequate immunosuppression and possibly lead to increased risk of graft versus host disease. Two specific drugs that alter the metabolism of CSP and tacrolimus in different ways are caspofungin and sirolimus. These interactions are described in more detail under those separate sections.
Mycophenolate Following oral administration, mycophenolate is rapidly hydrolyzed by esterases found within the intestinal wall, blood, and liver, to the active moiety, mycophenolic acid (MPA). MPA is subsequently metabolized by UGT enzymes to its major inactive metabolite, 7-O-MPA glucuronide (MPAG) in the liver, gastrointestinal tract, and kidneys. Upon excretion into the bile, MPAG may be converted back to MPA by intestinal bacteria before it is reabsorbed into the colon. MPAG is primarily excreted into the urine via active tubular secretion and, to a lesser extent, via the multidrug resistance protein 2 (MRP-2) transport system [60]. The main mechanisms behind mycophenolate’s drug interactions are chelation, inhibition of enterohepatic recycling, and competition for renal tubular secretion, which can result in changes in MPA exposure.
1530
Chapter 99
Table 99.6 Selected drug interactions with cyclosporine (CSP) Drug or class
Mechanism
Effect
Management
Azoles Fluconazole [45]
(−) CYP3A4
↑ CSP [ ] 21%
Frequent monitoring of CSP trough [ ] during concomitant administration and after the discontinuation of fluconazole. Adjust dose accordingly ↓ CSP dose by ~50% when starting itraconazole. Monitor CSP [ ] and adjust dose accordingly ↓ CSP dose by 25% when starting posaconazole. Monitor CSP [ ] and adjust dose accordingly ↓ CSP dose by 50% when starting voriconazole. Monitor CSP [ ] and adjust dose accordingly
Itraconazole [12]
↑ CSP [ ] 80%
Posaconazole [33]
↑ CSP [ ]
Voriconazole [46]
↑ CSP AUC 1.7-fold and [ ] 2.5-fold
Calcium channel blockers Diltiazem [47] (−) CYP3A4 Carbamazepine [48] (+) CYP3A4 Caspofungin [36] (−) CYP3A4
↑ CSP AUC 28–62% and [ ] 49–88% ↓ CSP [ ] 50% ↑ caspofungin AUC 35% → ↑ ALT and AST
Digoxin [49]
↑ digoxin [ ] and ↓ Cl 53% → arrhythmias
unknown
HMG-CoA Reductase Inhibitors [50] Atorvastatin (−) CYP3A4 Lovastatin Pravastatin Simvastatin Phenobarbital [51] (+) CYP3A4 Phenytoin [51] (+) CYP3A4 Rifampin [52] (+) CYP3A4
↑ ↑ ↑ ↑ ↓ ↓ ↓
Sirolimus
See Table 99.9
(−) CYP3A4
atorvastatin AUC 6-fold lovastatin AUC 5-fold pravastatin AUC 5-fold simvastatin AUC 6-fold CSP [ ] CSP [ ] CSP AUC
Monitor CSP [ ] and adjust dose accordingly Monitor CSP [ ] and adjust dose accordingly Combination therapy should only be given if the potential benefits outweigh the risks. Monitor liver function tests Use with caution. Consider ↓ digoxin dose by ~50% when starting CSP. Monitor digoxin [ ] Avoid concomitant use if possible or ↓ dose of statin. Monitor for signs/symptoms of skeletal muscle toxicity
Monitor CSP [ ] and adjust dose accordingly Monitor CSP [ ] and adjust dose accordingly Avoid concomitant use if possible. Monitor CSP [ ] and adjust dose accordingly See Table 99.9
(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; →, leading to; CYP3A4, cytochrome P450 3A4 isoenzyme; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC, area under the curve; Cl, clearance.
Reductions in MPA exposure can lead to therapeutic failures, while increases in MPA concentrations can result in increased levels of acylglucuronide, leading to toxicities such as hypersensitivity, gastrointestinal disturbances, and cytokine release [60]. Drugs interacting with mycophenolate and their clinical implications are discussed below and in Table 99.8 [60–66]. Co-administration of agents such as oral divalent electrolyte supplements and phosphate binders with mycophenolate have been shown to reduce the extent of its absorption, leading to lower concentrations of MPA [61,63,66]. CSP, tacrolimus, and sirolimus are common immunosuppressants used in combination with mycophenolate. Decreased MPA concentrations observed with CSP are related to the inhibition of the MRP-2 transport system, which inhibits the enterohepatic recycling of mycophenolate [62]. In contrast, increased concentrations of MPA are seen with tacrolimus and sirolimus because enterohepatic recycling of MPA is not inhibited by these agents [67,68]. Other inhibitors of enterohepatic recycling include bile acid sequestrants, rifampin, and, to some extent, sevelamer [61,65]. Competition for renal excretion of mycophenolate is also a possible mechanism for drug interactions. In a prospective, randomized, openlabel, single-dose, crossover study of healthy volunteers, the pharmacokinetics of mycophenolate and valacyclovir or acyclovir were studied [60]. Concurrent use of mycophenolate with acyclovir resulted in an
increase in the Cmax and AUC of acyclovir and an increase in the AUC of MPAG. The authors concluded that this was not clinically significant in healthy patients, but such increases in drug exposure to acyclovir and MPAG could result in neutropenia in patients with renal impairment. Pharmacokinetic changes were not observed with valacyclovir in this study. Furthermore, MPA concentrations were unaffected by either agent when combined with mycophenolate. Other potential drugs that may compete for renal excretion include ganciclovir, valganciclovir, and probenecid [64].
Sirolimus Like calcineurin inhibitors, sirolimus is removed from the bloodstream by P-gp. It is extensively metabolized by CYP3A4 enzymes in the liver and small intestine [69]. Inducers of CYP3A4 and P-gp, such as carbamazepine, phenytoin, and rifampin, increase the metabolism of sirolimus, leading to decreased sirolimus blood levels, efficacy, and potentially inadequate therapy. Conversely, inhibitors such as calcium channel blockers, cimetidine, macrolide antibiotics, cisapride, and metoclopramide reduce the metabolism of sirolimus, leading to increased sirolimus levels and
Common Potential Drug Interactions Following Hematopoietic Cell Transplantation
1531
Table 99.7 Selected drug interactions with tacrolimus Drug or class
Mechanism
Effect
Management
Azoles Fluconazole [45]
(−) CYP3A4
↑ tacrolimus [ ] 16%
Frequent monitoring of tacrolimus trough [ ] during concomitant administration and after the discontinuation of fluconazole. Adjust dose accordingly ↓ tacrolimus dose by ~50% when starting itraconazole. Monitor tacrolimus [ ] and adjust dose accordingly ↓ tacrolimus dose by 66% when starting posaconazole. Monitor tacrolimus [ ] and adjust dose accordingly ↓ tacrolimus dose by 66% when starting voriconazole. Monitor tacrolimus [ ] and adjust dose accordingly
Itraconazole [12]
↑ tacrolimus [ ] 83%
Posaconazole [33]
↑ tacrolimus AUC 358% and Cmax 121%
Voriconazole [53]
↑ tacrolimus [ ] 10-fold
Calcium channel blockers Diltiazem [54] (−) CYP3A4 Felodipine [55] Caspofungin [36] unknown Clarithromycin [56] (−) CYP3A4 Phenobarbital [57] (+) CYP3A4
↑ tacrolimus [ ] ↑ tacrolimus [ ] 3-fold ↓ tacrolimus AUC 20%, Cmax 16%, and [ ] 26% ↑ tacrolimus [ ] ↓ tacrolimus [ ] 73%
Phenytoin [58]
(+) CYP3A4
↓ tacrolimus [ ]
Rifampin [59] Sirolimus
(+) CYP3A4 (−) CYP3A4
↓ tacrolimus [ ], requiring a 10-fold ↑ in tacrolimus dose See Table 99.9
Monitor tacrolimus [ ] and adjust dose accordingly Monitor tacrolimus [ ] and adjust dose accordingly Monitor tacrolimus [ ] and adjust dose accordingly Monitor tacrolimus [ ] and adjust dose accordingly. Monitor for ↑ risk of GVHD Monitor tacrolimus [ ] and adjust dose accordingly. Monitor for ↑ risk of GVHD Avoid concomitant use See Table 99.9
(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; CYP3A4, cytochrome P450 3A4 isoenzyme; AUC, area under the curve; Cmax, maximum concentration; GVHD, graft-versus-host disease.
Table 99.8 Selected drug interactions with mycophenolate Drug or class
Mechanism
Effect
Management
Acyclovir [65]
Competes for renal tubular secretion
Monitor neutrophil count closely
Antacids [60]
↓ MPA absorption
↑ acyclovir Cmax by 40% and AUC; ↑ MPAG AUC by 10.6% → ↑ risk of neutropenia in setting of renal impairment ↓ extent of absorption → ↓ MPA [ ]
Bile acid binders [60] Cholestyramine Colestipol
(−) enterohepatic recycling
↓ MPA AUC by 40%
CSP [63]
(−) enterohepatic recycling
↓ MPAG → ↓ MPA [ ]
Divalent oral electrolyte supplement [61]
↓ MMF absorption via chelation Competes for renal tubular secretion Unknown
↓ extent of absorption → ↓ MPA [ ]
Avoid co-administration of bile acid binders with MMF Monitor CSP [ ] and adjust dose accordingly Monitor for MMF therapeutic failure Monitor blood counts
(+) glucuronidation activity and (−) enterohepatic recycling ↓ MMF absorption and (−) enterohepatic recycling
↓ total MPA AUC → ↓ MMF efficacy
Ganciclovir [66] Oral contraceptives [66] (ethinyl estradiol, levonorgesterol, norethindrone, norgestrel) Rifampin [64] Sevelamer [62]
↑ plasma ganciclovir [ ] in setting of renal impairment ↓ oral contraceptive exposure
↓ AUC of MPA by 25% and Cmax by 30%
Monitor for MMF therapeutic failure
Monitor for breakthrough bleeding Monitor for MMF therapeutic failure Administration should be separated by at least 2 hours
(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; → = leading to; AUC, area under the curve; Cmax, maximum concentration; CSP, cyclosporine; MMF, mycophenolate mofetil; MPA, mycophenolic acid; MPAG, 7-O-MPA glucuronide.
1532
Chapter 99
Table 99.9 Selected drug interactions with sirolimus Drug or class
Mechanism
Effect
Management
Azoles [70] Fluconazole
(−) CYP3A4 and P-gp
Sirolimus troughs tripled after 7 days of concurrent therapy despite empiric dosage adjustments See Table 99.3
Monitor sirolimus troughs and signs of toxicity. Adjust dose as needed. Refer to text for empiric dose adjustment for azole Use is contraindicated by manufacturer. Refer to text for small case study describing concurrent use
Itraconazole
↑ sirolimus AUC by 7-fold and Cmax by 11-fold → ↑ risk of sirolimus toxicity
Voriconazole
Calcium channel blockers [69] Diltiazem (−) CYP3A4 and P-gp Verapamil
Calcineurin inhibitors Cyclosporine (CSP) [71]
(−) CYP3A4 and P-gp
Tacrolimus [72] Erythromycin [69]
(−) CYP3A4 and P-gp
Micafungin [69]
Unknown
Rifampin [69]
(+) CYP3A4 and P-gp
↑ sirolimus Cmax by 1.4-fold, tmax by 1.3-fold, and AUC by 1.6-fold → ↑ risk of sirolimus toxicity ↑ sirolimus Cmax by 2.3-fold and AUC by 2.2-fold → ↑ risk of sirolimus toxicity. ↓ Cmax and AUC of the active S(−) enantiomer of verapamil by 1.5-fold
Monitor sirolimus troughs and signs of toxicity. Adjust dose as needed
Concurrent administration → ↑ sirolimus Cmax by 116% and AUC by 230%. When sirolimus is given 4 hours apart → ↑ Cmax by 37% and AUC by 80% No effect on sirolimus pharmacokinetcs; however, tacrolimus troughs decreased ↑ sirolimus Cmax by 4.4-fold, AUC by 4.2-fold, and tmax by 0.4 hour → ↑ risk of sirolimus toxicity. ↑ erythromycin Cmax by 1.6-fold, AUC by 1.7-fold, and tmax by 0.3 hour ↑ sirolimus AUC by 21% with no effect on Cmax
Administer sirolimus 4 hours after CSP
↓ sirolimus AUC by 82% and Cmax by 71% → ↓ sirolimus efficacy and therapeutic failure
Monitor tacrolimus levels with sirolimus ≥2 mg daily Monitor sirolimus troughs and signs of toxicity. Adjust dose as needed
Monitor sirolimus troughs and signs of toxicity. Adjust dose as needed Monitor sirolimus troughs and signs of toxicity. Adjust dose as needed. Consider using an alternative agent that has less enzyme inducing potential
(−), inhibits; (+), induces; ↑, increases; ↓, decreases; →, leading to; CYP3A4, cytochrome P450 3A4 isoenzyme; AUC, area under the curve; Cmax, maximum concentration; P-gp, P-glycoprotein; tmax, time to maximum concentration.
subsequently increased risk of toxicity. Monitoring sirolimus trough concentrations for signs of toxicity and treatment failure is recommended when it is combined with these and any other agents utilizing CYP3A as their metabolic route of elimination. Some clinically important drug interactions are further discussed below or described in Table 99.9 [69–72]. Reports of drug interactions that resulted in decreased metabolism and supratherapeutic sirolimus levels have been documented with the azole antifungals. Despite recommendations to empirically reduce the dose of ketoconazole by 80–90% and fluconazole by 50–70% (for doses ≥200 mg daily), caution should be used in reducing the azole dose as interpatient variability can widely affect the incidence, magnitude, and significance of the interaction [70]. Close monitoring of sirolimus levels and adjustments in sirolimus dosage are recommended to optimize therapy during and upon the discontinuation of azole therapy. The concurrent use of voriconazole and sirolimus is contraindicated by the manufacturer based on a sevenfold increase in the Cmax and an 11-fold increase in the AUC of sirolimus seen in healthy volunteers [69]. Recent small case reports, however, have described the safe administration of this combination [73]. Mathis et al. cited a 75–87% dosage reduction in sirolimus during co-administration with voriconazole in two
renal transplant patients. Both patients tolerated therapy well. Similarly, Marty et al. reported the use of sirolimus and voriconazole in 11 patients undergoing allogeneic HCT [74]. In this case series, the sirolimus dose was empirically reduced by 90% upon initiation of voriconazole. All patients received at least 30 days of concurrent therapy. The authors of both studies concluded that the combination is feasible and safe. Frequent monitoring of sirolimus blood levels upon initiation and discontinuation of voriconazole was also recommended. Data regarding drug interactions between sirolimus and posaconazole are currently unavailable. The timing of sirolimus administration in relation to CSP was shown to affect the systemic exposure of sirolimus in healthy volunteers [71]. When both agents were administered concomitantly, a statistically significant increase in the bioavailability of sirolimus was demonstrated, as seen by an increase in the minimum concentration, maximum concentration, time to maximum concentration, and AUC. In contrast, when sirolimus was administered 4 hours after CSP, these measures were only modestly increased. The pharmacokinetics of CSP was unaffected by sirolimus in this study. The authors advised that sirolimus should be administered 4 hours after CSP to avoid high systemic sirolimus levels. Changes in sirolimus levels were not demonstrated in a similar study
Common Potential Drug Interactions Following Hematopoietic Cell Transplantation
with tacrolimus. However, when sirolimus was administered with tacrolimus, a reduction in tacrolimus levels occurred compared with when tacrolimus was administered alone [75].
Supportive care agents Narcotic analgesics Morphine, hydromorphone, fentanyl, and oxycodone are common narcotics used for the treatment of pain in transplant. Since the CYP isoenzyme system plays a minimal role in the metabolism of morphine and hydromorphone, the impact of CYP inhibitors and inducers on their pharmacokinetics is limited. Both drugs are metabolized via glucuronidation by various UGT enzymes in the liver [76,77]. Very few studies have been published that examine the effects of hydromorphone concentrations when administered with UGT inhibitors or inducers. The role of P-gp inhibition to enhance the pharmacologic effects of morphine continues to be studied. However, it has been proposed that concurrent use of agents that inhibit or induce UGT enzymes can change the AUC of morphine and consequently affect its pharmacologic potency or adverse effect profile [76]. Co-administration of metoclopramide with controlled-release morphine was shown to result in a faster time of onset and increase in sedation in a double-blinded study of 20 patients [76]. The pharmacologic and adverse effects of morphine should be monitored when these agents are used together. CYP3A4 is the major pathway of metabolism for phenylpiperidine opiates (alfentanil, fentanyl, and sufentanil). The effect of CYP3A4 inhibitors, especially azole antifungals, on phenylpiperidine metabolism is well documented in the literature. In a study of nine healthy volunteers, fluconazole was reported to double the half-life and reduce the clearance of alfentanil by 55% [78]. An increase in pharmacologic response as well as increased respiratory depression was also demonstrated. The authors of this study recommended caution when using these agents concurrently and suggested a 60% dosage reduction in alfentanil for maintenance analgesia when used with CYP3A4 inhibitors. Co-administration of voriconazole with alfentanil resulted in a similar outcome in which alfentanil clearance was reduced by 85% [79]. The AUC of alfentanil was also increased sixfold by voriconazole. The pharmacokinetic profile of fentanyl was not affected by itraconazole in a study of 10 healthy volunteers [80]. As a result, the authors stated that itraconazole can be safely used with normal doses of fentanyl, although they warned that an interaction is still possible with higher doses of fentanyl. In general, since the interaction can result in a fatal outcome, caution should be used when phenylpiperidine opiates are coadministered with any azole antifungals and known inhibitors of CYP3A4. Oxycodone is primarily metabolized by CYP2D6 to its active moiety, oxymorphone, while CYP3A is responsible for the formation of its inactive metabolite, noroxycodone [81]. Higher concentrations of oxymorphone may occur if oxycodone is given in conjunction with agents such as selective serotonin reuptake inhibitors (SSRIs), venlafaxine, or cimetidine, which all inhibit oxycodone metabolism. As a guideline, patients receiving agents that affect the metabolic pathways of these narcotics should be carefully monitored for altered pharmacologic activity or enhanced toxicity.
Sedatives, hypnotics, and anxiolytics Benzodiazepines are frequently used in transplant for a variety of indications, including sedation, anxiety, and antiemesis. Alprazolam, loraze-
1533
pam, midazolam, oxazepam, temazepam, and triazolam are examples of some of these benzodiazepines. Alprazolam, midazolam, and triazolam are all metabolized by CYP3A4 [82]. Concurrent administration of CYP3A4 inhibitors, such as azole antifungals or inhibitors of CYP2C19, such as SSRIs, will decrease benzodiazepine clearance. Increases in benzodiazepine concentrations can result in prolonged sedation and psychomotor impairment. Conversely, administration of CYP3A4 or CYP2C19 inducers (Table 99.1) may lead to increased benzodiazepine clearance that results in decreased therapeutic effect. Dosage adjustments may be required when combining these benzodiazepines with CYP3A4 or CYP2C19 inducers or inhibitors. Patients should be carefully monitored for therapeutic failures in the case of combination therapy with inducers such as rifampin, or prolonged adverse effects when used with inhibitors. The pharmacokinetic interactions observed with oral midazolam are more pronounced when administered with the same inducers or inhibitors than when it is given intravenously. This is mainly due to the intestinal and hepatic metabolism associated with first-pass effects with oral dosing. Alprazolam, midazolam, and triazolam are not substrates of P-gp and thus are not affected by inducers or inhibitors of that transport system [82]. Agents such as lorazepam, temazepam, and oxazepam may be reasonable alternatives for HCT patients since these agents undergo glucuronidation and are not metabolized by CYP3A4 [1].
Antidepressants SSRIs, serotonin–norepinephrine reuptake inhibitors, and other agents such as bupropion have replaced tricyclic antidepressants as the preferred agents for the treatment of depression. Potential SSRIs encountered in transplant include fluoxetine, fluvoxamine, paroxetine, sertraline, citalopram, and escitalopram. Similarly, examples of serotonin–norepinephrine reuptake inhibitors include venlafaxine and duloxetine. Bupropion and mirtazapine have also been effective. Many of these antidepressants are dependent on one or more CYP isoenzymes for their metabolic conversions (Table 99.1). They are also inhibitors, in varying degrees, of CYP and UGT enzymes as well as P-gp. Fluoxetine, fluvoxamine, nefazodone (acutely), norfluoxetine (the active metabolite of fluoxetine), paroxetine, sertraline, and venlafaxine are known to inhibit P-gp. Sertraline differs in that it inhibits UGT metabolism [83]. As such, the metabolism of these antidepressants can affect and be affected by the concomitant administration of other drugs that rely on the different enzyme and transport systems. Elevated concentrations of serotonin by drugs that inhibit SSRIs can result in the development of serotonin syndrome, a potentially fatal adverse effect. Linezolid, a weak, reversible, nonselective monoamine oxidase inhibitor has been reported to interact with several SSRIs and other antidepressants in various studies [84]. The authors of these studies favor the use of alternative antibiotic agents in patients taking SSRIs or venlafaxine. If linezolid therapy is necessary, they recommend discontinuing or tapering the antidepressant, if possible, prior to initiating antibiotic treatment. Patients should also be followed closely for signs and symptoms of serotonin syndrome. If serotonin syndrome occurs, linezolid therapy should be discontinued. A minimum washout period for linezolid of at least 2 weeks should occur before the antidepressant can be resumed. Additional interactions with antidepressants are described in Table 99.10 [83,84].
1534
Chapter 99
Table 99.10 Selected drug interactions with antidepressants [83,84] Drug
Interacting drug
Mechanism
Effect
Management
Bupropion
Carbamazepine Duloxetine
(+) CYP2B6 (−) CYP2D6
Fluoxetine
(−) CYP2B6
Monitor for bupropion failure Monitor for side-effects and adjust dose as needed Monitor for seizures
MAOI
Paroxetine
(−) norepinephrine metabolism by MAOI (−) reuptake of norepinephrine by duloxetine (−) CYP2B6
↓ bupropion [ ] ↑ duloxetine [ ] → ↑ side-effects ↑ bupropion [ ] → ↑ seizure risk ↑ norepinephrine → ↑ serotonin syndrome
Phenytoin Sertraline
(+) CYP2B6 (−) CYP2B6
Bupropion Carbamazepine Fluoxetine
See above (+) CYP1A2 (−) CYP2D6
MAOI
Paroxetine
MAOI (−) serotonin and norepinephrine metabolism; duloxetine (−) reuptake of serotonin and norepinephrine (−) CYP2D6
Alprazolam
Bupropion Carbamazepine
Duloxetine
Fluoxetine
Paroxetine
↑ bupropion [ ] → ↑ seizure risk ↓ bupropion [ ] ↑ bupropion [ ] → ↑ seizure risk
Monitor blood pressure, consider using alternative agents Monitor for seizures Monitor for bupropion failure Monitor for seizures
See above ↓ duloxetine [ ] ↑ duloxetine [ ] → ↑ side effects ↑ serotonin → ↑ serotonin syndrome risk
See above Monitor for duloxetine failure Monitor for side-effects and adjust dose as needed Monitor for serotonin syndrome
↑ duloxetine [ ] → ↑ side-effects
Monitor for side-effects and adjust dose as needed
(−) clearance of alprazolam
↑ alprazolam [ ] by 30% and ↑ pyschomotor impairment See above ↓ fluoxetine [ ]
Duloxetine Linezolid MAOI
See above (+) of CYP1A2, CYP2B6, CYP2C9, and CYP3A4 See above (−) serotonin metabolism (−) serotonin metabolism
Monitor for psychomotor impairment and adjust dose as needed See above Monitor for fluoxetine failure
Phenytoin
(−) CYP2C9, CYP3A4, and P-gp
↑ phenytoin [ ] → ↑ phenytoin toxicity
Risperidone
(−) CYP2D6, CYP3A4, and P-gp
↑ risperidone [ ] → risk for extrapyramidal symptoms
See above See text for recommendations In patients already on SSRI: discontinue SSRI 5 weeks prior to initiating MAOI In patients already on MAOI: discontinue MAOI therapy at least 2 weeks prior to initiating SSRI Monitor phenytoin [ ] and adjust phenytoin dose accordingly Monitor for extrapyramidal symptoms
Bupropion Duloxetine Linezolid
See above
See above
See above
(−) serotonin metabolism
See text for recommendations
MAOI
(−) serotonin metabolism
↑ serotonin → ↑ serotonin syndrome risk ↑ serotonin → ↑ serotonin syndrome risk
Risperidone
(−) CYP3A4, CYP2D6 and (−) P-gp
See above (−) serotonin metabolism (−) serotonin metabolism
↑ risperidone [ ] by 43% → risk for extrapyramidal symptoms
In patients on SSRI: discontinue SSRI 2 weeks prior to initiating MAOI In patients on MAOI: discontinue MAOI therapy at least 2 weeks prior to initiating SSRI Monitor for extrapyramidal symptoms
Common Potential Drug Interactions Following Hematopoietic Cell Transplantation
1535
Table 99.10 Continued Drug
Interacting drug
Mechanism
Effect
Management
Sertraline
Bupropion Carbamazepine Lamotrigine
See above (+) CYP2B6, CYP2C9, and CYP3A4 (−) UGT
See above Monitor for sertraline failure Monitor for rash
Linezolid
(−) serotonin metabolism
MAOI
(−) serotonin metabolism
Phenytoin
(+) CYP2B6, CYP2C9, CYP2C19, and CYP3A4
See above ↓ sertraline [ ] Doubling of lamotrigine [ ] → ↑ risk for Stevens–Johnson rash or toxic epidermal necrolysis ↑ serotonin → ↑ serotonin syndrome risk ↑ serotonin → ↑ serotonin syndrome risk ↓ sertraline [ ]
Azoles antifungals
(−) CYP3A4
↑ citalopram [ ]
Carbamazepine Cimetidine
(+) CYP3A4 (−) citalopram metabolism
↓ citalopram [ ] ↑ citalopram [ ] by 43%
Linezolid
(−) serotonin metabolism
MAOI
(−) serotonin metabolism
Metoprolol
Unknown
Omeprazole
(−) CYP2C19
↑ serotonin → ↑ serotonin syndrome risk ↑ serotonin → ↑ serotonin syndrome risk ↑ metoprolol [ ] by 2-fold → ↓ cardioselectivity, no change in blood pressure ↑ citalopram [ ]
Phenytoin
(+) CYP3A4 and CYP2C19
↓ citalopram [ ]
Carbamazepine Fluvoxamine
(+) CYP1A2 and CYP3A4 (−) CYP1A2, CYP2D6, and CYP3A4
Linezolid MAOI
(−) serotonin metabolism ↑ release of serotonin and norepinephrine by mirtazapine and (−) serotonin metabolism by MAOI
↓ mirtazapine [ ] ↑ mirtazapine [ ] by up to 4-fold → ↑ serotonin syndrome risk ↑ serotonin → ↑ serotonin syndrome risk
Linezolid
(−) serotonin metabolism
MAOI
MAOI (−) serotonin, norepinephrine, and sometimes dopamine metabolism; venlafaxine (−) reuptake of serotonin, norepinephrine, and dopamine
Citalopram or escitalopram
Mirtazapine
Venlafaxine
↑ serotonin → ↑ serotonin syndrome risk
See text for recommendations Same recommendations as paroxetine Monitor for sertraline failure Monitor for citalopram toxicity and reduce dose as necessary Monitor for citalopram failure Monitor for citalopram toxicity and reduce dose as necessary See text for recommendations Same recommendations as paroxetine Adjustment in metoprolol dose unnecessary Monitor for citalopram toxicity and reduce dose as necessary Monitor for citalopram failure Monitor for mirtazapine failure Monitor for serotonin syndrome
See text for recommendations Monitor for serotonin syndrome
See text for recommendations Monitor for serotonin syndrome
(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; → = leading to; CYP1A2, cytochrome P450 1A2 isoenzyme; CYP2B6, cytochrome P450 2B6 isoenzyme; CYP2D6, cytochrome P450 2D6 isoenzyme; CYP2C9, cytochrome P450 2C9 isoenzyme; CYP2C19, cytochrome P450 2C19 isoenzyme; CYP3A4, cytochrome P450 3A4 isoenzyme; MAOI, monoamine oxidase inhibitor; P-gp, P-glycoprotein; SSRI, selective serotonin reuptake inhibitor; UGT, uridine 5′-diphosphate glucuronosyltransferase.
Food Grapefruit juice Since the accidental finding of an interaction between grapefruit juice and felodipine, increasing attention has been focused on this food–drug combination [85,86]. Grapefruit juice contains flavonoids such as naringin, and furanocoumarins such as 6′,7′-dihydroxybergamottin, which may contribute to the interaction. In vitro studies have shown that these components inhibit CYP3A4, but in vivo trials have not proven a defin-
itive agent(s). The proposed mechanism of action appears to be decreased presystemic drug metabolism of oral medications caused by inhibition of CYP3A4 in the small intestine, as well as a reduction in CYP3A4 protein concentrations [85]. This results in higher drug levels, increased absorption, and perhaps exaggerated drug effects. Several factors influence whether an interaction with grapefruit juice might happen. These include using drugs with low bioavailability because of first-pass metabolism, being a substrate for CYP3A4, and having high amounts of CYP3A4 in the intestines [87]. Grapefruit juice does not usually affect
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Chapter 99
Table 99.11 Selected drug interactions with grapefruit juice Drug
Mechanism
Effect
Management
Amiodarone [88]
(−) CYP3A4
↑ amiodarone AUC 50% and Cmax 84% → QTc changes
Avoid concomitant use
Calcineurin inhibitors CSP [89] (−) CYP3A4, ?(−) P-gp Tacrolimus [90] Carbamazepine [91] (−) CYP3A4
↑ CSP [ ] 2.1-fold and AUC 1.4 -fold ↑ tacrolimus [ ] ↑ carbamazepine AUC 41%, Cmax 40%, and [ ] 39%
Avoid concomitant use
Cisapride [92] Felodipine [93]
↑ cisapride AUC ~2.4-fold and Cmax 1.8-fold ↑ felodipine AUC 2.9-fold and Cmax 4-fold → ↓ BP, ↑ HR
(−) CYP3A4 (−) CYP3A4
HMG-CoA reductase inhibitors Lovastatin [94] (−) CYP3A4 Simvastatin [95] Midazolam [96] (−) CYP3A4 Terfenadine [97] (−) CYP3A4
↑ lovastatin AUC 15-fold and Cmax 12-fold ↑ simvastatin AUC 13.5-fold and Cmax 12-fold ↑ midazolam AUC 2.4-fold and Cmax 2.3-fold ↑ unmetabolized terfenadine [ ], ↑ acid metabolite AUC 55% → QTc prolongation
Avoid concomitant use if possible. Monitor carbamazepine [ ] Avoid concomitant use Monitor blood pressure and heart rate. May require dosage adjustment Avoid concomitant use Avoid concomitant use Avoid concomitant use
(−), inhibits; ↑, increases; ↓, decreases; [ ], concentration; →, leading to; CYP3A4, cytochrome P450 3A4 isoenzyme; AUC, area under the curve; Cmax, maximum concentration; CSP, cyclosporine; P-gp, P-glycoprotein.
hepatic CYP3A4 activity, but possibly could if consumed in large quantities. Grapefruit juice has been shown to interact with a broad range of medications, from immunosuppressants to cholesterol-lowering agents to antihypertensives. Clinically relevant drug interactions are summarized in Table 99.11 [88–97]. These studies demonstrated highly variable results, leading to difficulty in predicting the degree of an interaction on an individual basis. The dose of a drug, duration and schedule of administration, formulation of a drug, and the source and composition of grapefruit juice, as well as individual metabolism, can all have an effect on the magnitude of the interaction [98,99]. Patients may be more susceptible if they have liver dysfunction or a condition that predisposes
them to more adverse effects from a particular agent. Additionally, for drugs that have a narrow therapeutic index, it is even more important to avoid grapefruit juice so that toxicities do not occur.
Conclusion As new drugs are being developed and used in different combinations, the risk for drug interactions will always be a concern. Having a firm understanding of the mechanisms by which drugs are metabolized and eliminated is a powerful tool to help predict and possibly minimize the occurrence of these interactions in the HCT setting.
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60. Staatz CE, Tett SE. Clinical pharmacokinetics and pharmacodynamics of mycophenolate in solid organ transplant recipients. Clin Pharmacokinet 2007; 46: 13–58. 61. Kato R, Ooi K, Ikura-Morii M et al. Impairment of mycophenolate mofetil absorption by calcium polycarbophil. J Clin Pharmacol 2002; 42: 1275– 80. 62. Pieper AK, Buhle F, Bauer S et al. The effect of sevelamer on the pharmacokinetics of cyclosporine A and mycophenolate mofetil after renal transplantation. Nephrol Dial Transplant 2004; 19: 2630– 3. 63. Hesselink DA, van Hest RM, Mathot RA et al. Cyclosporine interacts with mycophenolic acid by inhibiting the multidrug resistance-associated protein 2. Am J Transplant 2005; 5: 987– 94. 64. Naesens M, Kuypers DR, Streit F et al. Rifampin induces alterations in mycophenolic acid glucuronidation and elimination: implications for drug exposure in renal allograft recipients. Clin Pharmacol Ther 2006; 80: 509–21. 65. Gimenez F, Foeillet E, Bourdon O et al. Evaluation of pharmacokinetic interactions after oral administration of mycophenolate mofetil and valaciclovir or acyclovir to healthy subjects. Clin Pharmacokinet 2004; 43: 685–92. 66. Cellcept Package Insert. New Jersey: Roche Laboratories, October 2005. 67. Undre NA. Pharmacokinetics of tacrolimus-based combination therapies. Nephrol Dial Transplant 2003; 18(Suppl 1): i12–15. 68. Picard N, Premaud A, Rousseau A, Le Meur Y, Marquet P. A comparison of the effect of cyclosporine and sirolimus on the pharmacokinetics of mycophenolate in renal transplant patients. Br J Clin Pharmacol 2006; 62: 477–84. 69. Rapamune Package Insert. Pennsylvania: Wyeth Pharmaceutical, March 2007. 70. Saad AH, DePestel DD, Carver PL. Factors influencing the magnitude and clinical significance of drug interactions between azole antifungals and select immunosuppression. Pharmacotherapy 2006; 26: 1730–44. 71. Zimmerman JJ, Harper D, Getsy J, Jusko WJ. Pharmacokinetic interactions between sirolimus and microemulsion cyclosporine when orally administered jointly and 4 hours apart in healthy volunteers. J Clin Pharmacol 2003; 43: 1168–76. 72. Filler G, Womiloju T, Feber J, Lepage N, Christians U. Adding sirolimus to tacrolimus-based immunosuppression in pediatric renal transplant recipients reduces tacrolimus exposure. Am J Transplant 2005; 5: 2005–10. 73. Mathis AS, Shah NK, Friedman GS. Combined use of sirolimus and voriconazole in renal transplantation: a report of two cases. Transplant Proc 2004; 36: 2708–9. 74. Marty FM, Lowry CM, Cutler CS et al. Voriconazole and sirolimus coadministration after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2006; 12: 552–9. 75. McAlister VC, Mahalati K, Peltekian KM, Fraser A, MacDonald AS. A clinical pharmacokinetic study of tacrolimus and sirolimus combination immunosuppression comparing simultaneous to separated administration. Ther Drug Monit 2002; 24: 346–50. 76. Armstrong SC, Cozza KL. Pharmacokinetic drug interactions of morphine, codeine, and their
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100
Gundeep S. Dhillon & Norman W. Rizk
Critical Care of the Hematopoietic Cell Transplant Recipient
Introduction More than 50,000 patients receive hematopoietic cell transplantation (HCT) worldwide each year, and the number is growing. High levels of complications relating to the conditioning regimens, cytopenias, and immunosuppression associated with HCT eventuate in roughly 11–40% of the recipients requiring transfer to a critical care unit [1]. Many critical illnesses involving HCT patients are unique and reflect the combination of myelosuppression, allogeneic reactivity, and eventual marrow recovery. Graft-versus-host disease (GVHD), diffuse alveolar hemorrhage (DAH), and transplant-associated thrombotic microangiopathy are examples of these disorders. Optimal care of HCT patients must be based on a working knowledge of their unique illnesses and vulnerabilities, the natural history of these disorders, and the degree to which modern critical care can rescue them from these otherwise frequently fatal illnesses. This chapter reviews the organization of critical care for HCT patients, the diagnosis and treatment of specific organ system diseases, the prognosis of critically ill HCT recipients, and decisions about the goals of care involving HCT patients considered for life support modalities.
Organization of critical care for HCT recipients There are approximately 5980 intensive care units (ICUs) in the United States with an average census of about 55,000 patients per day [2]. Most of these are combined medical–surgical units, and most have trouble providing enough skilled staffing to achieve optimal results. Systematic reviews have suggested that full-time intensivist care within critical care units may in fact lower risk-adjusted mortality for the general ICU population by as much as 40%. Sadly, only about 4% of United States hospitals can meet the high standards of critical care specialist availability and high-intensity ICU staffing necessary to achieve these results [2]. Current projections for the supply of physician intensivists, critical care nurses, and pharmacists indicate an increasingly severe shortage of them, beginning in 2007, when demand is expected to exceed supply. Many hospitals already have a 20% vacancy rate in critical care nursing positions, with this expected to worsen over time [3]. Prevalence levels of burnout in critical care nursing staff and physicians as high as 33–50% [4,5] have been well documented, and contribute to the scarcity of these
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
resources. Because HCT recipients represent special challenges and benefit from use of these scarce human resources, their care is best provided in referral centers with adequate means to sustain 24-hour full physician and nurse staffing, preferably with health-care practitioners skilled in the critical care of these patients. High-intensity units capable of providing this care are more common in teaching and larger hospitals. Because of the shortage of critical care specialists, and because of volume considerations, very few hospitals for the foreseeable future will be able to provide specialized critical care units dedicated to HCT recipients. The most practical model in most centers with transplant programs will continue to be critical care specialists teaming with transplant professionals in general medical–surgical units. The team approach is essential for developing protocols and treatment guidelines, for monitoring and improving infection control associated with neutropenia and invasive devices, and for controlling metabolic derangements like hyperglycemia. These tasks touch on both the unique needs of HCT patients and the special environment of critical care units. Considerable evidence indicates that very morbid and mortal conditions such as ventilator-associated pneumonia can be prevented successfully by simple measures, like elevating the head of the bed by 30 degrees, and decontamination of oral cavities by use of chlorhexidine mouthwashes [6]. Similarly, strict adherence to Center for Disease Control decontamination guidelines for controlling blood stream infections associated with central lines can dramatically reduce or nearly eliminate these infections [7], which now number about 80,000 cases per year in ICUs [8]. Neutropenic HCT recipients have two to three times the rate of blood stream infections of other ICU patients [9] and are clearly at increased risk. Evidence also indicates that prophylaxis against gastrointestinal bleeding is required for patients on mechanical ventilators [10]. Well-run ICUs develop metrics to follow the use of these processes and measure their patient outcomes, using tools like prognostic scales, observed-to-expected mortality ratios, and specific complication rates per 1000 patient days. Organizations such as the University Health System consortium provide benchmark data for like institutions, as a form of report card on overall function; infection control data may be pooled in national initiatives such as the National Nosocomial Infection Surveillance project, which can inform intensive care medical directors about their performance in preventing infection and in controlling nosocomial spread. Increasingly, these metrics are being publicly reported, which will provide referring physicians with an objective database about the safety and efficacy of critical care units their patients may require. Optimal care requires engagement by many specialties in improving processes
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Chapter 100
Bacterial Fungal Herpes simplex/?RSV
Herpes varicella-zoster
Pneumocystis Cytomegalovirus
DAH/ARDS
Idiopathic interstitial pneumonitis
CHF
Bacterial (sinopulmonary) Obstructive airways disease/BO Restrictive ventilatory defect Acute GVHD 1
2
Chronic GVHD 3
4
5
6
7
8
9
10
11
Months after BMT Neutropenic phase
Day 100 post-BMT
in ICUs, particularly in the large majority of units that are designed as general medical–surgical ones.
Specific organ complications of HCT Pulmonary complications Approximately 60% of HCT recipients develop pulmonary complications after transplantation, and 30% of deaths are related to pulmonary complications [1,11–13]. Respiratory compromise is the most common indication for admission to the ICU. The different pulmonary complications seen in HCT recipients occur during predictable time periods, and an understanding of this temporal sequence can be very helpful in developing a differential diagnosis (Fig. 100.1) [12]. In the immediate postHCT period, extending from day 0 to day 30, the recipients are usually neutropenic and at high risk for the development of bacterial or fungal infections. Other common pulmonary complications during this period include pulmonary edema, drug toxicities, DAH, and idiopathic pneumonia syndrome (IPS). The acute neutropenic phase is followed by an early post-engraftment period that extends from day 30 to day 100. During this phase, recipients are no longer neutropenic, but use of immunosuppressive drugs for prophylaxis and treatment of GVHD results in both cellular and humoral immunity deficits. The recipients are at risk for development of viral infections, interstitial pneumonitis, acute GVHD, and delayed pulmonary toxicity syndrome. The late post-engraftment starts at post-HCT day 100 and extends indefinitely. At this time, noninfectious complications are more common, although patients, especially allogeneic HCT recipients, remain at risk for infectious complications. The noninfectious pulmonary complications seen during this period include bronchiolitis obliterans with or without organizing pneumonia. The pulmonary complications in HCT recipients depend upon pre-existing pulmonary conditions, pretransplant conditioning regimens, and level of immune reconstitution. These
12
Fig. 100.1 Pulmonary complications occurring after hematopoietic stem cell transplantation. ARDS, acute respiratory distress syndrome; BMT, bone marrow transplantation (i.e., hematopoietic stem cell transplantation); BO, bronchiolitis obliterans; CHF, congestive heart failure; DAH, diffuse alveolar hemorrhage; GVHD, graft-versushost disease; RSV, respiratory syncytial virus. (Reproduced from [12], with permission.)
pulmonary complications can lead to respiratory failure in a significant percentage of patients, with the incidence ranging from 10% to more than 50%. Infectious pulmonary complications Pneumonia is the leading infectious cause of death after HCT. The factors leading to pneumonia include neutropenia, immunosuppressive state, GVHD, and mucositis. Bacterial pneumonias are more frequent during the first month after HCT (see Chapter 88). The incidence of bacterial pneumonia in this patient population may be as high as 15%, with half the cases occurring in the first 100 days [14]. Bacterial pneumonias are more common in patients undergoing high-intensity rather than reduced-intensity HCT, probably related to longer periods of neutropenia in the former [15]. During the neutropenic phase, presentation of bacterial pneumonia can be subtle. Patients may present with fever only, without any respiratory symptoms [13]. In addition, chest radiographs can also be completely normal. Chest computed tomography (CT) scans are significantly more sensitive than plain radiographs for diagnosis. The treatment consists of early institution of broad-spectrum antibiotics, followed by narrowing of the antimicrobial coverage once culture data become available. Bacterial pneumonia can rapidly lead to acute respiratory failure and multiorgan system failure (MOSF). Cytomegalovirus (CMV) is an important pathogen in HCT recipients (see Chapter 90). The vast majority of CMV infections in these patients are related to activation of latent disease, rather than new infections [13]. CMV pneumonitis occurs within the first 100 days; however, use of CMV prophylaxis may delay the onset of active disease [16]. CMV pneumonitis patients present with dyspnea, dry cough, and hypoxemia, and can progress to respiratory failure. The demonstration of CMV inclusion bodies in lung tissue is diagnostic. In the absence of lung tissue, diagnosis can be made by detection of CMV in bronchoalveolar lavage (BAL) fluid and/or in serum by the polymerase chain reaction,
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Table 100.1 Diagnostic criteria for idiopathic pneumonia syndrome I. Evidence of widespread alveolar injury, including: Multilobar infiltrates on routine chest radiographs or CT scans Symptoms and signs of pneumonia Evidence of abnormal pulmonary physiology Increased alveolar-to-arterial oxygen gradient New or increased restrictive pulmonary function test abnormality
Fig. 100.2 Chest tomograph showing right upper lobe consolidation and the air-crescent sign in a recipient of hematopoietic cell transplantation who had invasive aspergillosis.
in the appropriate clinical setting. However, a negative serum polymerase chain reaction or failure to detect CMV antigenemia does not rule out CMV disease [17]. Treatment consists of combination therapy with intravenous ganciclovir and immunoglobulin infusion [18,19]. The survival rates of HCT recipients with CMV pneumonitis have improved from 15% to 50–70%. This improvement in survival has been attributed to use of the combination therapy with ganciclovir and immunoglobulin, although controlled trials are lacking. Ganciclovir-resistant strains of CMV may require treatment with foscarnet. The respiratory viral infections, such as respiratory syncytial virus (RSV), parainfluenza, influenza, and rhinovirus are also a major cause of morbidity and mortality in this patient population. RSV is the most common respiratory viral isolate in HCT recipients, and is associated with poor outcomes [20]. A comprehensive strategy to reduce personto-person transmission of RSV can reduce the incidence of this infection in HCT recipients. The treatment of RSV consists of nebulized or intravenous ribavirin and administration of RSV-specific immunoglobulin. Invasive aspergillosis is a particularly serious pulmonary complication of HCT (see Chapter 89). It is more common in patients undergoing allogeneic HCT, probably as a result of long-term immunosuppressive therapy for GVHD [21]. Currently, there is no effective prophylaxis for invasive pulmonary aspergillosis (IPA), although low-dose intravenous amphotericin B, nebulized amphotericin B, voriconazole, and itraconazole have been used. Invasive disease is usually limited to the lungs, although it can involve the sinuses, central nervous system, and other organs, reflecting hematogenous spread from angioinvasion [13]. Patients usually present with dyspnea and cough. Pleuritic chest pain and hemoptysis are not uncommon. The chest radiographs and CT scan may show pulmonary nodules, consolidation or cavitation. The “halo sign,” indicating hemorrhage around the lesion, and the “air-crescent sign” (Fig. 100.2), signifying cavitation, are characteristic signs of IPA. In HCT recipients, detection of Aspergillus in respiratory secretions has a positive predictive value greater than 80% for IPA, but the sensitivity of this test is low [22,23]. Pulmonary nodules may require transthoracic needle biopsy or resection for diagnosis. An enzyme-linked immunosorbent assay for galactomannan, a fungal cell wall component, has been used for surveillance for IPA. It has a sensitivity of 0.71 and a specificity of 0.89 [24]. Intravenous voriconazole, a triazole antifungal agent,
II. Absence of active lower respiratory tract infection. Appropriate evaluation includes: Bronchoalveolar lavage negative for significant bacterial pathogens and/or lack of improvement with broad-spectrum antibiotics Bronchoalveolar lavage negative for pathogenic nonbacterial microorganisms Routine bacterial, viral, and fungal cultures Shell-vial CMV culture Cytology for CMV inclusions, fungi, and Pneumocystis jiroveci Detection methods for respiratory syncytial virus, parainfluenza virus, and other organisms Transbronchial biopsy if condition of the patient permits Ideally, a second confirmatory negative test for infection is done. This usually is performed 2–14 days after the initial negative bronchoalveolar lavage, and it may consist of a second bronchoalveolar lavage or a surgical lung biopsy CMV, cytomegalovirus; CT, computed tomography. Reproduced with permission from Clark et al. [28].
appears to be more efficacious and better tolerated than amphotericin B for the treatment of IPA [25]. Caspofungin, an echinocandin, has also been approved for salvage therapy of IPA. Pneumocystis jiroveci pneumonia (formerly Pneumocystis carinii pneumonia; PCP) is now an uncommon opportunistic infection in HCT recipients, secondary to effective prophylaxis with trimethoprim– sulfamethoxazole. In the absence of prophylaxis, the incidence of PCP in allogeneic HCT recipients is as high as 15% [26]. For HCT recipients who are on immunosuppressive therapies and are not receiving optimal prophylaxis, suspicion of this should remain high. BAL is the procedure of choice for diagnosis, with a diagnostic yield in excess of 90% [27]. The treatment of choice consists of high-dose trimethoprim–sulfamethoxazole. The role of corticosteroids in the treatment of PCP in this patient population, unlike in patients with acquired immune deficiency syndrome, is unclear. Noninfectious pulmonary complications IPS, as defined by a National Heart, Lung, and Blood Institute panel in 1993, encompasses a wide range of clinical presentations of lung injury in HCT recipients, which cannot be attributed to infectious or cardiogenic causes (see Chapter 96) [28]. The diagnostic criteria (Table 100.1) include evidence of pneumonia, nonlobar infiltrates on chest radiograph, and absence of infectious etiology. The histopathologic findings associated with IPS include interstitial pneumonitis, diffuse alveolar damage, bronchiolitis obliterans organizing pneumonia (BOOP), and lymphocytic bronchitis. The term “interstitial pneumonitis” has been used interchangeably with IPS [29]. The reported incidence of IPS after HCT ranges from 3% to 15% [30]. It is more common in patients receiving allogeneic rather than autologous HCT. Among allogeneic HCT recipients, IPS is more common among patients receiving high-dose preparative regimens. Patients usually present with dyspnea, hypoxemia, cough, and fever. The onset
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Fig. 100.3 Bilateral lower lobe air space disease secondary to diffuse alveolar hemorrhage in a recipient of hematopoietic cell transplantation.
of symptoms is within the first 100 days following transplantation, with more recent studies suggesting an earlier onset around 2–3 weeks post transplant [31,32]. The treatment for IPS is supportive. Patients are commonly treated with high-dose corticosteroids, although the evidence for their efficacy remains scant. The clinical course is one of progressive respiratory failure leading to MOSF and death. DAH is a pulmonary complication of HCT characterized by a progressively bloodier return of BAL aliquots. It is seen in 5% of HCT recipients, and is slightly more common in patients receiving autologous rather than allogeneic HCT [30]. The risk factors for DAH include intensive conditioning chemotherapy, total body irradiation, renal insufficiency, older age, and previous acute GVHD [33,34]. Patients usually present within the first 30 days after HCT with progressive dyspnea, fever, cough, hypoxemia, and bilateral ground glass infiltrates on highresolution CT of the chest (Fig. 100.3). Hemoptysis is uncommon in patients with DAH. The diagnosis is usually made with BAL that shows a progressively bloodier return of BAL aliquots and hemosiderin-laden macrophages. The following criteria are suggested for the diagnosis of DAH: (1) evidence of bilateral widespread alveolar injury; (2) absence of infection; and (3) BAL showing progressively bloodier return from three different subsegmental bronchi or the presence of 20% or more of hemosiderin-laden macrophages [33]. Most patients with DAH develop respiratory failure requiring mechanical ventilatory support. In addition to supportive care, treatment with high-dose corticosteroids (125–250 mg of methylprednisolone every 6 hours for 4–5 days, with taper over the next 4 weeks) may be helpful [35,36]. The overall prognosis remains poor, with earlier reported mortality rates as high as 80–100% [34–36], but more recent studies indicating it closer to 60% at 6 months [37]. BOOP, a pathologic entity with small airway injury and interstitial inflammation with granulation tissue plugs in small airways, is another manifestation of acute lung injury seen in patients with GVHD following allogeneic HCT. It develops 1–3 months after the transplant, and the usual presentation consists of fever, dry cough, dyspnea, hypoxemia, and bilateral pulmonary infiltrates. These patients can progress to acute respiratory failure requiring mechanical ventilation. BOOP usually responds to corticosteroid therapy, although patients may require it for prolonged periods of time [30]. A delayed pulmonary toxicity syndrome has been described in patients who have undergone high-dose chemotherapy with carmustine-
containing regimens followed by HCT. The median time to onset of symptoms is 10 weeks from HCT. Symptoms are usually mild and consist of exertional dyspnea, dry cough, and fever. Pulmonary function tests shows mild restriction and severe reduction in diffusion capacity. High-resolution CT scans of the chest show patchy or diffuse ground glass infiltrates. Symptoms are responsive to early and aggressive corticosteroid therapy [38]. However, carmustine-related pulmonary toxicity syndrome could lead to progressive respiratory failure and death in up to 8% of the affected patients [39]. Bronchiolitis obliterans (without organizing pneumonia) is a late pulmonary complication seen in allogeneic HCT recipients. The onset of symptoms is beyond 3 months after HCT and is strongly associated with chronic GVHD [40]. The clinical picture resembles progressive chronic obstructive lung disease. Besides GVHD, other risk factors for the development of bronchiolitis obliterans include pretransplant obstructive lung defect, recipient age, methotrexate use, and history of respiratory viral illness within the first 100 days after transplant [40]. The clinical course is variable, with a small subset of patients developing progressive obstructive lung defect leading to respiratory failure [41]. Treatment consists of augmentation of immunosuppression in an attempt to stabilize lung function [42]. Lung transplantation may be an option in select patients who are deemed cured of their underlying initial disease process [13]. The other uncommon pulmonary complications seen after HCT include pulmonary veno-occlusive disease and posttransplant lymphoproliferative disorder [13]. Respiratory failure Respiratory failure is present in half of the HCT patients admitted to the ICU [1]. The risk factors for development of respiratory failure after HCT include age above 21 years, active malignancy at the time of transplant, and nonidentical human leukocyte antigen (HLA) donor [43]. Earlier studies suggested that, among HCT recipients who required mechanical ventilation, mortality rates approached 100% [43]. However, more recent studies have suggested survival rates up to 25%. This improved survival is probably related to an increased use of peripheral blood hematopoietic cells [44,45], improvements in ventilatory strategies [46,47], and better supportive care. In general, HCT patients with respiratory failure should be managed using the same general principles that have been developed for patients with lung injury related to other diseases, while recognizing their unique vulnerabilities. In view of the rapidity with which pulmonary complications can lead to respiratory failure in HCT recipients, these patients should undergo rapid and thorough diagnostic evaluation. The choice of diagnostic procedures is guided by clinical history and radiographic appearance. CT scanning is useful in the evaluation of these patients, as it may suggest diagnoses and is a sensitive tool for defining the extent of disease. Fiberoptic bronchoscopy with BAL is the diagnostic test of choice in HCT patients with pulmonary opacities. Diagnostic yield for infectious etiologies, with the notable exception of focal diseases like invasive aspergillosis, is high [48]. Transbronchial biopsies do not improve yield and are potentially dangerous because of coagulopathies. Patients with discrete pulmonary nodules may need transthoracic needle aspiration. A surgical lung biopsy, either by thoracotomy or thoracoscopy, is safe but rarely impacts management and hence is rarely performed. HCT patients with acute lung injury, as defined in Table 100.2, should be managed according to lung-protective ventilatory strategies that have been shown to improve outcomes in patients with acute lung injury [46]. The goal tidal volume for mechanical ventilation should be less than 6 mL/kg of predicted body weight, airways plateau pressures should be maintained at less than 30 cmH2O, and the partial pressure of oxygen in arterial blood (PaO2) should be maintained between 55 and 80 mmHg, using a combination of fraction of inspired oxygen and positive end-
Critical Care of the Hematopoietic Cell Transplant Recipient Table 100.2 Definition of acute lung injury
Table 100.3 Diagnostic criteria for sepsis
Acute onset Bilateral infiltrates on chest radiograph PaO2: FiO2 38.3ºC) Hypothermia (core temperature >36ºC) Heart rate >90 min−1 or >2 sd above the normal value for age Tachypnea Altered mental status Significant edema or positive fluid balance (>20 mL/kg over 24 hrs) Hyperglycemia (plasma glucose >120 mg/dL or 7.7 mmol/L) in the absence of diabetes Inflammatory variables Leukocytosis (WBC count >12,000 μL−1) Leukopenia (WBC count 10% immature forms Plasma C-reactive protein >2 sd above the normal value Plasma procalcitonin >2 sd above the normal value Hemodynamic variables Arterial hypotension (SBP 3 L⋅min−1⋅M−2 Organ dysfunction variables Arterial hypoxemia (Pao2/Fio2 1.5 or aPTT >60 secs) Ileus (absent bowel sounds) Thrombocytopenia (platelet count 4 mg/dL or 70 mmol/L) Tissue perfusion variables Hyperlactatemia (>1 mmol/L) Decreased capillary refill or mottling aPTT, activated partial thromboplastin time; Fio2, fraction of inspired oxygen; INR, international normalized ratio; MAP, mean arterial blood pressure; Pao2, partial pressure of oxygen in arterial blood; SBP, systolic blood pressure; sd, standard deviation; S v¯o2, mixed venous oxygen saturation; WBC, white blood cell. Reproduced with permission from Levy et al. [55].
to maintain mean arterial pressure greater than 65 mmHg and urine output greater than 0.5 mL/kg/hour [58]. Vasopressin is useful in patients with refractory shock, but as an adjunctive rather than a primary agent [59]. Patients with refractory hypotension, worsening vasopressor requirements, low urine output or cardiac dysfunction may benefit from further evaluation of hemodynamic status with echocardiography or placement of a pulmonary artery catheter. Patients with low cardiac output despite fluid resuscitation may benefit from inotropic support with dobutamine. Diagnostic work-up for infections should be initiated during the initial resuscitation. If possible, appropriate cultures for both communityacquired and health-care-associated infections should be taken prior to antimicrobial therapy. Cultures from blood, urine, respiratory secretions, and other body fluids should be obtained as clinically indicated. The antimicrobial agents should be administered as soon as possible, with the current standard being within 4 hours of presentation. The initial antibiotic choice should be guided by the patient’s clinical situation. However, the antimicrobial coverage should be broad enough to cover
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all likely pathogens. A delay in initiation and/or inappropriate antimicrobial therapy has been associated with worse outcomes in patients with severe sepsis [60,61]. All patients should undergo a thorough evaluation for any possible infection source, which may be amenable to drainage, debridement or removal. Patients with severe sepsis and septic shock may have absolute or relative adrenal insufficiency. The risk of adrenal insufficiency may be higher in HCT recipients, as they have substantial exposure to exogenous corticosteroids. To evaluate adrenocortical reserve, patients with septic shock should undergo a cosyntropin stimulation test. Patients with either a low random cortisol level (30 days post transplant
A large body of information indicates that HCT recipients have a worse prognosis than other medical patients in critical care units. On average, medical patients in critical care units in the Untied States have a 5–15% mortality rate; however, in a large series in 1992, the hospital mortality rate for HCT patients in critical care units was as high as 96% for transplant recipients who required mechanical ventilation [43]. In that series, 348 patients were ventilated for an average of 8 days, and only 3% survived 6 months. Older age, active malignancy at the time of transplantation, and HLA nonidentity were determined to be risk factors for requiring mechanical ventilation. The authors suggested this should be considered when counseling patients about this level of support [43]. Following this provocative report, the literature has evolved considerably and has focused on predictors of mortality in HCT recipients, on whether critical care prognostic scales are useful for counseling, and on adoption of newer treatment modalities for MOSF. Mechanically ventilated patients following HCT have continued to fare particularly poorly. In a 1996 series comprised of 865 patients mechanically ventilated, only 53 survived to hospital discharge. Among the estimated 398 patients from this cohort who had lung injury and who required either more than 4 hours of vasopressor support or who had hepatic and renal failure, there were no survivors [109]. Compared with earlier studies from the same institution, patients reported in 1996 had a slightly better prognosis, with 16% surviving mechanical ventilation as long as MOSF was not present. Shortly thereafter, a study from M.D. Anderson Cancer Center found an 18.8% survival rate following intubation for respiratory failure, compared with a 65.7% survival for those not requiring mechanical ventilation. Multiple regression risk analysis pointed to allogeneic rather than autologous transplants, infection, bleeding, and prolonged time from transplant to ICU admission as other predictors of a fatal outcome [45]. The hope was that these kinds of studies might assist in providing a basis for deciding whether intubation and mechanical ventilation were warranted in patients with severe complications from HCT. In this light, formal evaluation of the most commonly used prognostic scale is of interest. A study of the APACHE, versions II and III, revealed it to be only moderately discriminating in determining whether HCT patients would survive in the Mayo Clinic critical care units. The receiver operating characteristic curve for APACHE III was 0.704, indicating that it probably cannot be used with a high degree of confidence for predicting the outcome of individual patients. Once again, allogeneic status, mechanical ventilation, vasoactive medication use, sepsis, and MOSF were associated with increased mortality [56]. Table 100.4 lists the prognostic factors now known to be associated with high mortality. In the last few years, the prognosis of HCT recipients in critical care units is continuing to make gains. Whether this reflects selection bias in patients referred to critical care units [110] as a result of prior studies encouraging forms of triage, or whether improved modalities of care are responsible, is not entirely clear; probably both are operative. However, the prognosis is still relatively dismal and much worse than that of general medical ICU patients. In two recent studies, the 12-month sur-
Probability of hospital survival in patients mechanically ventilated is approximately 15–30% Probability of hospital survival in patients mechanically ventilated who require pressors is most commonly 50 kg body weight
Usual dose for children* and adults† 5.6 kPa) is uncommon. Patients with pre-existing respiratory disease, those with sleep apnea-like symptoms, and those receiving other CNS-active drugs, especially benzodiazepines, are most at risk of respiratory depression. The degree of respiratory depression parallels the slowing respiratory rate and degree of sedation; these signs can be monitored if respiratory depression is a concern. Opioid tolerance and dependence Tolerance
Genitourinary effects Increased tone and contractility of the ureters and increased detrusor tone may lead to urinary retention. Pruritus Pruritus is a well documented yet poorly understood occasional sideeffect of opioid therapy. Antihistamines, rotation of opioids, and opioid dose reduction are common treatment strategies. In addition, mixed opioid agonists–antagonists can reverse itching, but this may reduce analgesic effects as well [68]. Sedation Sedation is an almost universal side-effect of opioids and usually occurs at onset of therapy or with dose escalation. Opioid induced sedation typically resolves within 72 hours of initiating therapy. It is important to remember that the differential diagnosis of sedation in the HCT
Tolerance is the progressively decreasing effect (analgesic or other effects) of a given dose of drug when that dose is administered repetitively. Tolerance may be seen when a patient who has been achieving satisfactory analgesia with a given dose of drug progressively fails to achieve analgesia without evidence of an increase in nociception. If it occurs in the HCT patient, tolerance may be overcome by increasing the dose of drug or changing to a different opioid. Tolerance is a drugrelated effect only and has little or no relation to addiction. Incomplete crosstolerance When changing from one opioid to another, it often takes less than an equianalgesic dose of the second opioid (Table 102.4). This phenomenon is observed frequently, but the mechanisms are not completely understood. If there is a need to switch opioid medications, a good rule of thumb is to give approximately two-thirds of an equianalgesic dose and provide liberal amounts of breakthrough medication until a new
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Table 102.4 Dose equivalents and starting doses for opioid analgesics (Adapted and modified from [52].) Usual starting dose Approximate equianalgesic dose (mg)
>50 kg
>50 kg
GH –0.8
–1 GHD to FAH
Fig. 104.5 Final height standard deviation (SD) score for 42 children with growth hormone (GH) deficiency (GHD) treated with GH when less than 10 years of age (black box) or more than 10 years of age (lighter shaded box), and final height SD score for 48 children with GH deficiency not treated with growth hormone when less than 10 years of age (open box) or more than 10 years of age (darker shaded box). FAH, final adult height.
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especially children less than 6 years of age at the time of irradiation to the head and neck, have the greatest risk for development of subsequent craniofacial and dental abnormalities [90–93]. Enamel and dentin formation are disturbed by TBI due to destruction of cells during the mitotic phase. Chemotherapy drugs are selectively toxic to actively proliferating cells by disruption of DNA synthesis and replication, RNA transcription, and cytoplasmic transport mechanisms [94]. The effects of chemotherapy and irradiation on dental development include tooth agenesis, complete or partial arrest of root development with thin, tapered roots, early apical closure, globular and conical crowns, dentin and enamel opacities and defects, microdontia, enlarged pulp chambers, taurodontism (prismshaped molars), and abnormal occlusion [92,95,96]. Hence, the development of secondary teeth is often affected, with delayed or arrested tooth formation, shortening and blunting of tooth roots, incomplete calcification, premature closure of apices, and dental caries. However, some children with hematologic malignancies who have received preparative regimens with chemotherapy alone do not have abnormalities in dental maturity or eruption of their permanent dentition [97]. Reduction in lower face height in HCT patients correlated with impaired dental development [93]. Vertical condyle growth and the alveolar and molar heights were adversely affected by pretransplant preparative regimens. Cephalometric measurements of facial bones to evaluate facial growth before and after 10 Gy TBI and HCT have resulted in a significant reduction in the maxilla length and mandible growth compared with healthy age-matched nontransplant children. These differences were most pronounced in mandibular growth [93]. Compared with the control group, the children and adolescents in the HCT group also had significantly reduced mouth opening capacity with reduced translation movement of the condyles diagnosed in 53% of children treated with TBI, compared with 5% in the control group [91]. Signs of craniomandibular dysfunction were found in 84% of children in the HCT children, compared with 58% in the control group. The long-term alterations in connective and muscle tissues result in changes in tissue inflammation and eventually fibrosis. An evaluation of craniofacial development in 16 prepubertal children (age range 1.7–11.0 years) with growth failure and GH deficiency following CY plus 10.0 Gy TBI and HCT demonstrated a significant positive effect on growth among the nine GH-treated patients compared with seven non-GH-treated patients [90]. Another study demonstrated improvement in vertical growth of the condyles, suggesting that condylar cartilage is the most likely site of mandibular growth activity [98]. These observations support the hypothesis that GH most likely encourages longitudinal bone growth both directly, by stimulating differentiation of epiphyseal growth plate precursor cells, and indirectly, by increasing the responsiveness to IGF-I [99]. Treatment with GH, however, did not improve the disturbed root development of the teeth. Thus, there does not appear to be a stimulating effect of dental development on growth of the alveolar process. Few data are available for the use of orthodontic treatment for children who have dental growth disturbances after TBI and HCT. A retrospective study of 10 children has demonstrated that orthodontic treatment plans were modified to reflect the patient’s medical condition, but in general the orthodontic treatment did not produce any harmful sideeffects, even though most treated children exhibited severe pre-existing disturbances in dental development [100]. Nine of the 10 patients had severe disturbances in dental development with short V-shaped roots, premature apical closure, enamel disturbances, microdontia, and aplasia. The most severe disturbances were found in children less than 5 years of age at the time of 10 Gy TBI. The strategies used to cope with the severe problems of dental growth disturbances included using appliances that minimized the risk of root resorption, using weaker forcers, terminating treatment earlier than normal, and choosing the simplest
method for treatment needs. In general, the lower jaw was not treated. The treatment was judged as unsatisfactory in four of the 10 patients.
Puberty Puberty, a transitional stage from a sexually immature to a mature sexual state, is accompanied by significant changes in gonadal and growth hormonal activity, development of secondary sexual characteristics, and increased growth velocity. There is considerable variation in the timing and sequence of pubertal events that makes it difficult to assess a child’s pubertal development based only on chronologic age. In the normal individual, pubertal development is usually closely related with osseous maturation as measured by bone age. An intact hypothalamic–pituitary–gonadal axis is required for initiation and completion of puberty. The normal pubertal growth rate is 1.5–2 times greater than prepubertal growth rates [101]. In the absence of pubertal sex hormone secretion, the increased growth velocity associated with the pubertal growth spurt is substantially blunted, and the development of secondary sexual characteristics is delayed or absent. Gonadal hormone production and germ cell viability are affected by high doses of alkylating agents and irradiation, with variables related to patient age, sex, and type and dose of therapy [102]. Azoospermia develops in prepubertal boys who have received a cumulative dose of more than 350 mg/kg CY, whereas doses of 200 mg/kg or less result in minimal alteration of spermatogenesis. The total dose of BU impacting future developmental potential for the prepubertal testes is not known. Irradiation to the prepubertal testes results in damage to the germinal epithelium that does not become apparent until after puberty [103]. Boys who have received more than 24 Gy testicular irradiation have delayed or arrested development of secondary sexual characteristics, with elevated gonadotropin and low testosterone values. Primary ovarian failure usually occurs following total cumulative doses of more than 500 mg/kg CY to prepubertal girls [102]. No data are available regarding BU or irradiation on the prepubertal ovary. Sex hormones indirectly stimulate linear growth by increasing endogenous GH secretion [104]. This stimulation leads to increasing circulating and tissue levels of IGF-I, which activates growth at the level of bone and cartilage. Treatment with low-to-moderate doses of sex hormones promotes increased linear growth, but physiologic adult doses of sex hormones result in a greater influence on skeletal maturation, with resultant compromise in final adult height via the mechanism of premature epiphyseal closure. Children with idiopathic GH deficiency usually have a late, but normal, pubertal growth spurt. Children with hypogonadotropic hypogonadism have an absence of sex hormone production, delayed puberty, delayed pubertal growth spurt, and a decrease in final adult height [67]. Thus, interruption in either production of pubertal GH or sex hormone production is likely to result in both delayed pubescence and decreased linear height. Preparative regimens containing only chemotherapy Following 200 mg/kg CY and BMT for aplastic anemia, 31 of 32 girls and 28 of 31 boys who were prepubertal at time of administration of CY have now been followed long enough to be more than 12 years of age and evaluable for pubertal development [105]. Nearly all of these children demonstrated normal age-appropriate progression through puberty (Tables 104.2 and 104.3). Three girls with delayed pubertal development had Fanconi’s syndrome and eventually developed normal secondary sexual characteristics and normal luteinizing hormone (LH), folliclestimulating hormone (FSH), and estradiol levels, and two girls showed delay due to chronic GVHD therapy. Among the 26 girls with normal age-appropriate development, menarche occurred at a median of 12.5
Growth and Development after Hematopoietic Cell Transplantation Table 104.2 Pubertal development in girls
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Preparative regimens
Current age Development
≥12 years Normal Delayed
CY*
BUCY†
10.0 Gy TBI‡
12–15.75 Gy TBI§
31 26 (84%) 5 (16%)
61 17 (28%) 44 (72%)
24 7 (29%) 17 (71%)
114 48 (42%) 66 (57%)
BU, busulfan; CY, cyclophosphamide; TLI, total lymphoid irradiation. References: * [44,108], † [50,112,113], Sanders, unpublished data, 2007; ‡ [44,63,112,113]; § [9,50,63,112,113], Sanders, unpublished data, 2007.
Table 104.3 Pubertal development in boys
Preparative regimens
Current age Development
≥12 years Normal Delayed
CY*
BUCY†
10.0 Gy TBI1‡
12–15.75 Gy TBI1§
28 25 (89%) 4 (14%)
54 28 (52%) 26 (48%)
32 6 (19%) 26 (81%)
77 32 (42%) 45 (58%)
1
No additional testicular irradiation. References: * [44,108], † [50,112,113], Sanders, unpublished data, 2007; ‡ [44,63,112,113]; § [9,50,63,112,113], Sanders, unpublished data, 2007.
(range 11–16) years of age, and the five with delayed development had menarche occur between 16 and 19 years of age. Twenty-eight of these formerly prepubertal girls have given birth to 49 normal children [106, Sanders, unpublished data, 2007]. The three boys with delayed pubertal development had chronic GVHD and did ultimately develop normal secondary sexual characteristics with normal LH, FSH, and testosterone values. Twenty-eight of these formerly prepubertal boys have fathered 49 normal children. Gonadal function after 14 mg/kg BU plus 200 mg/kg Cy and allogeneic BMT for thalassemia has been reported for 64 prepubertal patients (31 girls and 33 boys) who ranged in age from 9.3 to 17.2 years [107– 110]. Twenty-five girls had evidence of primary ovarian failure, with elevated gonadotropin levels, and six girls had hypogonadotropic hypogonadism. All girls had low estradiol levels both before and after transplantation and required hormone supplementation for development of secondary sexual characteristics. Among the 33 boys, post-transplant LH and FSH concentrations were within normal limits for 23, but abnormal with either elevated LH and FSH or low LH and FSH in 10 boys. However, after gonadotropin-releasing hormone stimulation, three had normal responses, two had elevated FSH responses, and 10 had low responses. These gonadal function results must be interpreted with caution, however, because patients with thalassemia treated with chelation and transfusion therapy frequently show delayed or absent puberty. Pubertal development has been evaluated among children transplanted for malignancy who received 16 mg/kg BU plus 120–200 mg/kg CY [107,111,112, Sanders, unpublished data, 2007]. One hundred and fifteen children have now been followed long enough to be evaluable for the progress of spontaneous pubertal development (Tables 104.2 and 104.3). Seventeen of 61 (28%) evaluable girls and 28 of 54 (52%) evaluable boys have developed normally through puberty. Whereas the girls have normal LH, FSH, and sex hormone production, many of the boys do not, with some having elevated LH and FSH values, indicating gonadal dysfunction. These data demonstrate that BU is highly toxic to
prepubertal, particularly female, gonads. These children must be carefully followed, with gonadal function evaluation beginning about 12 years of age. Supplementation with appropriate gonadal hormones administered in gradually increasing doses under supervision of a pediatric endocrinologist is needed for these children to develop secondary sexual characteristics. Preparative regimens containing irradiation The development of secondary sexual characteristics among children who were prepubertal at the time of TBI administration but more than 12 years of age at follow-up was evaluated in 24 girls and 31 boys after 10 Gy single-fraction TBI, and 77 girls and 77 boys after 12–15.75 Gy fractionated-exposure TBI (Tables 104.2 and 104.3). Following 10.0 Gy single-exposure TBI, 71% of girls and 83% of boys had delayed development of secondary sexual characteristics, elevated LH and FSH levels, and low sex hormone levels. After fractionated-exposure TBI, 49% of girls and 58% of boys had delayed development. The majority of these girls and boys have received appropriate sex hormone therapy for promotion of secondary sexual characteristic development. As larger numbers of prepubertal children are becoming evaluable for pubertal development, the amount of testicular irradiation received and patient age at the time of TBI are emerging as important factors in subsequent normal spontaneous pubertal development (Table 104.4). Thirty of the 36 (84%) boys who had received more than 10 Gy testicular irradiation for testicular leukemia in addition to 12–15.75 Gy TBI have primary gonadal failure and require testosterone therapy to promote the development of secondary sexual characteristics. However, about half of the boys who receive 400 cGy prophylactic testicular irradiation in addition to fractionated TBI develop normally through puberty [Sanders, unpublished data, 2007]. Among children receiving 14.40 Gy fractionated TBI, one study reported that, among 17 boys evaluated, those with increased levels of LH were significantly younger at BMT (5.4 ± 0.8 versus 7.8 ± 0.8 years;
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p = 0.024), but among the 16 girls evaluated, those with ovarian failure were significantly older at the time of TBI than those with spontaneous puberty (8.6 ± 2.3 versus 6.1 ± 1.8 years; p = 0.03) [29]. Another study did not observe an effect of age on subsequent gonadal function [54]. A third analysis suggested that an effect of age may be linked to the total dose of TBI [107]. Among 30 evaluable children given 12.0 Gy TBI, 15 of 16 (94%) less than 10 years of age and 11 of 14 (78%) more than 10 years of age developed normally. However, among the 46 evaluable children given 14.40–15.75 Gy TBI, 23 of 30 (77%) less than 10 years of age and three of 16 (19%) more than 10 years of age developed normally. Larger numbers of patients are needed to draw firm conclusions regarding the impact of patient age and total dose of TBI administered on development of puberty. It is recommended that the development of secondary sexual characteristics be monitored carefully after patients reach 10–11 years of age and that Tanner Developmental Scores be determined annually. Because the production of sex hormones is necessary for promotion of the pubertal growth spurt, in addition to promoting sexual maturation, children with evidence of gonadal failure and delayed development of secondary sexual characteristics may benefit from supplemental hormones. This supplementation should be administered under the guidance of a pediatric endocrinologist, and doses of sex hormone treatment should begin low with gradual increase to simulate natural hormone production, to prevent premature advancement of bone age, and to promote pubertal growth spurts. Patients with normal pubertal development, gonadotropins, and sex hormone production should receive appropriate sexual behavior counseling as pregnancy may occur.
Conclusion Evaluations of endocrine function following HCT demonstrate that the occurrence of abnormalities that may influence subsequent growth and development are related to the type of HCT preparative regimen received. Table 104.4 Pubertal development in boys given testicular irradiation
Children who receive CY only usually do not have endocrine function abnormalities. These children have normal thyroid function, normal growth rates, and normal development through puberty. Individuals who develop normally through puberty or in whom gonadal function returns to normal may be fertile. Offspring of these patients, in general, do not differ from the general population. Children who receive BU plus CY preparative regimens have normal thyroid function, and the majority have normal prepubertal growth rates. Pubertal growth may be affected. Data from the patients evaluable for pubertal development suggest that a substantial portion of these children may not develop secondary sexual characteristics at a normal age, which is particularly true for girls but not boys. Gonadal function, however, is likely to be abnormal in both boys and girls. In contrast, multiple endocrine function abnormalities that affect normal growth and development frequently occur after HCT regimens that contain TBI. Patients are at risk for development of thyroid function abnormalities for many years after TBI. These patients are also at risk for development of thyroid malignancy. Growth rates are usually blunted, especially if the child has received prior CNS irradiation. GH deficiency, which frequently occurs, and growth failure should be diagnosed and treated early with synthetic GH for best response. Without GH treatment, young children have significantly compromised final adult heights. Gonadal function damage results in a significant fraction of prepubertal patients having delayed pubertal development. Children with delayed development of secondary sexual characteristics may benefit from careful administration of appropriate sex hormone therapy. Children who do develop normally through puberty are potentially fertile. All patients who receive HCT should continue to undergo longterm follow-up evaluations; a suggested outline of studies is shown in Table 104.5. Table 104.6 outlines suggested treatment, but details of the management of hormone therapy are best performed by a pediatric endocrinologist. Table 104.6 Suggested treatment for post-transplant endocrine function disorders
Testicular irradiation
Current age Development
≥12 years Normal Delayed
4.0 Gy
≥10 Gy
51 28 (55%) 23 (45%)
36 6 (16%) 30 (84%)
Abnormality
Treatment*
Hypothyroid Growth failure with growth hormone deficiency Delayed puberty (girls)
Thyroxine Growth hormone therapy Ethinyl/estradiol and medroxyprogesterone testosterone enanthate
Sanders, unpublished data, 2007. * Details of doses and dose adjustments are best managed by a pediatric endocrinologist.
Table 104.5 Post-transplant evaluations of endocrine effects
Thyroid gland Growth
Puberty
Tests
Frequency
Thyroid stimulating hormone, triiodothyronine, thyroxine Height *Bone age *†Growth hormone measurement Tanner Development Score Luteinizing hormone, follicle stimulating hormone Estradiol (girls) Testosterone (boys)
Annual Annual Annual until epiphysis closure Annual until deficiency presents or epiphysis closure Annual age 8–18 years Annual after age 10 years Annual after age 10 years Annual after age 10 years
* Not needed for those receiving a cyclophosphamide-only preparative regimen. † Not needed if the patient is already receiving growth hormone therapy.
Growth and Development after Hematopoietic Cell Transplantation
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105
Mary E.D. Flowers & H. Joachim Deeg
Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation
Introduction Hematopoietic cell transplantation (HCT) provides curative therapy for a variety of diseases. The development of less toxic pretransplant conditioning regimens, more effective prophylaxis of acute graft-versushost-disease (GVHD), improved infection control, and other advances in transplant technology have resulted in a rapidly growing number of transplant recipients surviving long term free of the disease for which they were transplanted. Current data show, indeed, that patients transplanted for aplastic anemia and patients without chronic GVHD have life expectancies similar to age-matched controls. Patients with advanced malignant diseases, however, and those who develop chronic GVHD after allogeneic HCT may experience late disease recurrence or incur delayed complications that may prove fatal [1]. With the increase in numbers of transplants over the past three decades and the considerable improvement in early post-transplant survival (Fig. 105.1), an increasing population of survivors is at risk of developing late complications (Fig. 105.2) [2,3]. This chapter will focus on nonmalignant delayed complications, while secondary malignancies, also a late complication after HCT, will be discussed in Chapter 106.
Etiology and spectrum of delayed complications Several factors impact on recovery from and late effects of HCT, including prior therapy for the underlying disease, pretransplant comorbidities and psychosocial status, intensity of the transplant conditioning regimen, and development of chronic GVHD. Major nonmalignant delayed complications are listed in Table 105.1. Late complications develop also in patients given high-dose therapy who are not transplanted; however, those patients do not experience the typical transplant-related complications such as GVHD, which is affected by donor type, stem cell source, patient age, and intensity of the conditioning regimen. Chronic GVHD with the associated immunodeficiency, and the effects of glucocorticoids and other immunosuppressive treatments used to control GVHD, represent the most frequent late complications after allogeneic HCT. Infertility is dependent upon the type of the transplant conditioning regimen. Other complications are related to the prolonged use of glucocorticoids and other immunosuppressive drugs, and many (e.g. chronic pulmonary disease, endocrine complications, and secondary malignancies) are mul-
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5
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tifactorial in etiology. Some delayed complications are due directly to therapy-induced trauma and scar formation (e.g. destruction of skeletal growth plates and bladder dysfunction); others are related to immunologic graft–host interactions (e.g. lymphoproliferative disorders). Sideeffects of the treatment of acute complications early after HCT (e.g. the use of steroids) may also contribute to long-term complications. Nearly all organ systems can be affected (Table 105.2). To what extent delayed nonmalignant complications differ in patients conditioned with reduced-intensity or nonmyeloablative transplant regimens remains to be seen, as follow-up is still rather short compared with high-dose transplant regimens. Nonetheless, rates of late fungal and viral infections and chronic GVHD appear to be similar after reducedintensity and high-dose conditioning regimens [4,5].
Chronic GVHD The major cause of delayed morbidity and mortality after allogeneic HCT is chronic GVHD, related to its associated immune deficiency, and the toxicities of glucocorticoids and other immunosuppressive treatment given for control of GVHD (see Chapter 87). The incidence of chronic GVHD is influenced by the source of stem cells, donor–recipient gender mismatch, human leukocyte antigen compatibility, and age. The risk of nonrelapse mortality is higher for patients with chronic GVHD with direct progression from acute GVHD, for patients with platelet counts below 100,000/μL, hyperbilirubinemia, extensive skin disease or multiple organ involvement, and low clinical performance score [6,7]. If not treated adequately and in severe cases, chronic GVHD can result in major disability related to keratoconjunctivitis sicca (KCS), pulmonary insufficiency due to bronchiolitis obliterans (BO), or restrictive lung disease related to scleroderma or fasciitis, as well as joint contractures, skin ulcers, esophageal and vaginal stenosis, and others [8,9]. Metabolic, muscular/skeletal and chronic endocrine problems are complications of chronic GVHD or are due to toxicities of treatment given to control GVHD (Table 105.3). Most patients require glucocorticoids or other agents for at least 2 years from the initial diagnosis of chronic GVHD [6]. Approximately 10% of patients require continued immunosuppressive treatment beyond 5 years from the initial diagnosis of chronic GVHD, and 40% die or experience a recurrence of their malignancy before chronic GVHD resolves [6]. The remaining 50% of patients discontinue immunosuppressive treatment within 5 years of the initial diagnosis of chronic GVHD [6]. Therefore, it is not surprising that corticosteroid and other immunosuppressive therapies are major contributors of late complications after allogeneic HCT. Ancillary and supportive care directed at organ-specific control of symptoms or
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Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation
Table 105.2 Organs affected by late complications according to etiology
Organ Skin Eyes Sinuses Gastrointestinal tract Liver Lungs Muscles Connective tissues Bones Endocrine glands Gonads Kidneys
Regimen-related toxicity • • • • • • • • • • •
Chronic graftversus-hostdisease • • • • • • • •
Infections • • • • • •
• •
Fig. 105.1 Allogeneic hematopoietic cell transplantation at the Fred Hutchinson Cancer Research Center. Upper curve: cumulative number of allogeneic transplants. Lower curve: cumulative number of patients alive at the end of the indicated calendar year. Table 105.3 Metabolic, skeletal and endocrine complications related to chronic graft-versus-host disease or its treatment Metabolic problems Hyperlipidemia Hyperglycemia Hypertension Obesity Skeletal/muscular problems Osteoporosis/osteopenia Avascular bone necrosis Myopathy Endocrine problems Pancreatic insufficiency Adrenal insufficiency Autoimmune thyroid disease Delayed puberty and growth hormone deficiency Fig. 105.2 Proportion of patients given an allogeneic hematopoietic cell transplantation at the Fred Hutchinson Cancer Research Center who survived at least 100 days post transplant. Table 105.1 Nonmalignant delayed complications after hematopoietic cell transplantation Chronic graft-versus-host disease Autoimmune disorders/hematologic complications Late infections Airway and pulmonary disease Neuroendocrine dysfunction Impairment of growth and development Infertility Cardiovascular disease Ocular problems Musculoskeletal problems Dental problems Dysfunction of the genitourinary tract Gastrointestinal and hepatic complications Metabolic problems Central and peripheral nervous system impairment Psychosocial effects
complications resulting from GVHD and its therapy are central to the management of chronic GVHD. However, more than half of the recommendations in the current ancillary and supportive care guidelines for patients with chronic GVHD are based only on expert consensus opinion (level III) rather than controlled studies, which highlights the need for more clinical research [10]. Long-term clinical monitoring is necessary to identify early signs of GVHD, assess activity versus sequelae caused by GVHD, determine treatment-related toxicity, and, most importantly, prevent late complications associated with severe morbidity. We find it useful to apply the 0–3 scoring system (Table 105.4) proposed by the National Cancer Institute Consensus Group to assess chronic GVHD manifestations in each organ [9]. We use this assessment tool at the time of initial diagnosis, at any time when changes occur in the therapy to control GVHD (e.g. due to an increase in the dose of corticosteroid of 1 mg/kg or more every other day, substitution of one therapy for another or additional therapy), and at 6–12-month intervals if manifestations of GVHD persist or immunosuppressive treatment continues.
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Table 105.4 Assessment scores for monitoring patients with or at risk of chronic graft-versus-host disease (GVHD) Score 0
Score 1
Score 2
Score 3
ⵦ
Asymptomatic and fully active (ECOG 0; KPS or LPS 100%)
Symptomatic, fully ambulatory, restricted only in physically strenuous activity (ECOG 1, KPS or LPS 80–90%)
Symptomatic, ambulatory, capable of self-care, >50% of waking hours out of bed (ECOG 2, KPS or LPS 60–70%)
Symptomatic, limited self-care, >50% of waking hours in bed (ECOG 3–4, KPS or LPS 50% BSA OR deep sclerotic features “hidebound” (unable to pinch) OR impaired mobility, ulceration or severe pruritus
Mouth Diagnostic/distinctive features Present Absent
No symptoms
Mild symptoms with disease signs but not limiting oral intake significantly
Moderate symptoms with disease signs with partial limitation of oral intake
Severe symptoms with disease signs on examination with major limitation of oral intake
Performance Score:
KPS ECOG LPS
signs but NO sclerotic features
ⵦ
Abnormality present but NOT thought to represent GVHD Eyes Mean tear test (mm): >10 6–10 ≤5 Not done
No symptoms
Mild dry eye symptoms not affecting ADL (requiring eyedrops ≤3 × per day) OR asymptomatic signs of keratoconjunctivitis sicca
Moderate dry eye symptoms partially affecting ADL (requiring drops >3 × per day or punctal plugs), WITHOUT vision impairment
Severe dry eye symptoms significantly affecting ADL (special eyeware to relieve pain) OR unable to work because of ocular symptoms OR loss of vision caused by keratoconjunctivitis sicca
Gastrointestinal tract
No symptoms
Symptoms such as nausea, vomiting, anorexia, dysphagia, abdominal pain or diarrhea without significant weight loss (15%, requires nutritional supplement for most calorie needs OR esophageal dilation
Liver
Normal LFTs
Elevated bilirubin, AP, AST or ALT 3 mg/dL or bilirubin and enzymes 2–5 × ULN
Bilirubin or enzymes >5 × ULN
Lungs* PFTs not done
No symptoms
Mild symptoms (shortness of breath after climbing one flight of steps) FEV1 60–79% OR LFS 3–5
Moderate symptoms (shortness of breath after walking on flat ground)
Severe symptoms (shortness of breath at rest; requiring oxygen)
FEV1 40–59% OR LFS 6–9
FEV1 ≤39% OR LFS 10–12
FEV1
ⵦ ⵦ
DLCO
FEV1 > 80% OR LFS = 2
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Table 105.4 (Continued) Score 0
Score 1
Score 2
Score 3
Joints and fascia
No symptoms
Mild tightness of arms or legs, normal or mild decreased ROM AND not affecting ADL
Tightness of arms or legs OR joint contractures, erythema thought due to fasciitis, moderate decrease in ROM AND mild-to-moderate limitation of ADL
Contractures WITH significant decrease of ROM AND significant limitation of ADL (unable to tie shoes, button shirts, dress self, etc.)
Genital tract Diagnostic/distinctive features Present Absent Not examined
No symptoms
Symptomatic with mild signs on exam AND no effect on coitus and minimal discomfort with gynecologic exam
Symptomatic with moderate signs on exam AND with mild dyspareunia or discomfort with gynecologic exam
Symptomatic WITH advanced signs (stricture, labial agglutination or severe ulceration) AND severe pain with coitus or inability to insert vaginal speculum
Other indicators, clinical manifestations or complications related to chronic GVHD (check all that apply): ⵧ Weight loss ⵧ Bronchiolitis obliterans ⵧ Bronchiolitis obliterans with organizing pneumonia ⵧ Esophageal stricture or web ⵧ Pericardial effusion ⵧ Pleural effusion(s) ⵧ Ascites (serositis) ⵧ Nephrotic syndrome ⵧ Peripheral neuropathy ⵧ Myasthenia gravis ⵧ Polymyositis ⵧ Malabsorption ⵧ Cardiac conduction defects ⵧ Coronary artery involvement ⵧ Cardiomyopathy ⵧ Eosinophilia >500/μL ⵧ Other: ______________________________________________ ⵧ None Biopsy obtained: ⵧ Yes ⵧ No Organ system(s) biopsied: _____________________GVHD confirmed by histology: ⵧ Yes ⵧ No OVERALL severity of GVHD: ⵧ None ⵧ Mild ⵧ Moderate ⵧ Severe Change from previous evaluation: ⵧ No prior or current GVHD ⵧ Improved ⵧ Stable ⵧ Worse ⵧ N/A (baseline) ADL, activities of daily living; ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; BSA, body surface area; DLCO, diffusing capacity of the lung for carbon monoxide, corrected for hemoglobin; ECOG, Eastern Cooperative Oncology Group; FEV1, forced expiratory volume of the lung in 1 second; KPS, Karnofsky Performance Status; LFS, Lung Function Score; LFT, liver function test; LPS, Lansky Performance Status; PFT, pulmonary function test; ROM, range of motion; ULN, upper limit of normal. * Pulmonary scoring should be performed using both the symptom and PFT scale whenever possible. If discrepancy exists between pulmonary symptom or PFT scores, the higher value should be used for final scoring. Scoring using the LFS is preferred, but if DLCO is not available, grading using FEV1 should be used. The LFS is a global assessment of lung function after the diagnosis of bronchiolitis obliterans has already been made. The percent predicted FEV1 and DLCO (adjusted for hematocrit but not alveolar volume) should be converted to a numeric score as follows: >80% = 1; 70–79% = 2; 60–69% = 3; 50–59% = 4; 40–49% = 5; 3 years) of standard dose estrogen and progesterone in postmenopausal women. Late thrombotic events after HCT have been reported in 1–4% of patients, but these numbers may be underestimates (Flowers, unpublished data). Anticoagulation should be considered in patients at high risk (e.g. prior history of thrombosis or hypercoagulability status).
Ocular problems Late ocular complications after HCT can involve the anterior (i.e. cataracts and KCS) or the posterior (i.e. microvascular retinopathy, optic disc edema, infectious retinopathy, and hemorrhage) segment of the eyes. The most common problems affecting the eyes after HCT are cataracts and KCS, the latter often associated with chronic GVHD. Cataracts Glucocorticoids and gamma irradiation are the primary causes of cataracts after HCT. After TBI, posterior capsular cataracts are noticed approximately 1 year after HCT [70]. Following single-dose TBI (usually 920–1000 cGy), the incidence of cataracts was 60–80% at 5–6 years. After fractionated TBI, the incidence was approximately 50% at cumulative doses above 1200 cGy, 30–35% at doses of 1200 cGy, and as low as 10% in patients given hyperfractionated TBI (more than six fractions) or treated at low exposure rates (0.04 cGy/min) [34]. Eye shielding might prevent cataracts, but relapse of malignancy in the ocular bulb can occur. Among patients who have not received TBI or cranial irradiation, the incidence of cataracts is less than 20% and is
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almost exclusively due to corticosteroids [70]. The incidence of cataracts overall is higher in patients who also received cranial irradiation. Approaches to cataract prevention in the setting of HCT are experimental. One report suggests that heparin given, for example, in patients with veno-occlusive disease has a protective effect on the lens [34]. The treatment of choice of cataracts is lens extraction and implantation of an artificial lens. Although surgery should be done preferably after discontinuation of all systemic immunosuppressive treatment, procedures have been performed successfully in patients receiving low doses of prednisone (less than 0.25 mg/kg). The improved quality of modern lenses has allowed successful (long-surviving) implants even in children. Ocular sicca Chronic dry eyes are frequent after HCT and can result in acute conjunctivae inflammation, pseudomembranous and cicatricial conjunctivitis, and KCS. KCS occurs in approximately 40–60% of patients with chronic GVHD [71] and is often irreversible (see Chapter 87) [72]. Chronic KCS can result in scar formation (e.g. in the tarsus) and lead to synechiae, ectropion, corneal damage, and potentially perforation if not treated meticulously and aggressively. KCS also occurs in patients without chronic GVHD, although the possibility that it represents a sequel of prior GVHD or a forme fruste of GVHD must be considered [2,72]. Long-term management includes supportive care with artificial tears, long-acting ocular lubricants, punctal occlusion or cauterization, ophthalmic cyclosporine, topical glucocorticoids, autologous serum eye drops, and oral administration of cholinergic agents [10]. Occlusive eyeglasses can significantly improve the symptoms of dry eyes. For patients with severe KCS refractory to supportive care, the use of the liquid corneal bandage provided by a fluid-ventilated, gas-permeable scleral lens (Boston Scleral Lens) has been effective in mitigating symptoms and resurfacing corneal erosions [73]. Transplantation of limbal epithelial cells reported for other conditions [74] has been performed in some patients with GVHD-related KCS (using the original donor cells) with mixed results (Bensinger and Flowers, unpublished data). Ocular complications of the posterior segment Late retinitis is infrequent after HCT and usually occurs in patients with delayed immune reconstitution such as those with chronic GVHD, conditioned with Campath-1G, or recipients of T-cell-depleted or cord blood transplants. Late retinitis is often related to infections with CMV, herpes zoster or Toxoplasma gondii. Ischemic retinopathy with cottonwool spots and optic disc edema has been reported in 10% of patients after HCT [34]; atypical retinal microvasculopathy, without cotton-wool spots, has also been described [75]. The TBI regimens alone cannot explain the ischemic retinopathy reported after HCT, and additional factors such as cyclosporine may contribute to lower the threshold for radiation retinopathy [76]. Ischemic retinopathy has been reported also in patients conditioned without TBI. In most cases of ischemic retinopathy, retinal lesions resolve following withdrawal or reduction of immunosuppressive therapy, as described in a case with complete blindness [77]. The development of ischemic retinal lesions is a multifactorial process leading to capillary damage of the eye fundus.
Musculofascial problems Myopathy is common after HCT. One of the most common causes of myopathy is treatment with corticosteroids. The high rates of fatigue and lack of energy observed in transplant survivors, which lead to inactivity, are important contributors to muscle weakness and loss of muscle and
bone mass. It is important, therefore, to maintain and gradually increase the level of physical activity to counter a progressive decline in physical function. Occasionally patients with chronic GVHD have involvement of muscle, fascia, and serous membranes including the synovia [8]; joint effusions may occur in patients without any other sign of GVHD. Diagnostic procedures and management are discussed under GVHD (see Chapter 87). Involvement of fascia or tendons by an eosinophilic infiltrate (early) or fibrosis (late), frequently preceded by edema and often resulting in joint contractures of the wrists (most common), fingers, shoulders, elbows, ankles, and occasionally knees, may be manifestations of chronic GVHD. Because of the significant morbidity associated with joint contractures, range of motion assessment, including the extension of hands and wrists (i.e. palms together with the elbows extended, referred to by the author as “Praying Buddha position”), is necessary when examining allogeneic transplant recipients to rule out fibrotic fasciitis, panniculitis or tendonitis. Patients with limitations of wrist extension often use the back of the hands (rather than the palms) when, for example, standing up from the sitting position. Some patients may be misdiagnosed as having carpal tunnel syndrome. Stretching exercises are important to improve the range of motion of affected joints and restore functions of daily living. Physicotherapy and deep myofascia massage (Hellerwork) are critically important in the management of joint contractures [10]. Muscle cramps are common in long-term HCT recipients. Approximately 30% of allogeneic and 40% of autologous HCT recipients reported muscle cramps on the 5–15 years annual health questionnaires sent to all patients transplanted in Seattle. The etiology of muscle cramps remains unclear. Muscle cramps of the extremities and severe carpopedal spasms, with impairment of fine motor function, are often, but not always, observed in patients with a history of chronic GVHD. Generally, conventional muscle relaxants and analgesics are ineffective. Adequate hydration, correction of electrolyte imbalances (i.e. hypomagnesaemia), and regular stretching represent baseline interventions. Clonazepam or baclofen has been used with some success in a few patients. While treatment with quinine sulfate has been used in the past, the Food and Drug Administration has issued a cautionary statement (P06–195) regarding the off-label use of this drug to treat leg cramps because of serious adverse effects.
Skeletal complications Osteoporosis and osteopenia Dual-energy X-ray absorptiometry, a semiquantitative method to assess bone mineral density (BMD), is a validated test used to detect osteoporosis (t-score of equal to or less than −2.5 standard deviations below age-related mean BMD) and osteopenia (t-score of −1.0 to −2.4 standard deviations below age-related mean BMD). Other markers of bone loss include elevated alkaline phosphatase level, particularly in women, as well as high C-terminal propeptide. Increased urinary excretion of hydroxyproline can also be used to assess bone loss and response to treatment [78]. Bone loss is a frequent complication after HCT and results from impaired bone mineralization through disturbances of calcium and vitamin D homeostasis, osteoblast and osteoclast dysfunction, and deficiencies in growth or gonadal hormone secretion. Factors associated with bone loss after HCT include irradiation, corticosteroid treatment, inactivity, and iatrogenic hypogonadism [34,61]. Both the cumulative dose and duration of corticosteroid treatment and the duration of calcineurin inhibitor treatment have shown significant associations with loss of BMD. In a large study, approximately 40% of men and women were
Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation
found to have reduced BMD by 1 year after HCT [79]. In another study, osteopenia was found in 33% and osteoporosis in 18% of women after HCT [80]. Increased risk of bone loss without fracture risk was reported in a cohort of 79 patients after HCT with a median follow-up of 6.5 years [81]. Few studies on the safety and efficacy of bisphosphonate for prevention of bone loss after HCT have been reported. Results of a randomized study in adult allogeneic HCT recipients showed less bone loss in patients receiving additional pamidronate (60 mg before and 1, 2, 3, 6, and 9 months after HCT) compared with patients receiving 1000 mg calcium carbonate and 800 IU vitamin D daily, and estrogen (women) or testosterone (men) alone [82]. In a retrospective study of pediatric HCT recipients, treatment with bisphosphonate was well tolerated and was associated with improvement in BMD [83]. Measures to prevent bone loss after HCT are indicated. Many experts recommend the use of antiresorptive treatments (gonadal hormonal replacement or bisphosphonates) in patients with gonadal failure and with chronic GVHD requiring treatment with glucocorticoid [10]. Physicians and patients should be aware of osteonecrosis of the jaw, a rare but well-recognized potentially serious toxicity of bisphosphonate, which is reported more frequently with intravenous formulations [84]. As part of long-term follow-up, a detailed nutritional assessment of calcium and vitamin D needs to be recorded for each patient prior to HCT, between 80 and 100 days after HCT, and at least yearly thereafter if at risk. Measures to prevent osteoporosis (Table 105.5) are recommended to all women and all patients receiving long-term corticosteroid treatment. Patients should pursue an exercise program that combines aerobic weight bearing and resistive exercises, and should follow fall prevention strategies (conditioning, removing trip hazards from the home, and eye examination and correction of vision). Also, patients should stop smoking and limit alcohol consumption. In women, supplementation with estrogens with or without medroxyprogesterone can increase bone mass after HCT. Thus, if not contraindicated, gonadal hormonal replacement is a reasonable consideration after HCT, particularly in patients receiving corticosteroids and in premenopausal women [10,34].
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MRI and structurally abnormal radiographs), which occur prior to collapse of the femoral head, from more advanced lesions that are identified by abnormal MRI and radiography showing loss of the femoral head contours, and often loss of joint space [87]. In summary, the major risk factors of AVN are glucocorticoid use followed by single-dose (10 Gy) or fractionated higher-dose TBI. An attempt to minimize the dose and duration of glucocorticoids is advisable. For patients with early-stage AVN, core decompression can result in prompt relief of hip pain [88]. However, the only satisfactory treatment for advanced AVN, particularly in weight-bearing joints, is joint replacement.
Dental problems An oral sicca syndrome related to conditioning therapy or chronic GVHD may lead to poor oral hygiene with recurrent infections and periodontitis. Dental decay occurs because of a lack of cleansing by saliva, which is of altered consistency and reduced volume. Also, the mouth is painful, and patients are hesitant to take care of their teeth and mucosa (see Chapter 103). The incidence of dental decay is not higher in children after HCT than in those given chemotherapy only, suggesting that tooth decay is mostly related to the effects of high-dose cytotoxic therapy rather than transplant-specific factors [89]. The management is similar to that in nontransplanted patients who have received head or neck irradiation, and includes diligent hygiene, fluoride treatment, artificial saliva, capping of teeth, and other supportive measures. In children, irradiation interferes with dental and facial development (see Chapters 103 and 104). There may be poor calcification, micrognathia, mandibular hypoplasia, root blunting, and apical closure [90]. The changes are most severe in children less than 7 years of age at the time of HCT. No effective prophylactic measures are currently available; careful planning (and where possible timing) of the preparative regimen may minimize these side-effects. A detailed discussion of oral complications is provided in Chapters 103 and 104.
Genitourinary dysfunction Avascular necrosis
Bladder
Avascular necrosis (AVN), especially in weight-bearing joints, is a classic side-effect of corticosteroid therapy, reported in approximately 4–10% of allogeneic HCT survivors at a median of 12 (range 2–132) months after HCT [85,86]. The hip is the most frequently affected joint (in up to 88% of individuals), with bilateral involvement in more than 60% of cases. Most patients have more than one joint affected. In addition to corticosteroid therapy, male gender (relative risk 4.2) and age 16 years or over (relative risk 3.8) have been identified as risk factors. The largest multi-institutional study to date, including 4388 patients, reported AVN in 77 patients for a 5-year incidence of 4.3% [85]. Symptoms developed at 2–132 months, and 1–7 (mean 1.9) joints were affected; hip joint replacement was required in nearly 50% of the patients. Time to joint replacement was 2–42 months from initial diagnosis of AVN. Older age, a diagnosis of aplastic anemia or acute leukemia as opposed to other diagnoses, an irradiation-based conditioning regimen, certain types of GVHD prophylaxis, and acute or chronic GVHD were associated with an increased risk of AVN. In a case-control study of 87 patients with AVN, post-transplant glucocorticoid use and a TBI-containing regimen pre HCT were significant risk factors [86]. Cyclosporine, shown by others to stimulate osteoclasts, was not a significant risk factor in that study. Magnetic resonance imaging (MRI) is the gold standard test to diagnose early AVN. Findings in the MRI can be used to differentiate AVN stage I (abnormal MRI with normal radiographs) and stage II (abnormal
Hemorrhagic cystitis, common in the early posttransplant period, is often related to the toxicity of CY conditioning (see Chapter 97). However, patients with protracted hemorrhagic cystitis may later develop bladder wall scarring with volume loss, resulting in severe morbidity with dysuria and increased urinary frequency, recurrent infections, and occasionally hydronephrosis. In severe cases, cystectomy with the surgical reconstruction of a new bladder from a bowel loop is necessary. Supportive care includes management of pain with antispasmodic drugs and topical anesthetic. Prophylaxis and treatment of acute hemorrhagic cystitis is discussed in Chapter 97. Late hemorrhagic cystitis is often associated with infections with adenovirus, polyoma virus, BK or other viruses. Adenovirus strains with a tropism for the genitourinary organs also involve ureters and kidneys and may cause renal failure. Late-onset cystitis associated with polyoma virus has been treated successfully with vidarabine infusion [91]. Women with gonadal failure and those affected by chronic GVHD involving the vulva or vagina are at risk for recurrent urinary tract infections, which require prompt antibiotic therapy. Kidneys Renal complications after HCT are discussed in detail in Chapter 97. The true incidence of late renal impairment and end-stage renal disease
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has not been well established. Renal impairment after HCT can result from nephrotoxins, TBI, be related to the underlying disease (e.g. multiple myeloma), arise from treatment with intravenous bisphosphonates or be related to chronic GVHD. The most common nephrotoxins used in the HCT setting are calcineurin inhibitors and antibiotics, which often result in early renal dysfunction. In contrast to observations in solid-organ transplant recipients, late renal failure is infrequent after HCT. However, particularly in patients treated aggressively with nephrotoxic chemotherapy (e.g. with platinum compounds) or irradiation before HCT, some investigators reported a 20–25% incidence of renal insufficiency by 2 years after HCT [92,93]. The term “marrow transplant nephropathy” has been coined for this syndrome, which comprises azotemia, hypertension, and a disproportionate degree of anemia [93,94]. Some patients may also show features of a hemolytic–uremic syndrome, which is more common in patients receiving calcineurin inhibitors combined with sirolimus (see below). Renal biopsies show changes of radiation nephritis including mesangial and endothelial drop-out, expansion of the glomerular basement membrane, and widening of the glomerular capillary loops. Analysis of clinical and animal studies indicates that TBI can lead to long-term renal function impairment, as determined by creatinine levels, blood urea nitrogen, residual activity on chromium-51 EDTA, proteinuria or declining hematocrit [95]. In one clinical study, renal dysfunction was strongly related to the total TBI dose delivered and to the dose per fraction; the presence of GVHD was another risk factor [96]. The use of angiotensin-converting enzyme inhibitors such as captopril may be beneficial [97]. Once nephropathy has developed, control of hypertension is the mainstay of therapy. Renal failure associated with hemolytic–uremic syndrome or microangiopathic hemolytic anemia can occur even in patients who are not heavily pretreated and are not conditioned with TBI. The mechanism of hemolytic–uremic syndrome is not fully understood, and it may become manifest either during or after discontinuation of treatment with calcineurin inhibitors [98]. The mechanism involves altered pathways of prostaglandin synthesis, which, in conjunction with endothelial damage, might interfere with coagulation homeostasis. Discontinuation of the presumptive causative agent is essential. Plasmapheresis and the use of glucocorticoids have been suggested as therapeutic options [99]. In some patients, the disease stabilizes upon blood pressure control. Nephrotic syndrome has been reported in few case series after HCT, often in association with GVHD [100]. A complete response of 45% has been reported with immunosuppression treatment, with a significant reduction in proteinuria observed in 55% of the treated cases. Nephrotoxicity of intravenous bisphosphonates should be ruled out as the cause of nephrotic syndrome in patients at risk [101]. Genital organs Chronic GVHD may also involve the genital organs, in particular the glans penis and vagina, and can lead to late complications. Vaginitis may be severe and cause considerable distress and dyspareunia. In addition to topical immunosuppressive therapy, topical estrogens, if indicated, are often used to better control symptoms and prevent the development of adhesions. The vulva has also been a site of posttransplant malignancies. Post-transplant reproductive function is discussed in Chapters 98 and 104.
Gastrointestinal and hepatic complications Chapter 95 provides a detailed discussion of gastrointestinal and hepatic complications. A description focusing on late problems follows below.
Gastrointestinal tract The gastrointestinal tract is a frequent target of acute transplant-related complications. Chronic problems are less common. Involvement of the esophagus by chronic GVHD may lead to strictures and web formation. Repeated dilator treatments are often required to allow for normal food intake. The most common cause of late-onset and recurrent diarrhea not explained by GVHD, oral magnesium supplementation or infections (e.g. CMV, adenovirus, parvovirus, Clostridium difficile, etc.) is malabsorption related to pancreatic insufficiency. Oral enzyme supplementation is recommended, with good responses reported in adults and children. Pneumatosis cystoides intestinalis has been described in patients post transplant, generally while receiving glucocorticoid therapy. The diagnosis is usually made 2–3 months after HCT. No specific therapy is available, and the bowel normalizes spontaneously or with the resolution of other underlying problems. Liver Late liver function abnormalities can be caused by GVHD, drug effects (e.g. antifungal drugs, calcineurin inhibitors, and TMP/SMX), iron overload, fat liver, and viral infections (see Chapter 95) [102]. At 3 or more months after HCT, the most frequent cause for enzyme or bilirubin elevations is chronic GVHD. However, viral hepatitis has to be considered at any time after HCT. Hepatitis may be due to hepatitis B or C (or other) viruses and may be transmitted from transplant or transfusion donors or occur by reactivation of host virus. Some cases of hepatitis B after HCT have been diagnosed upon tapering of immunosuppressive drugs given for GVHD prophylaxis or therapy. In an occasional patient, liver function abnormalities may be related to VZV which may cause hepatitis without showing cutaneous manifestations. Prompt institution of therapeutic doses of acyclovir is indicated. Of considerable concern are the late sequelae of hepatitis, i.e. cirrhosis, related to hepatitis C virus (HCV), and hepatoma, related to HCV or hepatitis B virus [103]. A report by the transplant team in Pesaro, Italy, summarized the effects of iron overload and HCV on liver fibrosis in patients transplanted for thalassemia [104]. Among 211 patients followed for a median of more than 5 years, 46 (22%) showed progressive fibrosis of the liver. The risk was related to the hepatic iron concentration and HCV positivity. None of the HCV-negative patients with an iron content of less than 16 mg/g dry weight showed progression, whereas all patients who were HCV positive and had an iron level of over 22 mg/g progressed. Thus, attempts should be made at treating both iron overload (with phlebotomy and chelation, oral agents now being available) and HCV (with interferon and antiviral antibiotics). Several hepatic malignancies, including unusual fibrous histiocytomas, have been observed (see Chapter 106). Cases of hemosiderosis of the liver have occurred post transplant and are thought to be related to altered iron absorption (see Chapter 95). Iron overload has been suggested to mimic exacerbation of liver GVHD with subsequent normalization of abnormal liver tests in response to phlebotomy [105].
Metabolic problems Several studies have shown that survivors of childhood cancer and pediatric HCT are at an increased risk for developing insulin resistance and the metabolic syndrome [106]. Similarly, survivors of adult cancer have a high prevalence of metabolic syndrome and are at an increased risk for cardiovascular mortality [107]. The definition of metabolic syndrome by the Adult Treatment Panel III with modifications proposed by
Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation Table 105.7 Definition of metabolic syndrome Measure (presence of any 3 of 5 will constitute the diagnosis of metabolic syndrome) Elevated waist circumference Elevated triglycerides
Reduced HDL-C
Elevated blood pressure
Elevated fasting glucose
Categorical Cut Points ≥102 cm (≥40 inches) in men ≥88 cm (≥35 inches) in women ≥150 mg/dL (≥1.7 mmol/L) or Receiving medication for hypertriglyceridemia