Allogeneic Stem Cell Transplantation (Second Edition)

Allogeneic Stem Cell Transplantation

C O N T E M P O R A R Y H E M AT O L O G Y

Judith E. Karp, Series Editor

For other titles published in the series, go to www.springer.com/7861

Allogeneic Stem Cell Transplantation Second Edition

Edited by

Hillard M. Lazarus University Hospitals Case Medical Center Cleveland, OH USA

Mary J. Laughlin Case Western Reserve University Cleveland, OH USA

Editors Hillard M. Lazarus University Hospitals Case Medical Center Cleveland, OH USA [email protected]

Mary J. Laughlin Case Western Reserve University Cleveland, OH USA [email protected]

ISBN 978-1-934115-33-6 e-ISBN 978-1-59745-478-0 DOI 10.1007/978-1-59745-478-0 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2009930362 © Springer Science+Business Media, LLC 2003, 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Dr. Hillard M. Lazarus dedicated his contributions to his wife Joan and his sons Adam and Jeffrey for their unwavering encouragement and support.

Preface

Allogeneic hematopoietic stem cell (HSC) transplantation has undergone fast-paced changes after our original publication of Allogeneic Stem Cell Transplantation: Clinical Research and Practice, first published more than 5 years ago. In this second edition, the editors have focused on topics relevant to evolving knowledge in the field in order to better guide clinicians in decisionmaking and management of their patients, as well as help lead laboratory investigators in new directions emanating from clinical observations. Some of the most respected clinicians and scientists in this discipline have responded in this second edition by providing state-of-the-art discussions addressing these topics. Important advances have been recognized in HLA disparity between HSC donor and recipient triggers for T-cell and NK-cell allorecognition; such may induce the graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects and may cause an engraftment failure. This text covers the scope of human genomic variation, the methods of HLA typing, and interpretation of high-resolution HLA results. Durable GVL responses may be the result of the elimination of leukemia stem cells or the establishment of a durable immune control on their progeny. Alternative sources of donor HSC continue to be used for transplantation at an increased frequency and include HLA-matched unrelated donor and umbilical cord blood; overall patient outcome has improved steadily using these diverse stem cell sources. The administration of reduced-intensity as well as non-myeloablative conditioning has also brought forth new concepts in the management of hematologic malignancies, thought to be of emerging importance in patients with lower grade malignant disorders such as chronic lymphocytic leukemia, multiple myeloma, and low-grade non-Hodgkin lymphoma. The elderly or those with comorbid conditions who have acute leukemia in complete remission also may benefit by using this lower-intensity therapy. The reduced toxicity of these novel conditioning regimens has also raised new possibilities in the application of allogeneic HSC transplantation for patients with non-malignant hematologic disorders such as sickle cell anemia and selected solid tumors such as renal cell carcinoma. Allogeneic SCT remains the only available curative therapy for hematologic malignancies and some inborn errors such as beta-thalassemia. Its application, however, may result in significant morbidity and mortality, predominantly as a consequence of opportunistic infections and GVHD. While differences in HLA between donor and recipient make a crucial contribution to the alloreactivity vii

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driving the donor-mediated GVL response, the cytokine milieu both promotes and regulates the allogeneic response after transplantation. As such, genetic studies correlating donor, host, or the combination of cytokine polymorphisms with disease outcomes have provided useful insight into disease pathogenesis, often confirming effects that have been determined in pre-clinical studies. It is now clear that the polymorphic expression of key cytokines (particularly tumor necrosis factor and interleukin 10) has a demonstrable effect on disease outcome and overall transplant-related mortality. Many challenges in allogeneic SCT remain and include the risk of graft failure, recurrent disease, acute GVHD, opportunistic infections and longterm sequelae such as chronic GVHD, increased risk of second malignancies, endocrinopathies, and iron overload. The editors hope that this new information, well summarized by the authors in this text, will be of significant benefit to clinicians and researchers in allogeneic HSC transplantation. We envision that the generation of further knowledge and clinical studies to be of ultimate benefit to our patients. Cleveland, Ohio, USA

Hillard M. Lazarus, MD Mary J. Laughlin, MD

Contents

  1 Allogeneic Stem Cell Transplantation: The Last Century................ John M. Goldman

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  2 Full Intensity and Reduced Intensity Allogeneic Transplantation in AML.................................................................... Charles Craddock

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  3 Allogeneic Stem Cell Transplantation for Adult Acute Lymphoblastic Leukemia (ALL)...................................................... Bella Patel, Anthony H. Goldstone, and Adele K. Fielding

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  4 Hematopoietic Progenitor Cell Transplantation for Treatment of Chronic Lymphocytic Leukemia........................... Leslie A. Andritsos, John C. Byrd, and Steven M. Devine

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  5 The Role of Allogeneic Hematopoietic Stem Cell Transplantation for Chronic Myelogenous Leukemia Patients in the Era of Tyrosine Kinase Inhibitors............................. Richard T. Maziarz   6 Allogeneic Transplantation for Hodgkin’s Lymphoma.................... William Broderick and Patrick Stiff   7 Myeloablative Allogeneic Stem Cell Transplantation for Non-Hodgkin’s Lymphoma......................................................... J. Kuruvilla, P. Mollee, and J.H. Lipton

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  8 Non-Hodgkin’s Lymphoma: Allogeneic Reduced Intensity Conditioning..................................................................................... 109 Sonali M. Smith and Ginna G. Laport   9 The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults................................................................. 127 Heidi D. Klepin and David D. Hurd

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10 Single Versus Tandem Autologous Hematopoietic Stem Cell Transplant in Multiple Myeloma..................................... 143 David H. Vesole 11 Treatment Strategies for Follicular Center Cell Non-Hodgkin’s Lymphoma.............................................................. 159 Frank Heinzelmann, Hellmut Ottinge, and Claus Belka 12 The Role of Transplantation in Favorable-Risk Acute Myeloid Leukemia............................................................................ 177 Mickey Liao and Gary J. Schiller 13 Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia in Adults................................................ 193 David I. Marks 14 Allogeneic Transplantation for Myelodysplastic Syndromes........... 203 Geoffrey L. Uy and John F. DiPersio 15 Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Acute Lymphoblastic Leukemia.................................... 219 Adriana Balduzzi, Lucia Di Maio, Mary Eapen, and Vanderson Rocha 16 Allogeneic Transplantation for the Treatment of Multiple Myeloma........................................................................ 261 Rebecca L. Olin, Dan T. Vogl, and Edward A. Stadtmauer 17 Blood Vs. Marrow Allogeneic Stem Cell Transplantation............... 281 Brian McClune and Daniel Weisdorf 18 Hematopoietic Cell Transplantation from Partially HLA-Mismatched (HLA-Haploidentical) Related Donors.............. 299 Ephraim J. Fuchs and Heather J. Symons 19 Unrelated Donor Transplants............................................................ 345 Andrea Bacigalupo 20 Update on Umbilical Cord Blood Transplantation........................... 363 Karen Ballen 21 Selection of Cord Blood Unit(s) for Transplantation........................ 375 Donna A. Wall and Ka Wah Chan 22 Mobilization of Hematopoietic Cells Prior to Autologous or Allogeneic Transplantation........................................................... 387 Steven M. Devine

Contents 

23 Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation................................................................. 413 Martin Stern, Sandrine Meyer-Monard, Uwe Siegler, and Jakob R. Passweg 24 Cryopreservation of Allogeneic Stem Cell Products........................ 427 Noelle V. Frey and Steven C. Goldstein 25 Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic Stem Cell Transplantation....................... 441 Steven C. Goldstein and Selina Luger 26 Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer Cell Alloreactivity............................................... 459 Franco Aversa and Andrea Velardi 27 Therapeutic Potential of Mesenchymal Stem Cells in Hematopoietic Stem Cell Transplantation........................... 477 Luis A. Solchaga and Hillard M. Lazarus 28 Hematopoietic Stem Cell Transplantation for Thalassemia............. 491 Javid Gaziev and Guido Lucarelli 29 Viral Infections in Hematopoietic Stem Cell Transplant Recipients.......................................................................................... 505 Per Ljungman 30 Fungal Infections.............................................................................. 533 John R. Wingard 31 Immune Reconstitution and Implications for Immunotherapy Following Hematopoeitic Stem Cell Transplantation....................... 545 Kirsten M. Williams and Ronald E. Gress 32 Acute Graft Versus Host Disease: Prophylaxis................................. 565 Corey Cutler, Vincent T. Ho, and Joseph H. Antin 33 Chronic Graft-Versus-Host Disease.................................................. 577 Madan Jagasia and Steven Pavletic 34 Post-transplant Lymphoproliferative Disorder.................................. 597 Ran Reshef, Alicia K. Morgans, and Donald E. Tsai 35 Psychological Care of Adult Allogeneic Transplant Patients........... 619 Flora Hoodin, Felicity W.K. Harper, and Donna M. Posluszny 36 Second Allogeneic Transplantation: Outcomes and Indications.................................................................................. 657 Koen van Besien, Dan Pollyea, and Andrew Artz

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37 Minimal Residual Disease................................................................ 667 Mehmet Uzunel 38 Functional Assessment Tools and Co-morbidity Scoring in Hematopoietic Progenitor Cell Transplantation........................... 687 Sergio Giralt and Uday Popat 39 Unique Thrombotic and Hemostatic Complications Associated with Allogeneic Hematopoietic Stem Cell Transplantation................................................................. 695 Amber A. Petrolla, Hillard M. Lazarus, and Alvin H. Schmaier 40 How Much Isolation Is Enough for Allografts?................................ 717 Brandon Hayes-Lattin 41 Monoclonal Antibodies in Allogeneic Hematopoietic Stem Cell Transplantation for Hematologic Malignancies............... 733 Maria Corinna Palanca-Wessels and Oliver Press 42 Treatment of Acute Graft-vs-Host Disease....................................... 747 Steven C. Goldstein, Sophie D. Stein, and David L. Porter 43 The Importance of Non-Human Primate Models for Pre-clinical Studies in Hematopoiesis......................................... 767 Erzsebet Szilagyi, Nadim Mahmud, and Amelia Bartholomew 44 In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation.................................................................................. 789 Lisbeth A. Welniak and William J. Murphy 45 Dendritic Cells.................................................................................. 807 Jacalyn Rosenblatt and David Avigan 46 Augmentation of Hematopoietic Stem Cell Transplantation with Anti-cancer Vaccines................................................................ 855 Edward D. Ball and Peter R. Holman Erratum ................................................................................................... .

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Index......................................................................................................... 871

Contributors

Leslie A. Andritsos, MD Division of Hematology & Oncology, The Ohio State University Medical Center, Columbus, OH, USA Joseph H. Antin, MD Harvard Medical School and Dana Farber Cancer Institute, Boston, MA, USA Andrew Artz, MS, MD Section of Hematology and Oncology, Department of Medicine, University of Chicago, Chicago, IL, USA Franco Aversa, MD Section of Haematology and Clinical Immunology, Department of Clinical and Experimental Medicine, HSCT Unit, University of Perugia, Perugia, Italy David Avigan, MD Division of Hematological Malignancies/Bone Marrow Transplantation, Beth Israel Deaconess Medical Center, Boston, MA, USA Andrea Bacigalupo Ospedale San Martino, Genova, Italy Adriana Balduzzi, MD Hematopoeitic Transplant Unit, Clinica Pediatrica, Università degli Studi di Milano, Bicocca Ospedale, San Gerardo, Italy Edward D. Ball, MD Division of Blood and Marrow Transplantation, Department of Medicine and the Moores UCSD Cancer Center, University of California, San Diego, La Jolla, CA, USA Karen Ballen, MD Division of Hematology/Oncology, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA Amelia Bartholomew, MD Division of Transplant Surgery, Department of Surgery, University of Illinois at Chicago College of Medicine, Chicago, IL, USA

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Claus Belka, MD Department of Radiation Oncology, University of Tuebingen, Tuebingen, Germany William Broderick, MD Division of Hematology-Oncology, Department of Medicine, Bone Marrow Transplant Program, Loyola University Stritch School of Medicine, Maywood, IL, USA John C. Byrd, MD Division of Hematology & Oncology, The Ohio State University Medical Center, Columbus, OH, USA Ka Wah Chan, MD Pediatric Blood and Marrow Transplantation Program, Texas Transplant Institute, San Antonio, TX, USA Charles Craddock Centre for Clinical Haematology, Queen Elizabeth Hospital, Edgbaston, Birmingham, UK Corey Cutler, MD, MPH, FRCP Harvard Medical School and Dana Farber Cancer Institute, Boston, MA, USA Steven M. Devine, MD Division of Hematology & Oncology, The Ohio State University Medical Center, Columbus, OH, USA Lucia Di Maio, MD Hematopoeitic Transplant Unit, Clinica Pediatrica, Università degli Studi di Milano, Bicocca Ospedale, San Gerardo, Italy John F. DiPersio, MD, PhD. Section of BMT and Leukemia, Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA Mary Eapen, MD Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, WI, USA Adele K. Fielding, MD Department of Haematology, Royal Free and University College Medical School, London, UK Noelle V. Frey, MD Division of Hematology-Oncology and Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PA, USA Ephraim J. Fuchs, MD Divisions of Pediatric Oncology, Cancer Immunology and Hematologic Malignancies, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA Javid Gaziev, MD International Centre for Transplantation in Thalassemia and Sickle Cell Anemia, Mediterranean Institute of Hematology, Rome, Italy

Contributors 

Sergio Giralt, MD Department of Stem Cell Transplant and Cellular Therapy, University of Texas MD Anderson Cancer Center, Houston, TX, USA John M. Goldman, MD Department of Hematology, Imperial College Faculty of Medicine and World Marrow Donor Association, London, UK Steven C. Goldstein, MD Division of Hematology-Oncology and Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PA, USA Anthony H. Goldstone Department of Haematology, University College London Hospitals, London, UK Ronald E. Gress, MD Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Felicity W.K. Harper, PhD Communication and Behavioral Oncology Program, Barbara Ann Karmanos Cancer Institute and Department of Family Medicine and Public Health Sciences, Wayne State University School of Medicine, Detroit, MI, USA Brandon Hayes-Lattin, MD Center for Hematologic Malignancies, OHSU Cancer Institute, Oregon Health and Science University, Portland, OR, USA Frank Heinzelmann, MD Department of Radiation Oncology, University of Tuebingen, Tuebingen, Germany Vincent T. Ho, MD Harvard Medical School and Dana Farber Cancer Institute, Boston, MA, USA Peter R. Holman, MD Division of Blood and Marrow Transplantation, Department of Medicine and The Moores UCSD Cancer Center, University of California, San Diego, La Jolla, CA, USA Flora Hoodin, PhD Department of Psychology, Eastern Michigan University, Ypsilanti, MI, USA David D. Hurd, MD Section of Hematology-Oncology, Department of Internal Medicine, School of Medicine, Wake Forest University, Winston-Salem, NC, USA Madan Jagasia, MBBS, MS Division of Hematology-Oncology, Department of Medicine, Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA Heidi D. Klepin, MD Section of Hematology-Oncology, Department of Internal Medicine, School of Medicine, Wake Forest University, Winston-Salem, NC, USA

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Contributors

John Kuruvilla, MD Division of Medical Oncology and Hematology, Princess Margaret Hospital and University of Toronto, Toronto, ON, Canada Ginna G. Laport, MD Division of Blood and Marrow Transplantation, Stanford University Medical Center, Stanford, CA, USA Hillard M. Lazarus, MD Case Comprehensive Cancer Center, University Hospitals Case Medical Center, Cleveland, OH, USA Mickey Liao, MD Hematologic Malignancies Unit/Stem Cell Transplant Unit, University of California at Los Angeles, Los Angeles, CA, USA Jeffrey H. Lipton, MD Division of Medical Oncology and Hematology, Princess Margaret Hospital and University of Toronto, Toronto, ON, Canada Per Ljungman, MD Department of Hematology, Karolinska University Hospital, Stockholm, Sweden Guido Lucarelli, MD International Centre for Transplantation in Thalassemia and Sickle Cell Anemia, Mediterranean Institute of Hematology, Rome, Italy Selina Luger, MD Division of Hematology-Oncology and Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PA, USA Nadim Mahmud, MD, PhD Division of Hematology-Oncology, University of Illinois at Chicago, Chicago, IL, USA David I. Marks, MD University Hospitals of Bristol, Oncology Day Beds, Bristol Children’s Hospital, Bristol, UK Richard T. Maziarz, MD Center for Hematologic Malignancies, Adult Bone Marrow Transplantation Program, Oregon Health Science Cancer Institute, Oregon Health & Science University, Portland, OR, USA Brian McClune, DO Blood and Marrow Transplantation Program, University of Minnesota, Minneapolis, MN, USA Keith McCrae, MD Division of Hematology and Oncology, Case Western Reserve University School of Medicine, Cleveland, OH, USA Sandrine Meyer-Monard, MD Division of Hematology, Basel University Hospital, Basel, Switzerland

Contributors 

Peter Mollee, MD Department of Haematology, Princess Alexandra Hospital and University of Queensland, Brisbane, QLD, Australia Alicia K. Morgans, MD Abramson Cancer Center, Hematologic Malignancies Program, University of Pennsylvania Medical Center, Philadelphia, PA, USA William J. Murphy, MD Department of Dermatology, University of California, Davis Sacramento, CA 95817 Rebecca L. Olin, MD Abramson Cancer Center, Bone Marrow and Stem Cell Transplant Program, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Hellmut Ottinger, MD Department of Bone Marrow Transplantation, University of Essen, Essen, Germany Maria Corinna Palanca-Wessels, MD Fred Hutchinson Cancer Research Center and Department of Medicine, University of Washington, Seattle, WA, USA Jakob R. Passweg, MD Division of Hematology, Geneva University Hospitals, Geneva, Switzerland Bella Patel Department of Haematology, Royal Free and University College Medical School, London, UK Steven Pavletic, MD Graft-versus-Host and Autoimmunity Unit, Experimental Transplantation and Autoimmunity Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Amber A. Petrolla, MD Department of Pathology, Case Western Reserve University and University Hospitals Case Medical Group, Cleveland, OH, USA Dan Pollyea, MD Divisions of Hematology and Oncology, Stanford University School of Medicine, Palo Alto, CA, USA Uday Popat, MD Department of Stem Cell Transplant and Cellular Therapy, University of Texas MD Anderson Cancer Center, Houston, TX, USA David L. Porter, MD Allogeneic Stem Cell Transplantation, University of Pennsylvania Medical Center, Philadelphia, PA, USA Donna M. Posluszny, PhD Department of Medicine, University of Pittsburgh School of Medicine and Behavioral Medicine Clinical Service, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

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Contributors

Oliver W. Press, MD Fred Hutchinson Cancer Research Center and University of Washington School of Medicine, Seattle, WA, USA Ran Reshef, MD Abramson Cancer Center, Hematologic Malignancies Program, University of Pennsylvania Medical Center, Philadelphia, PA, USA Vanderson Rocha, MD, PhD Acute Leukemia Working Party of the European Blood and Marrow Transplant Group, Hopital Saint Antoine and Hematopoeitic Transplant Unit and Eurocord Registry, Hopital Saint Louis, Assistance Publique des Hopitaux de Paris, University of Paris, Paris, France Jacalyn Rosenblatt, MD Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA Gary J. Schiller, MD Hematologic Malignancies Unit/Stem Cell Transplant Unit, University of California at Los Angeles, Los Angeles, CA, USA Alvin H. Schmaier, MD Division of Hematology and Oncology, Case Western Reserve University and University Hospital Case Medical Group, Cleveland, OH, USA Uwe Siegler, MD Division of Hematology, Basel University Hospital, Basel, Switzerland Sonali M. Smith, MD Section of Hematology/Oncology, The University of Chicago Medical Center, Chicago, IL, USA Luis A. Solchaga, PhD Case Comprehensive Cancer Center, University Hospitals Case Medical Center, Cleveland, OH, USA Edward A. Stadtmauer, MD Abramson Cancer Center, Bone Marrow and Stem Cell Transplant Program, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Sophie D. Stein, MD Department of Hematology-Oncology, University of Pennsylvania Medical Center, Philadelphia, PA, USA Martin Stern, MD Division of Hematology, Basel University Hospital, Basel, Switzerland Patrick Stiff, MD Division of Hematology-Oncology, Department of Medicine, Bone Marrow Transplant Program, Loyola University Stritch School of Medicine, Maywood, IL, USA Heather J. Symons, MD Divisions of Pediatric Oncology, Cancer Immunology and Hematologic Malignancies, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA

Contributors 

Erzsebet Szilagyi, MD Division of Hematology-Oncology, University of Illinois at Chicago, Chicago, IL, USA Donald E. Tsai, MD Abramson Cancer Center, Hematologic Malignancies Program, University of Pennsylvania Medical Center, Philadelphia, PA, USA Geoffrey L. Uy, MD Section of BMT and Leukemia, Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA Mehmet Uzunel, PhD Karolinska University Hospital, Stockholm, Sweden Koen van Besien, MD Section of Hematology/Oncology, University of Chicago, Chicago, IL, USA Andrea Velardi, MD Section of Haematology and Clinical Immunology, HSCT Unit, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy David H. Vesole, MD, PhD, FACP Attending Physician, St. Vincent’s Comprehensive Cancer Center, New York, NY, USA Dan T. Vogl, MD Abramson Cancer Center, Bone Marrow and Stem Cell Transplant Program, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Donna A. Wall, MD Cancer Care Manitoba, Winnipeg, MB, Canada Daniel Weisdorf, MD Blood and Marrow Transplantation Program, University of Minnesota, Minneapolis, MN, USA Lisbeth Welniak, PhD Department of Dermatology, University of California, Davis Sacramento, CA 95817 Kirsten M. Williams, MD Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA John R. Wingard, MD Division of Hematology-Oncology, Bone Marrow Transplant Program, University of Florida Shands Cancer Center, Gainesville, FL, USA

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Chapter 1 Allogeneic Stem Cell Transplantation: The Last Century John M. Goldman

Sporadic and always unsuccessful attempts to reconstitute bone marrow function by transfusion of blood, bone marrow or fetal liver cells collected from normal individuals were made in the nineteenth and first half of the twentieth centuries, but in practice the notion that hematopoietic stem cell transplantation could prove to be of clinical value proceeded only slowly in the last century [1]. One of the earlier important observations was made by a Dutch scientist in 1922, who noted that the expected thrombocytopenia and hemorrhage in guinea pigs following total body irradiation (TBI) could be prevented by shielding from irradiation the animal’s legs [2]. The studies were not pursued at that time, but in 1949, Jacobson and colleagues reported that mice exposed to lethal doses of irradiation could be protected by shielding the spleen, which functions as a hematopoietic organ in the mouse [3]. These workers went on to show that this protection could also be provided by transfusion of spleen cells into the mouse peritoneum [4]. In the same year, Lorenz and coworkers showed that “lethally” irradiated mice and guinea pigs could be protected by intravenous injections of bone marrow cells collected from syngeneic animals [5]. One possible interpretation of these findings was simply that a humoral factor transferred from the healthy animal was able to “stimulate regeneration” of hematopoiesis in the irradiated animal, but experiments using a variety of histochemical and genetic markers showed convincingly that this prevention of lethality after irradiation was due to transfer of donor cells rather than of components of the plasma [6–9]. In 1956, Ford introduced the term “radiation chimaera” to describe an animal whose hematopoiesis was derived from a donor animal after TBI [8]. Subsequently, it was shown that such chimerism could be established in animals whose own hematopoiesis was destroyed by combinations of cytotoxic drugs without any irradiation. Also, in 1956, Barnes and colleagues studied a lymphoid leukemia that could be transmitted in mice by passage of cells intravenously or subcutaneously [10, 11]; when these leukemic mice were subjected to 1,500 rad of TBI followed by transfusion of cells from a normal donor animal, the majority survived. This was the first convincing demonstration that leukemia cells could be killed by high dose irradiation and laid the foundation for the use of high dose cytotoxic drugs

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_1, © Springer Science + Business Media, LLC 2003, 2010

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treatment (with or without irradiation) followed by marrow infusion as therapy for leukemia in man.

1.  Early Clinical Studies Some of the earliest interpretable studies of stem cell transplantation in man involved autografting. In 1958, Kurnick and colleagues collected and cryopreserved bone marrow cells from two patients with metastatic malignant disease; the patients were then treated with high dose irradiation and their thawed marrow cells were infused intravenously [12]. Though the authors could not be certain that the subsequent marrow recovery was indisputably due to the autologous infusion of marrow cells, this seemed very probable. The following year Thomas and coworkers reported the results of treating two patients with acute leukemia, both of whom had identical twins, by high dose irradiation followed by transfusion of nucleated cells from their respective twins [13]. Both patients engrafted but in both cases the leukemia recurred and was the cause of death. Also, in 1959, Mathé reported the results of his attempts to treat six persons accidentally exposed to high dose irradiation in Vinca in Yugoslavia [14]; there was transient evidence of engraftment in some of the patients. Subsequently, his group in Paris reported the first case of complete engraftment with survival beyond 1 year; in the event the patient developed both acute and chronic graft-versus-host disease (GvHD) and died eventually of varicella encephalitis [15]. In 1968, Mathé summarized his experience in treating 21 patients by bone marrow transplantation, of which 6 had failed to engraft and 8 had sustained GvHD [16]. The first truly successful use of allogeneic hematopoietic cells in man was reported by Gatti and coworkers from Minneapolis in 1968 [17]. They treated a five-month-old male with a sexlinked lymphopenic immunological deficiency by transfusion of cells from blood buffy coat and bone marrow collected from an immunologically competent sibling donor. The patient was clinically well and the continued success of the procedure was confirmed by a follow-up report published 25 years later [18]. This was apparently the first patient cured by infusion of hematopoietic cells collected from an allogeneic donor, using of course contemporary HLA matching techniques. The first successful allograft for aplastic anemia was reported by Thomas and coworkers in 1972 [19]. In 1975, the status of syngeneic and allogeneic transplantation in man was well summarized by a two-part review published by Thomas and coworkers in the New England Journal of Medicine [20]. Techniques for conditioning the individual patient were discussed and methodology for collecting and transplanting bone marrow cells was described in detail. The experience with transplants performed for 37 patients with aplastic anemia and 73 patients with leukemia was summarized. Attention was drawn to the problems of GvHD, slow engraftment and opportunistic infection. This was followed by the first definitive paper describing the use of allogeneic stem cell transplantation to treat a large series of patients with “end-stage” acute leukemia in Seattle [21]; Thomas and colleagues reported that of 100 poor-risk patients, 13 had become very long-term leukemia-free survivors. It was clear that the risk of transplant-related mortality was substantial and some of those who survived the procedure relapsed with leukemia, but the notion that even a minority of

Chapter 1  Allogeneic Stem Cell Transplantation: The Last Century 

these poor-risk patients might be cured was seen by many as “exciting.” The workers reasoned that if some patients with advanced leukemia could be cured by allografting, the incidence of cure might be considerably higher if the transplants were performed in complete remission. This assumption proved subsequently to be correct.

2.  Allografting for Aplastic Anemia The first systematic approach to the use of allogeneic stem cell transplantation started in Seattle in the early 1970s. By 1974, the group was able to report results of transplanting 38 patients with aplastic anemia, most of whom had been conditioned with cyclophosphamide 50  mg/kg daily for 4  days [22], which was a modification of a regimen proposed originally by Santos in 1970. One major problem with these early transplants for aplastic anemia was failure of sustained engraftment, which was attributed to sensitization of the host to minor histocompatibility antigens present on donor cells. This could be overcome to some extent by increasing the intensity of the conditioning. In 1976, Camitta reported results of a randomized prospective study showing that allogeneic stem cell transplantation resulted in survival superior to that achieved with conventional nontransplant therapy [23].

3.  Allografting for Acute Leukemia In 1979, the Seattle group was able to report preliminary data on results of allogeneic stem cell transplant using HLA-identical siblings for 19 patients with “acute nonlymphoblastic leukemia” in remission [24]. A follow-up paper 4  years later reported that 10 of the first 19 patients were alive and free of leukemia at more than 5 years after their transplants [25]. These results led specialist groups on both sides of the Atlantic to initiate programs for transplanting adult patients with both acute myeloid and acute lymphoblastic leukemia (ALL) in remission. Clearly, children with ALL in first remission continued to be candidates for maintenance chemotherapy rather than allografting, but the notion of offering a transplant to a child in second remission of ALL gained support. A further important step in the use of allogeneic SCT for acute leukemia was the first successful use of an unrelated donor – as reported by Hansen and colleagues in 1980 [26]. Since that time it has been generally accepted that the best donor for a given patient is an HLA-matched sibling (or possibly a genetically identical twin), but HLA matched volunteer donors can in some series yield clinical results comparable to those achieved with sibling donors. For children cord blood stem cells, as originally demonstrated in 1988 by Gluckman and coworkers in Paris [27], have become an important alternative source of stem cells, in the absence of a matched sibling. The extent to which cells in the graft is depleted played an important role in control and eradication of leukemia was a matter of considerable debate in the 1970s. In the late 1979 and 1981, the Seattle group published two important papers that showed convincingly that relapse of acute leukemia was much rarer in patients who sustained acute or chronic GvHD compared with those who had little or no GvHD [28, 29]. This was impressive if indirect evidence that a

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graft-versus-leukemia (GvL) effect which segregated with a GvHD effect was important in suppressing or eradicating leukemia at least in some cases. The later demonstration that depletion of T-cells from the donor inoculum increased the incidence of leukemia relapse, most especially in chronic myeloid leukemia (CML) [30, 31], helped to establish the concept that a GvL effect did indeed play a crucial role in the cure of leukemia by allogeneic SCT. Final proof of the efficacy of the GvL effect came from the observation by Kolb and coworkers that relapse after allogeneic stem cell transplantation for CML could be readily reversed by transfusion of lymphocytes of donor origin [32].

4.  Allografting for Chronic Myeloid Leukemia It is difficult in the modern era to imagine that CML was generally regarded until the 1980s as an inexorably fatal disease for which no cure could usefully be contemplated. It was therefore very exciting when in 1979, Fefer and co­workers in Seattle published a report of four patients with CML in chronic phase that had been treated with high dose chemoradiotherapy followed by transplantation of marrow cells collected from their respective identical twins [33]. All four patients were well without evidence of Ph-positive marrow metaphases at follow-up periods from 22 to 31 months after transplantation. Though it was entirely possible that each of these patients would still relapse, it gave impetus to the notion that allogeneic stem cell transplantation performed with marrow cells from HLA-identical siblings might be a useful approach to managing and hopefully curing this form of leukemia. Transplant programs for CML using matched sibling donors were therefore initiated in Seattle and elsewhere [34–36]. Preliminary results with small numbers of patients confirmed that the principal risks were indeed GvHD and opportunistic infection, whereas relapse was rare in survivors. In the early 1980s, Prentice and colleagues in London showed that depletion of donor marrow cells by incubation with anti-T cells monoclonal antibodies prior to infusion to the patient very greatly reduced the incidence of GvHD [37]. Unfortunately, T-cell depletion was associated with an increased risk of nonengraftment and impaired immune reconstitution. It appeared also to abrogate the GvL effect, most prominently in CML, since the actuarial relapse for CML patients allografted in chronic phase who received sibling marrow cells treated with a pan-lymphoid monoclonal antibody (CD52, Campath, now known as alemtuzumab) approached 70%. Various methods of T-cell depletion have been explored subsequently and its use is undoubtedly valuable in selected cases, most especially where later relapse of leukemia is amenable to management with donor lymphocyte infusions.

5.  Graft-Versus-Host Disease In the 1950s, Barnes and Loutit reported that irradiated mice that received spleen cells from syngeneic donors engrafted and survived without significant problems, whereas irradiated mice transfused with spleen cells from a different murine strain died within 100 days of the transplant [38]. These observations were extended by Cohen and coworkers who noted that the affected animals had severe diarrhea, weight loss and skin lesions, and a syndrome that they

Chapter 1  Allogeneic Stem Cell Transplantation: The Last Century 

designated “secondary disease” [39]. Gradually it became clear that this secondary disease was probably caused by immunological incompatibility between donor lymphoid cells and specific organs in the recipient, a syndrome now designated “graft-versus-host disease.” The requirements for establishment of GvHD are: (1) that the graft consists of immunologically competent cells; (2) that the host cells express antigens that are absent on the donor cells; and (3) that the recipient is incapable of mounting an effective immunological reaction against the graft. The requirements can, of course, be met in patients who have not been subjected to conditioning prior to transplant, for example in the patient with severe combined immunodeficiency or the patient who has received extensive chemotherapy for Hodgkins’s disease. It is conventional today to ensure that all blood products administered to patients post transplant are irradiated in vitro to at least to 1,500 cGy to prevent allogeneic GvHD.

6.  Histocompatibility In the late 1930s, Gorer described alloantigens in the sera of mice which comprise part of a murine major histocompatibility complex (MHC), subsequently named H2. These alloantigens were shown to play a central role in tissue rejection [40, 41]. In 1958, Dausset described the first analogous human antigen, then named Mac and now known as HLA-A2 [42]. A number of scientific workshops were then convened at regular intervals and by 1968, it had become clear that human leukocyte antigens (HLA) A and B were closely linked on the short arm of human chromosome 6. HLA-C was identified in 1971. The HLA D locus which is defined in part by expression of alloreactivity in the mixed lymphocyte culture was characterized by Dupont in 1980 [43]. The term haplotype was introduced to describe a sequence of genes on one or other chromosome 6 that together comprise the major MHC in man [44]. In more recent years restriction fragment length polymorphisms were used to characterize more precisely the various HLA genes and this technique has, in turn, given way to a variety of molecular methods, which include direct sequencing of polymorphic regions and allele specific oligonucleotide PCR (Table 1-1).

7.  Evolution of Conditioning Regimens Therapeutic regimens were developed originally as immunosuppression to permit engraftment in patients with nonmalignant conditions (i.e., genetic diseases or aplastic anemia) and were at the time termed “conditioning regimens.” When the use of allogeneic stem cell transplantation was adapted for patients with leukemia, the regimen was intended to provide immunosuppression but also to eradicate residual leukemia cells, yet the term “conditioning regimen” was retained. Initially, the Seattle group concentrated on the use of TBI delivered from two opposing 60Cobalt sources while Santos in Baltimore performed transplants after administration of high dose cyclophosphamide. Because the leukemia relapse rate was relatively high with both approaches to conditioning, both were changed. The Seattle group adopted the regimen of high dose cyclophosphamide followed by 1,000 cGy of TBI (cyclo-TBI); initially the TBI was administered as a single dose but subsequently it was given as fractions over three consecutive days. Later, the use of gamma irradiation

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J.M. Goldman Table 1-1.  Chronology of selected important developments in hematopoietic stem cell transplantation in the last century. 1930s   Gorer lays the foundation for the major histocompatibility complex in mice 1950s   Description of the first “transplantation antigen” in man   Description of secondary disease, later termed “graft-versus-host disease” 1960s   Identification of the major histocompatibility complex in man   Syngeneic transplants for AA   Allografts for immune deficiency diseases 1970s   Technology refined for performed human stem cell allografts   SD allo-SCT to treatment AML in relapse   SD allo-SCT to treat AML in remission   SD-allo-SCT to treat CML in advanced phase   Syngeneic transplants to treat CML in chronic phase   Establishment of the first volunteer donor panel 1980s   Allo-SCT to treat CML in chronic phase   MUD-SCT to treat acute leukemia   Recognition that hematopoietic stem cells were present in the peripheral blood   Introduction of T-cell depletion with monoclonal antibodies   Identification of umbilical cord as source of hematopoietic stem cells 1990s   Demonstration of the efficacy of DLI, most notably in CML   First use of G-CSF to mobilize stem cell from peripheral blood   Introduction of reduced intensity conditioning allo-SCT (mini-transplants) SCT stem cell transplantation, AA aplastic anemia, AML acute myeloid leukemia, CML chronic myeloid leukemia, SD sibling donor, MUD matched unrelated donor, DLI donor lymphocyte infusions, G-CSF granulocyte colony-stimulating factor

was replaced by X-rays from a linear accelerator. Meanwhile the Baltimore group designed a combination of high dose busulfan with cyclophosphamide (BuCy) and despite many modifications tested subsequently by specialist groups on both sides of the Atlantic, most conditioning regimens for leukemia today still comprise either “cyclo-TBI” or “BuCy.”

8.  Donor Selection It is interesting to note that the progression in the choice of donors for the different diseases treated by allogeneic transplantation over the years has followed essentially the same sequence. Thus with some exceptions the first success was achieved with syngeneic donors, and this, in turn, set the scene

Chapter 1  Allogeneic Stem Cell Transplantation: The Last Century 

for studies with genetically HLA-identical sibling donors. It was for some while believed that human hematopoietic stem cell transplants could not be successful without this level of matching, but it appeared subsequently that one or even two antigens HLA mismatched family members could be used as transplant donors. The observation that matched unrelated donors could also be used with success (mentioned above) prepared the way for establishment of unrelated donor registries worldwide, such that today more than 13 million volunteers have been tissue typed with varying levels of resolution, each of whom could theoretically serve as a hematopoietic stem cell donor. Hematopoietic stem cells with marrow regenerating capacity can be collected either from the marrow or from the peripheral blood of selected donors. Cord blood stem cells are also valuable as source of stem cells in children and are now being used with increasing success also for adult patients. The use of stem cell transplantation has developed far over the last 40 years. There is every hope that this pace of development will be maintained, such that transplant-related mortality may fall below its current level and transplants may safely be offered on a more routine basis to patients with both malignant and nonmalignant conditions.

References   1. Santos GW (1983) History of bone marrow transplantation. Clin Haematol 12:611–639   2. Fabricius-Moeller J (1922) Experimental studies of hemorrhagic diathesis from X-ray sickness. Levin and Munksgaard, Copenhagen   3. Jacobson LO, Marks EK, Gaston EO, Zirkle RE (1949) Effect of spleen protection on mortality following X-irradiation. J Lab Clin Med 34:1538–1543   4. Jacobson LO, Simmons EL, Marks EK, Eldredge JH (1951) Recovery from irradiation injury. Science 113:510–511   5. Lorenz E, Uphoff DE, Reid TR, Shelton E (1951) Modification of irradiation injury in mice and guinea pigs by bone marrow injections. J Nat Cancer Inst 12:197–201   6. Lindsley DL, Odell TT, Tausche FG (1955) Implantation of functional erythropoietic elements following total body irradiation. Proc Soc Exp Biol Med 90:512–515   7. Nowell PC, Cells LJ, Habermeyer JG, Roan PL (1956) Growth and continued function of rat marrow cells in X-irradiated mice. Cancer Res 16:256–261   8. Ford CE, Hamerton JL, Barnes DWH, Loutit JF (1956) Cytological identification of radiation chimaeras. Nature 177:452–454   9. Mitchison NA (1956) The colonisation of irradiated tissue by transplanted spleen cells. Br J Exp Pathol 37:239–247 10. Barnes DWH, Corp MJ, Loutit JL et  al (1956) Treatment of murine leukaemias with x-rays and homologous bone marrow. Br Med J 2:626–627 11. Barnes DWH, Loutit JF (1957) Treatment of murine leukaemia with X-rays and homologous bone marrow. II. Br Med J 3:241–252 12. Kurnick NB, Montano A, Gerdes JC et al (1958) Preliminary observations on the treatment of postirradiation hematopoietic depression in man by the infusion of stored autogenous bone marrow. Ann Intern Med 49:973–986 13. Thomas ED, Lochte HL Jr, Cannon JH et al (1959) Supralethal whole body irradiation and isologous marrow transplantation in man. J Clin Invest 38:1709–1716 14. Mathé G, Amiel JL, Schwarzenberg L et al (1963) Hematopoietic chimera in man after allogeneic (homologous) bone marrow transplantation. BMJ 2:1633–1635 15. Mathé G, Jammet H, Pendic B et  al (1967) Transfusions et greffes de moelle osseuse homologue chez des humains irradiés à haute dose accidentellement. Rev Fr Etud Clin Biol 4:226–238

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J.M. Goldman 16. Mathé G (1968) Bone marrow transplantation. In: Rappaport FT, Dausset J (eds) transplantation. Grune and Stratton, New York, pp 284–303 17. Gatti RA, Meuwissen HJ, Allen HD (1968) Immunological reconstitution of sexlinked lymphopenic immunological deficiency. Lancet ii:1366–1369 18. Bortin MM, Bach FH, van Bekkum DW, Good RA, van Rood JJ (1994) 25th anniversary of the first successful bone marrow transplants. Blood 73:603–613 19. Thomas ED, Buckner CD, Storb R et al (1972) Aplastic anemia treated by marrow transplantation. Lancet 1:284–289 20. Thomas ED, Storb R, Clift RA et al (1975) Bone marrow transplantation. N Engl J Med 292:832–843 895–902 21. Thomas ED, Buckner CD, Banaji M et al (1977) One-hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 49:511–533 22. Storb R, Thomas ED, Buckner CD et  al (1974) Allogeneic marrow grafting for treatment for aplastic anemia. Blood 43:157–180 23. Camitta BM, Thomas ED, Nathan DG et  al (1957) Severe aplastic anemia: a prospective study of the effect of early marrow transplantation on acute mortality. Blood 48:63–70 24. Thomas ED, Buckner CD, Clift RA et al (1979) Marrow transplantation for acute nonlymphoblastic leukemia in first remission. N Engl J Med 301:597–599 25. Thomas ED (1983) Marrow transplantation for acute nonlymphoblastic leukemia in first remission. N Engl J Med 309:1539 (letter) 26. Hansen JA, Cift RA, Thomas ED et al (1980) Transplantation of marrow from an unrelated donor to a patient with acute leukaemia. N Engl J Med 303:565–567 27. Gluckman E, Broxmeyer HE, Auerbach AD et al (1989) Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLAidentical sibling. N Engl J Med 321:1174–1178 28. Weiden PL, Flournoy N, Thomas ED et  al (1979) Antileukemic effect of graftversus-host disease in human recipients of allogeneic grafts. N Engl J Med 300:1068–1073 29. Weiden PL, Sullivan KM, Flournoy N, Storb R, Thomas ED (1981) Antileukemic effect of chronic graft-versus-host disease : contribution to improved survival after allogeneic marrow transplantation. N Engl J Med 304:1529–1533 30. Apperley JF, Jones L, Hale G et al (1986) Bone marrow transplantation for chronic myeloid leukaemia: T-cell depletion reduces the risk of graft-versus-host disease but may increase the risk of leukaemic relapse. Bone Marrow Transplant 1:53–66 31. Goldman JM, Gale RP, Horowitz MM et al (1988) Bone marrow transplantation for chronic myelogenous leukemia in chronic phase: increased risk of relapse associated with T-cell depletion. Ann Intern Med 108:806–814 32. Kolb HJ, Mittermuller J, Clemm CH et  al (1990) Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 76:2462–2465 33. Fefer A, Cheever MA, Thomas ED et al (1979) Disappearance of the Ph1-positive cells in four patients with chronic granulocytic leukemia after chemotherapy, irradiation and marrow transplantation from an identical twin. N Engl J Med 300:333–337 34. Clift RA, Buckner CD, Thomas ED et al (1982) Treatment of chronic granulocytic leukaemia in chronic phase by allogeneic marrow transplantation. Lancet 2:621–623 35. Goldman JM, Baughan ASJ, McCarthy DM, Worsley AM et  al (1982) Marrow transplantation for patients in the chronic phase of chronic granulocytic leukaemia. Lancet 2:623–625 36. McGlave PB, Arthur DC, Kim TH et al (1982) Successful allogeneic bone-marrow transplantation for patients in the accelerated phase of chronic granulocytic leukaemia. Lancet 2:625–627

Chapter 1  Allogeneic Stem Cell Transplantation: The Last Century  37. Prentice HG, Blacklock HA, Janossy G et al (1984) Depletion of T lymphocytes in donor marrow prevents significant graft-versus-host disease in matched allogeneic leukaemic marrow transplant recipients. Lancet 1:472–476 38. Barnes DWH, Loutit JF (1955) Spleen protection: the cellular hypothesis. In: Bacq ZM (ed) Radiobiology Symposium Liege. Butterworths, London, pp 134–135 39. Cohen JA, Vos O, van Bekkum DW (1957) The present status of radiation protection by chemical and biological agents in mammals. In: de Hevesy GC, Forssberg AG, Abbott JD (eds) Advances in Radiobiology. Oliver & Boyd, Edinburgh, pp 134–144 40. Gorer RA (1936) The detection of antigenic differences in mouse erythrocytes by the employment of immune sera. Br J Exp Pathol 17:42–50 41. Gorer RA (1937) The genetic and antigenic basis of tumour transplantation. J Pathol Bacteriol 44:691–697 42. Dausset J (1958) Iso-leuko-anticorps. Acta Haematol 20:156–166 43. Dupont B (1980) HLA factors and bone marrow grafting. In: Burchenal JH, Oettgen HF (eds) Cancer: achievements, challenges and prospects for the 1980s. Grune & Stratton, New York, pp 683–693 44. Ceppellini R, van Rood JJ (1974) The HLA system. I. Genetic and molecular biology. Semin Hematol 11:233–251

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Chapter 2 Full Intensity and Reduced Intensity Allogeneic Transplantation in AML Charles Craddock

1.  Introduction Acute myeloid leukemia (AML) is now the commonest indication for allogeneic stem cell transplantation (SCT) in adults [1]. This reflects the continued inability of conventional chemotherapeutic regimens to deliver long-term disease-free survival in most adults -- a failure which is particularly marked in patients over the age of 50 years whose outcome has barely improved in the last three decades [2, 3]. Whilst it has been clear for a number of years that the allogeneic transplantation delivers a more potent anti-leukemic effect than chemotherapy, the toxicity of myeloablative conditioning regimens has precluded its use in precisely the group of patients who urgently need new therapeutic options. However, the recent demonstration that the use of reduced intensity conditioning regimens substantially reduces the transplant-related mortality (TRM) has provided the prospect of delivering a potentially curative graft-versus-leukemia (GVL) effect in patients in whom it was previously contraindicated [4, 5]. Importantly, this has provided a new treatment option for a group of patients whose outcome if treated with chemotherapy alone would be very poor.

2.  Biology of AML and Impact on Future Development of Therapeutic Strategies in AML The demonstration that AML originates from a population of mitotically quiescent leukemic stem cells (LSC) is likely to transform treatment strategies in the coming decade [6]. Since the markedly reduced sensitivity of LSC to a number of currently used chemotherapeutic agents is likely to underlie the high rate of disease relapse in AML, there is an urgent need to develop distinct therapeutic approaches which target this cellular compartment. The notable clinical activity of the tyrosine kinase inhibitor, imatinib, in patients with chronic myeloid leukemia (CML) led to a initial enthusiasm that targeted inhibition of dysregulated signaling pathways might be effective in AML [7]. However, this was not substantiated by the initial experience with drugs From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_2, © Springer Science + Business Media, LLC 2003,2010

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designed to inhibit constitutively active FLT3 and Ras pathways, and clinical responses to these agents in AML are rare and usually of short duration [8–10]. Furthermore, it is now clear from cytogenetic analysis and, more recently from microarray studies that AML is a highly heterogeneous disease at a molecular level whose pathogenesis is dependent on the acquisition of mutations in a number of different cellular pathways [11, 12]. This model is supported by data from transgeneic mouse models which demonstrate that mutations in at least two distinct cellular pathways are required for the pathogenesis of AML [13, 14]. As a consequence, it appears unlikely that a pharmacological strategy which targets a single dysregulated pathway will be of sustained therapeutic benefit. Attention is instead switching to the development of agents which can target the LSC and approaches are under investigation that include inhibitors of pathways mediating self-renewal and proliferation, such as NFkB, Wnt, and hox genes [15–17]. 2.1.  Emergence of Immunotherapeutic Strategies as an Important form of Targeted Therapy The challenges associated with the development of new drug therapies in AML provides a compelling case for the extension of immunotherapeutic approaches which target dysregulated cell surface antigens expressed on the surface of LSC rather than abnormalities in intracellular signaling pathways. The GVL effect in which the donor immune system targets “foreign” antigens expressed on the leukemic blast is the most widely exploited form of immunotherapy in clinical practice, but there is growing interest in the clinical benefit of strategies by which the patient’s own immune system can be manipulated to exert an anti-leukemic effect. A number of lines of evidence attest the presence of a potent GVL effect in patients allografted for AML. These include the demonstration that relapse risk is reduced in patients who develop GVHD and, a compelling observation by Bacigalupo that reduction in the level of post-transplant immunosuppression, achieved by reducing the cyclosporine dose to 1  mg/kg in the first 20 days post-transplant, markedly reduces the risk of disease relapse (Fig. 2-1) [18–20].

Fig.  2-1.  Impact of post-transplant cyclosporine (CyA) dose on disease relapse in patients allografted for AML in first CR [18]

Chapter 2  Full Intensity and Reduced Intensity Allogeneic Transplantation in AML 

The major factor limiting the effective exploitation of GVL effect in patients with high-risk AML is our continued inability to develop transplant protocols which effectively dissociate GVL from severe, potentially fatal, acute, and chronic GVHD. Putative targets of the GVL reaction include minor histocompatibility antigens such as HA-1, HA-2 and H-Y, and the leukemic-specific antigens WT1 and proteinase 3 [21–25]. However, attempts to target mHAgs such as HA-1 have been hampered by the rare frequency of this allele and its HLA restriction. In contrast, antigens such as WT1 represent an attractive option given its over-expression in up to 70% of patients with AML [26, 27]. It is clear, however, that further characterization of the immunogenicity of the AML stem cell will be vital if we are to improve the current immunotherapeutic strategies in high-risk AML [25]. The prospect of exploiting an autologous immune response for clinical benefit has been supported by the demonstration that gemtuzumab ozogamicin (GO), a monoclonal antibody to the putative LSC antigen CD33, can salvage patients with relapsed AML [28]. Based on this encouraging experience, humanized antibodies to a number of other antigens expressed on leukemic progenitors and LSC are currently under clinical trials in high-risk AML. Recent studies demonstrating the presence of an immune response to leukemiaspecific antigens such as WT1 and proteinase 3 has led to the development of strategies designed to induce an autologous T-cell response using either peptide vaccination or TCR gene transfer [29]. Early phase clinical trials studying the effect of peptide vaccination with immunodominant epitopes of WT1 or proteinase 3 confirm that it is possible to augment these responses in  vitro although compelling evidence of clinical benefit is awaited. In the future it will be important to define how such a strategy can be utilized to improve the outcome of allogeneic transplantation.

3.  Preparative Regimens in AML The clinical studies which, over the past three decades, have established a central role for allogeneic transplantation in AML therapy have been achieved using myeloablative conditioning regimens. It was initially unclear whether reduced intensity allografts would possess the capacity to deliver long-term disease-free survival in patients with AML. Whilst it is now evident that such regimens are also capable of delivering durable long-term remissions, a myeloablative conditioning regimen should continue to be viewed as the “gold standard” preparative regimen at least until the results of prospective studies of reduced intensity transplant regimens are available.

4.  Outcome in Patients with AML Transplanted Using a Myeloablative Conditioning Regimen 4.1.  Patients in First Complete Remission (CR1) Randomized controlled trials assessing the role of allogeneic transplantation in the management of patients in CR1 have been difficult to perform. Obstacles have not only included the randomization biases introduced because of the fixed perceptions of either physicians or patients, but also the very real problem

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that the allograft arm may include a smaller proportion of patients with highrisk disease, given the propensity of such patients to relapse before they reach transplant. The only effective approach which allows these biases to be removed is the use of a “donor versus no-donor” analysis which exploits the availability of an HLA identical sibling donor as a form of biological randomization. Although weakened by the fact that not all patients with an available donor will be transplanted, “donor versus no-donor” analyses have proved the only unbiased statistical methodology by which the benefit of a sibling allograft can be assessed [30]. A number of international co-operative groups have reported the results of “donor versus no-donor” analyses in the management of younger adults with AML in CR1 [31–35]. All but one study reported an improvement in the disease-free survival in patients with a donor (Tables 2.1 and 2.2). Although no individual study showed a statistically significant survival benefit – probably because a proportion of patients in the “no-donor” arm could be effectively salvaged by a transplant in CR2 – a recent meta-analysis has demonstrated both an improved disease-free and overall survival in the donor group [32]. Importantly, these studies demonstrate that presentation karyotype and patient age are powerful tools in identifying who will benefit from a sibling allograft. Allogeneic transplantation delivers a clear survival advantage in patients with intermediate or adverse risk cytogenetics [32]. Indeed the risk of relapse is so high in patients with adverse risk cytogenetics that allogeneic transplantation using an unrelated donor is indicated in all CR1 patients with an available donor [36]. In contrast, allogeneic transplantation should not be performed in CR1 patients with good risk cytogenetics whose outcome with chemotherapy can be predicted to be relatively good. This approach is also supported by the 90% salvage rate achievable in patients with good Table  2-1.  A donor-vs.-no-donor analysis of impact of sibling allogeneic transplantation on overall survival (OS) and disease-free survival (DFS) in patients with AML in CR1. OS(%)

DFS(%)

Study

Donor no-donor

p

Donor

no-donor

GOELAM [31]

53

53

NS

44

38

0.6

HOVON [32]

54

46

NS

48

37

30 × 109/l and for T lineage >100,000 × 109/l, and the presence of the Philadelphia chromosome t(9;22) or BCR/ABL fusion gene. In recent years, other cytogenetic subgroups associated with poor outcomes have been identified and include leukemia which harbor the t(4;11) or MLL/AF4 fusion gene, complex karyotypes, and

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_3, © Springer Science + Business Media, LLC 2003, 2010

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Table 3-1.  Prognostic factors in adult ALL. Age WBC

>35 years

  B-lineage

>30 × 109/l

  T-lineage

>100,000 × 109/l

Immunophenotype

B-lineage ALL Cortical T-ALL

Cytogenetics

t(9;22)/BCR/ABL t(4;11)/MLL/AF4 Low hypodiploidy/near triploidy Complex karyotype (>5 abnormalities)

Treatment response

Late achievement of CR: >4 weeks Minimal Residual Disease late into therapy

low hypodiploidy/near triploidy [10, 11]. Immunophenotypic subgroups such as early T-ALL and pro-B ALL are also regarded by some groups as poor prognostic features [8, 11]. The speed and magnitude of treatment response has also been shown to predict treatment outcome. Some studies showing a late achievement of CR >4 weeks identifies a subset of patients with a high risk of relapse, although the recent UKALLXII/ECOG2993 study did not show a worse outcome for patients who took two cycles of induction therapy to achieve remission [9]. The involvement of the central nervous system during diagnosis probably has a worse prognosis, although long-term disease free survival can be achieved in this setting [12]. More recently, the demonstration of residual disease or occult leukemia cells detected by sensitive methods at various times in therapy has been shown to be of prognostic value, predicting a worse outcome [13, 14]. Importantly, this assessment appears to be independent of age and WBC. In adult ALL, demonstration of residual disease at later time points in therapy (~16 weeks) appears to be most predictive. A summary of prognostic factors is given in Table 3-1. Due to the poor prognostic outcome of patients in whom the Philadelphia chromosme is detected, physicians have for long followed an aggressive approach with the use of allo-HSCT in this group. Hence, the main body of this chapter will focus on the use of allo-HSCT in Ph negative ALL. A smaller section at the end will comment specifically on the use of allo-HSCT in Ph pos ALL.

3.  Sibling Allogeneic Hematopoietic Stem Cell Transplantation in First Remission (CR1) In adult patients lacking the Ph chromosome, the role of allo-HSCT to consolidate remission is controversial and has been the focus of intense study. It is generally accepted that randomization between allogeneic HSCT and no HSCT is neither feasible nor desirable, so trials which seek to determine the role of sibling allo-HSCT have generally employed a so-called “biological randomization.” Sibling allo-HSCT is assigned to those with an HLA compatible family donor and where a donor is not available, allocation to either chemotherapy or autologous hematopoietic stem cell transplantation

Chapter 3  Allogeneic Stem Cell Transplantation for Adult Acute Lymphoblastic Leukemia (ALL) 

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Table 3-2.  Definitions of “high risk” for stratification in adult ALL trials. Trial

High risk features

LALA 87 Pethema

Ph +/Common ALL with age >35/WBC > 30 × 109/l, CR >4/52, Null ALL Age 30–50,WBC ³ 25 × 109/l, t(4;11)/11q23 rearrangement, t(1,19)

GMALL

WBC > 30,000 × 109/l, CR > 4/52, immunophenotype

JALSG 93

Any patient with Ph+, age >30 or WBC over 30,000 × 109/l

LALA 94

Any patient with CNS disease at diagnosis, CR beyond first induction; B lineage ALL with 11q23 rearrangements, t(1;19), WBC > 30,000 × 109/l,or myeloid markers

GOELAL02

Ph+, t(4;11), t(1,19) WBC > 30 × 109/l. CR after first induction, age >35

UKALL XII/ECOG 2993 Ph+

(auto-HSCT) or a randomization between the latter two treatments is carried out. The results for such studies are compared in a “donor versus no-donor” intention-to-treat basis. A summary of the available trials, comparing the results of allo-SCT in first CR in patients lacking the Philadelphia chromosome are given in Table 3-2. Generally, results indicate that allo-HSCT provides the most potent antileukemic therapy, significantly reducing the risk of relapse to a greater magnitude than either conventional chemotherapy or autoSCT. However, an overall survival benefit is not always demonstrated due to the high incidence of toxicity associated with the procedure. In the LALA 87 study, patients under the age of 40 years with a donor received an HLA-identical sibling HSCT, those over 50 years were treated with chemotherapy, and the remaining patients were randomized between chemotherapy and auto-HSCT. At 10 years there was a significant survival benefit for allo-SCT in patients with high-risk features [15]. The BGMT study randomized patients in remission; those with an HLA matched sibling donor to allo-HSCT and the others to auto-HSCT. The 3 year disease free survival was significantly higher in allo-SCT recipients [16]. Recent prospective studies have focused on applying allo-HSCT to highrisk patients alone. Table 3-2 shows the features that the various studies have used to categorize “high-risk.” In the LALA 94 trial [17], high-risk patients (i.e., those falling outside the standard risk criteria or those with CNS disease at presentation) were assigned allo-HSCT if a HLA matched donor was available while others were randomized between auto-SCT and standard chemotherapy. Those receiving a sibling allo-HSCT had significantly better outcomes (5 year overall survival, 45 vs. 23%) [17]. In the Goelam-02 study [18], only high-risk patients under the age of 50 years were offered allo-SCT in first remission if a HLA-matched sibling donor was available, those lacking a donor were assigned to auto-HSCT. A clear survival advantage was observed in the allo-HSCT group at 6 years. Other studies have failed to demonstrate improved outcomes from allo-SCT in first remission. Two case-controlled studies performed by the IBMTR showed no overall advantage for transplant over chemotherapy even when stratifying for risk [19, 20] However, owing to the retrospective nature of these studies and inherent selection biases, it is difficult to arrive at any definitive conclusions from these studies. The largest donor vs. no-donor analysis comes from the UKALL12/ECOG2993 study, where almost 500 patients were included in

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the analysis. There was a clear and statistically significant survival advantage to having a matched sibling donor. Indeed, a number of patients with a donor did not receive allo-HSCT so the advantage may even have been underestimated. However, this study introduced a cautionary note in those patients who were older than 40 years;even though there was a considerable decrease in relapse risk among those who had a donor, there was no survival advantage, due to a very high treatment-related mortality, approaching 39% at 2 years. Thus, there is likely to be an upper age limit for patients who can benefit from myeloablative allo-HSCT, and this will continue to limit the applicability of the procedure. Overall, one can conclude that despite differences in the criteria applied for high-risk features between studies, there is a large body of data to support the use of allo-HSCT in first remission in adult ALL patients with high-risk features other than old age. However, the inclusion of patients with Ph + ALL in the overall results of many studies does not allow specific assessment of the value of allo-HSCT in patients with high-risk features outside this cytogenetic group. For patients with standard-risk disease, the role of allo-HSCT is less well studied. However, results from the largest study to compare post-remission therapies in adult ALL, the UKALL XII/ECOG 2993 study has helped to answer this question. Patients younger than 50 years (55 since 2004) in the first CR were assigned to allo-HSCT if a matched sibling donor was available. The other patients were randomized to auto-HSCT or standard chemotherapy. A survival advantage was observed for the allogeneic transplant group as a whole but this was largely due to the improved survival in standard risk patients; EFS was 59% with a donor, compared to 41% without a donor.

4.  Allo-HSCT Beyond CR1 Allogeneic stem cell transplantation has the best potential for long-term OS when applied in CR1 compared to CR2. However, in a proportion of patients with relapsed disease, long-term remission can be gained by receipt of an allogeneic stem cell transplant. Analysis based on IBMTR data showed that the probability of survival in patients achieving second remission is 30% at 5 years [21]. In the largest reported study of 609 patients, with recurring ALL, the overall estimated survival at 5 years was 7% [22]. Receipt of a hematopoietic stem cell transplantation from either a related or unrelated source in patients who had previously been treated with chemotherapy resulted in a better outcome compared to chemotherapy alone (23 vs. 4%). The same study showed the outcome after relapse was very poor and was not influenced by prior therapy. However, when treatment received post-relapse was evaluated in patients with an equal chance of receiving allo-HSCT (i.e., those alive at the median time to transplant and not having had allo-HSCT in CR1), a better outcome was observed in those receiving allo-HSCT than in those receiving chemotherapy alone [22]. Among the few published systematic studies examining factors responsible for achieving long-term remission following allogeneic stem cell transplantation for relapsed ALL, the receipt of a transplant

Chapter 3  Allogeneic Stem Cell Transplantation for Adult Acute Lymphoblastic Leukemia (ALL) 

at second remission compared to a sustained relapse, appears to be associated with better results [22, 23].

5.  Unrelated Donor Stem Cell Transplantation (UD-SCT) In Philadelphia chromosome positive disease UD-SCT in first remission is regarded as standard therapy in the absence of a matched sibling donor. The alternative for those lacking a sibling donor in this situation is an auto-HSCT. A retrospective comparison performed by the IBMTR of patients treated with either of these therapeutic modalities for ALL did not demonstrate a superior survival for UD-HSCT despite a demonstrated lower relapse risk due to an extremely high TRM which approached >40% [21]. With improvements in supportive care, HLA matching and GVHD prophylaxis, the TRM in UD-HSCT is improving. Three published studies have examined the role of UD-HSCT performed in the first remission in adult patients with ALL [24–26]. The results reported from these studies and from registry data are encouraging and do not show wide differences in outcomes from UD-HSCT (DFS ~40–50%) compared to sibling allogeneic transplantation. Sincethe analysis of these studies include Ph + cases, the role of UD-HSCT in first remission in patients outside this cytogenetic group have not been exclusively studied. As less than one-third of patients will have a HLA compatible sibling donor, UD-HSCT could pose a reasonable therapeutic option and is currently the subject of ongoing study.

6.  Other Stem Cell Sources Despite increasing size of donor pools, it is not possible to find an eight of eight allelic matched unrelated donor for all individuals. Using donors who are not fully matched or turning to alternative sources of stem cells are options which may be considered by transplant units. In children, a recent, and a very extensive retrospective study comparing the outcomes of HSCT using marrow or cord blood was carried out in patients receiving MUD or umbilical cord blood HSCT for leukemia [27]. Greater than 60% of those included had a diagnosis of ALL, mostly beyond CR1. TRM was statistically significantly higher after transplant of HLA-antigen mismatched umbilical cord blood compared to fully matched bone marrow stem cells (relative risk 2.31 for two antigen mismatch, 1.88 for one antigen mismatch), although relapse rates were lower after two-antigen HLA-mismatched umbilicalcord-blood transplants (RR 0.54, p = 0.0045). The authors conclude that the data support the use of HLA-matched and even one- or two-antigen HLAmismatched umbilical cord blood in children with acute leukemia who need transplantation. These data do not specifically address the issue of children with Ph + ALL in CR1, but the conclusion from this study may be relevant to this situation. The current pan-European trial in pediatric Ph + ALL allows the use of umbilical cord blood as a source of stem cells for HSCT and is recommended for all children with donors (Table 3-3).

33

Treatment compared

Chemo/auto-SCT

Auto-SCT

Chemo/auto-SCT

Chemo

Chemo

Chemo

Chemo

Chemo/auto-SCT

Group

LALAb 87 [15, 54, 55]

BGMTb [16]

PETHEMAb ALL 93 [56]

JALSG 1998 [20]

GMALL 1981 and 1984 [19]

Gupta et al. [57]

JALSGb 93 [5]

EORTCb ALL3 2004 [58]

68 vs. 116

34 vs. 108

48 vs. 39

234 vs. 484

214 vs. 76

84 vs. 98

43 vs. 77

116 vs. 141

N donor vs. no-donor

6 year RFS/OS 38 vs. 37%/41 vs. 39% p = not sig

6 year OS 40 vs. 46% p = 0.58

3 year RFS 40 vs. 39% p = 0.74

9 year RFS 34 vs. 32% p > 0.2 standard risk/high risk groups p³05

Age >30 years: 30 vs. 26% p = 0.70

Age  35%)

Less than CCgR

Less than MMolR

18 months after diagnosis

Less than CCgR

Less than MMolR

NA

Anytime

Loss of CHRa, loss of CCgRb, mutationc

ACA in Ph+ cellsd, loss of MMolRd, mutatione

Any rise in transcript level; other chromosome abnormalities in Ph- cells

a

High risk, del9q+, ACAs in Ph+ cells NA

To be confirmed on two occasions unless associated with progression to AP/BC To be confirmed on two occasions, unless associated with CHR loss or progression to AP/BC c High level of insensitivity to IM d To be confirmed on two occasions, unless associated with CHR or CCgR loss e Low level of insensitivity to IM Failure implies that the patient should be moved to other treatments whenever available. Suboptimal response implies that the patient may still have a substantial benefit from continuing IM treatment but that the long-term outcome is not likely to be optimal, so the patient becomes eligible for other treatments. Warnings imply that the patient should be monitored very carefully and may become eligible for other treatments. The same definitions can be used to define the response after IM dose escalation PCgR indicates partial CgR; and NA, not applicable This research was originally published in Blood. Ref. [90] © American Society of Hematology b

Chapter 5  The Role of Allogeneic Hematopoietic Stem Cell Transplantation 

Table 5-4.  An institutional perspective of CML patients and indications for hematopoietic stem cell transplantation: 2008. Phase

Clinical setting

CP1

Primary hematologic failure to IM therapy

CP1

Primary cytogenetic failure to IM therapy

CP1

Progression after primary imatinib therapy

CP1

Imatinib resistant, mutations at BCR-ABL 315 locus

CP1

Imatinib resistant, partial response to second-generation inhibitors

CP1

Intolerance to tyrosine kinase inhibitors

CP1

Clonal evolution, in setting of TKI resistance

CP1

Outgrowth of alternate Philadelphia-negative cytogenetically abnormal clones with myelodysplasia

AP

New diagnosis, after primary imatinib therapy, in patients with low EBMT score

AP

Imatinib responsive but later progression in patients with high EBMT score

BC

After induction with TKI ± combination chemotherapy

AP accelerated phase, BC blast crisis, CP1 first chronic phase, EBMT European Group for blood and marrow transplantation

[90]. In the first chronic phase, at our institution, potential HSCT scenarios are reviewed and considered for patients with primary hematologic or cytogenetic failure to IM therapy, as well in those with progressive disease (see Table 5-4). Described mechanisms of resistance such as tyrosine kinase mutations are more frequent in patients with secondary failure, and thus the concern is that over time such “unstable” clonal disease may evolve further and evade salvage options as well, thus warranting HSCT after re-establishing control in such patients [77]. A small number of patients are intolerant of IM and can be considered for second-generation TKI therapy with transplantation at progression. Clearly, patients who are resistant to IM and exhibiting a partial response only to second-generation TKI treatment must consider HSCT, particularly if they are young and have excellent matched options, either sibling or unrelated donor. Finally, there are some circumstances for which most patients are not monitored that would influence the decision to pursue HSCT. These include the identification of mutations at the 315 site of BCR-ABL, which are universally associated with resistance to the known first-generation and second-generation TKIs [27, 28]. Novel kinase inhibitors are being tested in clinical trials that target this particular mutant BCR-ABL site. Our policy, as in many centers, is to offer HSCT to CML patients with T315I mutations, either directly or after being treated in a clinical trial. Finally, there are relatively uncommon circumstances for consideration for HSCT when early clonal evolution is seen in patients failing IM [92] or when myelodysplasia is found in patients with TKI-induced PH– hematopoiesis [93]. For patients with advanced phase CML, early transplantation for accelerated phase patients is considered, but preferably for those with lower EBMT scores (see Table 5-2 for HSCT outcomes impacted by the EBMT score) [34]. For those patients who are in an accelerated phase with significant co-morbid conditions or with a high EBMT score, transplant options are outlined, but alternatively, one may choose to closely monitor the TKI failure before pursuing

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HSCT in those patients. For patients with blast crisis who are age eligible, HSCT is universally recommended after disease control is achieved, generally by induction chemotherapy in combination with IM or another TKI. The transplant community recognizes that these considerations will change. As new agents are developed, for instance those that may overcome the resistance associated with BCR/ABL T315I mutations, it will create circumstances where management algorithms will also evolve. Certainly, there are questions that remain that have attracted significant attention without clear answers or without clear agreement of the transplant community such as the role of transplantation in the very young patient with CML. We all hope and expect that the role of small molecule targeted therapy may impact and change the natural history of many malignancies. Certainly, there has been a dramatic alteration in the treatment strategies and improvement in clinical outcomes in adults with Ph+ ALL (rev in [94]). Early HSCT still remains the primary goal in this setting in adults, but IM therapy has entered all aspects of management from primary treatment to long-term maintenance, and appears to have changed the natural history with markedly improved PFS and OS reported. But, even in this clinical setting which had been universally considered a non-debatable indication for HSCT in the ageappropriate patient, novel trials are being considered within the United States to compare HSCT in CR1 to primary therapy with second-generation TKIs alone.

9.  Conclusions HSCT is a complex and morbid procedure that requires significant resources and technical skills in those whom perform it. The advent of molecular targeted therapy for CML has led to the dramatic decline in HSCT procedures, but the need remains for HSCT for patients who fail TKI therapies or who present with more aggressive disease. In the interim, ongoing advances in HSCT research may continue to improve outcomes for CML patients either by (a) decreasing transplant-related mortality, perhaps by the development of novel conditioning regimens [95] or developing technologies to identify those at highest risk of organ damage [96] or (b) by determining novel means to reduce relapse rates, for example by optimizing natural killer cell grafts [97] or by using selected tumor-specific, vaccine strategies [98]. Alternatively, autologous HSCT may reemerge in therapeutic algorithms, given the ability to collect large numbers of PH– peripheral blood stem cells [99–101]. Currently, one fact that we do know is that the future of HSCT for CML will change and will be determined by clinical investigations of novel therapeutics with close attention to the economics of cancer care, and it will be interesting to observe in this current age of cellular and regeneration therapy whether HSCT for CML takes the path of the Irish Elk toward extinction [102] or evolves dramatically and adapts to become more functional, similar to the development of the Panda’s thumb [103].

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R.T. Maziarz 57. Or R, Shapira MY, Resnick I et al (2003) Nonmyeloablative allogeneic stem cell transplantation for the treatment of chronic myeloid leukemia in the first chronic phase. Blood 101(2):441–445 58. Okamoto S, Watanabe R, Takahaski S et al (2002) Long-term follow-up of allogeneic bone marrow transplantation after reduced-intensity conditioning in patients with chronic myelogenous leukemia in the chronic phase. Int J Hematol 75(7):493 59. Kerbauy FR, Storb R, Hegenbart U et al (2005) Hematopoietic cell transplantation from HLA-identical sibling donors after low-dose radiation-based conditioning for treatment of CML. Leukemia 19(6):990–997 60. Kebriaei P, Detry MA, Giralt S et al (2007) Long-term follow-up of allogeneic hematopoietic stem-cell transplantation with reduced-intensity conditioning for patients with chronic myeloid leukemia. Blood 110(9):3456–3462 61. Bornhauser M, Kroger N, Schwerdtfeger R et al (2006) Allogeneic haematopoietic cell transplantation for chronic myelogenous leukaemia in the era of imatinib: a retrospective multicentre study. Eur J Haematol 76(1):9–17 62. Crawley C, Szydlo R, Lalancette M et al (2005) Outcomes of reduced-intensity transplantation for chronic myeloid leukemia: an analysis of prognostic ­factors from the Chronic Leukemia Working Party of the EBMT. Blood 106(9): 2969–2976 63. Faber E, Koza V, Vitek A et al (2007) Reduced-intensity conditioning for allogeneic stem cell transplantation in patients with chronic myeloid leukemia is associated with better overall survival but inferior disease-free survival when compared with myeloablative conditioning – a retrospective study of the Czech National Hematopoietic Stem Cell Transplantation Registry. Neoplasma 54(5):443–444 64. Giralt SA, Arora M, Goldman JM et al (2007) Impact of imatinib therapy on the use of allogeneic haematopoietic progenitor cell transplantation for the treatment of chronic myeloid leukaemia. Br J Haematol 137(5):461–467 65. Ruiz-Arguelles GJ, Gomez-Almaguer D et al (2005) the early referral for reduced-intensity stem cell transplantation in patients with PH1(+) chronic myelogenous leukemia in chronic phase in the imatinib era: results of the Latin American Cooperative Oncohematolgoy Group (LACOHG) prospective, multicenter study. Bone Marrow Transplant 36(12):1043–1047 66. Ruiz-Arguelles GJ, Tarin-Arzaga LC, Gonzalez-Carrillo ML et al (2008) Therapeutic choices in patients with PH-positive CML living in Mexico in the tyrosine kinase inhibitor era: SCT or TKIs? Bone Marrow Transplant 42(1): 23–28 67. Krejci M, Mayer J, Doubek M et al (2006) Clinical outcomes and direct hospital costs of reduced-intensity allogeneic transplant 38(7):483–491 68. Millot F, Guilhot J, Nelken B et al Imatinib mesylate is effective in children with chronic myelogenous leukemia in late chronic and advanced phase and in relapse after stem cell transplantation. Leukemia 20(2):187–192 69. Hehlmann R, Berger U, Pfirrmann M et al (2007) Drug treatment is superior to allografting as first-line therapy in chronic myeloid leukemia. Blood 109(11):4686–4692 70. Zaucha JM, Prejzner W, Giebel S et al (2005) Imatinib therapy prior to myeloablative allogeneic stem cell transplantation. Bone Marrow Transplant 36(5): 417–424 71. Shimoni A, Kroger N, Zander AR et al (2003) Imatinib mesylate (STI571) in preparation for allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusions in patients with Philadelphia-positive acute leukemia. Leukemia 17(2):290–297 72. Oehler VG, Gooley T, Snyder DS et al (2007) The effects of imatinib mesylate treatment before allogeneic transplantation for chronic myeloid leukemia. Blood 109(4):1782–1789

Chapter 5  The Role of Allogeneic Hematopoietic Stem Cell Transplantation  73. Jabbour E, Cortes J, Kantarijian H et al (2007) Novel tyrosine kinase inhibitor therapy before allogeneic stem cell transplantation in patients with chronic myeloid leukemia: no evidence for increased transplant-related toxicity. Cancer 110(2):340–344 74. Deininger M, Schleuning M, Greinix H et al (2006) The effect of prior exposure to imatinib on transplant-related mortality. Haematologica 91(4):452–459 75. Lee SJ, Kukreja M, Wang T et al (2008) Impact of prior imatinib mesylate on the outcome of hematopoietic cell transplantation for chronic myeloid leukemia. Blood 112(8):3500–3507 76. Weisser M, Schmid C, Schoch C et al (2005) Resistance to pretransplant imatinib therapy may adversely affect the outcome of allogeneic stem cell transplantation in CML. Bone Marrow Transplant 36(11):1017–1018 77. Jabbour E, Cortes J, Kantarjian HM et al (2006) Allogeneic stem cell transplantation for patients with chronic myeloid leukemia and acute lymphocytic leukemia after BCR-ABL kinase mutation-related imatinib failure. Blood 108(4): 1421–1423 78. Marin D, Kaeda J, Szydlo R et al (2005) 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 19(4):507–512 79. Kaeda J, O'Shea D, Szydlo RM et al (2006) Serial measurement of BCR-ABL transcripts in the peripheral blood after allogeneic stem cell transplantation for chronic myeloid leukemia: an attempt to define patients who may not require further therapy. Blood 107(10):4171–4176 80. Goldman JM, Sobocinski KA, Zhang MJ et al (2006) Long-term outcome after allogeneic hematopoietic cell transplantation (HCT) for CML. Bio Blood Marrow Transpl 12(2):17 81. Olavarria E, Ottmann OG, Deininger M et al (2003) Response to imatinib in patients who relapse after allogeneic stem cell transplantation for chronic myeloid leukemia. Leukemia 17(9):1707–1712 82. Kantarjian HM, O'Brien S, Cortes JE et al (2002) Imatinib mesylate therapy for relapse after allogeneic stem cell transplantation for chronic myelogenous leukemia. Blood 100(5):1590–1595 83. DeAngelo DJ, Hochberg EP, Alyea EP et al (2004) Extended follow-up of patients treated with imatinib mesylate (Gleevec) for chronic myelogenous leukemia relapse after allogeneic transplantation: during cytogenetic remission and conversion to complete donor chimerism without graft-versus-host disease. Clin Cancer Res 10(15):5065–5071 84. Ullmann AJ, Hess G, Kolbe K et al (2003) Current result of the use of imatinib mesylate in patients with relapsed Philadelphia chromosome positive leukemia after allogeneic or syngeneic hematopoietic stem cell transplantation. Keio J Med 52(3):182–188 85. Kim YJ, Kim DW, Lee S et al (2004) Cytogenetic clonal evolution alone in CML relapse post-transplantation does not adversely affect response to imatinib mesylate treatment. Bone Marrow Transplant 33(3):237–242 86. Savani BN, Montero A, Kurlander R et al (2005) Imatinib synergizes with donor lymphocyte infusions to achieve rapid molecular remission of CML relapsing after allogeneic stem cell transplantation. Bone Marrow Transplant 36(11): 1009–1015 87. Simula MP, Markel S, Fozza C et al (2007) Response to donor lymphocyte infusions for chronic myeloid leukemia is dose-dependent: the importance of escalating the cell dose to maximize therapeutic efficacy. Leukemia 21(9):943–948 88. Carpenter PA, Snyder DS, Flowers ME et al (2007) Prophylactic administration of imatinib after hematopoietic stem cell transplantation for high-risk Philadelphia chromosome-positive leukemia. Blood 109(7):2791–2793

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R.T. Maziarz 89. Olavarria E, Siddique S, Griffiths MJ et al (2007) Posttransplantation imatinib as a strategy to postpone the requirement for immunotherapy in patients undergoing reduced-intensity allografts for chronic myeloid leukemia. Blood 110(13):4614–4617 90. Baccarani M, Saglio G, Goldman J et al (2006) Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. Blood 108(6):1809–1820 91. Jabbour E, Kantarjian H, O'Brien S et al (2006) Sudden blastic transformation in patients with chronic myeloid leukemia treated with imatinib mesylate. Blood 107(2):480–482 92. O'Dwyer ME, Mauro MJ, Blasdel C et al (2004) Clonal evolution and lack of cytogenetic response are adverse prognostic factors for hematologic relapse of chronic phase CML patients treated with imatinib mesylate. Blood 103(2):451–455 93. Deininger MW, Cortes J, Paquette R et al (2007) The prognosis for patients with chronic myeloid leukemia who have clonal cytogenetic abnormalities in Philadelphia chromosome-negative cells. Cancer 110(7):1509–1519 94. Kovacsovics T, Maziarz RT (2006) Philadelphia chromosome positive acute lymphoblastic leukemia: impact of imatinib treatment on remission induction and allogeneic stem cell transplantation. Curr Onc Rep 8:343–351 95. Holowiecki J, Giebel S, Wojnar J et al (2008) Treosulfan and fludarabine lowtoxicity conditioning for allogeneic haemotopoietic stem cell transplantation in chronic myeloid leukaemia. Br J Haematol 142(2):284–292 96. Mohty M, Szydlo RM, Yong AS et al (2008) Association between BMI-1 expression, acute graft-versus-host disease and outcome following allogeneic stem cell transplantation from HLA-identical siblings in chronic myeloid leukemia. Blood 112(5):2163–2166 97. Van der Meer A, Schaap NP, Schattenberg AV et al (2008) KIR2D55 is associated with leukemia free survival after HLA identical stem cell transplantation in chronic myeloid leukemia patients. Mol Immunol 45:3631–3638 98. Yong AS, Keyvanfar K, Eniafe R et al (2008) Hematopoietic stem cells and progenitors of chronic myeloid leukemia express leukemia-associated antigens: implications for the graft-versus-leukemia effect and peptide vaccine-based immunotherapy. Leukemia 22(9):1721–1727 99. Gordon MK, Sher D, Karrison T et al (2008) Successful autologous stem cell collection in patients with chronic myeloid leukemia in complete cytogenetic response, with quantitative measurement of BCR-ABL expression in blood, marrow, and apheresis products. Leuk Lymphoma 49(3):531–537 100. Olavarria E (2007) Autologous stem cell transplantation in chronic myeloid leukemia. Semin Hematol 44(4):252–258 101. CML Autograft Trials Collaboration (2007) Autologous stem cell transplantation in chronic myeloid leukemia: a meta-analysis of six randomized trials. Cancer Treat Rev 33(10):39–47 102. Gould SJ (1977) Ever since Darwin: Reflections in natural history. W.W. Norton & Company, New York 103. Gould SJ (1980) The Panda's thumb. W. W. Norton & Company, New York

Chapter 6 Allogeneic Transplantation for Hodgkin’s Lymphoma William Broderick and Patrick Stiff

1.  Introduction There are only approximately 7,500 cases of Hodgkin’s lymphoma diagnosed in the United States annually. While there is a bimodal distribution in incidence, the majority of cases occur in young adults 15–30 years of age [1]. Initial therapy is highly successful with progression-free survival (PFS) rates at 10 years of 70–90% for early stage disease and 60–70% for advanced disease. For those relapsing after initial therapy, 30–50% are long-term survivors after an autologous stem cell transplant, making this one of the most curable adult malignancies. However, a small minority of these typically young and otherwise healthy patients will relapse even after an autologous stem cell transplantation (ASCT) and it is for these rare patients that allogeneic transplantation has been increasingly considered. Initially discarded in the early 1990s as being too toxic utilizing myeloablative conditioning, allogeneic transplantation for this disease is increasing again based on the availability of reduced intensity regimens and therapies that have decreased mortality due to regimen-related toxicities, graft versus host disease (GVHD), and opportunistic infections. Whether or not this will be an effective approach for multiply resistant patients remains to be determined, making this one of the most controversial areas in 2008 in allogeneic transplantation. This chapter outlines modern therapy for this group of lymphomas and the potential utility of allogeneic transplantation in select patients.

2.  Conventional Therapy Initial therapy for patients with Hodgkin’s lymphoma is based primarily on stage, with early stage disease (stage I and II) without high risk features (B symptoms, bulky adenopathy, age >50 years) treated with radiation alone (stage I disease) or, short course chemotherapy combined with localized radiation therapy (stage II disease). Radiation alone in the past was usually reserved for patients with stage IA or IIA disease confirmed by laparotomy, however, From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_6, © Springer Science + Business Media, LLC 2003, 2010

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as PET scanning has recently replaced laparotomy as a staging tool, those with localized disease and a negative PET scan can receive mantle radiation with or without periaortic fields and expect a complete response (CR) >95%. Of these, approximately 70% of patients obtain a long-term remission and >90% of patients are alive 10 years from diagnosis [2]. To decrease relapses in early stage disease, the combination of abbreviated course chemotherapy with involved field radiation therapy (IFRT) is being increasingly used. Several studies have demonstrated that 2–4 cycles of ABVD chemotherapy followed by IFRT induces response rates >90% with 2–3 year follow up [2–4]. Patients presenting early stage disease with high risk features require systemic chemotherapy for optimal outcome. Patients with mediastinal masses >10 cm generally receive both full-course chemotherapy and IFRT. The addition of radiation to residual disease after chemotherapy, has also been shown to convert partial remissions (PR) in many to durable CRs with again a long-term survival of 70% [3, 4]. Patients with Stage III and IV disease require chemotherapy for optimal outcome. MOPP (nitrogen mustard, vincristine, procarbazine, prednisone) was developed for use in Hodgkin’s disease in the 1960s and achieved high response rates and long-term survival rates of 50–60%. ABVD (doxorubicin, bleomycin, vinblastine DTIC) demonstrated activity, first in MOPP resistant patients, then as a first-line regimen with less gonadal toxicity and a lower risk of secondary malignancies than MOPP. It still remains the standard regimen for advanced Hodgkin’s disease in adults with CR rates reaching 70% (Table  6-1.) [5–7]. Failure-Free Survival rates (FFS) of 65%, and overall survival (OS) reaching 89% at 3 years. There is general agreement that IFRT even for responders is needed in the 25–30% of patients who are present with mediastinal masses >1/3 of the chest diameter. Studies have also shown that IFRT is effective in consolidating PRs as well [8].

Table 6-1.  Selected results of standard therapy for newly diagnosed Hodgkin’s disease. Stage

Treatment

Freedom from treatment failure

Long-term survival (%)

Early stage [4]

EFRT alone

67% (7 years)

92

ABVD + EFRT

88% (7 years)

94

ABVD

81% (5 years)

90

ABVD + IFRT

86% (5 years)

97

ABVD + EFRT

91.4% (10 years)

90.4

EBVM + EFRT

80% (10 years)

90.3

MOPP + EFRT

62.8% (7 years)

67.9

ABVD + EFRT

82.8% (7 years)

77.4

ABVD

63% (5 years)

82

MOPP/ABV

66% (5 years)

81

IA–IIIA [3] IA–IIIB [6] Advanced stage [5] IIIA, IIIB, IV [7]

Early stage: stage I or II disease, no bulky disease Advanced stage: stage III or IV disease, bulky stage II disease EFRT extended field radiation

Chapter 6  Allogeneic Transplantation for Hodgkin’s Lymphoma 

77

3.  High-Dose Therapy with Autologous Stem Cell Transplantation For those who relapse after an initial chemotherapy-based treatment regimen, ASCT is generally the considered standard of care, particularly for those who relapse early. Patients who relapse >12 months after an initial CR, however, may be treated with combination chemotherapy and if they achieve a second CR, 30–50% will remain disease-free at 4 years without any further therapy [9, 10]. Most relapsing patients do so less than 12 months after a CR, and for these patients and those without a CR after initial therapy, standard dose second-line therapy is not likely to lead to a durable remission. In fact, longterm PFS is 0% for this group (Table 6-2.) [6]. For these patients, high-dose chemotherapy with ASCT has emerged as the treatment of choice. Overall 30–50% of such patients are alive and disease-free for 5+ years suggesting superiority of this approach for otherwise eligible patients. Several randomized trials have been performed to validate the efficacy of this approach. In 1993, the British National Lymphoma Investigation (BNLI) group published a 3-year event-free survival rate of 53% compared to 10% in their standard combination chemotherapy arm [11]. There was no difference in OS, but the follow up appeared not to have been long enough to demonstrate a survival benefit. The German Hodgkin’s Lymphoma Study Group (GHSG) in conjunction with the European Bone Marrow Transplant Registry (EBMTR) randomized heavily treated patients with relapse to chemotherapy or high-dose chemotherapy with ASCT [12]. They demonstrated a 55%

Table 6-2.  Results of autologous bone marrow transplant in Hodgkin’s disease. Importance of risk factors. Outcome by # of high risk features Study

n

Reece [13]

  58

High risk features

0

1

2

3+

(1) B-symptoms

3 years PFS 100%

3 years PFS 81%

3 years PFS 40%

3 years PFS 0%

43 months EFS 27%

43 months EFS 10%

3 years FFP 41%

3 years FFP 50 associated with increased TRM compared to age 50 had comparable survival to younger patients

No difference in OS, DFS, GVHD by age group. Patients >45 with advanced leukemia had increased TRM

Age >50 associated with increased GVHD, TRM, and decreased OS

Comments

CML chronic myelogenous leukemia, AML acute myelogenous leukemia, MDS myelodysplastic syndrome, MSD matched sibling donor, MUD matched unrelated donor, GVHD graft versus host disease, TRM treatment related mortality, OS overall survival, DFS disease free survival

Year

Author (reference)

Table 9-1.  Myeloablative allogeneic transplantation for patients above age 50.

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treated for various hematologic malignancies were analyzed, including 389 patients above age 50. Age above 50 was found to be independently associated with TRM and decreased overall survival. Estimated 1-year TRM was 34.7 and 22.7% in the older and younger groups, respectively. The probability of overall survival at 4 years was 35.6% for patients over 50 and 53.5% for those younger than 50.

5.  Potential Barriers to Allogeneic Transplantation in Older Adults There are multiple potential barriers to successful allogeneic transplantation in older adults. These include concerns relate to the toxicity of the preparative regimens, GVHD, donor availability, and patient selection. Optimizing the preparative regimens to maximize efficacy but minimize morbidity is critical to successful transplantation in older adults. There are no prospective studies comparing preparative regimens in older adults. Retrospective data suggest that regimens containing total body radiation may increase TRM in this population [26]. RIC regimens designed to minimize the toxicity of myeloablation have been explored in older adults and will be discussed in the next section. GVHD represents another potential barrier to transplantation in the elderly and has been identified as a major contributing factor to transplant-related mortality in older adults [19]. Increasing age has been identified as a risk factor for development of acute GVHD [27, 28]. Analysis of 2,036 recipients of HLA-identical sibling transplants from the IBMTR demonstrated an increased risk for acute GVHD in older patients. The age gradient was modest and the association was no longer significant after excluding female-to-male transplants [27]. Similar findings were reported by Weisdorf et  al. using an age cutoff of 18 while analyzing 469 patients with histocompatible sibling donors at a single institution [28]. The question of whether increasing age poses an incremental increased risk is unclear. One study supports an increased risk of acute GVHD in adults >50 years of age compared to younger adults [23] while others do not [20–22]. There is some evidence, however, that age is associated with increased severity of acute GVHD [29]. Several studies have also documented an increase in chronic GVHD associated with increasing age [30–32]. Analysis of 2,534 recipients of HLA-identical sibling transplants conducted by the IBMTR showed that the strongest risk factor for chronic GVHD was a history of acute GVHD [30]. When patients with a history of acute GVHD were excluded, age over 20 years became an independent risk factor. Again the question of incremental risk with increasing age remains unclear. There are conflicting results from single institution studies regarding the impact of age >50 years on risk of chronic GVHD [20–23]. A study from Aschan et  al. of 182 leukemia patients reported that adults >30 years benefited more significantly from double GVHD prophylaxis than younger patients with decreased chronic GVHD and improved survival [33]. Continued advances in GVHD prophylaxis will likely improve transplant outcomes in older adults. Donor availability represents another potential obstacle in allogeneic transplantation for older adults. The possibility of finding a matched family donor

Chapter 9  The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults 

tends to be lower in older adults due to aging of the family. Matched sibling donors are also likely to be older which is associated with an increased risk of developing acute GVHD and potentially increased TRM [34, 35]. Additional evidence regarding the significance of increased donor age comes from the National Marrow Donor Program. Analysis of 6,978 unrelated-donor marrow transplantations showed that increasing donor age was independently associated with increased risk of GVHD and decreased overall survival [36]. Optimal donor selection for older adults is unclear and may require investigation of the potential trade-off between use of older sibling grafts and younger matched unrelated grafts. Advances in HLA matching using high-resolution typing result in improved survival in unrelated donor transplantation [37] and may translate into improved options and outcomes for older adults. Finally, patient selection remains a major obstacle in myeloablative transplantation for older adults. There are two issues related to patient selection to consider in this context. The first relates to disease status and the second relates to the fitness of the individual patient. It is clear from retrospective studies that transplantation in earlier stages of disease results in superior outcomes for adults >50 years of age [19, 21]. One study of 215 adults >50 years of age who underwent myeloablative allogeneic transplantation for either early (41%) or advanced (59%) hematologic malignancies reported significantly decreased TRM and improved overall survival in the early stage group [19]. The timing of transplantation remains an unresolved issue particularly in the setting of multiple myeloma where multiple treatment options are now available. Finally, a systematic approach to the assessment of biologic age has not yet been developed making selection of “fit” older adults a challenge in the clinical setting. Older adults represent a heterogeneous population. They are more likely to present with diagnosed comorbid disease [5, 38, 39] or may have subclinical changes in organ function resulting in decreased physiologic reserve. Age-related changes in drug metabolism may also impact toxicity risk. The development of evidence-based patient selection algorithms to identify older adults who are most likely to tolerate and benefit from allogeneic transplantation is critical to successful application of this modality to an older population.

6.  Reduced Intensity Conditioning Transplantation RIC or non-myeloablative allogeneic transplantation approaches were designed to achieve engraftment without marrow ablation. This method may be efficacious in settings where the graft versus tumor effect is sufficient to eradicate or control underlying disease. Due to the non-myeloablative approach this method has been investigated in patients who would have been considered ineligible for traditional myeloablative transplantation including older adults. Data from the CIBMTR demonstrate that the majority of RIC transplants reported in 2005–2006 were performed in adults >50 years of age (Fig. 9-3). Multiple retrospective studies have compared outcomes between standard myeloablative conditioning and RIC regimens. Overall there appears to be a decrease in TRM and in the incidence of GVHD favoring the reduced intensity regimens despite increased comorbidity in many of the RIC patients [40–50]. The difference in overall survival appears less clear due in part to an increased risk of relapse seen with RIC particularly in the setting of leukemia [41, 44].

133

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H.D.Age Klepin and D.D.ofHurd Distribution Patients Receiving Allogeneic Transplants by Conditioning Regimen Intensity, 2005-2006

60 years of age. Additional negative prognostic factors included prior autologous transplant and Eastern Cooperative Oncology Group Performance Score >1. Alternatively, Corradini et  al. compared outcomes of 90 patients younger than 55 years with 60 patients older than 60 years who were treated with RIC transplantation from a sibling donor [55]. There was no difference in TRM or overall survival between the two groups. However, in the subset of patients who had a prior autologous transplant, older age was associated with increased TRM. Overall, RIC regimens may offer a viable alternative to myeloablative transplantation in selected older adults.

7.  Improving Patient Selection Older patients with hematologic malignancies represent a very heterogeneous population requiring more detailed assessment of health status prior to treatment

Chapter 9  The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults 

135

determination. Eligibility criteria remain a central issue in future trials focused on older adults particularly with regard to allogeneic transplantation. Chronologic age alone is a poor surrogate marker for tolerance to therapy. Developing a set of measurable clinical characteristics which better reflect physiologic age is necessary to critically evaluate the role of allogeneic transplantation in the heterogeneous older adult population. Comorbidity and functional status represent measurable patient-specific characteristics that can refine evaluation of older adults in clinical trials and practice. Older adults are more likely to present with increased comorbid disease [5, 7]. Comorbidity assessment with the Charlson Comorbidity Index has been shown to be predictive of increased toxicity and mortality in allogeneic transplantation [49, 57]. Sorror et al. refined this index to improve sensitivity and documented the reliability and validity of this tool in allogeneic transplantation (Table 9-2) [58]. The new transplant-specific index showed better survival prediction than the Charlson Comorbidity Index in this population. Low scores on the transplant-specific comorbidity index appear predictive of improved survival in both myeloablative and non-myeloablative transplants and in patients with both low and high risk disease [59]. Similarly, Artz et al.

Table 9-2.  Hematopoietic cell transplantation (HCT)-specific comorbidity index (HCT–CI). Comorbidity

Definition

Score

Cardiac

Coronary artery disease, congestive heart failure, myocardial infarction, EF £ 50%

1

Arrhythmia

Atrial fibrillation or flutter, sick sinus or ventricular arrhythmia

1

Cerebrovascular disease

Transient ischemic attack or cerebrovascular accident

1

Diabetes

Requiring treatment with medication

1

Inflammatory bowel disease

Crohn’s disease or ulcerative colitis

1

Obesity

Body mass index >35 kg/m2

1

Infection

Requiring use of antimicrobial treatment

1

Psychiatric disturbance

Depression or anxiety requiring psychiatric consult or treatment

1

Peptic ulcer

Requiring treatment

2

Hepatic disease (mild)

Chronic hepatitis, bilirubin > ULN to 1.5 X the ULN, or AST/ ALT > ULN to 2.5 X ULN

2

Pulmonary (moderate)

DLCO and or FEV1 66–80% or dyspnea with slight activity

2

Rheumatologic

SLE, RA, polymyositis, mixed CTD, or polymyalgia rheumatica

2

Renal (moderate/severe)

Serum creatinine >2 mg/dL, on dialysis, or prior renal transplant

2

Prior solid tumor

Treated at any point in patient’s past history, excluding nonmelanoma

3

Hepatic (moderate/severe)

Liver cirrhosis, bilirubin >1.5X ULN, or AST/ALT > 2.5 X ULN

3

Heart valve disease

Except mitral valve prolapse

3

Severe pulmonary

DLCO and/or FEV1 £65% or dyspnea at rest or requiring oxygen

3 Total score

This table was adapted from Ref. [58] EF ejection fraction, ULN upper limit of normal, SLE systemic lupus erythmatosis, RA rheumatoid arthritis, CTD connective tissue disease, DLCO diffusion capacity of carbon monoxide

136 

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reported that a simple scale combining the Kaplan–Feinstein Comorbidity Scale and the Eastern Cooperative Oncology Group Performance Status Scale enabled separation of high and low risk patients with 6-month cumulative incidences of TRM of 50 and 15%, respectively [60]. Prospective assessment of comorbidity using established or transplant-specific indices may provide important information for the development of evidence-based risk stratification in older adults evaluated for allogeneic transplantation. Comorbidity assessment alone may not provide sufficient information regarding the physiologic reserve of older patients. In clinical practice it is apparent that older patients with similar age and comorbidity may differ substantially with regard to functional status. Specific measures of functional status can provide more prognostic information than comorbidity alone in older adults [61]. In older cancer patients, Extermann et al. demonstrated that comorbidity and functional assessment were not well correlated and provided independent information [62]. Careful functional assessment during pretransplant evaluation may provide added information for risk stratification. It will be important to prospectively assess the predictive value of task-specific functional assessment tools such as activities of daily living [63] and instrumental activities of daily living [64]. These self-report measures are able to identify functional impairment in cancer patients with good performance scores on the Eastern Cooperative Oncology Group scale [65]. This added discriminatory capacity may be useful to detect subtle changes reflective of decreased functional reserve. Finally, objective measures of physical performance and cognition may be particularly useful in developing an evaluation protocol for older patients being considered for allogeneic transplantation. Physical performance measures such as walking speed and lower extremity function are predictive of future disability, hospitalizations, and mortality in the geriatric population [66–68]. These objective measures may be sensitive to subclinical disability which could be closely associated with morbidity and mortality outcomes in the setting of allogeneic transplantation.

8.  Applications in Multiple Myeloma Despite the increased use of dose-intensive therapy and autologous transplantation in older patients with multiple myeloma, there remains a dearth of information on the use of allogeneic transplantation in this patient population. Refined assessment of physiologic age may facilitate the evaluation of allogeneic transplantation techniques in the treatment of older adults with multiple myeloma. There is recent evidence which supports a potential role for RIC allogeneic transplantation in multiple myeloma treatment. A prospective study of 162 consecutive patients (median age 55; range 30–65 years) compared RIC allogeneic transplantation from a sibling donor with tandem autologous transplantation in newly diagnosed multiple myeloma [69]. All patients received vincristine, doxorubicin, and dexamethasone followed by high-dose melphalan with autologous stem cell rescue. After recovery, patients with an

Chapter 9  The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults 

HLA-identical sibling underwent non-myeloablative allogeneic transplantation. Patients without an HLA-matched sibling underwent a second autologous transplant. Overall survival was 80 months versus 54 months (p = 0.01) favoring the allograft group. Among patient who completed their assigned treatments, TRM was not significantly different between the two groups while disease-related mortality was higher in the double autologous transplant group. These findings suggest that allogeneic transplantation may offer longer term disease control for selected fit patients with multiple myeloma. However, this conclusion should be interpreted with caution due to the disproportionately poor outcomes seen in the tandem autologous transplant arm compared to other published studies. It is currently unclear which older adults might benefit from allogeneic transplantation and where transplantation fits in the sequencing of treatment options currently available. Additional clinical trials are underway evaluating tandem autologous transplantation followed by RIC transplantation for multiple myeloma. Similarly, the BMT Clinical Trials Network Protocol 0102 is a trial of tandem autologous stem cell transplants versus a single autologous stem cell transplant followed by a matched sibling non-myeloablative allogeneic stem cell transplant in patients up to the age of 70 (with a Karnofsky performance status of ³70). All autologous transplants utilize melphalan 200 mg/m2 while the RIC allogeneic transplant gives only a single fraction of 200  cGy of total body irradiation. GVHD prophylaxis is cyclosporine and mycophenolate mofetil. The primary objective of this study is to compare progression-free survival at 3 years between the two strategies. Approximately 600 subjects have been enrolled on this study, including 150 allografts, which should provide representative data to help define the role of RIC allogeneic transplantation for fit adults up to age 70 years with multiple myeloma. While these studies begin to address the role of RIC transplantation in the younger, older patients with multiple myeloma, the majority of patients with this disease will still be excluded secondary to age (no studies for patients >70 years of age) and the lack of a suitable sibling donor for the majority of patients.

9.  Future Directions Allogeneic transplantation may offer a potentially curative treatment option for selected older adults with hematologic malignancies including multiple myeloma. However, substantial concerns regarding treatment toxicity persist which will continue to limit application of this treatment modality in the true elderly or frail population. Future advances in this field will need to build upon a better understanding of the relationship between physiologic aging and allogeneic transplantation rather than relying on chronologic age cutoffs in research and practice. Future research goals include (1) prospective evaluation of elderly specific transplantation strategies; (2) development of refined patient selection criteria which incorporate comorbidity, physical function, and cognition; and (3) incorporation of additional outcome measures into clinical trials to evaluate transplantation in the context of its impact on quality of life and disability as well as overall survival (Table 9-3).

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Table 9-3.  Future research directions for allogeneic transplantation in older adults. Research goal

Topics for investigation

Development of elderly specific transplantation regimens

Reduced intensity conditioning regimens Optimal donor selection (sibling vs. younger matched unrelated) Optimal GVHD prophylaxis Optimal timing of transplantation

Development and validation of an elderly specific patient selection algorithm

Transplant-specific comorbidity assessment Predictive value of self-report functional status (e.g., Activities of daily living, instrumental activities of daily living) Predictive value of baseline physical performance measures (e.g., walking speed, lower extremity strength/balance) Evaluation of cognitive assessment

Incorporation of additional outcome measures into clinical trials

Quality of life measures Disability assessment

References 1. SEER Cancer Statistics Review 1975–2003. http://seer.cancer.gov/publications/ 2006 2. Kyle RA, Gertz MA, Witzig TE, Lust JA, Lacy MQ, Dispenzieri A et al (2003) Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc 78(1):21–33 3. Blade J, Munoz M, Fontanillas M, San Miguel J, Alcala A, Maldonado J et  al (1996) Treatment of multiple myeloma in elderly people: Long-term results in 178 patients. Age Ageing 25(5):357–361 4. Clavio M, Casciaro S, Gatti AM, Spriano M, Bonanni F, Poggi A et  al (1996) Multiple myeloma in the elderly: Clinical features and response to treatment in 113 patients. Haematologica 81(3):238–244 5. Rodon P, Linassier C, Gauvain JB, Benboubker L, Goupille P, Maigre M et  al (2001) Multiple myeloma in elderly patients: Presenting features and outcome. Eur J Haematol 66(1):11–17 6. Nilsson T, Hoglund M, Lenhoff S, Rylander L, Turesson I, Westin J et al (2003) A pooled analysis of karyotypic patterns, breakpoints and imbalances in 783 cytogenetically abnormal multiple myelomas reveals frequently involved chromosome segments as well as significant age- and sex-related differences. Br J Haematol 120(6):960–969 7. Mileshkin L, Prince HM (2005) The adverse prognostic impact of advanced age in multiple myeloma. Leuk Lymphoma 46(7):951–966 8. Cohen HJ, Bartolucci A (1985) Age and the treatment of multiple myeloma. Southeastern Cancer Study Group experience. Am J Med 79(3):316–324 9. Jantunen E, Kuittinen T, Penttila K, Lehtonen P, Mahlamaki E, Nousiainen T (2006) High-dose melphalan (200  mg/m2) supported by autologous stem cell transplantation is safe and effective in elderly (>or  =  65 years) myeloma patients: Comparison with younger patients treated on the same protocol. Bone Marrow Transplant 37(10):917–922 10. Palva IP, Ahrenberg P, Ala-Harja K, Almqvist A, Hanninen A, Ilvonen M et  al (1989) Treatment of multiple myeloma in old patients. Finnish Leukaemia Group. Eur J Haematol 43(4):328–331 11. Wisloff F, Andersen P, Andersson TR, Brandt E, Eika C, Fjaestad K et al (1991) Has the incidence of multiple myeloma in old age been underestimated? The myeloma project of health region I in Norway. Eur J Haematol 47(5):333–337

Chapter 9  The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults  12. Barlogie B, Kyle RA, Anderson KC, Greipp PR, Lazarus HM, Hurd DD et  al (2006) Standard chemotherapy compared with high-dose chemoradiotherapy for multiple myeloma: Final results of phase III US Intergroup Trial S9321. J Clin Oncol 24(6):929–936 13. Frassoni F, Labopin M, Gluckman E, Prentice HG, Vernant JP, Zwaan F et  al (1996) Results of allogeneic bone marrow transplantation for acute leukemia have improved in Europe with time – a report of the acute leukemia working party of the European group for blood and marrow transplantation (EBMT). Bone Marrow Transplant 17(1):13–18 14. Goldman JM, Gale RP, Horowitz MM, Biggs JC, Champlin RE, Gluckman E et al (1988) Bone marrow transplantation for chronic myelogenous leukemia in chronic phase. Increased risk for relapse associated with T-cell depletion. Ann Intern Med 108(6):806–814 15. Thomas ED, Clift RA, Fefer A, Appelbaum FR, Beatty P, Bensinger WI et  al (1986) Marrow transplantation for the treatment of chronic myelogenous leukemia. Ann Intern Med 104(2):155–163 16. Clift RA, Appelbaum FR, Thomas ED (1993) Treatment of chronic myeloid leukemia by marrow transplantation. Blood 82(7):1954–1956 17. de la Camara R, Alonso A, Steegmann JL, Arranz R, Granados E, RodriguezMacias G et  al (2002) Allogeneic hematopoietic stem cell transplantation in patients 50 years of age and older. Haematologica 87(9):965–972 18. Deeg HJ, Shulman HM, Anderson JE, Bryant EM, Gooley TA, Slattery JT et  al (2000) Allogeneic and syngeneic marrow transplantation for myelodysplastic syndrome in patients 55 to 66 years of age. Blood 95(4):1188–1194 19. Ditschkowski M, Elmaagacli AH, Trenschel R, Steckel NK, Koldehoff M, Beelen DW (2006) Myeloablative allogeneic hematopoietic stem cell transplantation in elderly patients. Clin Transplant 20(1):127–131 20. Du W, Dansey R, Abella EM, Baynes R, Peters WP, Klein J et al (1998) Successful allogeneic bone marrow transplantation in selected patients over 50 years of age – a single institution’s experience. Bone Marrow Transplant 21(10):1043–1047 21. Farag SS, Elder PJ, Marcucci G, Penza S, Mrozek E, Molina A et  al (2003) Radiation-free regimens result in similar outcomes of allogeneic hematopoietic progenitor cell transplantation in patients aged >or  =  50 years compared to younger adults with low-risk disease. Bone Marrow Transplant 31(2):87–93 22. Hansen JA, Gooley TA, Martin PJ, Appelbaum F, Chauncey TR, Clift RA et  al (1998) Bone marrow transplants from unrelated donors for patients with chronic myeloid leukemia. N Engl J Med 338(14):962–968 23. Klingemann HG, Storb R, Fefer A, Deeg HJ, Appelbaum FR, Buckner CD et  al (1986) Bone marrow transplantation in patients aged 45 years and older. Blood 67(3):770–776 24. Wallen H, Gooley TA, Deeg HJ, Pagel JM, Press OW, Appelbaum FR et al (2005) Ablative allogeneic hematopoietic cell transplantation in adults 60 years of age and older. J Clin Oncol 23(15):3439–3446 25. Ringden O, Horowitz MM, Gale RP, Biggs JC, Gajewski J, Rimm AA et al (1993) Outcome after allogeneic bone marrow transplant for leukemia in older adults. JAMA 270(1):57–60 26. Yanada M, Emi N, Naoe T, Sakamaki H, Iseki T, Hirabayashi N et  al (2004) Allogeneic myeloablative transplantation for patients aged 50 years and over. Bone Marrow Transplant 34(1):29–35 27. Gale RP, Bortin MM, van Bekkum DW, Biggs JC, Dicke KA, Gluckman E et  al (1987) Risk factors for acute graft-versus-host disease. Br J Haematol 67(4):397–406 28. Weisdorf D, Hakke R, Blazar B, Miller W, McGlave P, Ramsay N et al (1991) Risk factors for acute graft-versus-host disease in histocompatible donor bone marrow transplantation. Transplantation 51(6):1197–1203 29. Nash RA, Pepe MS, Storb R, Longton G, Pettinger M, Anasetti C et  al (1992) Acute graft-versus-host disease: Analysis of risk factors after allogeneic marrow

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H.D. Klepin and D.D. Hurd transplantation and prophylaxis with cyclosporine and methotrexate. Blood 80(7):1838–1845 30. Atkinson K, Horowitz MM, Gale RP, van Bekkum DW, Gluckman E, Good RA et al (1990) Risk factors for chronic graft-versus-host disease after HLA-identical sibling bone marrow transplantation. Blood 75(12):2459–2464 31. Carlens S, Ringden O, Remberger M, Lonnqvist B, Hagglund H, Klaesson S et al (1998) Risk factors for chronic graft-versus-host disease after bone marrow transplantation: A retrospective single centre analysis. Bone Marrow Transplant 22(8):755–761 32. Ochs LA, Miller WJ, Filipovich AH, Haake RJ, McGlave PB, Blazar BR et  al (1994) Predictive factors for chronic graft-versus-host disease after histocompatible sibling donor bone marrow transplantation. Bone Marrow Transplant 13(4):455–460 33. Aschan J, Ringden O (1994) Prognostic factors for long-term survival in leukemic marrow recipients with special emphasis on age and prophylaxis for graft-versushost disease. Clin Transplant 8(3 Pt 1):258–270 34. Doney K, Fisher LD, Appelbaum FR, Buckner CD, Storb R, Singer J et al (1991) Treatment of adult acute lymphoblastic leukemia with allogeneic bone marrow transplantation. Multivariate analysis of factors affecting acute graft-versus-host disease, relapse, and relapse-free survival. Bone Marrow Transplant 7(6):453–459 35. Mehta J, Gordon LI, Tallman MS, Winter JN, Evens AM, Frankfurt O et al (2006) Does younger donor age affect the outcome of reduced-intensity allogeneic hematopoietic stem cell transplantation for hematologic malignancies beneficially? Bone Marrow Transplant 38(2):95–100 36. Kollman C, Howe CW, Anasetti C, Antin JH, Davies SM, Filipovich AH et al (2001) Donor characteristics as risk factors in recipients after transplantation of bone marrow from unrelated donors: The effect of donor age. Blood 98(7):2043–2051 37. Lee SJ, Klein J, Haagenson M, Baxter-Lowe LA, Confer DL, Eapen M et al (2007) High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood 110(13):4576–4583 38. Popplewell LL, Forman SJ (2002) Is there an upper age limit for bone marrow transplantation? Bone Marrow Transplant 29(4):277–284 39. Shapira MY, Tsirigotis P, Resnick IB, Or R, Abdul-Hai A, Slavin S (2007) Allogeneic hematopoietic stem cell transplantation in the elderly. Crit Rev Oncol Hematol 64(1):49–63 40. Alyea EP, Kim HT, Ho V, Cutler C, Gribben J, DeAngelo DJ et  al (2005) Comparative outcome of nonmyeloablative and myeloablative allogeneic hematopoietic cell transplantation for patients older than 50 years of age. Blood 105(4):1810–1814 41. Aoudjhane M, Labopin M, Gorin NC, Shimoni A, Ruutu T, Kolb HJ et al (2005) 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 19(12):2304–2312 42. Couriel DR, Saliba RM, Giralt S, Khouri I, Andersson B, de Lima M et al (2004) Acute and chronic graft-versus-host disease after ablative and nonmyeloablative conditioning for allogeneic hematopoietic transplantation. Biol Blood Marrow Transplant 10(3):178–185 43. Diaconescu R, Flowers CR, Storer B, Sorror ML, Maris MB, Maloney DG et  al (2004) Morbidity and mortality with nonmyeloablative compared with myeloablative conditioning before hematopoietic cell transplantation from HLA-matched related donors. Blood 104(5):1550–1558 44. Martino R, Iacobelli S, Brand R, Jansen T, van Biezen A, Finke J et  al (2006) Retrospective comparison of reduced-intensity conditioning and conventional

Chapter 9  The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults  high-dose conditioning for allogeneic hematopoietic stem cell transplantation using HLA-identical sibling donors in myelodysplastic syndromes. Blood 108(3):836–846 45. Massenkeil G, Nagy M, Neuburger S, Tamm I, Lutz C, le Coutre P et  al (2005) Survival after reduced-intensity conditioning is not inferior to standard high-dose conditioning before allogeneic haematopoietic cell transplantation in acute leukaemias. Bone Marrow Transplant 36(8):683–689 46. Mielcarek M, Martin PJ, Leisenring W, Flowers ME, Maloney DG, Sandmaier BM et al (2003) Graft-versus-host disease after nonmyeloablative versus conventional hematopoietic stem cell transplantation. Blood 102(2):756–762 47. Perez-Simon JA, Diez-Campelo M, Martino R, Brunet S, Urbano A, Caballero MD et al (2005) Influence of the intensity of the conditioning regimen on the characteristics of acute and chronic graft-versus-host disease after allogeneic transplantation. Br J Haematol 130(3):394–403 48. Shimoni A, Hardan I, Shem-Tov N, Yeshurun M, Yerushalmi R, Avigdor A et al (2006) Allogeneic hematopoietic stem-cell transplantation in AML and MDS using myeloablative versus reduced-intensity conditioning: The role of dose intensity. Leukemia 20(2):322–328 49. Sorror ML, Maris MB, Storer B, Sandmaier BM, Diaconescu R, Flowers C et al (2004) Comparing morbidity and mortality of HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative and myeloablative conditioning: Influence of pretransplantation comorbidities. Blood 104(4):961–968 50. Valcarcel D, Martino R, Sureda A, Canals C, Altes A, Briones J et  al (2005) Conventional versus reduced-intensity conditioning regimen for allogeneic stem cell transplantation in patients with hematological malignancies. Eur J Haematol 74(2):144–151 51. Bertz H, Potthoff K, Finke J (2003) Allogeneic stem-cell transplantation from related and unrelated donors in older patients with myeloid leukemia. J Clin Oncol 21(8):1480–1484 52. Falda M, Busca A, Baldi I, Mordini N, Bruno B, Allione B et  al (2007) Nonmyeloablative allogeneic stem cell transplantation in elderly patients with hematological malignancies: Results from the GITMO (Gruppo Italiano Trapianto Midollo Osseo) multicenter prospective clinical trial. Am J Hematol 82(10):863–866 53. Kroger N, Shimoni A, Zabelina T, Schieder H, Panse J, Ayuk F et  al (2006) Reduced-toxicity conditioning with treosulfan, fludarabine and ATG as preparative regimen for allogeneic stem cell transplantation (alloSCT) in elderly patients with secondary acute myeloid leukemia (sAML) or myelodysplastic syndrome (MDS). Bone Marrow Transplant 37(4):339–344 54. Tsirigotis P, Bitan RO, Resnick IB, Samuel S, Ackerstein A, Eladi S et al (2006) A non-myeloablative conditioning regimen in allogeneic stem cell transplantation from related and unrelated donors in elderly patients. Haematologica 91(6):852–855 55. Corradini P, Zallio F, Mariotti J, Farina L, Bregni M, Valagussa P et  al (2005) Effect of age and previous autologous transplantation on nonrelapse mortality and survival in patients treated with reduced-intensity conditioning and allografting for advanced hematologic malignancies. J Clin Oncol 23(27):6690–6698 56. Gomez-Nunez M, Martino R, Caballero MD, Perez-Simon JA, Canals C, Mateos MV et al (2004) Elderly age and prior autologous transplantation have a deleterious effect on survival following allogeneic peripheral blood stem cell transplantation with reduced-intensity conditioning: Results from the Spanish multicenter prospective trial. Bone Marrow Transplant 33(5):477–482 57. Alamo J, Shahjahan M, Lazarus HM, de Lima M, Giralt SA (2005) Comorbidity indices in hematopoietic stem cell transplantation: A new report card. Bone Marrow Transplant 36(6):475–479 58. Sorror ML, Maris MB, Storb R, Baron F, Sandmaier BM, Maloney DG et al (2005) Hematopoietic cell transplantation (HCT)-specific comorbidity index: A new tool for risk assessment before allogeneic HCT. Blood 106(8):2912–2919

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H.D. Klepin and D.D. Hurd 59. Sorror ML, Sandmaier BM, Storer BE, Maris MB, Baron F, Maloney DG et  al (2007) Comorbidity and disease status based risk stratification of outcomes among patients with acute myeloid leukemia or myelodysplasia receiving allogeneic hematopoietic cell transplantation. J Clin Oncol 25(27):4246–4254 60. Artz AS, Pollyea DA, Kocherginsky M, Stock W, Rich E, Odenike O et al (2006) Performance status and comorbidity predict transplant-related mortality after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 12(9):954–964 61. Inouye SK, Peduzzi PN, Robison JT, Hughes JS, Horwitz RI, Concato J (1998) Importance of functional measures in predicting mortality among older hospitalized patients. JAMA 279(15):1187–1193 62. Extermann M, Overcash J, Lyman GH, Parr J, Balducci L (1998) Comorbidity and functional status are independent in older cancer patients. J Clin Oncol 16(4):1582–1587 63. Katz S (1983) Assessing self-maintenance: Activities of daily living, mobility, and instrumental activities of daily living. J Am Geriatr Soc 31(12):721–727 64. Lawton MP, Brody EM (1969) Assessment of older people: Self-maintaining and instrumental activities of daily living. Gerontologist 9(3):179–186 65. Repetto L, Fratino L, Audisio RA, Venturino A, Gianni W, Vercelli M et al (2002) Comprehensive geriatric assessment adds information to Eastern Cooperative Oncology Group performance status in elderly cancer patients: An Italian Group for Geriatric Oncology Study. J Clin Oncol 20(2):494–502 66. Cesari M, Kritchevsky SB, Penninx BW, Nicklas BJ, Simonsick EM, Newman AB et al (2005) Prognostic value of usual gait speed in well-functioning older people – results from the Health, Aging and Body Composition Study. J Am Geriatr Soc 53(10):1675–1680 67. Guralnik JM, Ferrucci L, Simonsick EM, Salive ME, Wallace RB (1995) Lowerextremity function in persons over the age of 70 years as a predictor of subsequent disability. N Engl J Med 332(9):556–561 68. Studenski S, Perera S, Wallace D, Chandler JM, Duncan PW, Rooney E et  al (2003) Physical performance measures in the clinical setting. J Am Geriatr Soc 51(3):314–322 69. Bruno B, Rotta M, Patriarca F, Mordini N, Allione B, Carnevale-Schianca F et al (2007) A comparison of allografting with autografting for newly diagnosed myeloma. N Engl J Med 356(11):1110–1120

Chapter 10 Single Versus Tandem Autologous Hematopoietic Stem Cell Transplant in Multiple Myeloma David H. Vesole

1.  Single Autologous Transplantation It has been known for over 20 years, that high-dose therapy with melphalan can produce a profound anti-myeloma effect. Initially introduced to help overcome the native resistance of myeloma cells to conventional chemotherapy, highdose therapy requiring autologous hematopoietic stem cell support was first evaluated in patients with refractory disease [1]. This resulted in improved response rates and overall survival (OS). This approach was then extended to newly diagnosed patients. In 1996, the seminal randomized study on symptomatic stage II and III patients by the French Myeloma Intergroup, showed conclusively that high-dose therapy yielded a superior disease-free and OS outcome compared to conventional therapy [2]. The 5-year projected survival for the transplant group was 52 versus 12% for the standard therapy (SDT) group (Table 10-1). Whereas, complete responses were observed in only 5% of the conventional therapy group, 22% of the high-dose therapy group achieved complete remissions (CR). The transplant-related mortality was only 2.7% in the setting of bone marrow transplant without hematopoietic stem cell growth factors. Other randomized and nonrandomized comparisons have also demonstrated that high-dose therapy is superior to conventional chemotherapy [3–5]; this included a second large randomized trial by the Medical Research Council Myeloma VII trial also showing an improvement in median eventfree survival (EFS) and OS of approximately 12 months [3]. In an Arkansas study comparing tandem hematopoietic stem cell transplantation (HSCT) to conventional VAD chemotherapy, patients achieving complete response had a median disease-free survival of 50 months and median OS of more than 7 years [4]. Based on these results, multiple myeloma is currently the most common indication for HSCT in North America, with over 5,000 transplants performed yearly (Center for International Blood and Marrow Transplant Research [CIBMTR] estimates). Indeed, national and international guidelines consider upfront HSCT in transplant eligible patients as one of the standard treatment options for newly diagnosed myeloma (http://www.nccn.org).

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_10, © Springer Science + Business Media, LLC 2003, 2010

143

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Table 10-1.  Randomized trials of conventional chemotherapy compared to single ASCT as upfront therapy. Author

Group

N

Age years

CR% SDT vs. HSCT

p

Criteria for defining CR

OS benefit for HSCT

Comments

Attal et al. [2]

IFM90

200

£65

  5 vs. 22

S

Electrophoresis

Significant benefit

BMT. No GF. TRM 2.7%

Child et al. [3]

MRC7

401

£64

  8 vs. 44

S

EBMT– IBMTR (IF)

Significant benefit

TRM 3%

Blade et al. [6]

PETHEMA

216

£65

11 vs. 30

S

EBMT– IBMTR (IF)

No OS/EFS benefit

Only PR and CR included

Barlogie et al. [7]

USIG

899

90% reduction in paraprotein) to the first transplant. Additional randomized clinical trials from Italy (Bologna 96), Netherlands (HOVON 24), Germany (GMMG-HD2), and France (MAG 95) have compared double intensive therapy to single intensive therapy (Table  10-2) [25–28].

147

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D.H. Vesole

Table 10-2.  Randomized trials of single vs. double autologous ASCT. Age Conditioning regimen CR% single EFS% single OS% single limit first → second vs. double vs. double vs. double

Author

N

IFM 94 [23]

399 60

Mel140 + TBI → Mel 140 42 vs. 50

25 vs. 30*

48 vs. 58*

Follow-up 75M

HOVON 24 [25]

304 65

56M

Mel 140 → Cy 120 + TBI 13 vs. 32*

21 vs. 22*

55 vs. 50

GMMG HD2 [26] 261 65

Mel 200 → Mel 200

23 vs. 29*

No difference NR

MAG 95 [27]

227 55

Mel140 + CCNU/VP16/ 39 vs. 37 CY-TBI vs. MEL 140 → Mel 140 + TBI

31 vs. 33

49 vs. 73**

53M

Bologna 96 [24]

228 60

Mel 200 → Mel 120 + BU 12

35 vs. 48

22 vs. 35*

59 vs. 73

55M

Tunisia [28]

195 60

Mel 200 → Mel 200 vs. Mel 200 → *thal 100×6 mo

68 vs. 54*

85 vs. 57*

85 vs. 65*

33M

NR

*Statistically significant difference (p 12 months remission duration and normal beta-2 microglobulin [30]. Unfortunately, there are no randomized studies that compare a salvage second transplant at relapse to a planned upfront tandem second transplant. The European Bone Marrow Transplant Registry (EBMT) published a registry-based analysis suggesting the performance of a second planned autologous transplant before relapse and within 6–12 months of the first transplant resulted in superior outcomes [36]. The Arkansas group performed serial landmark analyses to define the optimal timing of a planned second transplant in their original total therapy patient population [20]. EFS and OS both were longer among the patients who had received a second transplant within 13 months (85% of second transplants had been completed at that time) compared with the others receiving their second cycle of high-dose therapy later or not at all. A later analysis with a larger (1,000 patients) group undergoing melphalan-based tandem transplants suggested that timely application of a second transplant was significant for EFS and OS [37].

6.  Maintenance Therapy Following Transplant Interferon, a mainstay for maintenance therapy after transplantation, has been shown to be ineffective in the United States Intergroup S9321 trial [8]. Although corticosteroids have been shown to improve EFS following conventional therapy, there has not been a clinical trial to evaluate their efficacy post-HSCT [38, 39]. Other maintenance strategies are outlined in Table 10-3 [45–51]. Thalidomide, an effective agent in MM for relapsed disease and as induction therapy, has been evaluated as maintenance therapy post-transplant. Three randomized studies have demonstrated the benefit of thalidomide, either as a single agent or in combination, as maintenance therapy after autologous transplantation [22, 40, 41]. In a French trial, thalidomide maintenance improved the 3-year EFS compared to observation (52 vs. 36%) and 4-year OS (87 vs. 77%) [40]. Of note, only those patients who did not achieve at least a very good partial remission were the patients who benefited from thalidomide maintenance. Similar findings were observed in the Tunisian study [29]. An Arkansas trial showed superior CR rates (62 vs. 43%) and 5-year EFS with thalidomide (56 vs. 44%) but no improvement in OS [22]. The preliminary report by the Australian ALLG MM6 study of thalidomide plus prednisone for 12 months versus observations post-HSCT also showed an improvement in PFS (90 vs. 69%, p = 0.005) and OS (91 vs. 80%, p = 0.21) although this was observed until beyond the first year from transplant [41]. Recently, BMT CTN completed a large trial of over 500 autologous transplant recipients randomized to either thalidomide plus dexamethasone (for 1 year) or observation post-tandem HSCT. The results of these trials

Chapter 10  Single Versus Tandem Autologous Hematopoietic Stem Cell 

151

Table 10-3.  Maintenance after primary myeloma therapy. Study

N

Maintenance

PFS (months)

OS

US Intergroup Trial S9321 [7]

249

IFN

25 vs. 21 (p = 0.05)

58 vs. 53 (p = 0.8)

Cunningham [45]

  84

IFN

NSa

NSa

EBMT [46]

892

IFN

29 vs. 20 (p = 0.006)

78 vs. 47 (p = 0.007)

HOVON-50b [47]

128

IFN

13.5 vs. 8.5 (p = 0.04)

41 vs. 38.4 NSa

SWOGb [48]

125

Pred

14 vs. 5 (0.03)

37 vs. 26 (p = 0.05)

NCIC MY7b [49]

307

Dex

33 vs. 23

46.3 vs. 43.8

Arkansas TT2 vs. TT1 [50]

668 (TT2) 231 (TT1)

IFN

50 vs. 25 (p 30, non-T cell, poor-risk cytogenetics, no CR at day 35 b Donor vs. No-donor analysis (chemotherapy only-this arm was superior to autograft)

reduced relapse rate in allograft patients, it is surprising that many haematologists and even transplanters deny or underrate this effect [29, 31]. Harnessing the allogeneic GvL effect and using the positive benefits of acute and chronic GVHD are essential for curing the high-risk patient or patients with positive MRD prior to transplant. Passweg and colleagues showed 10 years ago that patients with acute, chronic, or both acute and chronic GVHD had a 2.5fold reduction in relapse risk on multivariate analysis (RR = 0.40) [4]. Other studies have shown that chronic GVHD has more of an effect than acute GVHD. Some forms of T cell depletion prevent grade II–IV acute GVHD but there may still be a significant incidence of chronic GVHD; this may be a strategy worth further investigation. Further evidence will come from trials of RIC allografting where there is greater reliance on the GVL effect. There are no large-scale mature data available, but the CIBMTR will be analysing the outcome of >200 RIC allografts for ALL in 2009.

3.  Conditioning Regimens There are no randomised studies comparing conditioning regimens and many large studies have missed opportunities to compare regimens. There are very few data on non-TBI-containing regimens and the survival data does not recommend their use. The City of Hope and Stanford groups have a 20 year experience with etoposide (60 mg/kg) and 13.2 Gy of total body irradiation given in 9 fractions and have excellent survival data [5]. Marks and colleagues from the CIBMTR compared this regimen with standard cyclophosphamide and TBI. Cyclophosphamide and 12  Gy of TBI produced markedly inferior survival compared to etoposide-containing or higher-dose TBI regimens [6]. However, if >13 Gy TBI was given, etoposide/ TBI was not superior to cyclophosphamide/TBI. Transplant-related mortality was (perhaps surprisingly) not higher in the etoposide TBI arm although this is undoubtedly a more toxic regimen. The issue of mucositis will be discussed later in this chapter.

4.  Sibling Allografting in First Remission There are methodological difficulties establishing the efficacy of allograft in adults with ALL. A simple comparison of outcome in patients who received an allograft with those who did not would not suffice, as the allograft group

Chapter 13  Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia in Adults 

195

are on average fitter and had to survive a certain time in remission in order to have an allograft. To overcome these biases, donor vs. no-donor analyses were devised. These too are flawed in that they may underestimate the potential benefit of allograft as many patients with matched sibling donors do not proceed to allograft and indeed in many it was never the intention to do so. Nonetheless, they have become widely accepted and if they do show an advantage one can be confident that such a difference exists [7]. Sibling allografting has long been established as the therapy of choice in high risk ALL (Hoelzer criteria). Two French studies compared allografting with chemotherapy and autografting, and there were significant differences in overall survival [21]. A meta-analysis support this [30]. The results of the recent very large international ALL study have provided clarity. All patients 35 years. The donor arm is 6% better at 5 years but the difference was not significant. There was still excellent protection against relapse (35 vs. 67%) but survival was not improved because of a high non-relapse mortality (29% at 1 year and 39% at 2 years). The investigators concluded that if the allogeneic effect could be harnessed more safely in this group allograft in high-risk patients with ALLl in CR1 might be worth pursuing. The TRM in low-risk (younger) patients was a disappointing 20% at 2 years; improving this should also be the focus of research efforts.

5.  Unrelated Donor SCT in First Remission Encouraging results of sibling allografting and studies showing that UD SCT can produce similar results to sibling allografts for leukaemia have lead to many investigating the role of UD SCT in high-risk CR1 ALL (Table 13-2). Marks and colleagues from the CIBMTR recently described 169 adult patients with a median age of 33 years who underwent UD SCT. One hundred and fifty-seven were at highrisk and 93 had multiple high-risk factors. Overall survival was 40%. Multivariate analysis showed that the following factors affected survival: WCC at diagnosis, HLA mismatch, >8 weeks to CR1 and T cell depletion. This latter finding was a surprise and as there were only Table 13-2.  Studies of the outcome of unrelated donor stem cell transplantation for adult patients with acute lymphoblastic leukemia in CR1. Author

Patient no

Age (years)

Survival (%)

TRM

Grade 2–4 acute/chronic (%)

Marks [22]

169

33

39

42%

50/43

Dahlke [8]

  38

23

44

NK

36/23

Kiehl [7]

  45

29

45

NK

33/NK

NK not known or not specifically stated

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16 patients with T cell depletion (with a variety of techniques) it cannot be regarded as a definite finding. Patel and colleagues have analysed 55 patients (median age 25 years) who had T-depleted UD SCT for high-risk ALL in CR1 mainly using in vivo alemtuzamab (Patel et al. in press). About half of these patients were taken out of UKALL XII to have an “off-protocol” allograft. Survival was an excellent 59% at 3 years and there was a clear plateau with no events after 2 years. The incidences of grade II–IV, grade III–IV and chronic GVHD were 25, 7 and 22%, respectively. It is difficult to compare the two series but the UK series had a low TRM (19%) and good survival, albeit in a younger population. Kiehl and colleagues from Germany reported 97 patients, 87 of whom received TCD, who had a TRM of 31% and grades III–IV acute GVHD in only 15% [8]. On a similar note, Dahlke and colleagues compared sibling and unrelated donor allografts in 38 and 46 patients in CR1, respectively and found that survival was the same in the 2 groups (44 vs. 46%, p = NS) [9]. Unrelated donor SCT clearly has a growing role in this disease. The promising results from (albeit) limited data and the finding that survival is now similar to allografts with sibling donors make it reasonable to perform prospective trials of this therapeutic modality. However, patients should be entered in studies so that we learn how to optimise this procedure and the data do not support the use of unrelated donor SCT as standard therapy for ALL in CR1.

6.  The Role of RIC Allografting We have been slow to investigate this transplant modality in adult ALL. The mistaken notion that the GvL effect was less important in this disease and the view that conditioning regimens had to contain TBI may have led to this attitude. Consequently, it has initially been performed in patients who could not tolerate myeloablative conditioning because of comorbidity or in elderly patients who have little prospect of cure with chemotherapy (Table 13-3). There are no large scale prospective studies of RIC allografting in this disease. The data with us are relatively small retrospective series of patients with various stem cell sources and heterogeneous disease states. The largest series from the European Blood and Marrow Transplantation Group (EBMT), reported in 2008 by Mohty and colleagues describes 97 patients, 70 of whom had died at the time of analysis [10]. Two-thirds received stem cells from sibling Table 13-3.  Studies of the outcome of reduced intensity conditioning allogeneic stem cell transplantation for adult ALL. Author a

Patient no

Sibs/UDs (%)

CR1/CR2/other (%)

Regimens

Survival

Mohty  [9]

97

67/33

29/26/45

Various

21% at 2 year

Martino [23]

27

56/44

15/41/44

Various

31% at 2 year

Massenkeil [24]

 9

NK

NK

Flu/Bu/ATG

40% at 3 year

Forman [25]

22

33/67

48/19/33

Flu/Mel

77% at 1 year

Hamaki [26]

33

20/13

19/0/14

Flu/Bu/ATG

30% EFS 1 year

a

41% Philadelphia positive. Survival was 52% at 2 years if patient was in CR1 Reported transplant-related mortality ranged from 4 to 27% Flu fludarabine, Bu busulfan, ATG antithymocyte globulin, Mel melphalan, UD unrelated donor

Chapter 13  Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia in Adults 

donor and one-third from unrelated donors. Survival at 2 years was 21% in the whole group but promisingly exceeded 50% in those with high-risk disease. Patients in CR1 did significantly better with 52% surviving disease free at 2 years. Patients with chronic GVHD had superior OS (RR 0.4, p 5% shift in whole blood chimerism) was in the absence of intervention associated with universal relapse [16].

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Chimerism was monitored very intensively in their study, weekly in the first year. Their intervention was a program of escalating DLI which resulted in about a third of such patients surviving. Acute GVHD (of some grade) were seen in 31% of such patients. This approach deserves evaluation in an adult population. Some RIC regimens result in a high incidence of mixed chimerism and it is standard practice to correct this mixed chimerism with DLI but there are no data available to assess if this approach reduces relapse.

9.  Allografting for Refractory Disease About 10% of adult patients with ALL are refractory to primary chemotherapy and a very small percentage of these patients can be cured by allogeneic SCT either with a sibling or unrelated donor. Similarly, some patients who relapse fail to respond to reinduction chemotherapy. These patients are often extremely unwell due to prolonged neutropenia and severe infections. The author knows of few such patients who have survived and prefers to attempt to achieve remission with novel agents such as clofarabine, nelarabine or monoclonal antibodies prior to a curative allograft.

10.  Haploidentical and Cord Blood Transplantation for High Risk ALL Patients with ALL, who have a low chance of cure with chemotherapy but have no sibling or suitably matched unrelated donor, are candidates for allografts with haploidentical or cord blood stem cells. The data are small scale and heterogeneous, and come from a small number of centres of excellence. Aversa described 62 patients with ALL transplanted in remission who had 25% event-free survival [17]. TRM was substantial but there was no chronic GVHD, so longer-term quality of life was good. Survival data for patients purely with ALL cannot be gleaned from the reports from Henslee Downey’s group in South Carolina but TRM was seen in 15 of 49 patients and acute and chronic GVHD in about 15%. The data for cord blood transplant is limited. Many studies do not separate this disease from other diseases. Rocha on behalf of Eurocord reported 98 patients (34% in CR1) who achieved 36% 2 year survival and a 26% incidence of grade II–IV acute GVHD and 30% incidence of chronic GVHD [18]. The Minneapolis group have some excellent survival data in small number of adults with ALL but these data cannot be used to make decisions for individual patients. Further larger-scale studies are required, and infection and slow engraftment remain major hurdles.

11.  Other Issues and Supportive Care 11.1.  CNS Disease Patients with CNS disease at diagnosis have an inferior outcome to those without CNS disease (29 vs. 39% survival, p  =  0.03). However, they can achieve long-term DFS with a sibling allograft. In the Lazarus study, 11 of

Chapter 13  Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia in Adults 

25 such patients remain alive 21–102 months post-allograft. The issue of whether the dose of cranial radiotherapy that is part of TBI is sufficient for the control of CNS disease remains uncertain. CNS prophylaxis is a major issue for RIC allografting and additional therapy (such as post-transplant intrathecal injections) should be contemplated but there is no evidence to inform practice. 11.2.  Palifermin Grade 4 mucositis is a major problem with VP/TBI allografts with cyclosporin and minidose prophylaxis. Typical patients have severe symptoms requiring prolonged narcotic analgesia and frank bleeding from the mouth. This may prevent the delivery the four doses of methotrexate which, in turn, may affect the chance of acute and chronic GVHD. Some investigators have used mycophenolate mofetil but data showing this to be as effective as methotrexate are lacking. Palifermin (keratinocyte growth factor 1), which has level-one evidence after chemotherapy for autologous SCT has been the subject of phase I studies [19]. 11.3.  The Future Allografting for adults for ALL is currently too toxic and the TRM is too high. Exploration of reduced intensity conditioning in clinical trials will determine whether this is a viable approach and will answer the biologic question of how important conditioning is in obtaining a negative MRD status before the effects of an allogeneic GvL effect. It seems likely that patients with positive MRD prior to transplant and those with resistant disease will not be cured by less conditioning. TBI conditioning may be made less toxic by drugs such as palifermin and velafermin and this may improve the outcome. Selecting the right patients for allogeneic SCT is also a major issue. Gene profiling may add to our abilities to discriminate using the existing prognostic factors. Further trials are needed to determine if allografting can overcome the adverse prognostic impact of biologic factors such as a high WCC and adverse cytogenetics. If allografting can do this, the use of allografting will expand if unrelated donor allografting can be safely performed on a multicentre basis. If it can, then there will be exploration of the use of cord blood as a stem cell source but, again, this is mainly performed in certain specialist centres and there are no data to suggest that it can safely be “rolled out” to large number of transplant centres worldwide (Table 13-4).

Table 13-4.  Likely future developments. • Better selection of patients (who will benefit from allograft) • Increased role for reduced intensity allografting, particularly in older patients • Expanded role for alternative donor allografting including cord blood SCT • Recombinant keratinocyte growth factors to mitigate toxicity of TBI

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References 1. Goldstone AH, Richards SM, Lazarus HM, Tallman MS, Buck G, Fielding AK, Burnett AK, Chopra R, Wiernik PH, Foroni L, Paietta E, Litzow MR, Marks DI, Durrant J, McMillan A, Franklin IM, Luger S, Ciobanu N, Rowe JM (2008) In adults with standard-risk acute lymphoblastic leukemia, the greatest benefit is achieved from a matched sibling allogeneic transplantation in first complete remission, and an autologous transplantation is less effective than conventional consolidation/ maintenance chemotherapy in all patients: final results of the International ALL Trial (MRC UKALL XII/ECOG E2993). Blood 111:1827–1833 2. Moorman AV, Harrison CJ, Buck GA, Richards SM, Secker-Walker LM, Martineau M, Vance GH, Cherry AM, Higgins RR, Fielding AK, Foroni L, Paietta E, Tallman MS, Litzow MR, Wiernik PH, Rowe JM, Goldstone AH, Dewald GW, Adult Leukaemia Working Party, Medical Research Council/National Cancer Research Institute (2007) Karyotype is an independent prognostic factor in adult acute lymphoblastic leukemia (ALL): Analysis of cytogenetic data from patients treated on the Medical Research Council (MRC) UKALLXII/Eastern Cooperative Oncology Group (ECOG) 2993 trial. Blood 109:3189–3197 3. Brüggemann M, Raff T, Flohr T, Gökbuget N, Nakao M, Droese J, Lüschen S, Pott C, Ritgen M, Scheuring U, Horst HA, Thiel E, Hoelzer D, Bartram CR, Kneba M, German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (2006) Clinical significance of minimal residual disease quantification in adult patients with standard-risk acute lymphoblastic leukemia. Blood 107:1116–1123 4. Passweg JR, Cahn J-Y, Tiberghien P, Vowels MR, Camitta BM, Gale RP, Herzig RH, Hoelzer D, Horowitz MM, Ifrah N, Klein JP, Marks DI, Ramsey NKC, Rowlings PA, Weisdorf DJ, Zhang M-J, Barrett AJ (1998) Graft versus leukaemia effect in T-lineage and cALLa+ (B-lineage) acute lymphoblastic leukaemia. Bone Marrow Transplant 21:153–158 5. Snyder DS, Chao NJ, Amylon MD, Taguchi J, Long GD, Negrin RS, Nademanee AP, O'Donnell MR, Schmidt GM, Stein AS et al (1993) Fractionated total body irradiation and high-dose etoposide as a preparatory regimen for bone marrow transplantation for 99 patients with acute leukemia in first complete remission. Blood 82:2920–2928 6. Marks DI, Aversa F, Lazarus H (2006) Alternative donors transplants for adult acute lymphoblastic leukaemia: A comparison of the three major options. Bone Marrow Transplant 38:467–475 7. Frassoni F (2000) Randomised studies in acute myeloid leukaemia: The double truth. Bone Marrow Transplant 25:471–473 8. Kiehl MG, Kraut L, Schwerdtfeger R et al (2004) Outcome of allogeneic hematopoietic stem-cell transplantation in adult patients with acute lymphoblastic leukemia: No difference in related compared with unrelated transplant in first complete remission. J Clin Oncol 22:2816–2825 9. Dahlke J, Kröger N, Zabelina T, Ayuk F, Fehse N, Wolschke C, Waschke O, Schieder H, Renges H, Krüger W, Kruell A, Hinke A, Erttmann R, Kabisch H, Zander AR (2006) Comparable results in patients with acute lymphoblastic leukemia after related and unrelated stem cell transplantation. Bone Marrow Transplant 37:155–163 10. Mohty M, Labopin M, Tabrizzi R et  al (2008) Reduced intensity conditioning allogeneic stem cell transplantation for adults with acute lymphoblastic leukaemia: A retrospective study of the European BMT group. Haematologica 93:303–306 11. Stein A, O’Donnell M, Parker P, Nademanee A, Falk P, Rosenthal J, Palmer J, Tsai N, Forman S (2007) Reduced-intensity stem cell transplantation for high-risk acute lymphoblastic leukemia. Biol Blood Marrow Transplant 13:134

Chapter 13  Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia in Adults  12. Fielding AK, Richards SM, Chopra R, Lazarus HM, Litzow M, Buck G, Durrant IJ, Luger SM, Marks DI, McMillan AK, Tallman MS, Rowe JM, Goldstone AH (2006) Outcome of 609 adults after relapse of acute lymphoblastic leukaemia (ALL); an MRC UKALL12/ECOG 2993 study. Blood 113:4489–96, 2009 13. Kolb H-J, Mackinnon S (2004) Adoptive cellular immunotherapy for treatment or prevention of relapse of hematologic malignancy posttransplant. In: Atkinson K (ed) Chapter 65 in clinical bone marrow and blood stem cell transplantation, vol 3. Cambridge University Press, New York, pp 992–1008 14. Tomblyn M, Lazarus HM (2008) Donor lymphocyte infusions: The long and winding road: How should it be traveled? Bone Marrow Transplant 42:569–78, 2008 15. Shaw BE, Mufti GJ, Mackinnon S, Cavenagh JD, Pearce RM, Towlson KE, Apperley JF, Chakraverty R, Craddock CF, Kazmi MA, Littlewood TJ, Milligan DW, Pagliuca A, Thomson KJ, Marks DI, Russell NH (2008) Outcome of second allogeneic transplants using reduced-intensity conditioning following relapse of haematological malignancy after an initial allogeneic transplant. Bone Marrow Transplant 42:783–9, 2008 16. Bader P, Kreyenberg H, Hoelle W, Dueckers G, Handgretinger R, Lang P, Kremens B, Dilloo D, Sykora KW, Schrappe M, Niemeyer C, Von Stackelberg A, Gruhn B, Henze G, Greil J, Niethammer D, Dietz K, Beck JF, Klingebiel T (2004) Increasing mixed chimerism is an important prognostic factor for unfavorable outcome in children with acute lymphoblastic leukemia after allogeneic stem-cell transplantation: possible role for pre-emptive immunotherapy? J Clin Oncol 22:1696–1705 17. Aversa F (2008) Haploidentical haematopoietic stem cell transplantation for acute leukemia in adults: experience in Europe and the United States. Bone Marrow Transplant 41:473–481 18. Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A, Jacobsen N, Ruutu T, de Lima M, Finke J, Frassoni F, Gluckman E (2004) Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 351:2276–2285 19. Spielberger R, Stiff P, Bensinger W, Gentile T, Weisdorf D, Kewalramani T, Shea T, Yanovich S, Hansen K, Noga S, McCarty J, LeMaistre CF, Sung EC, Blazar B R, Elhardt D, Chen MG, Emmanouilides C (2004) Palifermin for oral mucositis after intensive therapy for hematologic cancers. N Engl J Med 351:2590–2598 20. Attal M, Blaise D, Marit G, Payen C, Michallet M, Vernant JP, Sauvage C, Troussard X, Nedellec G, Pico J et al (2005) 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 86:1619–1628 21. Hunault M, Harousseau JL, Delain M, Truchan-Graczyk M, Cahn JY, Witz F, Lamy T, Pignon B, Jouet JP, Garidi R, Caillot D, Berthou C, Guyotat D, Sadoun A, Sotto JJ, Lioure B, Casassus P, Solal-Celigny P, Stalnikiewicz L, Audhuy B, Blanchet O, Baranger L, Béné MC, Ifrah N, GOELAMS (Groupe Ouest-Est des Leucémies Airguës et Maladies du Sang) Group (2004) Better outcome of adult acute lymphoblastic leukemia after early genoidentical allogeneic bone marrow transplantation (BMT) than after late high-dose therapy and autologous BMT: A GOELAMS trial. Blood 104:3028–3037 22. Marks DI, Pérez WS, He W, Zhang MJ, Bishop MR, Bolwell BJ, Bredeson CN, Copelan EA, Gale RP, Gupta V, Hale GA, Isola LM, Jakubowski AA, Keating A, Klumpp TR, Lazarus HM, Liesveld JL, Maziarz RT, McCarthy PL, Sabloff M, Schiller G, Sierra J, Tallman MS, Waller EK, Wiernik PH, Weisdorf DJ (2008) Unrelated donor transplants in adults with Philadelphia-negative acute lymphoblastic leukemia in first complete remission. Blood 112:426–434

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D. I. Marks 23. Martino R, Giralt S, Caballero MD, Mackinnon S, Corradini P, Fernández-Avilés F et  al (2003) Allogeneic hematopoietic stem cell transplantation with reducedintensity conditioning in acute lymphoblastic leukemia: A feasibility study. Haematologica 88:555–560 24. Massenkeil G, Nagy M, Neuburger S, Tamm I, Lutz C et al (2005) Survival after reduced-intensity conditioning is not inferior to standard high-dose conditioning before allogeneic haematopoietic cell transplantation in acute leukemias. Bone Marrow Transplant 36:683–689 25. Marks DI, Forman SJ, Blume KG, Perez WS, Weisdorf DJ, Keating A, Gale RP, Cairo MS, Copelan EA, Horan JT, Lazarus HM, Litzow MR, McCarthy PL, Schultz KR, Smith DD, Trigg ME, Zhang MJ, Horowitz MM (2006) A comparison of cyclophosphamide and total body irradiation with etoposide and total body irradiation as conditioning regimens for patients undergoing sibling allografting for acute lymphoblastic leukemia in first or second complete remissi. Biol Blood Marrow Transplant 12:438–453 26. Hamaki T, Kami M, Kanda Y, Yuji K, Inamoto Y, Kishi Y et al (2005) Reduced intensity stem-cell transplantation for acute lymphoblastic leukemia: A retrospective study of 33 patients. Bone Marrow Tranplant 35:549–556 27. Collins RH, Shpilberg O, Drobyski WR et al (2000) Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 15:433–444 28. Horowitz M, Gale RP, Sondel PM, Goldman JM, Kersey J, Kolb HJ et al (1990) Graft versus leukemia reactions after bone marrow transplantation. Blood 75: 555–562 29. Yanada M, Matsuo K, Suzuki T, Naoe T (2006) Allogeneic hematopoietic stem cell transplantation as part of post-remission therapy improves survival for adult patients with high-risk acute lymphoblastic leukemia: A meta-analysis. Cancer 106:2657–2663 30. Weiden PL, Flournoy N, Thomas ED et  al (1979) Antileukemic effect of graftversus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 300:1068–1073

Chapter 14 Allogeneic Transplantation for Myelodysplastic Syndromes Geoffrey L. Uy and John F. DiPersio

1.  Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders characterized by peripheral cytopenias and marrow dysplasia with a variable propensity to evolve into acute myeloid leukemia. MDS is primarily a disease of the elderly with the median age of 76 years at diagnosis and 86% of patients diagnosed over the age of 60 [1]. Although the true incidence is unknown, reports from Germany and the United States estimate the incidence of MDS to be approximately 3 to 4 per 100,000 [1, 2]. While the majority of cases of MDS arise de novo, exposure to alkylating agents and ionizing radiation during treatment for other conditions are important etiological factors. Compared to de  novo MDS, secondary MDS is associated with higher rates of adverse cytogenetics abnormalities, poor treatment response, and a worse overall prognosis [3, 4]. Currently, allogeneic stem cell transplantation is considered the only curable treatment modality for MDS. However, the advanced age and the associated comorbidities typical for this patient population have limited the utility of this approach for most patients.

2.  Classification and Prognostic Factors in MDS The French–American–British classification of 1982, recognizes five distinct subgroups of MDS: (1) refractory anemia (RA), (2) refractory anemia with ringed sideroblasts (RARS), (3) refractory anemia with excess blasts (RAEB), (4) refractory anemia with excess blasts in transformation (RAEB-T), and (5) chronic myelomonocytic leukemia (CMML). These subtypes are based on morphological features in the bone marrow and peripheral blood including the presence of ringed sideroblasts, bone marrow blasts, and peripheral blood monocytes [5].

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_14, © Springer Science + Business Media, LLC 2003, 2010

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Table 14-1.  Comparison of French American British (FAB) and World Health Organization (WHO) classifications of MDS. FAB category

WHO category

Definition

Refractory anemia (RA)

Refractory anemia (RA)

Dysplasia in erythroid series, 3.7 × 10(7) NC/kg, and matching at 4/6 or more HLA loci with each other and with the patient, were infused on the same day. Twenty-one patients received the GVHD prophylaxis of

Table 20-4.  Reduced intensity double cord blood transplantation in adults. Investigator (citation)

N

Conditioning regimen

Diseases

Median follow-up Disease-free (months) survival (%)

Brunstein et al. [41]

110

Fludarabine, cyclophospha- AML, ALL, MDS, mide, low dose TBI lymphoma

19

38

Ballen et al. [43]

  21

Fludarabine, melphalan, thymoglobulin

AML, ALL, MDS, lymphoma

18

55

Cutler et al. [42]

  32

Fludarabine, melphalan, thymoglobulin

AML, ALL, MDS, lymphoma

15

54

Chapter 20  Update on Umbilical Cord Blood Transplantation 

cyclosporine and mycophenolate mofetil, and 32 patients received sirolimus and tacrolimus. The median days to neutrophil engraftment were 21 days and median days to platelet engraftment to 20 × 109/l were 42 days. The 100-day transplant-related mortality was 12%. The risk of acute GVHD was less in the sirolimus/tacrolimus patients (10% vs. 40%). With a median follow-up of 20 months, the 1-year overall and disease-free survival was 74% and 59%, respectively. Double cord blood transplantation offers unique challenges in the interpretation of post transplantation chimerism, or the contribution of each cord blood donor and recipient to hematopoiesis. Brunstein reported the presence of only one unit contributing to hematopoiesis [41]. In our Boston experience, by Day +100, 72% of patients had hematopoiesis derived from a single cord blood unit [44]. A higher post-thaw nucleated cell count and CD34+ cell dose were associated with cord predominance; in 68% patients, the predominant cord blood unit was the first unit infused. The post-thaw CD34+ dose of the predominant unit predicted time to neutrophil and platelet engraftment. However, other programs have not reported an association between the order of infusion and predominant unit; the issues affecting the cord unit predominance remain unclear. The presence of only one cord contributing to long-term hematopoiesis raises the controversy over the importance of the second cord blood unit, and the benefit of double cord blood transplants in general. Verneris et  al. reported a lower relapse rate in patients who received double, rather than single, cord blood unit transplants, perhaps related to a stronger immunologic attack [45]. These questions have not been fully answered but the superior results of double cord blood transplantation, in comparison to historical controls receiving single cord blood units, have fostered the growth of double cord blood transplants, particularly in the heavier US population.

6. Other Strategies for Adult Cord Blood Transplantation Ex vivo expansion is another strategy to improve the infused progenitor cell doses, and decrease the risk of graft failure. Early attempts of ex vivo expansion failed to show improvement in engraftment, probably due to the expansion of mature cells. Shpall and colleagues expanded CD34+ cord blood cells with a mixture of stem cell factor, G-CSF, and megakaryocyte growth and differentiation factor [46]. The median time to neutrophil engraftment was 28 days and the overall survival was 37%, with a median follow-up of 30 months. Current strategies for expansion include targeting the Notch signaling pathway that is involved in cellular differentiation and proliferation; this approach was used by the Seattle group [47, 48]. MD Anderson has initiated a trial comparing two unmanipulated cord blood units with one unmanipulated and one expanded cord blood unit. The expanded cells are prepared from the CD133+ fraction and incubated with G-CSF, stem cell factor, and thrombopoietin [49]. The Spanish group had adopted a novel approach, infusing haploidentical peripheral blood stem cells and a single cord blood unit [50]. Twenty-seven patients received a myeloablative conditioning regimen followed by co-infusion of a cord blood unit and peripheral blood stem cells from a third party. Neutrophil engraftment occurred at a median of 10 days, usually from the stem cells, but hematopoiesis

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converted to cord blood origin in all patients. The incidence of acute Grades II–IV graft vs. host disease was 15%. The 4-year overall survival was 69%.

7. Unique Challenges of Cord Blood Transplantation The emergence of better expansion techniques may help to improve engraftment after adult cord blood transplantation, but infection and poor immune reconstitution remain significant concerns even with expanded cells or double cord blood transplantation. A review of infections in 100 recipients of pediatric and adult cord blood transplants revealed 221 infections in the first 100 days post transplant [51]. These infections included 22 fungal infections, 105 bacterial infections, and 62 viral infections, including adenovirus, respiratory synctial virus, and influenzae. There were four cases of CMV end organ disease. Late infections included CMV reactivation, varicella zoster, staphylococcal and pseudomonas bacteremia, aspergillus, and mycobacterium. A survey of 128 cord blood recipients revealed 14 cases of invasive fungal infection, 13 related to aspergillus, with a mortality rate of 86% [52].

8. Future Trends in Cord Blood Transplantation During the next 5–10  years, the availability and applications of cord blood transplantation will continue to increase. Future trials are needed to determine the best donor choice for those patients without a matched sibling donor. There may be indications for transplantation in situations where graft vs. host disease is intolerable or unnecessary, such as with older patients or patients with nonmalignant disease. There are potential exciting avenues for cord blood for non-hematopoietic diseases. Cord blood cells are a more primitive population than adult marrow cells, and have increased capacity for multi-lineage differentiation [53]. Cord blood cells have been shown to improve the neurologic recovery in rats with strokes [54]. Preliminary investigation has analyzed the use of autologous CB for autoimmune disease, particularly childhood diabetes. Haller and colleagues infused seven children with Type I diabetes with autologous CB; these children had lower hemoglobin A1c and fewer insulin requirement than a randomly selected control population of severe diabetic children [55]. Repair of damaged cardiac tissue is another exciting avenue for cord blood stem cells. In a mouse model, cord blood cells injected into the tail vein migrated to infarcted myocardial tissue and reduced the infarct size [56]. Cord blood cells express connexin and stromal cell-derived factor (SDF)-1 alpha, which are proteins that are important for cardiovascular regeneration [57]. Cord blood cells were injected into a rat myocardial infarction model. Apoptotic cells were decreased and cardiac function improved in rats that received cord blood as opposed to rats that received a mock injection [58]. The next 10 years should prove exciting for the field of cord blood transplantation. Improved results in adult transplantation and strategies for advancing immune reconstitution will extend the application of cord blood in the oncology setting. The use of cord blood for indications outside of oncology will likely develop as we learn more about the multiple applications of umbilical cord blood.

Chapter 20  Update on Umbilical Cord Blood Transplantation 

References 1. Knudtzon S (1974) In vitro growth of granulocytic colonies from circulating cells in human cord blood. Blood 43:357–361 2. Broxmeyer HE, Kurtzberg J, Gluckman E et al (1991) Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation. Blood Cells 17:313–329 3. Lansdorp PM, Dragowska W, Mayani H (1993) Ontogeny-related changes in proliferative potential of human hematopoietic cells. J Exp Med 178:787–791 4. Gluckman E, Broxmeyer HA, Auerbach AD (1989) Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical cord blood from an HLA identical sibling. N Engl J Med 321:1174–1178 5. Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lansdorp PM (1994) Evidence for a mitotic clock in human hematopoeitic stem cells: Loss of telomeric DNA with age. Proc Natl Acad Sci USA 91:9857–9860 6. Szabolcs P, Park KD, Reese M, Marti L, Broadwater G, Kurtzberg J (2003) Coexistant naïve phenotype and higher cycling rate of cord blood T cells compared to adult peripheral blood. Exp Hematol 31:708–714 7. Kim YJ, Brutkiewicz PR, Broxmeyer HE (2002) Role of 4–1BB (CD137) in the functional activation of cord blood CD28-CD8+ T cells. Blood 100:3253–3260 8. Berthou C, Legros-Maida S, Soulie A et  al (2003) Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes. Blood 102:4608 9. Bone Marrow Donors Worldwide, Annual Report 2006 10. Smythe J, Armitage S, McDonald D, Pamphilon D, Guttridge M, Brown J et  al (2007) Directed sibling cord blood banking for transplantation: The 10-year experience in the national blood service in england. Stem Cells 25:2087–2093 11. Ballen KK, Kurtzberg J, Lane TA, Lindgren BR, Miller JP, Nagan D et al (2004) Racial diversity with high nucleated cell counts and CD34 counts achieved in a national network of cord blood banks. Biol Blood Marrow Transplant 10:269–275 12. Cairo MS, Wagner EL, Fraser J et al (2005) Characterization of banked umbilical cord blood hematopoietic progenitor cells and lymphocyte subsets and correlation with ethnicity, birth weight, sex, and type of delivery: A cord blood transplantation (COBLT) study report. Transfusion 45:856–866 13. Laskey LC, Lane TA, Miller JR, Lindgren B, Patterson H, Haley NR et al (2002) In utero or ex utero cord blood collection: Which is better?. Transfusion 42:1261–1267 14. Chow R, Nademanee A, Rosenthal J, Karanes C, Jaing TH, Graham ML (2007) Analysis of hematopoietic cell transplants using plasma-depleted cord blood products that are not red blood cell reduced. Biol Blood Marrow Transplant 13:1346–1357 15. Barker JN, Krepski TP, DeFor TE, Davies SM, Wagner JE, Weisdorf DJ (2002) Searching for unrelated donor hematopoietic stem cells: Availability and speed of umbilical cord blood versus bone marrow. Biol Blood Marrow Transplant 8:257–260 16. Atlas LD (2006) The national marrow donor program in 2006: Constants and challenges. Transfusion 46:1080–1084 17. American Academy of Pediatrics Section of Hematology/Oncology (2007) Cord blood banking for potential future transplantation. Pediatrics 119:165–170 18. American College of Obstetricians and Gynecology (1997) Routine storage of umbilical cord blood for potential future transplantation. ACOG Comm Opin 183:1–3 19. Ballen KK, Barker JN, Stewart SK, Greene MF, Lane TA (2008) Collection and preservation of cord blood for personal use. Biol Blood Marrow Transplant 14:356–363 20. Gluckman E, Auerbach BHA, AD FHS, Douglas GW, Devergie A et  al (1989) Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilicalcord blood from an HLA-identical sibling. N Engl J Med 321:1174–1178

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K. Ballen 21. Locatelli F, Rocha V, Chastang C et  al (1999) Factors associated with outcome after cord blood transplantation in children with acute leukemia: Eurocord-cord blood transplant group. Blood 93:3662–3671 22. Locatelli F, Rocha V, Reed W et al (2003) Related umbilical cord blood transplantation in patients with thalassemia and sickle cell disease. Blood 101:2137–2143 23. Kurtzberg J, Laughlin M, Graham ML et al (1996) Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 335:157–166 24. Eapen M, Rubinstein P, Zhang MJ, Stevens C, Kurtzberg J, Scaradavou A et  al (2007) Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukemia: A comparison study. Lancet 369:1947–1954 25. Hwang WY, Samuel M, Tan D, Koh LP, Lim W, Lim YC (2007) A meta-analysis of unrelated donor umbilical cord blood transplantation versus unrelated donor bone marrow transplantation in adult and pediatric patients. Biol Blood Marrrow Transplant 13:444–453 26. Staba SL, Escolar ML, Poe M, Kim Y, Martin PL, Szabolcs P et  al (2004) Cord-blood transplants from unrelated donors in patients with Hurler’s syndrome. N Engl J Med 350:1960–1969 27. Escolar ML, Poe MD, Provenzale JM et al (2005) Transplantation of umbilical-cord blood in babies with infantile Krabbe’s disease. N Engl J Med 352:2069–2081 28. Kobayashi R, Ariga T, Nonoyama S et al (2006) Outcome in patients with WiskottAldrich syndrome following stem cell transplantation: An analysis of 57 patients in Japan. Br J Hematol 135:362–366 29. Gluckman E, Rocha V, Ionescu I, Bierings M, Harris RE, Wagner J et  al (2007) Results of unrelated cord blood transplant in Fanconi anemia patients: Risk factor analysis for engraftment and survival. Biol Blood Marrow Transplant 13:1073–1082 30. Wall DA, Chan KW (2008) Selection of cord blood unit (s) for transplantation. Bone Marrow Transplant 42:1–7 31. Laughlin MJ, Barker J, Bambach B et  al (2001) Hematpoietic engraftment and survival in adult recipients on umbilical-cord blood from unrelated donors. N Engl J Med 344:1815–1822 32. Laughlin MJ, Eapen M, Rubinstein P, Wagner JE, Zhang MJ, Champlin RE et al (2004) Outcomes after transplantation of cord blood or bone marrow from unrelated donors in patients with leukemia. N Engl J Med 351:2265–2275 33. Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A et al (2004) Transplants of umbilical cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 351:2276–2285 34. Takahashi S, Ooi J, Tomonari A, Konuma T, Tsukada N, Monna MO et al (2007) Comparative single-institute analysis of cord blood transplantation from unrelated donors with bone marrow or peripheral blood stem-cell transplants from related donors in adult patients with hematologic malignancies after myeloablative conditioning regimen. Blood 109:1322–1330 35. Khouri IF, Saliba RM, Giralt SA et al (2001) Nonablative allogeneic hematopoietic transplantation as adoptive immunotherapy for indolent lymphoma: Low incidence of toxicity, acute graft vs host disease, and treatment-related mortality. Blood 98:3595–3599 36. Daly A, McAfee S, Dey B, Colby C, Schulte L, Yeap B et al (2003) Nonmyeloablative bone marrow transplantation: Infectious complications in 65 recipients of HLA-identical and mismatched transplants. Biol Blood Marrow Transplant 9:373–382 37. Yuji K, Miyakoshi S, Kato D et  al (2005) Reduced-intensity unrelated cord blood transplantation for patients with advanced lymphoma. Biol Blood Marrow Transplant 11:314–318

Chapter 20  Update on Umbilical Cord Blood Transplantation  38. Miyakoshi S, Kami M, Tanimoto T, Yamguchi T, Narimatsu H, Kusumi E et  al (2007) Tacrolimus as prophylaxis for acute graft versus host disease in reduced intensity cord blood transplantation for adult patients with advanced hematologic diseases. Transplantation 84:316–322 39. Chao NJ, Koh LP, Long GD et al (2004) Adult recipients of umbilical cord blood transplants after nonmyeloablative preparative regimens. Biol Blood Marrow Transplant 10:569–575 40. Barker JN, Weisdorf DJ, Defor TE, Blazar BR, McGlave PB, Miller JS et al (2005) Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 105:1343–1347 41. Brunstein CG, Barker JN, Weisdorf DJ, DeFor TE, Miller JS, Blazar BR et  al (2007) Umbilical cord blood transplantation after nonmyeloablative conditioning: Impact of transplantation outcomes in 110 adults with hematologic disease. Blood 110:3064–3070 42. Cutler C, Mitrovich R, Kao G, Ho V, Alyea E, Koreth J et al (2007) Double umbilical cord blood transplantation with reduced intensity conditioning and sirolimus-based GVHD prophylaxis. Blood 118:600a Abstract 43. Ballen KK, Spitzer TR, Yeap B, McAfee S, Dey BR, Attar E et al (2007) Double unrelated reduced-intensity umbilical cord blood transplantation in adults. Biol Blood Marrow Transplant 13:82–89 44. Haspel RL, Kao G, Yeap BY, Cutler C, Soiffer RJ, Alyea EP et  al (2008) Preinfusion variables predict the predominant unit in the setting of reduced intensity double cord blood transplantation. Bone Marrow Transplant 41:523–529 45. Verneris MR, Brunstein C, DeFor TE, Barker J, Weisdorf DJ, Blazar BR et al (2005) Risk of relapse after umbilical cord blood transplantation in patients with acute leukemia: Marked reduction in recipients of two units. Blood 106:305a Abstract 46. Shpall EJ, Quinones R, Giller R, Zeng C, Baron AE, Jones RB et  al (2002) Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 8:368–376 47. Hofmeister CC, Zhang J, Knight KL, Le P, Stiff PJ (2007) Ex vivo expansion of umbilical cord blood stem cells for transplantation: Growing knowledge from the hematopoietic niche. Bone Marrow Transplant 39:11–23 48. Delaney C, Varnum-Finney B, Aoyama K, Brashem-Stein C, Bernstein ID (2005) Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood 106:2693–2699 49. Shpall EJ, De Lima M, McManis JD, Robinson S, McNiece IK, Champlin RE (2005) Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 11:932–935 50. Magro E, Regidor C, Cabrera R, Sanjuan I, Fores R, Garcia-Marco JA et al (2006) Early hematopoietic recovery after single unit unrelated cord blood transplantation in adults supported by co-infusion of mobilized stem cells from a third party donor. Hematologica 91:640–648 51. Safdar A, Rodriguez G, DeLima M, Petropoulos D, Chemaly R, Worth L et  al (2007) Infections in 100 cord blood transplantations: Spectrum of early and late posttransplant infections in adult and pediatric patients 1996–2005. Medicine 86:324–333 52. Miyakoshi S, Kusumi E, Matsumura T, Hori A, Murashige N, Hamaki T et  al (2007) Invasive fungal infection following reduced-intensity cord blood transplantation for adult patients with hematologic diseases. Biol Blood Marrow Transplant 13:771–777 53. Goodwin HS, Bicknese AR, Chien SN, Bogucki BD, Quinn CO, Wall DA (2001) Multilineage differentiation activity by cells isolated from umbilical cord blood: Expression of bone, fat, and neural markers. Biol Blood Marrow Transplant 7:581–588

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K. Ballen 54. Vendrame M, Cassady J, Newcomb J et al (2004) Infusion of human umbilical cord blood cells in a rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke 35:2390–2395 55. Viener HL, Brusko T, Wasserfall C et  al (2007) Changes in regulatory T cells following autologous umbilical cord blood transfusion in children with type I diabetes. J Am Diab Assoc 7:0314 Abstract 56. Ma N, Stamm C, Kaminski A et  al (2005) Human cord blood cells induce angiogenesis following myocardial infarction in NOD/scid-mice. Cardiovasc Res 5:45–54 57. Prat-Vidal C, Roura S, Farre J, Galvez C, Llach A, Molina CE et  al (2007) Umbilical cord blood-derived stem cells spontaneously express cardiomyogenic traits. Transplant Proc 39:2434–2437 58. Wu KH, Zhou B, Yu CT, Cui B, Lu SH, Han ZC, Liu Y (2007) Therapeutic potential of human umbilical cord derived stem cells in a rat myocardial infarction model. Ann Thorac Surg 83:1491–1500 59. Wagner JE, Barker JN, DeFor TE et al (2002) Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: Influence of CD34 cell dose and HLA disparity n treatment-related mortality and survival. Blood 100:1611–1618

Chapter 21 Selection of Cord Blood Unit(s) for Transplantation Donna A. Wall and Ka Wah Chan

1. Introduction Over the past decade, cord blood (CB) has been established as an alternative source of donor cells for allogeneic hematopoietic stem cell transplantation. The outcome of CB transplants, particularly in children, is similar to the unrelated donor transplants using bone marrow cell or mobilized peripheral blood progenitor cells [1–4]. Early experience in adult CB transplantation was hampered by poor engraftment and immune recovery [5–7]. Recent experiences with better risk patients, double CB unit transplants, and submyeloablative preparative regimens have been more encouraging [8–10]. There are several important differences between bone marrow or GCSFmobilized peripheral blood progenitor cells and cord blood, that have to be taken into account when selecting donors/products for transplantation. Cord blood transplants are being performed with approximately a lot fewer hematopoietic progenitors than other stem cell sources [1]. The adult donor grafts deliver cell numbers well above the engraftment threshold such that a loss of even half of the product would not have a major impact on the transplant. CB as a donor source is not as tolerant. In general, clinical series have shown that the CB transplantation is associated with a higher incidence of graft failure and delayed count recovery [6, 11]. However, these risks are offset by a lower risk for acute and chronic GVHD despite major HLA disparity. This is due in part to the lower number of mature T-cells in the graft (functionally, CB is a partially T-cell depleted graft) and to the nature of the cord blood T-cell responsiveness to the allogenic stimulus [12–19]. The question that arises is how to apply these observations to one’s strategy for cord blood unit selection, especially when there are competing variables. In this review, we summarize the literature on selection strategy, comparing unrelated adult donor to CB searches, and provide our personal preferences on this issue.

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_21, © Springer Science + Business Media, LLC 2003, 2010

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1.1.  Selection of Unrelated Adult Donor HSC for Transplantation: General Principles The two critical components in selecting adult donors for transplantation are HLA compatibility and the ability to harvest hematopoietic progenitors (i.e., the availability of the donor). By and large, cell dose is not an issue. The numbers of hematopoietic progenitors delivered in adult donor HSC transplantation are well above the minimum threshold for engraftment and the size differential between donor and recipient is rarely greater than two-fold. It has been shown that a larger graft improves the outcome of transplants in bone marrow transplantation [20, 21]. Higher numbers of cells are required when there is a graft manipulation (such as T-cell depletion, CD34 selection) or when submyeloablative preparative regimens are planned. With adult unrelated donors, it is the HLA matching that is generally the most significant challenge. Current search algorithm recommends matching at least seven of eight high resolution at HLA-A, HLA-B, HLA-C, and HLADRB1 loci [22]. Recent data support that allelic (high resolution) mismatches are as significant as broad antigen mismatches. A recent National Marrow Donor Program review of 3,857 US transplantations performed from 1988 to 2003 with patient-donor pairs fully typed for HLA-A, B, C, DRB1, DQB1, DQA1, DPB1, and DPA1 alleles, demonstrated that high resolution DNA matching for HLA-A, B, C, and DRB1 [8/8 match] was the minimum level of matching associated with the highest survival [23]. A single mismatch detected by low or high resolution DNA testing at HLA-A, B, C, or DRB1 [7/8 match] was associated with higher mortality (relative risk 1.25, 95% CI 1.13–1.38, p 3.7 × 107/kg was required. Gluckman et  al. reported a log linear relation between cell dose and the probability of engraftment [32]. Rubinstein et  al. also noted a step-wise increase in cell dose correlated with the speed of myeloid recovery [33]. Many pediatric centers accept a minimum cell dose of 2 × 107 TNC/kg but most target cell doses above 5 × 107/kg with no upper cell dose limit. This approach does not work for adults where the cell doses achievable are rarely above 3 × 107/kg with

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Table 21-1.  Minimum cell doses based on hematopoietic measurement and the effect of higher cell dose on transplant outcomes for unrelated donor CB transplant. Author

Engraftment rate

Cell dose (/kg) recommended

Effect of higher cell dose noted on

Rubinstein [33] (n = 861)

93%

>2.5 × 107 TNC

↑Engraftment, ↓TRM

Wagner [34](n = 102)

88%

>1.7 × 10 CD34

↑Engraftment, ↓TRM,↑ survival

Migliaccio [28] (n = 204)

NA

>5 × 104 CFC

↑Engraftment, ↓TRM

Barker [35] (n = 608)

NA

5

7

>2.5 × 10 TNC

↓TRM

TRM transplant-related events/mortality; NA not reported

s­ ingle CB units. Only a small fraction of CB inventories have adequate cell numbers to support adult transplants. The institutional cellular therapy laboratory plays a critical role in the CB transplant process. Approximately 20% of CB cells may be lost in the thaw process – due to cell death arising out of thaw, institutional testing, loss in manipulation. Laboratories need to develop operating procedures that minimize the cell loss on thaw. Convention is to use the pre-cryopreservation cell dose and not the post-thaw cell dose in reporting transplant outcomes. Other measures of graft progenitor content, such as CD34 and hematopoietic colony-forming cell (CFC) enumeration, are likely to be equal or better predictors of successful engraftment (Table 21.1). Unique to CB is the high percentage of nucleated red blood cells (NRBC) in the cell product. The NRBC are included in the TNC. Our experience and that of the National Cord Blood Program support that the increased number of primitive progenitors that accompany higher NRBC offset any difference in cell content (i.e., we do not adjust the TNC for NRBC content [30]). For search situations where there are several CB units with comparable TNC and HLA matching, some authors advocated selecting units of higher CD34 or CFC [28, 34]. Given the variability of these results between banks, we use the CD34 or CFU assessments to screen the units that may have poor hematopoietic potential – avoiding selection of units with very low CFU/CD34 content. Similarly, if available, CB CD3 content may be used to screen units to avoid subsequent severe immunodeficiency syndromes. 1.4. Selection of Untreated Donor CB for Transplantation: HLA Matching CB inventories are only a small fraction of the 9 million HSC donors registered around the world. Convention is to define the HLA matching for CB transplantation as low resolution HLA-A and B matching and high resolution HLA-DR matching – a huge difference from the 8/8 high resolution HLA matching used in adult unrelated donor matching. Multiple HLA mismatches are tolerated with CB grafting. When high resolution matching of 10 alleles is looked at in the units selected for transplant, there are frequently many more mismatches present [11].

Chapter 21  Selection of Cord Blood Unit(s) for Transplantation 

Over half of our transplants are performed with 4/6 or less HLA matching. In practice, there is no difference in survival based on degree of HLA matching. The reason for this is that in a primarily pediatric population we usually achieve high cell doses, and treat many patients with high risk leukemia. A distinction needs to be made between adequate and ideal matching. Higher degrees of HLA matching were associated with improved engraftment and transplant outcome compared to 5/6, or 4/6 matches (Table  21-2) but the impact is relatively small [1, 33, 34]. This is because of the confounding impact of cell dose on engraftment and HLA mismatch on graft vs. leukemia effect. As yet, there is no consensus as to which specific HLA mismatches are better tolerated. Given that 90% of transplants are performed with at least one major HLA mismatch, it has not been possible to isolate the impact of HLA C or DQ mismatching on CB transplant outcomes. Recipients of two HLA mismatched grafts have fared surprisingly well and the limited data on 3/6 matches is surprisingly good. In general, 3/6 matching is reserved for small children with no other options [11]. Rubinstein and colleagues observed that any HLA disparity adversely affected the engraftment rate and increased the risk of acute GVHD; but there was no additive effect with increasing incompatibility [33]. There was, however, a step-wise increase in the incidence of transplant-related complications with increasing number of HLA mismatches. Gluckman et al. noted that co-existence of class I and class II disparities was associated with a higher incidence of severe GVHD and failure of platelet engraftment [32]. The effect of HLA mismatch is most apparent when the cell dose is low [35]. An important question is how much of the adverse effect of HLA disparity can be overcome by a higher cell dose. In malignant diseases, data from the Eurocord registry has demonstrated that with 2–4 HLA differences, the negative effect of delayed engraftment was abrogated by a higher cell dose [36]. However, a threshold of cell dose to overcome HLA disparities could not be defined. Based on the immunobiologic fundamentals, it seems logical to pick a unit with the most HLA alleles matching with the recipient for transplant. However, data to support this approach are scarce. Using allelic typing for HLA-A, -B, -C, -DR and -DQ loci, Kogler et al. showed retrospectively that three-quarter of CB transplants had three or more mismatches. Surprisingly, there was no improved survival in the subset of children receiving 10/10 allelematched CB units [37]. In children with leukemia, when an adequate cell dose could be administered, high-resolution HLA-A, -B, and -DRB1 matching was not found to improve survival [11]. There is some evidence that class II mismatching is less well tolerated [38]. However, this has not been a universal finding. In fact the reviews from the National Cord Blood Bank do not find an impact of location of mismatch and outcome in single major HLA antigen mismatched (5/6) transplants [35]. 1.5.  Selection of Untreated Donor CB for Transplantation: Non-inherited Maternal Allele Matching One area that needs further exploration and which is unique to CB is the potential exploitation of the non-inherited maternal (NIMA) and paternal (NIPA) alleles [39]. A fetus is a haplotype match with the mother. In utero

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Table 21-2.  Impact of HLA disparity on outcomes following unrelated donor CB transplant. Effect of ≠ mismatch noted on

Authors

HLA disparity

No effect noted on

Rubinstein [33] (n = 861)

0 vs. ³1

↓Engraftment, Relapse ↑aGVHD, ↑TRM, ↓event-free survival

Wagner [34] (n = 102)

0–1 vs. 2

None

Gibbons [55] (n = 755)

0 vs. 1 vs. 2

↓Engraftment, ↓survival

Gluckman [32] (n = 550)

0 vs. 1 vs. 2 vs. 3–4

↓Engraftment, aGVHD (II–IV), ↑aGVHD (III–IV), TRM, survival ↓ relapse

Barker [35] (n = 608)

0 vs. 1 vs. 2 vs. 3

↑TRM

GVHD, TRM, relapse, survival

the fetal lymphocytes, which are immunocompetent from 18-weeks gestation, are kept in a state of non-responsiveness to the mismatching maternal antigens. As most cord blood transplants are being performed with major HLA mismatches, a question that arises is whether matching the HLA mismatch to the NIMA would result in a transplant with less GVHD. There is evidence in the renal transplantation and partially HLA matched family member marrow transplants that there is less graft rejection or acute GVHD, respectively, if the HLA mismatch is a NIMA [40–42]. In fact in a small series of haploidentical sibling CB transplants, the haplotype mismatch was disparate at the NIMA, 0/10 recipients. However, grII-IV GVHD was observed in 4/5 transplants with the NIPA mismatch [43]. It is possible that targeting the mismatch in an unrelated donor setting to the NIMA may result in less GVHD – admittedly this will be difficult to test. Following this logic, one would postulate that transplanting CB from the donor infant to its mother should also have a lower risk of GVHD, given that those CB immune cells have been exposed to the mismatching maternal haplotype while in utero. Most banks store samples of maternal DNA so NIMA testing is feasible. 1.6.  Selection of Unrelated Donor CB for Transplantation: Other Factors As only a small fraction of CB transplants are performed with fully HLA matched CB units, it is difficult to tease out the impact of other factors conventionally associated with transplant outcome – gender or ABO mismatch or CMV status. Most CB units are CMV naive due to the placental barrier. There has repeatedly been an association between CMV positivity in the recipient and poorer transplant outcome which may be due to the lack of prior exposure of CB cells to CMV [11, 44, 45]. ABO mismatches have been associated with delays in red cell and platelet transfusion independence. In a study of 95 adults who underwent unrelated cord blood transplantation (CBT) (27 ABO-identical, 29 minor, 21 major, and 18

Chapter 21  Selection of Cord Blood Unit(s) for Transplantation 

bidirectional ABO-incompatible recipients), Tomonari and colleagues reported that neutrophil and red cell engraftment did not differ but that the cumulative incidence of platelet engraftment in ABO-identical/minor ABO-incompatible recipients was higher than in major/bidirectional ABO-incompatible recipients (HR 1.88, p = 0.013). In addition, fewer platelet and red cell transfusions were required in ABO-identical/minor ABO-incompatible recipients (HR 0.80, p = 0.040) [46]. 1.7. Selection of Unrelated Donor CB for Transplantation: Effect of the Underlying Diagnosis In analyzing the data from the Eurocord registry, Gluckman et al. showed that underlying disease affects the cord blood selection criteria [36, 47]. Transplant for malignant diseases can be successfully performed with a lower cell dose (down to 2 × 107 NC/kg infused) and that with HLA mismatching relapse was less frequent. Therefore, in the high-risk cases, a larger unit with greater HLA mismatch may actually be the preferred CB graft. In the setting of unrelated CBT for non-malignant disorders the needs are very different. It is in this population, where GVHD is not beneficial, one would expect to see the greatest impact of HLA matching. In a recent analysis of CBT for Fanconi’s anemia, Gluckman and colleagues retrospectively analyzed results of unrelated CB transplantation in 93 Fanconi anemia patients [45]. In this series, HLA mismatches were associated with poorer survival. 1.8. Selection of Unrelated Donor CB for Transplantation: Double Cord Blood Units To overcome the cell dose restriction, infusion of two separate cord blood units (double CB) have been used with encouraging results [8, 9, 48, 49]. When two or more CB units are used as the HPC source, there is only one unit contributing to hematopoiesis within 1 month of post transplant. Neither the total nucleated, CD34, and CD3 cell doses, HLA matching, nucleated cell viability, ABO typing, gender match, or order of unit infusion was predictive of which unit eventually dominated [9]. The potential benefits of the two units are transient hematopoiesis from the second unit ameliorating toxicity during the time of early post transplant period and immunologic synergy between the two units during the early post transplant period which is known to be important in the development of alloreactivity and possibly speeding hematopoietic recovery. The potential risks with double CBT are negative graft-graft interactions and possibly increased risk of chronic GVHD. Given the difficulty in determining tolerable mismatches in the single CBT setting, it is hard to be dogmatic about HLA matching in the double CBT setting. As a generalization, higher cell doses in at least one of the CB units is important (ideally >2.5 × 107/kg) and it is thought that there should be some matching between both patient-cord and cord-cord. A comparison between single vs. double CBT is being tested in a phase III trial in children with acute leukemia (BMT CTN 0501). In an extension of this approach in a small pilot trial, Lister and colleagues infused multiple CB units (5–7 units) at the time of transplant and found that in the patients evaluable there was always one unit becoming the sole source

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for long-term hematopoiesis [50]. In one long-term survivor, the CB source was only a single antigen match with the recipient. 1.9.  Selection of Unrelated Donor CB for Transplantation: Texas Transplant Institute Perspective The transplant population in South Texas is highly represented by Hispanic recipients, and as such HLA matching is frequently a challenge. Given the rapid availability of CB and the ability to use partially matched donors, most unrelated donor transplants in the pediatric program utilize CB as the donor source. The independent influences of HLA matching and cell dose on the outcome of CBT has driven our CB selection approach. Over the past 5  years, we have developed an algorithm for selection of CB units for transplant. As outlined in Table 21-3, we target a maximum cell dose, even at the expense of HLA matching. Only 10% of CB transplants at our center are performed with 6/6 antigen matched units. A single unit CB is used if there are more than 2 × 107 TNC/kg pre-cryopreservation with 6/6 antigen matched CB, and a minimum of 3 × 107 TNC/kg if there is 4/6 HLA-matching. High resolution HLA-A, -B, and DR typing is obtained on both donor and recipient. While we start with matching at low-antigen level for class I and high resolution for class II, if a 4/6 or 3/6 matched unit is being considered we would like to have the matches to be allelic matches. Matching at both HLA-DRB1 alleles is preferred, especially if there are multiple mismatches (i.e., 4/6 match). Mismatches at both class II (either antigen or allele) loci are not accepted. When several large CB units are available, and the cell dose is at least 3–5 × 107 TNC/kg, we would choose the unit with best HLA match. In situations of a small, well matched unit and a much larger, less well matched unit we will utilize the larger unit preferentially, especially when treating malignancies. In larger units, when the cell dose threshold is not met, we use two CB units for transplantation. This functionally includes all adult transplants. In young children with no other options, we will utilize 3/6 HLA matched units, with at least one match at HLA-A, B, and DR. We have not noticed a difference in engraftment, GVHD or survival with these less well matched units, but

Table 21-3.  Texas Transplant Institute algorithm for CB unit selection. HLA match 6/6

Cell dose (TNC/kg of recipient weight)

Location of mismatch

7

N/A

7

Class I (A or B) mismatch preferred over DR mismatch

>2 × 10

5/6

>3 ×10

4/6

>3 × 107

3/6

7

>5 × 10

A + B, A/B + DRB1 At least one DRB1 match Younger recipients

7

Target cell dose: TNC >5 × 10 nucleated cells/kg (no upper limit on cell dose) Acceptable cell dose is dependent on degree of HLA matching If no CB unit is identified that meets these criteria we consider double cord blood grafts or ex vivo expansion

Chapter 21  Selection of Cord Blood Unit(s) for Transplantation 

our numbers are small and these are a selected group of young children for whom we can generally achieve a very large cell dose. Up to 10% of CB transplants are complicated by primary graft failure. We monitor engraftment closely in the first few months post transplant and will proceed to an early second transplant utilizing a submyeloablative preparative regimen [51]. In these situations we target the largest CB unit(s) with a minimum of 4/6 HLA matches, accepting a DRB1 allele mismatch if necessary. Given the real risk for graft failure, it is advised to have an alternative stem cell source identified prior to start of transplant. At our center we have a second CB unit on reserve at the bank.

2. Summary Selection of CB units for transplantation involves combining both cell dose and HLA matching as independent yet overlapping variables. Cell dose and cell yield at the time of transplant are critical given that the transplants are being performed with minimal cells for reliable engraftment. In transplants for malignant disorders, the greater allogeneicity and lower relapse rate associated with the less well matched units balances any benefit that better HLA matching may have on transplant-related morbidity/mortality. The only factor that has repeatedly been associated with improved outcome post CB transplant is cell dose. The CB inventories are rapidly increasing in size and the quality of the CB units being banked (larger, better characterized) is improving. With this some of our current limitations in CB availability will soon become moot. Explorations into the CB expansion and multiple CB unit transplants address the limited cell doses attainable with a single CB collection [49, 52–54]. At this point one must conclude that bigger is better when selecting CB units for transplantation.

References 1. Grewal SS, Barker JN, Davies SM, Wagner JE (2003) Unrelated donor hematopoietic cell transplantation: marrow or umbilical cord blood? Blood 101: 4233–4244 2. Eapen M, Rubinstein P, Zhang MJ et al (2006) Comparable long-term survival after unrelated and HLA-matched sibling donor hematopoietic stem cell transplantations for acute leukemia in children younger than 18 months. J Clin Oncol 24:145–151 3. Bunin N, Carston M, Wall D et al (2002) Unrelated marrow transplantation for children with acute lymphoblastic leukemia in second remission. Blood 99:3151–3157 4. Hwang WY, Samuel M, Tan D, Koh LP, Lim W, Linn YC (2007) A meta-analysis of unrelated donor umbilical cord blood transplantation versus unrelated donor bone marrow transplantation in adult and pediatric patients. Biol Blood Marrow Transplant 13:444–453 5. Chao NJ, Liu CX, Rooney B et  al (2002) Nonmyeloablative regimen preserves "niches" allowing for peripheral expansion of donor T-cells. Biol Blood Marrow Transplant 8:249–256 6. Cornetta K, Laughlin M, Carter S et al (2005) Umbilical cord blood transplantation in adults: results of the prospective cord blood transplantation (COBLT). Biol Blood Marrow Transplant 11:149–160

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D.A. Wall and K.W. Chan 7. Laughlin MJ, Eapen M, Rubinstein P et al (2004) Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med 351:2265–2275 8. Ballen KK, Spitzer TR, Yeap BY et al (2007) Double unrelated reduced-intensity umbilical cord blood transplantation in adults. Biol Blood Marrow Transplant 13:82–89 9. Brunstein CG, Barker JN, Weisdorf DJ et al (2007) Umbilical cord blood transplantation after nonmyeloablative conditioning: impact on transplantation outcomes in 110 adults with hematologic disease. Blood 110:3064–3070 10. Takahashi S, Iseki T, Ooi J et  al (2004) Single-institute comparative analysis of unrelated bone marrow transplantation and cord blood transplantation for adult patients with hematologic malignancies. Blood 104:3813–3820 11. Kernan N, Carter S, Wagner J, Baxter-Lowe L, Wall D, Kapoor N (2006) Umbilical cord blood transplantation in pediatric patients: results of the prospective, multiinstitutional cord blood transplantation study (COBLT). Biol Blood Marrow Transplant 12:14 (abst 33) 12. Roncarolo MG, Bigler M, Martino S, Ciuti E, Tovo PA, Wagner J (1996) Immune functions of cord blood cells before and after transplantation. J Hematother 5:1 57–160 13. Han P, Hodge G (1999) Intracellular cytokine production and cytokine receptor interaction of cord mononuclear cells: relevance to cord blood transplantation. Br J Haematol 107:450–457 14. Rainsford E, Reen DJ (2002) Interleukin 10, produced in abundance by human newborn T cells, may be the regulator of increased tolerance associated with cord blood stem cell transplantation. Br J Haematol 116:702–709 15. Gardiner CM, Meara AO, Reen DJ (1998) Differential cytotoxicity of cord blood and bone marrow-derived natural killer cells. Blood 91:207–213 16. Joshi SS, Tarantolo SR, Kuszynski CA, Kessinger A (2000) Antitumor therapeutic potential of activated human umbilical cord blood cells against leukemia and breast cancer. Clin Cancer Res 6:4351–4358 17. El Marsafy S, Dosquet C, Coudert MC, Bensussan A, Carosella E, Gluckman E (2001) Study of cord blood natural killer cell suppressor activity. Eur J Haematol 66:215–220 18. Hodge S, Hodge G, Flower R, Han P (2001) Cord blood leucocyte expression of functionally significant molecules involved in the regulation of cellular immunity. Scand J Immunol 53:72–78 19. Nomura A, Takada H, Jin CH, Tanaka T, Ohga S, Hara T (2001) Functional analyses of cord blood natural killer cells and T cells: a distinctive interleukin-18 response. Exp Hematol 29:1169–1176 20. Gorin NC, Labopin M, Rocha V et al (2003) Marrow versus peripheral blood for geno-identical allogeneic stem cell transplantation in acute myelocytic leukemia: influence of dose and stem cell source shows better outcome with rich marrow. Blood 102:3043–3051 21. Sierra J, Storer B, Hanson J, Bjerke J, Martin P, Petersdorf E (1997) Transplantation of marrow cells from unrelated donor for the treatment of high-risk acute leukemia: the effect of leukemia burden, donor HLA matching, and marrow cell dose. Blood 89:4226–4235 22. Hurley C, Baxter-Lowe L, Logan B, Karanes C, Anasetti C, Weisdorf D (2003) National Marrow Donor Program HLA-matching guidelines for unrelated marrow transplants. Biol Blood Marrow Transplant 9:610–615 23. Lee SJ, Klein J, Haagenson M et al (2007) High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood 110:4576–4583 24. Peterdorf E, Kollman C, Hurley C, Dupont P, Nademannee A, Bogovich A (2001) Effect of HLA class II gene disparity on clinical outcome in unrelated donor

Chapter 21  Selection of Cord Blood Unit(s) for Transplantation  hematopoietic cell transplantation for chronic myeloid leukemia: the US national marrow donor program experience. Blood 98:2922–2929 25. Flomenberg N, Baxter-Lowe L, Confer D, Fernandez-Vina M, Filipovich A, Horowitz M (2004) Impact of HLA class I and class II high-resolution matching on outcomes of unrelated donor bone marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplantation outcome. Blood 104:1923–1930 26. Serna DS, Lee SJ, Zhang MJ et al (2003) Trends in survival rates after allogeneic hematopoietic stem-cell transplantation for acute and chronic leukemia by ethnicity in the United States and Canada. J Clin Oncol 21:3754–3760 27. Sasazuki T, Juji T, Morishima Y, Kinukawa N, Kashiwabara H, Inoko H (1998) Effect of matching class I alleles on clinical outcome after transplantation of hematopoietic stem cells from an unrelated donor. N Engl J Med 339:1177–1185 28. Migliaccio AR, Adamson JW, Stevens CE, Dobrila NL, Carrier CM, Rubinstein P (2000) Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity. Blood 96:2717–2722 29. Wall DA, Noffsinger JM, Mueckl KA et al (1997) Feasibility of an obstetricianbased cord blood collection network for unrelated donor umbilical cord blood banking. J Matern Fetal Med 6:320–323 30. Stevens CE, Gladstone J, Taylor PE et  al (2002) Placental/umbilical cord blood for unrelated-donor bone marrow reconstitution: relevance of nucleated red blood cells. Blood 100:2662–2664 31. Goodwin HS, Grunzinger LM, Regan DM et al (2003) Long term cryostorage of UC blood units: ability of the integral segment to confirm both identity and hematopoietic potential. Cytotherapy 5:80–86 32. Gluckman E, Rocha V, Arcese W et  al (2004) Factors associated with outcomes of unrelated cord blood transplant: guidelines for donor choice. Exp Hematol 32:397–407 33. Rubinstein P, Stevens CE (2000) Placental blood for bone marrow replacement: the New York Blood Center's program and clinical results. Baillieres Best Pract Res Clin Haematol 13:565–584 34. Wagner JE, Barker JN, DeFor TE et al (2002) 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 100:1611–1618 35. Barker J, Scaradavou A, Stevens C, Rubinstein P (2005) Analysis of 608 umbilical cord blood transplants: HLA-match is a critical determinant of transplant-related mortality in the post-engraftment period even in the absence of acute graft-versushost disease. Blood 106:abst 303 36. Gluckman E, Rocha V (2006) Donor selection for unrelated cord blood transplants. Curr Opin Immunol 18:565–570 37. Kogler G, Enczmann J, Rocha V, Gluckman E, Wernet P (2005) High-resolution HLA typing by sequencing for HLA-A, -B, -C, -DR, -DQ in 122 unrelated cord blood/patient pair transplants hardly improves long-term clinical outcome. Bone Marrow Transplant 36:1033–1041 38. van Heeckeren WJ, Fanning LR, MH J et al. Influence of HLA disparity and graft lymphocytes on allogeneic engraftment and survival after umbilical cord blood transplants in adults. Leukemia (in press) 39. Moretta A, Locatelli F, Mingrat G et al (1999) Characterisation of CTL directed towards non-inherited maternal alloantigens in human cord blood. Bone Marrow Transplant 24:1161–1166 40. van Rood JJ (2000) Double role of HLA in organ transplantation. World J Surg 24:823–827

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D.A. Wall and K.W. Chan 41. van Rood JJ, Roelen DL, Claas FH (2005) The effect of noninherited maternal antigens in allogeneic transplantation. Semin Hematol 42:104–111 42. van Rood JJ, Loberiza FR Jr, Zhang MJ et al (2002) Effect of tolerance to noninherited maternal antigens on the occurrence of graft-versus-host disease after bone marrow transplantation from a parent or an HLA-haploidentical sibling. Blood 99:1572–1577 43. Wagner JE, Kurtzberg J (1998) Allogeneic umbilical cord blood transplantation. In: Broxmeyer HE, Broxmeyer HE, Broxmeyer HE (eds) Cellular characteristics of cord blood and cord blood transplantation. AABB, Bethesda, MD, pp 113–145 44. Rocha V, Chastang C, Souillet G, Rocha V, Chastang C, Souillet G et  al (1998) Related cord blood transplants: the Eurocord experience from 78 transplants. Eurocord transplant group. Bone Marrow Transplant 21(Suppl 3):S59–S62 45. Gluckman E, Rocha V, Ionescu I et al (2007) Results of unrelated cord blood transplant in fanconi anemia patients: risk factor analysis for engraftment and survival. Biol Blood Marrow Transplant 13:1073–1082 46. Tomonari A, Takahashi S, Ooi J et  al (2007) Impact of ABO incompatibility on engraftment and transfusion requirement after unrelated cord blood transplantation: a single institute experience in Japan. Bone Marrow Transplant 40:523–528 47. Gluckman E (2006) Cord blood transplantation. Biol Blood Marrow Transplant 12:808–812 48. Barker JN, Weisdorf DJ, Defor TE et  al (2005) Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 105:1343–1347 49. Majhail N, Brunstein C, Wagner J (2006) Double umbilical cord blood transplantation. Curr Opin Immunol 18:571–575 50. Lister J, Gryn JF, McQueen KL, Harris DT, Rossetti JM, Shadduck RK (2007) Multiple unit HLA-unmatched sex-mismatched umbilical cord blood transplantation for advanced hematological malignancy. Stem Cells Dev 16:177–186 51. Chan K, Grimley M, Taylor C, Wall D (2006) Primary graft failure after unrelated donor cord blood transplant: risk factors and management. Blood 108:abst 44 52. Shpall EJ, McNiece IK, De Lima M et  al (2004) Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 10:738 53. Shpall EJ, Quinones R, Giller R et al (2002) Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 8:368–376 54. Peled T, Landau E, Mandel J et al (2004) Linear polyamine copper chelator tetraethylenepentamine augments long-term ex vivo expansion of cord blood-derived CD34+ cells and increases their engraftment potential in NOD/SCID mice. Exp Hematol 32:547–555 55. Gibbons R (2005) Appendix G: statistical report. Cord blood: establishing a national hematopoietic stem cell bank program. National Academy of Sciences, Washington, DC

Chapter 22 Mobilization of Hematopoietic Cells Prior to Autologous or Allogeneic Transplantation Steven M. Devine

1. Introduction The recruitment of hematopoietic progenitor/stem cells (HPCs) from the marrow into the peripheral blood is termed stem cell mobilization [1, 2]. These stem cells are capable of homing to the marrow cavity and regenerating a full array of hematopoietic cell lineages in a timely fashion after ablative and nonmyeloablative conditioning. Pioneering studies performed over 40 years ago, demonstrated that hematopoietic stem cells (HSC) circulate in the peripheral blood at low frequency [3]. In the 1980s, investigators demonstrated that the frequency of circulating hematopoietic stem and progenitor cells was greatly enhanced (10- to 100-fold) following recovery from myelosuppressive chemotherapy through a process termed mobilization [4–7]. These hematopoietic progenitor cells (HPC) could be collected by leukapheresis (LP) in sufficient quantities to promote hematopoietic reconstitution following myeloablative therapy. These seminal observations have revolutionized clinical stem cell transplantation. In current practice, mobilized peripheral blood (MPB) has essentially replaced BM (BM) as a source of autologous cells for hematopoietic rescue in patients undergoing high-dose chemotherapy/radiotherapy due to improved neutrophil and platelet engraftment, shortened hospital stay, and lower cost [8–11]. More recently, recipients of allogeneic HSC have been administered cytokine MPB in preference to BM based on recent randomized studies that have clearly demonstrated improved kinetics of neutrophil and platelet engraftment, somewhat higher rates of acute and chronic graft-versus-host disease (GVHD), and similar to improved overall survival rates following the use of MPB compared with BM [12–19]. Despite the fact that virtually all autologous transplants are now performed using MPB, the optimal method to mobilize HPC remains the subject of debate. While chemotherapy-based mobilization typically results in collection of greater numbers of CD34+ cells compared to granulocyte colony-stimulating factor

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_22, © Springer Science + Business Media, LLC 2003, 2010

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(G-CSF) alone, as a strategy, chemotherapy-based mobilization is associated with greater morbidity due to infectious complications and has not decisively improved outcomes following transplantation [20–23]. Efforts to improve the yield of CD34+ cells following G-CSF-based mobilization through combination with other hematopoietic cytokines, have met with limited success due either to lack of efficacy or unacceptable toxicity. Novel strategies continue to be sought given that a substantial proportion of patients who have been heavily pretreated have poor stem cell mobilization with current approaches. The development of innovative strategies has recently accelerated due to a more complete understanding of the mechanism underlying stem/progenitor cell mobilization.

2. The Mechanisms of Stem Cell Mobilization Several adhesion molecules, including LFA-1, VLA-4, CXCR4, c-kit, CD44, and Mac-1, are known to anchor stem cells to the bone marrow microenvironment, and disruption of the interactions between these adhesion molecules and their ligands by both chemotherapy and cytokines may promote stem and progenitor cell egress into the peripheral circulation. For instance, hematopoietic progenitor cells (HPCs) are mobilized after exposure to antibodies which interrupt the interaction of the b1 integrin VLA-4, expressed on HPCs, and its ligand, VCAM-1, expressed on endothelial and stromal cells [24, 25]. Chemokines have recently been identified as key regulators of HPC mobilization, particularly members of the CXC chemokine family, including stromalderived factor 1 (SDF-1, also known as CXCL-12), and GROb. The chemokine SDF-1 is constitutively expressed by bone marrow stromal cells, and its receptor CXCR4 is a transmembrane G-protein-coupled receptor expressed on CD34+ cells. Interaction between SDF-1 and CXCR4 critically regulates migration of bone marrow, umbilical cord blood, and G-CSF-mobilized peripheral blood cells transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice [26], and neutralizing antibodies to CXCR4 or SDF-1 significantly reduce HPC homing, migration, and mobilization [27]. As discussed below, a direct inhibitor of the SDF-1/CXCR4 interaction, AMD3100, is an effective mobilizing agent currently being tested in clinical trials. Binding of the chemokine GROb to its receptor CXCR2 also plays an important role in stem cell localization in bone marrow. The N-terminal truncated variant of GROb, SB-251353 (GROb-T) can rapidly mobilize HPCs in mice and monkeys, an effect that is enhanced by combination with G-CSF. A single injection of SB-251353 combined with 4 days of G-CSF results in fivefold greater HPC mobilization than G-CSF alone [28, 29]. These promising results in animal models suggest that the efficacy of SB-251353 for stem cell mobilization might be exploited clinically if this agent was well tolerated, but the fact that this agent may activate neutrophils is a real concern. The mechanisms by which cytokines trigger stem cell mobilization remain incompletely understood. Liu et al. have shown that G-CSF receptor expression is not required on HPCs for their mobilization by G-CSF, suggesting that G-CSF acts indirectly on HPCs [30]. Proteases have been implicated as the secondary signals which lead to HPC mobilization induced by G-CSF. G-CSF may activate neutrophils and other target cells, leading to the release of proteases which cleave the adhesive interactions between HPCs and the bone marrow microenvironment [31, 32]. In support of this model, Levasque et al.

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identified two serine proteases, neutrophil elastase and cathepsin G, which increase in concentration during G-CSF mobilization and are directly able to cleave VCAM-1 [33]. In addition, the activity of IL-8, a CXC chemokine that induces rapid mobilization of HPCs [34], appears to be mediated by neutrophil activation and subsequent release of matrix metalloproteinase (MMP-9) [35]. In rhesus monkeys, IL-8-induced mobilization is completely inhibited by antibodies that block MMP-9 activity [36]. GROb-induced mobilization also may be mediated by MMP-9 [37]. Regulation of the SDF-1/CXCR4 interaction may occur via proteolytic cleavage of SDF-1 or CXCR4 [2, 38, 39]. This mechanism may be operational whether mobilization is induced by G-CSF alone or when combined with the chemotherapeutic agent cyclophosphamide [40]. However, a recent study suggests that cyclophosphamide-based mobilization may differ fundamentally from G-CSF alone. Using various murine systems, Mayack and Wagers demonstrated that treatment with cyclophosphamide and G-CSF enhances first osteoblast proliferation followed by HSC proliferation which is mediated at least in part by the function of the ataxia telangiectasia mutated (ATM), the product of the ARM gene, which is induced in response to DNA damage and oxidative stress. These potential differences require further study [41]. Recent evidence has identified CD26, a membrane-bound extracellular peptidase, as the prime protease that cleaves SDF-1 within the bone marrow [42]. Of note, G-CSF-induced mobilization is inhibited in mice deficient in CD26 [43, 44]. Importantly, inhibition of this molecule may enhance the homing of HPC/HSC to the bone marrow and provide a means to enhance engraftment [42]. This could become a novel strategy to enhance engraftment of umbilical cord blood cells, for instance. Despite this evidence, important studies in protease-deficient mice may contradict a central role for proteases in cytokine-induced stem cell mobilization. Three studies have shown that MMP-9-deficient mice have a similar increase in peripheral HPCs following treatment with IL-8 or G-CSF as wild-type mice [45]. Similarly, HPC mobilization with IL-8 or G-CSF was intact in neutrophil elastase- cathepsin G-deficient mice, and SDF-1 protein levels were decreased in the protease-deficient mice, as is observed in wild-type mice [37, 46]. These studies suggest that nonproteolytic mechanisms may play a fundamental role in stem cell mobilization. One likely mechanism is transcriptional regulation. Semerad et al. have found that SDF-1 mRNA in bone marrow cells is significantly reduced during G-CSF mobilization, suggesting that SDF-1 expression is regulated at the mRNA level [47].

3.  Regulation of Stem Cell Mobilization by Neural and Osteolineage Derived Cells Recent studies have shed light on another pathway in which G-CSF may indirectly exert its effects on SDF-1/CXCR4 signaling. UDP-galactose ceramide galactosyltransferase-deficient (Cgt(−/−)) mice exhibit aberrant nerve conduction and display virtually no PBSC egress from BM following G-CSF or fucoidan administration. Using mice lacking Cgt, a known neurotransmitter, Katayama and colleagues demonstrated that G-CSF-dependent stem cell mobilization may be governed by neural signals such as those transmitted by norepinephrine (NE) [48]. Stem cell mobilization was deficient in mice lacking

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Cgt, and could also be diminished using beta-blockers. Beta-adrenergic drugs, on the other hand, stimulated mobilization. These data suggest a heretofore unknown neural control over stem cell trafficking and raise the intriguing possibility that currently available agents (e.g., beta agonists) may be exploited to enhance mobilization in certain subsets of patients. This concept of neural control of stem/progenitor cell trafficking was further developed in an intriguing study in which mice were observed to exhibit circadian secretion of noradrenaline by sympathetic nerves contained within the bone marrow. These adrenergic signals were transmitted to stromal cells within the marrow by beta-adrenergic receptors, leading ultimately to downregulation of CXCL12 levels through decreases in transcription factors regulating CXCL12 production [49]. These findings support the concept of an optimal time to mobilize and collect HPC for clinical uses. The hematopoietic stem cell niche is a complex multidimensional environment where HSC/HPC communicate with a variety of supporting elements such as osteoblasts, endothelial cells, and extracellular matrix, which collectively sustain stem cell function [50]. Osteoblasts, in particular, have been shown to provide critical signals (e.g. via notch) that can either maintain stem cell quiescence or direct self-renewal, differentiation, or both. Until recently, osteoclasts (OCLs) were not recognized to have an important role in regulating stem cell behavior, including mobilization. However, Kollet et al. demonstrated that specific stimulation of OCLs with RANK ligand (RANKL) recruited immature progenitors to the circulation in a CXCR4- and MMP-9dependent manner [51]. RANKL did not induce mobilization in mice with defective OCL bone adhesion and resorption. Inhibition of OCLs with calcitonin reduced progenitor egress during homeostasis, and following G-CSF stimulation. RANKL-stimulated bone-resorbing OCLs also reduced the stem cell niche components SDF-1, stem cell factor (SCF), and osteopontin, which were associated with progenitor mobilization. These findings indicate a potential involvement of OCLs in selective progenitor recruitment as part of homeostasis and host defense. Osteoclasts may therefore be an important mediator of mobilization induced by G-CSF and other cytokines, creating a possible link between bone remodeling and regulation of hematopoiesis. Taken together, recent insights seem to implicate the CXCR4/SDF-1 pathway as a critical determinant of HSC/HPC mobilization. This interaction therefore has become a logical target for manipulations designed to enhance mobilization or, conversely, improve homing of transplanted cells back into the hematopoietic microenvironment. A more complete understanding of the interactions between HPCs and the bone marrow microenvironment is needed to identify additional targets for the stimulation of HPC mobilization.

4.  Novel Agents Capable of Inducing Hematopoietic Stem/Progenitor Cell Mobilization HPC mobilization has been induced clinically in humans or experimentally in mouse models using variety of approaches including: chemotherapeutic agents such as cyclophosphamide or paclitaxel; cytokines such as G-CSF, GM-CSF, IL-7, IL-3, IL-12, SCF, and Flt-3 ligand, and chemokines such as SDF-1, IL-8, or GROb. Below, we review several different strategies used recently in patients to enhance HPC mobilization.

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4.1. Stem Cell Factor Recombinant human SCF (rHuSCF) is a cytokine that stimulates pre-lineagecommitted HPC. Most clinical studies of (SCF) report the use of this agent with other cytokines. Limited reports of SCF by itself are available and this cytokine appears to result in a dose-dependent 6- to 10-fold mobilization of CFU-GM [52]. One phase 2 study demonstrated enhanced mobilization when SCF was used in conjunction with G-CSF to mobilize stem cells from lymphoma patient undergoing auto-SCT [53]. Recently, rHuSCF (20 mg/kg/day) when combined with G-CSF (10 mg/kg/day) was shown to enhance mobilization of HPC in heavily pretreated patients who have failed a previous attempt with G-CSF alone [54]. In this study, 29/48 (60%) achieved a cumulative total of >2.0 × 106 CD34+ cells/kg following remobilization with SCF and G-CSF after initial failure with G-CSF alone. Owing to occasional anaphylactoid reactions to SCF, including angioedema, urticaria, pruritus, and laryngospasm [55], the FDA decided not to approve the agent for use as an agent to enhance autologous stem cell mobilization in the United States. SCF is approved for use in Canada and New Zealand. 4.2.  Recombinant Human Growth Hormone Growth hormone is a pleiotropic cytokine targeting a variety of nonhematopoietic and hematopoietic cells by binding to its specific receptor [56]. In vitro, recombinant human growth hormone (rhGH) increases colony formation by HPC (CFU-GM and BFU-E) [57]. In vivo, a 7-day course of rhGH in mice significantly induces HPC mobilization into peripheral blood [58]. Carlo-Stella and colleagues investigated rhGH administration associated with chemotherapy plus G-CSF (5 mg/kg/day × 5 days) for enhancing stem cell mobilization in 16 patients with relapsed or refractory hematological malignancies who had failed a first mobilization attempt with chemotherapy plus G-CSF [59]. Patients were then re-mobilized with chemotherapy, G-CSF (5 mg/kg/day × 5 days) and rhGH (100 mg/kg/day, maximum daily dose of 6  mg). This combination resulted in efficient mobilization and collection of ³5 × 106 CD34+ cells/kg in 87% of these poor mobilizers with a median of three leukapheresis (i.e., from 1.1 × 106/kg up to 6 × 106/kg). The exact mechanism by which rhGH restores stem cell mobilization capacity in heavily pretreated patients with relapsed or refractory hematological malignancies is not clear, but is probably related to the expansion of HSC or HPC which become susceptible to be released upon a subsequent or concomitant stimulus, such as G-CSF. No further clinical trials have been reported, however, possibly because of regulatory issues or safety concerns in using this molecule in cancer patients. 4.3.  Pegfilgrastim Polyethylene glycosolated filgrastim (Pegfilgrastim;Neulasta®) differs from filgrastim only by the addition of a 20-kDa polyethylene glycol (PEG) molecule covalently bound to the N-terminal methionyl residue [60]. PEG modification of proteins has been demonstrated to sustain the duration of action by reducing renal clearance of the protein and decreasing rates of cellular uptake and proteolysis. In contrast to filgrastim, it appears that the kidney does not play a significant role in the elimination of pegfilgrastim, which appears to be

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primarily cleared by neutrophils and neutrophil precursors. Because pegfilgrastim directly stimulates the production of neutrophils, it effectively regulates its own clearance from the body. In clinical trials, it was demonstrated that a single dose of pegfilgrastim is as safe and effective as daily injections of filgrastim for the prevention and treatment of chemotherapy-induced neutropenia in a phase 2 study [61]. On the basis of this and other trials, it received approval by the FDA to decrease the incidence of infection in patients with nonmyeloid malignancies receiving myelosuppressive chemotherapy. It has been compared to its parent compound, filgrastim, to determine its efficacy in the stimulation and collection of progenitor cells for autologous transplantation. In mice, higher peak numbers of progenitor cells were mobilized into the peripheral blood following pegfilgrastim (300-fold over baseline) compared to filgrastim (100-fold over baseline), and occurred more rapidly, with a peak occurring over days 2–4, as compared with days 4 and 5 for filgrastim [60]. In a trial of chemotherapy-naive subjects with nonmall cell lung cancer, 13 patients were randomized to receive daily filgrastim 5 mg/kg or a single injection of pegfilgrastim 30, 100, or 300 mg/kg 2 weeks before chemotherapy, and again 24  h after administration of carboplatin and paclitaxel. In the prechemotherapy cycle, the median peak CD34+ count was similar in the filgrastim and pegfilgrastim 30 mg/kg cohorts, with higher median peaks observed in the 100 and 300 mg/kg pegfilgrastim cohorts. Adverse events attributed to study drug were mild-to-moderate bone pain, and were similar for those receiving pegfilgrastim or filgrastim [62]. A multicenter trial in patients with Hodgkin’s disease and non-Hodgkins lymphoma eligible for autologous transplantation was performed comparing the safety and efficacy of pegfilgrastim to filgrastim for CD34+ cell mobilization. In this randomized, double-blinded phase 2 trial, patients received either daily filgrastim at the standard mobilization dose of 10 mg/kg or one of two fixed doses (6 or 12 mg) of pegfilgrastim. Safety and capacity to mobilize CD34+ cells were the primary endpoints. The trial was halted when it was demonstrated that in this setting G-CSF was more effective than pegfilgrastim in mobilizing CD34+ cells (Amgen database). This may have been due to the clearance of pegfilgrastim, given that the patients had normal neutrophil counts at the start of mobilization. This hypothesis is further substantiated by the data that demonstrate pegfilgrastim can be used effectively to mobilize HPC following myelosuppressive chemotherapy [63–65]. Some groups continue to use this agent following chemotherapy since it can be given as a single injection following chemotherapy. 4.4.  Thrombopoietin Thrombopoietin (TPO) is a cytokine that regulates megakaryocytopoiesis. Some studies have showed that this also induces mobilization of CD34+ [66], and it synergizes with G-CSF to enhance stem cell mobilization [67]. Currently, no thrombopoietins have been approved by the FDA for stem cell mobilization and there are no data currently available to determine whether the thrombopoiesis-stimulating peptibodies currently being evaluated in patients with immune thrombocytopenic purpura will have any mobilizing activity.

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4.5.  Parathyroid Hormone (hrPTH) Calvi and colleagues showed that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow [68]. They showed that PTH/PTHrP receptors-stimulated osteoblastic cells that are increased in number, produce high levels of the Notch ligand, Jagged-1, and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in  vivo. Furthermore, liganddependent activation of PTH/PTHrP receptors with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gammasecretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, they showed that osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function. Niche constituent cells or signaling pathways provide pharmacological targets with therapeutic potential for stem cell- based therapies. A clinical trial reported by Ballen and colleagues demonstrated a modest increase in CD34+ cell mobilization when combined with G-CSF in a phase 1 trial but no larger studies of this combination have been reported [69]. The major drawback of the 10–14-day time period for maximal effect with PTH remains, but further study to potentially exploit this pathway seems warranted [70]. 4.6.  CXCR4 Peptide CTCE-0021 is a novel cyclized CXCR4 agonist peptide (SDF-1a analog) developed to stabilize the SDF-1 a-helix to increase their bioactivity, and terminating the C-terminus as an amide to reduce its immunogenicity [29, 71]. This compound retains comparable CXCR4 receptor agonist activity. In mice, a single bolus administration of CTCE-0021 demonstrated a rapid dose-dependent mobilization of HPC between 5 min and 4 h post-dosing, with an increase in WBC resulting from an increase in granulocytes within 5 min post-dosing that persisted for approximately 24  h. The mechanisms involved in this CXCR4 agonist peptide mobilization remains unknown, but Pelus et al. suggested that CTCE-0021 mobilization is associated with downregulation of CXCR4 on HPC, and alteration in the plasma to marrow SDF-1 gradient [29, 37]. CTCE0021 is an efficient and rapid mobilizer of PMN and HPC when used alone and shows synergistic activity when used in combination with G-CSF. No clinical trials have been reported. 4.7.  GROb GRO is a member of the CXC chemokine family, which includes the related ligands GRO, GRO, ENA78, NAP-2, GCP-2, IP10, and interleukin-8 (IL-8), and it has biological activities related to specific binding to the CXCR2 receptor [29, 71]. SB-251353 is a recombinant N-terminal 4-amino acid truncated form of the human chemokine GRO specifically binding only to CXCR2 and with greater potency than full-length GRO [72]. The human CXCR2 selective ligand SB-251353 induces rapid mobilization of hematopoietic stem and progenitor cells in mice and monkeys and synergizes with G-CSF [28].

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Initially, chemokine administration is associated with a leukopenia within 5 min of injection followed by a period of neutrophilia 30–45 min later. The combination of SB-251353 with G-CSF resulted in augmented stem cell mobilization compared with the use of G-CSF alone. The mechanism of action of SB-251353-induced stem and progenitor cell mobilization appears similar to IL-8, which involves upregulation of MMP-9 activity [29]. Concerns that GRO-b may also activate neutrophils have dampened enthusiasm for its clinical development in this setting. 4.8.  AMD3100 AMD3100 is a bicyclam derivative that reversibly inhibits the binding of SDF-1 to its receptor CXCR4, promoting mobilization of CD34+ cells to the peripheral circulation [73]. Preclinical work in murine, canine, and nonhuman primate systems have suggested that AMD3100 alone can rapidly mobilize hematopoietic cells possessing both short- and long-term term repopulating capacity [74–76]. Broxmeyer and colleagues compared the SCID-repopulating capacity (SRC) of human hematopoietic cells mobilized by G-CSF to AMD3100-mobilized cells and found a greater frequency of SRC among the population of cells mobilized by AMD3100 [74]. Later, Burroughs used a well-described canine allogeneic transplant model to demonstrate the long-term repopulating capacity of cells contained within apheresis products collected from donors treated just 6  h previously with a single injection of AMD3100 [75]. The cells were able to maintain trilineage hematopoiesis and promote a full-donor chimeric state in three lethally irradiated recipients for up to 32 months. Further, Larochelle and colleagues collected CD34+ cells from rhesus macaques mobilized following either 5 days of G-CSF or a single dose of AMD3100 [76]. The CD34+ cells were retrovirally marked with the neomycin resistance gene (NeoR) and subsequently transplanted back into autologous recipients following myeloablative conditioning. AMD3100-mobilized cells engrafted gene marked myeloid and lymphoid cells up to 32 months following transplantation. The AMD3100mobilized CD34+ population contained a higher frequency of cells in the G1 phase of the cell cycle, with greater expression of CXCR4 and VLA-4 compared to G-CSF mobilized cells. Two recent studies revealed that G-CSF MPB CD34+ cells have higher levels of the pro-apoptotic genes caspase 3, 4 and 8 and reduction in inhibitors of apoptosis, such as anti-proteinase-2 compared to BM CD34+ cells [77, 78]. These data support the recent studies of Abkowitz in parabiotic mice which suggest that release of HSC into the circulation may also serve as an apoptotic pathway at steady state or following stress signals such as G-CSF stimulation [79]. DNA array technology will be useful to compare the gene expression profile between CD34+ cells mobilized by G-CSF with AMD3100. Together, these preclinical studies suggest fundamental differences in the characteristics of HSC/HPC mobilized by AMD3100 versus G-CSF and set the stage for initial clinical trials to evaluate the effects of transplanting cells mobilized by AMD3100 into humans. Recently, the repopulating capacity of CD34+ cells mobilized with AMD3100 was more fully characterized. Using leukapheresis products collected from 7 sibling donors treated on a trial of AMD3100 mobilization, Hess et al. compared the NOD/SCID-repopulating activity of the total mononuclear cell (MNC) fraction and purified CD34+ cells mobilized from each donor by

Chapter 22  Mobilization of Hematopoietic Cells Prior to Autologous  395

AMD3100 or G-CSF [80]. Comparison of paired samples from each patient eliminated inter-patient variability in the analysis. Bone marrow repopulation was found to be threefold greater with AMD3100-mobilized MNCs than with G-CSF-mobilized MNCs. Purified AMD3100-mobilized CD34+ cells also possessed strong repopulating capacity, which was still superior to that of G-CSF-mobilized CD34+ cells in most patients. These results demonstrate potentially important qualitative differences in the repopulating capacity of grafts mobilized by the two agents. Initial clinical trials of AMD3100 in healthy volunteers demonstrated a more than tenfold increase in PBSCs beginning at1  h and peaking at 9  h after subcutaneous injection of AMD3100 [81]. The addition of AMD3100 to G-CSF results in even greater increases in circulating CD34+ cells [82]. AMD3100 can mobilize PBSCs in patients who have received prior chemotherapy as well. In a phase 1 study, patients with multiple myeloma or nonHodgkin lymphoma had a sevenfold increase in circulating CD34+ cells 6 h after a single dose of AMD3100 240 mg/kg [83]. In autologous stem cell collection trials, AMD3100 160–240 mg/kg has been added to G-CSF on day 4, 6–12 h prior to pheresis. Flomenberg et al. reported use of this combination in 25 multiple myeloma and non-Hodgkin lymphoma patients each of whom underwent two mobilizations, one using G-CSF alone and the other with G-CSF + AMD3100 [84]. Given as either the first or second mobilization regimen, G-CSF + AMD3100 mobilized more CD34+ cells per leukapheresis. In addition, patients underwent fewer leukaphereses, and more patients attained the target collection of 5 × 106 CD34+ cells/kg with the combination of G-CSF and AMD3100. Eighteen of 19 patients undergoing transplant with the G-CSF/AMD3100-mobilized product had early, stable engraftment. Mobilization with G-CSF + AMD3100 is also efficacious in patients with Hodgkin disease (HD). Cashen et al. reported on ten HD patients mobilized with AMD3100 and G-CSF [85]. All patients collected the minimum 2 × 106 CD34+ cells/kg, and 60% of patients collected more than 5 × 106 CD34+ cells/kg, a significantly higher percentage than a historic control group mobilized with G-CSF alone. All eight patients who had been transplanted with AMD3100 + G-CSF mobilized grafts had stable engraftment. This proof-of-principle study paved the way for phase 3 trials. Two randomized phase 3 clinical trials with AMD3100 plus G-CSF in MM and NHL patients have recently been successfully completed. An initial analysis of the data indicates that the prospectively defined clinical endpoints were exceeded [86, 87]. A Compassionate Use Program (CUP) has allowed patients, who have previously failed mobilization with regimens such as cytokine or chemotherapy treatment, to be given access to AMD3100. An analysis of 115 patients with either nonHodgkin’s lymphoma, Hodgkin’s disease or multiple myeloma, who had been unable to collect enough HSC for transplant and were eligible for CUP, showed an overall >66% success of collecting ³2 × 106 CD34+ cells/kg with G-CSF plus AMD3100 [88]. Similarly, patients who failed to collect enough cells for transplant in the phase 3 non-Hodgkin’s lymphoma trial were eligible for rescue by AMD3100 plus G-CSF. Thirty-three of 52 patients who failed on the G-CSF plus placebo arm successfully mobilized with G-CSF plus AMD3100 [89]. These results support the initial observation in the phase 2 trial that AMD3100 treatment can enhance mobilization of HSC in the poor mobilizer patient population. Table 22-1 lists molecules implicated in stem/progenitor cell mobilization.

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Table 22-1.  Molecules implicated in hematopoietic stem/progenitor cell mobilization. Cytokines/chemokines

Adhesion molecules

Receptors

Proteases

Agents used/tested clinically

G-CSF

VLA-4/VCAM-1

cKit

NE

G-CSF

CXCR4

CG

GM-CSF

G-CSF

MMP-9

SCF

GM-CSF

CD26

PTH

GM-CSF CD44 SDF-1/CXCL12 IL-8

Il-3

GH

SCF

Flt-3

Pegfilgrastim

TPO

FL

FL

TPO

Gro-b

IL-3/GM-CSF (pixy321)

CTCE-0021 IL-3 agonist (daniplestim) Il-3/G-CSF (leridistim) G-CSF granulocyte-colony stimulating factor, GM-CSF granulocyte-macrophage colony stimulating factor, SDF-1 stromal derived factor-1, IL-8 interluekin-8, SCF stem cell factor, TPO thrombopoietin, FL fms-like tyrosine kinase ligand, VLA-4 very late antigen 4, VCAM-1 vascular cell adhesion molecule-1, Flt-3 fms-like tyrosine kinase 3 receptor, NE neutrophil elastase, CG cathepsin-G, MMP-9 matrix metalloproteinase-9, CD26 dipeptidylpeptidase IV, PTH parathyroid hormone, GH growth hormone, IL-3 interleukin-3

5.  Choosing a Regimen to Mobilize Autologous Stem/Progenitor Cells There are several factors which may determine the success of HPC ­mobilization, which include extent of prior cytotoxic chemotherapy, especially ­treatments with certain drugs such as alkylating agents (melphalan, carmustine) or fludarabine, radiotherapy, advanced age, and certain diseases such as Hodgkin’s disease, non Hodgkin’s lymphoma, and myelodysplasia [23, 90]. The mobilization capacity of patients with hematological malignancies is, in general, lower than in patients with solid tumors such as breast or testicular cancer [90]. Some authors reported higher probabilities of mobilization failure in woman than in men [91], but this could be related more to differences in ideal body weight between men and woman. The differential expression of diverse adhesion molecules and their cognate receptors, probably impact the characteristics of a specific mobilization. Consistent with this hypothesis, “good mobilizers” showed significantly lower CXCR4, SDF-1, and VLA-4 expression than “poor mobilizers” [92, 93]. SDF-1 gene polymorphism has been proposed as a conditional factor for CD34+ cell mobilization [94]. Autologous HPC/HSC may be mobilized by the administration of chemotherapy followed by hematopoietic growth factors (HGF) or by HGF alone, either as single agents or in combination. While it is generally believed that the combination of chemotherapy followed by HGF results in greater yields of HPC, not all data are in agreement [20–23]. G-CSF is the most commonly used HGF for HPC mobilization and has been demonstrated to be superior to GM-CSF when used as a single agent to mobilize HPC [95]. A number of variables may impact the decision to use a chemotherapy-based or a hematopoietic

Chapter 22  Mobilization of Hematopoietic Cells Prior to Autologous  397

growth factor only mobilization regimen and these include underlying disease and status of disease prior to transplantation, extent of prior chemotherapy and/ or radiation, anticipated morbidity to the patient, target CD34 dose intended for transplantation, intent to manipulate the HPC product, cost and resource utilization, and transplant center experience. Mobilization of HPC by G-CSF alone may be preferable to other techniques due to the ease of administration, decreased cost and morbidity to the patient, and the greater ability to predict the kinetics of mobilization. If G-CSF alone could consistently affect the mobilization of the required number of HPC to be used in patients treated for malignant diseases, it would likely be used preferentially in most circumstances because of its predictability and safety, but concerns remain regarding high rates of failure when used alone to mobilize CD34+ cells. Although some controversy exists regarding optimal number of CD34+ cells required to fully reconstitute hematopoiesis following autologous transplantation, the general consensus is that reinfusion of greater than 5 × 106 CD34+ cells/kg recipient weight will promote prompt trilineage reconstitution [23, 96–98]. Lesser numbers (2.5 to 4.9 × 106 CD34+ cells/kg) infused results in prompt neutrophil recovery, but platelet recovery and hospital stays may be prolonged. Below 2.0–2.5 × 106 CD34+ cells/kg re-infused, meaningful delays in platelet recovery may be observed. A recent study suggests that expression of aldehyde dehydrogenase (ALDH) in mobilized cells may serve as a functional marker for engrafting cells (both CD34+ and CD34 cells can be ALDH+) and correlates with engraftment kinetics better than CD34 [99]. Since ALDH is expressed in cells enriched with engraftment capacity, it may serve as a better marker than CD34 to designate the functional capacity of mobilized cells but this will need to be confirmed by other groups [100]. At any rate, current trends favor strategies designed to mobilize the greatest number of HPC in the fewest number of collections. Unfortunately, recent data suggest that a significant proportion of patients who have received prior chemotherapy fail to mobilize >5.0 × 106 CD34+ cells/kg with G-CSF alone [23, 87, 101]. In one large trial involving breast cancer patients, only one-third of patients given G-CSF as a single mobilizing agent achieved a 5.0 × 106 CD34+ cell dose per kilogram level [101]. In the recently completed phase 3 trial comparing G-CSF plus placebo to G-CSF plus AMD3100 (Optimize 1), a startling 53% of patients with NHL on the placebo-controlled arm failed to achieve a minimum CD34+ cell dose of 2.0 × 106/kg after four collections [87]. Thus, in previously treated patients with NHL (and probably HD) it is likely that G-CSF will need to be combined with other methods (additional HGF, chemotherapy, novel mobilizing agents such as AMD3100) in order to consistently induce mobilization of large number of HPC for transplantation. Ultimately, the development of predictive models to estimate the likelihood of an efficient HPC mobilization for any given mobilization strategy seems the most rational way to determine the likelihood of success as well as the need for novel approaches. Finally, some have argued that the use of chemotherapybased mobilization strategies are required since this additional chemotherapy will result in a lower risk of relapse during or after HPC mobilization. The randomized trials performed have not substantiated this claim and a recent French study even suggested that in multiple myeloma patients cyclophosphamide-based mobilization may skew the collection toward increased number of regulatory T-cells

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which emerge upon recovery from chemotherapy, which paradoxically when reinfused could raise the risk of disease recurrence [102]. This observation in a small number of patients requires further study by others. Studies have been conflicting on whether there are differences, particularly clinically relevant ones, in the mobilization of contaminating tumors cells based on the type of mobilization strategy employed but in general this if difficult to prove one way or the other. Many centers use chemotherapy-based mobilization simply because they believe that it will result in a higher yield of CD34+ cells mobilized compared to G-CSF alone, and this may indeed be the case. So, despite the potential drawbacks chemotherapy-based mobilization has its merits. The addition of AMD3100 to the list of agents capable of inducing mobilization may change the landscape toward a cytokine-chemokine antagonist approach but there are still concerns that the need to administer AMD3100 8–10 h prior to leukapheresis, to achieve optimal mobilization, will prove impractical and clearly further studies aimed at evaluating alternative schedules will be necessary before this agent is widely adopted. One practical approach may be to base the use of AMD3100 on a peripheral blood CD34+ cell count obtained on the fourth or fifth day of mobilization with G-CSF alone and to add it to G-CSF only in those individuals who do not appear to be mobilizing well.

6.  Mobilization of Stem Cells from Normal Donors In the allogeneic transplantation setting, the need for novel strategies to collect HPC from normal donors is less clear than in patients with hematological malignancies. In the vast majority of donors, G-CSF safely and effectively mobilizes HPC for transplantation. Seven randomized trials comparing HLAidentical BM to MPB have been published and uniformly confirm that MPB results in more rapid hematopoietic engraftment compared to BM [12, 13, 15–19, 103]. Increasingly, G-CSF is used for mobilization of HPC in unrelated donor transplantation and will be compared to BM in an upcoming randomized clinical trial supported by the NIH-sponsored Blood and Marrow Transplant Clinical Trials Network. Nevertheless, meta-analyses suggest a higher risk of either acute or chronic GVHD following transplantation of MPB [12, 16]. Further, a recent retrospective study in recipients of unrelated donor cells did not demonstrate an advantage to mobilized blood over marrow due to more GVHD [104]. Controversy exists regarding the optimal CD34+ cell dose to be transplanted. The general consensus is that cell doses greater than 4 × 106 CD34+ cells are necessary for prompt and durable engraftment [105, 106]. However, a small proportion of donors will not mobilize a graft containing at least 4.0 × 106 CD34+ cells/kg following G-CSF alone. In our institution, about 10–15% of donors given G-CSF at 10 mg/kg/daily mobilize a graft containing 38 years and use of a single rather than multiple daily G-CSF doses as factors associated with a low CD34 yield [120] Suzuzya et  al. [118] and Lysak et  al. [117] also found increased age to be a negative predictor for successful mobilization. Another study found

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that donors with higher baseline peripheral blood CD34+ cell levels prior to G-CSF mobilized better than those with lower levels [121]. Thus, donor age, steady-state CD34 level, and both the total dose and schedule of G-CSF may impact CD34+ cell mobilization. In addition to causing CD34+ cell mobilization by its indirect effects on CXCR4/SDF-1, G-CSF has pleiotropic effects on other important allograft constituents including T-, B-, NK-, and dendritic cells. G-CSF administration has been associated with a polarization of T-cells to a Th2 phenotype [122–124]. G-CSF effects circulating monocytes, leading to an increase in IL-10 secretion. Also, G-CSF administration may induce a plasmacytoid differentiation effect on donor dendritic cells [125–128]. Together, these immunomodulatory effects of G-CSF have been cited to explain the observation that rates of acute GVHD are not substantially different using mobilized cells compared to bone marrow despite the transplantation of about tenfold greater numbers of T-cells. In an effort to further understand and possibly exploit these immunomodulatory effects, G-CSF analogs have recently been studied to determine their influence on stem cell mobilization, GVHD, and graft versus leukemia (GVL). Pegylated filgrastim (Neulasta, Amgen) has a much longer half-life compared to native filgrastim and a single dose results in the mobilization of sufficient number of CD34+ cells to promote engraftment in allogeneic recipients following myeloablative conditioning [129]. One study in HLA-identical sibling donors suggested that a 12 mg dose may be superior to 6 mg in terms of CD34+ cell dose mobilized [129]. The allografts mobilized by pegfilgrastim appeared to result in similar kinetics of engraftment as well as comparable rates of GVHD compared to native filgrastim. The kinetics of mobilization following a single dose of pegylated filgrastim were roughly the same as that observed following daily G-CSF administration with peak CD34+ cell mobilization occurring by day 5–6. Recently, a chimeric molecule containing ligands for both G-CSF and Flt-3 has been shown to limit GVHD while retaining the capacity to induce GVL in murine models, possibly through the expansion of regulatory NK/T type cells [130, 131]. Further work is needed to validate these effects. It is unclear whether chimeric molecules will be further developed for clinical use, particularly in volunteer donors, due to concerns regarding enhanced toxicity associated with stimulating multiple receptors.

7.  Safety and Toxicity of G-CSF in Normal Donors G-CSF-based PBSC mobilization is generally well tolerated, and essentially all donors complete the mobilization and collection procedures. However, retrospective and prospective studies have identified transient, but not insignificant, morbidities experienced by G-CSF-mobilized donors. The most common symptoms are bone pain, headache, fatigue, and nausea, and the incidence of pain and anxiety are similar to that observed with bone marrow donation [119, 121, 132–135]. In a retrospective analysis of >1,300 donors registered with the International Bone Marrow Transplant Registry (IBMTR) or European Blood and Marrow Transplant Group (EBMT), the rate of serious complications from G-CSF mobilization and PBSC collection was 1.1%, as compared to 0.5% following bone marrow collection [132, 136].

Chapter 22  Mobilization of Hematopoietic Cells Prior to Autologous  401

Many of these complications were associated with central venous catheters, which were inserted in 20% of donors. More serious side effects, while rare, nonetheless can occur and represent a major issue when they affect normal individuals. Stroncek et  al. reported that 19 of 20 adult allogeneic blood donors given G-CSF 10 mg/kg/day had transient increases in spleen length by an average of 17% [137]. A prospective trial published by Platzbecker et  al. in 91 healthy allogeneic donors treated with G-CSF 7.5 mg/kg/day for 5 days evaluated changes in spleen size using serial ultrasound examinations [138]. Donors exhibited a mean 110% transient increase in spleen length; 20% had a 1.9 cm increase in length and 0.9  cm increase in spleen thickness. A similar study design communicated by Stroncek et  al. in 18 healthy subjects given G-CSF 10 mg/kg/day for 5 days revealed a transient mean enlargement of spleen length from 10.7 to 12.1 cm that returned to baseline 10 days after completion of apheresis [139]. Falzetti et  al. reported the development of splenomegaly and spontaneous splenic rupture in a 33-year-old male allogeneic blood stem cell donor given G-CSF 16 mg/kg/day for 6 days [140]. He recovered fully after an emergency splenectomy that revealed a 445  g spleen and a capsular tear with massive extra-medullary myelopoiesis. Becker et al. reported a 22-year-old man who donated bone marrow for a first cousin and 4 months later was mobilized with G-CSF 10 mg/kg/day for 6 days as a planned blood progenitor cell collection for relapse [141]. Four days after completion of collection, he underwent an emergency splenectomy for spontaneous splenic rupture. G-CSF therapy was not unequivocally the etiology as the donor has serologic findings consistent with convalescence after Epstein-Barr infection. Kröger et al. retrospectively reviewed data obtained from 90 healthy allogeneic donors given G-CSF 5 or 8 mg/kg twice daily [142]. One young subject had a nonfatal traumatic splenic rupture after 5 days of the G-CSF 5 mg/kg twice daily dosing, but this serious adverse event fully resolved without surgical intervention. Dincer and coworkers reported a 43-year-old man who received the same treatment experienced a spontaneous splenic rupture on the fifth day of therapy that resolved without surgical intervention [143]. Finally, Balaguer et al. reported a case of a 51-year-old man who developed spontaneous splenic rupture after G-CSF treatment for stem cell mobilization for his HLA-identical sibling [144]; he fully recovered after undergoing an emergency splenectomy. The Spanish National Donor Registry reported this case to be the only such occurrence in 1,240 registered PBSC donors [145]. Another potentially serious toxicity may be related to the procoagulant effects of G-CSF and include the risk of precipitating a myocardial infarction or causing cerebral ischemia in high-risk individuals. Both adverse events have been reported and therefore all donors treated with G-CSF should be carefully screened for any history of coronary artery or cerebrovacular disease [146– 149]. Owing to its potential to precipitate acute sickle crisis, G-CSF is contraindicated in patients with sickle cell disease; further, in donors with sickle cell trait, G-CSF has been associated with precipitation of sickle crisis [150–152]. Patients with underlying autoimmune disease have been treated with G-CSF alone to mobilize cells prior to planned autologous transplantation. In this setting, its use has been associated with flares of disease in patients with rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis [153–156]. Therefore, its use in donors with these disorders also is not recommended.

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Even short-term stimulation by G-CSF results initiation of the cell cycle by at least a fraction of primitive hematopoietic cells [157]. The effects of short-term G-CSF stimulation on genomic stability or chromatin remodeling are unclear [158–160]. A few studies raise concern that even short-term G-CSF treatment may affect genomic stability [158, 159]. The long-term ramifications of these changes, if any, are unknown and concerns remain largely speculative. That said, there has always been a theoretical concern that exogenous stimulation of hematopoiesis in a first-degree relative of a patient with a hematopoietic malignancy, particularly acute myeloid leukemia, may put the donor at risk for later development of leukemia. In fact, acute leukemia has been noted in G-CSF-stimulated siblings, including two cases of AML occurring 5–6 years after G-CSF administration to mobilize cells for transplant into recipients with AML [161, 162]. Given that the cases of leukemia may have just as easily occurred by chance and there is a higher risk of leukemia in first-degree relatives of patients with acute leukemia, the role that G-CSF short-term administration may have played in these cases is speculative [132, 163–165]. Clearly, such occurrences raise concern and justify the call for continued long-term follow-up of G-CSF stimulated donors. The use of G-CSF in pediatric siblings donating for individuals with malignant or non-malignant disorders also raises concern as to the long-term impact of this maneuver on the health in a young individual [166]. Further, this concern raises several ethical issues related to obtaining both parental consent and assent of the adolescent donor. In summary, collection of G-CSF-mobilized blood is associated with morbidity comparable to that experienced after bone marrow donation, and many donors must undergo more than one leukapheresis procedure. Donors may benefit from new mobilization strategies that minimize exposure to G-CSF injections and reduce the number of leukapheresis sessions. In addition, recipient outcomes could be improved with grafts that provide faster count recovery or that reduce the incidence of GVHD.

8.  Mobilizing Donor Cells without Using Cytokines Although G-CSF is an effective agent for the mobilization of stem cells from normal donors, such altruistic individuals could benefit from new mobilizing strategies which are more effective and/or less toxic. The agents currently in use to mobilize stem cells in donors have both unique and overlapping toxicities. Each has its own potential advantages and disadvantage. Notably, each product results in the mobilization of an allograft with unique cellular compositions that may alter the likelihood of graft failure, GVHD, immune reconstitution, and possibly relapse. The search for new mobilizing agents currently is driven by pre-clinical research which is elucidating the mechanisms of stem cell localization within the bone marrow and mobilization in response to cytokine signals. As our understanding of the interactions between stem cells and the bone marrow microenvironment improves, new mobilizing agents can be designed rationally. Given the promising results in autologous mobilization, AMD3100 is now being investigated for stem cell mobilization and transplantation from healthy donors. Devine et  al. have reported the preliminary results of a pilot study evaluating the safety and efficacy of AMD3100 mobilization and transplantation

Chapter 22  Mobilization of Hematopoietic Cells Prior to Autologous  403

Table 22-2.  Agents used to mobilize stem/progenitor cells in normal donors. Agent

Results

Unique aspects

G-CSF

Effective as single agent

Bone pain, requires 5 days for maximal effect

GM-CSF

Less effective than G-CSF and possibly more toxic

Causes fever, edema, bone pain; may be associated with less acute GVHD

Pegfilgrastim

Similar ability to mobilize compared to G-CSF

Same toxicity as G-CSF; requires only one dose; same kinetics as G-CSF

AMD3100

Mobilized functional cells capable of long-term engraftment

Mobilized functional cells in only 4 h; lower CD34+ cell doses mobilized compared to G-CSF; possibly less toxic than G-CSF

G-CSF granulocyte-colony stimulating factor, GM-CSF granulocyte-macrophage colony stimulating factor

in HLA-matched sibling donors [167]. Allografts from HLA-matched sibling donors were mobilized and collected without G-CSF using AMD3100. Donors (N = 25) were treated with AMD3100 at a dose of 240 mg/kg by subcutaneous injection and leukapheresis was then initiated just 4 h later. Two-thirds of the donors collected an allograft with a CD34+ cell dose, sufficient for transplantation after just one dose of AMD3100. No donor experienced more than grade one toxicity. The allografts collected after AMD3100 contained higher CD4+ T-cell doses compared to G-CSF mobilized grafts. Following a myeloablative regimen, twenty patients with hematological malignancies received allografts collected after AMD3100 alone. All patients engrafted neutrophils (median day +10) and platelets (median day +12) promptly. Acute GVHD grades 2–4 occurred in 35% of patients. Therefore, despite the infusion of higher T-cell doses, there was no appreciable increase in GVHD in comparison to G-CSFmobilized grafts. One patient died due to complications related to acute GVHD. No unexpected adverse events were observed in any of the recipients. All 14 patients surviving in remission had robust trilineage hematopoiesis and were transfusion-free with a median follow-up of 277 days (range 139–964 days). This small study suggests that direct antagonism of CXCR4 by AMD3100 may provide a more rapid and possibly less-toxic and cumbersome alternative to traditional G-CSF-based mobilization in normal donors and appears worthy of further pursuit in larger multi-center trials. Table 22-2 lists unique aspects of agents used to mobilize stem/progenitor cells in normal donors.

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Chapter 22  Mobilization of Hematopoietic Cells Prior to Autologous  411 130. MacDonald KPA, Rowe V, Filippich C et  al (2003) Donor pretreatment with progenipoietin-1 is superior to granulocyte colony-stimulating factor in preventing graft-versus-host disease after allogeneic stem cell transplantation. Blood 101: 2033–2042 131. Morris ES, MacDonald KPA, Hill GR (2006) Stem cell mobilization with G-CSF analogs: A rational approach to separate GVHD and GVL? Blood 107:3430–3435 132. Anderlini P, Champlin RE (2008) Biologic and molecular effects of granulocyte colony-stimulating factor in healthy individuals: Recent findings and current challenges. Blood 111:1767–1772 133. Fortanier C, Kuentz M, Sutton L et al (2002) Healthy sibling donor anxiety and pain during bone marrow or peripheral blood stem cell harvesting for allogeneic transplantation: Results of a randomised study. Bone Marrow Transplant 29:145–149 134. Murata M, Harada M, Kato S et al (1999) Peripheral blood stem cell mobilization and apheresis: Analysis of adverse events in 94 normal donors. Bone Marrow Transplant 24:1065–1071 135. Rowley SD, Donaldson G, Lilleby K, Bensinger WI, Appelbaum FR (2001) Experiences of donors enrolled in a randomized study of allogeneic bone marrow or peripheral blood stem cell transplantation. Blood 97:2541–2548 10.1182/ blood.V97.9.2541 136. Anderlini P, Przepiorka D, Korbling M, Champlin R (1998) Blood stem cell procurement: Donor safety issues. Bone Marrow Transplant 21:S35–S39 137. Stroncek D, Shawker T, Follmann D, Leitman SF (2003) G-CSF-induced spleen size changes in peripheral blood progenitor cell donors. Transfusion 43:609–613 138. Platzbecker U, Prange-Krex G, Bornhauser M et al (2001) Spleen enlargement in healthy donors during G-CSF mobilization of PBPCs. Transfusion 41:184–189 139. Stroncek D, Dittmar K, Shawker T, Heatherman A, Leitman S (2004) Transient spleen enlargement in peripheral blood progenitor cell donors given G-CSF. J Transl Med 2:25 140. Falzetti F, Aversa F, Minelli O, Tabilio A (1999) Spontaneous rupture of spleen during peripheral blood stem-cell mobilisation in a healthy donor. The Lancet 353:555 141. Becker P, Wagle M, Matous S et al (1997) Spontaneous plenic rupture following administration of granulocyte colony-stimulating factor (G-CSF): Occurrence in an allogeneic donor of peripheral blood stem cells. Biol Blood and Marrow Transplant 3:45–49 142. Kröger N, Renges H, Sonnenberg S et  al (2002) Stem cell mobilisation with 16 mg/kg vs 10 mg/kg of G-CSF for allogeneic transplantation in healthy donors. Bone Marrow Transplant 29:727–730 143. Dincer AP, Gottschall J, Margolis DA (2004) Splenic rupture in a parental donor undergoing peripheral blood progenitor cell mobilization. J Pediatr Hematol Oncol 26:761–763 144. Balaguer H, Galmes A, Ventayol G, Bargay J, Besalduch J (2004) Splenic rupture after granulocyte-colony-stimulating factor mobilization in a peripheral blood progenitor cell donor. Transfusion 44:1260–1261 145. de la Rubia J, Martínez C, Solano C et al (1999) Administration of recombinant human granulocyte colony-stimulating factor to normal donors: Results of the Spanish national donor registry. Bone Marrow Transplant 24:723–728 146. Dagia NM, Gadhoum SZ, Knoblauch CA et al (2006) G-CSF induces E-selectin ligand expression on human myeloid cells. Nat Med 12:1185–1190 147. Fukumoto Y, Miyamoto T, Okamura T et  al (1997) Angina pectoris occurring during granulocyte colony-stimulating factor-combined preparatory regimen for autologous peripheral blood stem cell transplantation in a patient with acute myelogenous leukaemia. Br J Haematol 97:666–668 148. Hill JM, Syed MA, Arai AE et  al (2005) Outcomes and risks of granulocyte colony-stimulating factor in patients with coronary artery disease. J Am Coll Cardiol 46:1643–1648

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S.M. Devine 149. Lindemann A, Rumberger B (1993) Vascular complications in patients treated with granulocyte colony-stimulating factor (G-CSF). Eur J Cancer 29:2338–2339 150. Adler BK, Salzman DE, Carabasi MH, Vaughan WP, Reddy VVB, Prchal JT (2001) Fatal sickle cell crisis after granulocyte colony-stimulating factor administration. Blood 97:3313–3314 151. Horowitz MM, Confer DL (2005) Evaluation of hematopoietic stem cell donors. Hematology 2005:469–475 152. Kang EM, Areman EM, David-Ocampo V et al (2002) Mobilization, collection, and processing of peripheral blood stem cells in individuals with sickle cell trait. Blood 99:850–855 153. Burt RK, Fassas A, Snowden J et al (2001) Collection of hematopoietic stem cells from patients with autoimmune diseases. Bone Marrow Transplant 28:1–12 154. Gottenberg JE, Roux S, Desmoulins F, Clerc D, Mariette X (2001) Granulocyte colony-stimulating factor therapy resulting in a flare of systemic lupus erythematosus: Comment on the article by Yang and Hamilton. Arthritis Rheum 44:2458–2460 155. Nash RA, Bowen JD, McSweeney PA et al (2003) High-dose immunosuppressive therapy and autologous peripheral blood stem cell transplantation for severe multiple sclerosis. Blood 102:2364–2372 156. Stricker RB, Goldberg B (1996) G-CSF and exacerbation of rheumatoid arthritis. Am J Med 100:665–666 157. Mahmud N, Devine SM, Weller KP et al (2001) The relative quiescence of hematopoietic stem cells in nonhuman primates. Blood 97:3061–3068 158. Hernandez JM, Castilla C, Gutierrez NC et al (2005) Mobilisation with G-CSF in healthy donors promotes a high but temporal deregulation of genes. Leukemia 19:1088–1091 159. Nagler A, Korenstein-Ilan A, Amiel A, Avivi L (2004) Granulocyte colonystimulating factor generates epigenetic and genetic alterations in lymphocytes of normal volunteer donors of stem cells. Exp Hematol 32:122–130 160. Pamphilon D, Mackinnon S, Nacheva E et  al (2006) The use of granulocyte colony-stimulating factor in volunteer blood and marrow registry donors. Bone Marrow Transplant 38:699–700 161. Bennett CL, Evens AM, Andritsos LA et al (2006) Haematological malignancies developing in previously healthy individuals who received haematopoietic growth factors: Report from the research on adverse drug events and reports (RADAR) project. Br J Haematol 135:642–650 162. Makita K, Ohta K, Mugitani A et  al (2004) Acute myelogenous leukemia in a donor after granulocyte colony-stimulating factor-primed peripheral blood stem cell harvest. Bone Marrow Transplant 33:661–665 163. Hasenclever D, Sextro M (1996) Safety of AlloPBPCT donors: Biometrical considerations on monitoring long term risks. Bone Marrow Transplant 17(Suppl 2) :S28–S30 164. Rauscher GH, Sandler DP, Poole C et  al (2002) Family history of cancer and incidence of acute leukemia in adults. Am J Epidemiol 156:517–526 165. Shpilberg O, Modan M, Modan B, Chetrit A, Fuchs Z, Ramot B (1994) Familial aggregation of haematological neoplasms: A controlled study. Br J Haematol 87:75–80 166. Pulsipher MA, Nagler A, Iannone R, Nelson RM (2006) 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 46:422–433 167. Devine SM, Vij R, Rettig M et al (2008) Rapid mobilization of functional donor hematopoietic cells without G-CSF using plerixafor, an antagonist of the CXCR4/ SDF-1 interaction. Blood 112:990–998

Chapter 23 Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation Martin Stern, Sandrine Meyer-Monard, Uwe Siegler, and Jakob R. Passweg

1. Background Natural killer cells (NK) reside in the bone marrow, spleen, and peripheral blood where they form approximately 10% of peripheral blood lymphocytes [1]. Unlike the B- and T-lymphocytes, NK cells do not express clonally rearranged receptors to detect antigens. Instead, activation is regulated by the integration of signaling from germline-encoded activating and inhibitory cell surface receptors [2]. These include inhibitory receptors for HLA class I antigens and activating receptors such as DNAM-1, NKG2D, and natural cytotoxicity receptors (NCRs) [3]. Inhibitory receptors for self HLA include Killer cell Immunoglobulin-like Receptors (KIR), the lectin-like receptor NKG2A, and LIR1/ILT-2 [4] (Table 23-1). Upon interaction with target cells expressing activating ligands, lack of involvement of inhibitory receptors results in predominance of activating signaling and target cell lysis. These systems form the basis of the “missing self” recognition and exemplify the mechanisms of the immune system to counteract the HLA downregulation induced by tumors and viral infection to escape the T-cell recognition. While initial data derived from clonally expanded NK cells had suggested that every NK cell expresses at least one inhibitory receptor for self MHC [5], more recent analyses in mice and humans have shown that subsets of NK cells do not express inhibitory receptors for self HLA [6–8]. The mechanism of tolerance in this subset is not completely understood. However, growing evidence exists for the role of KIR-HLA interactions in “licensing” of NK cells in a manner that only NK cells expressing inhibitory receptors for self HLA acquire full functional competence. The ligands for inhibitory KIRs are HLA class I antigens. The main inhibitory KIR/HLA pairs are KIR2DL1 recognizing HLA-C antigens with a lysine at position 80 (e.g. HLA-C 2, 4, 5, 6); KIR2DL2 and KIR2DL3 recognizing HLA-C antigens with asparagine at position 80 (e.g. HLA-C 1, 3, 7, 8), and KIR3DL1 recognizing HLA-B antigens with Bw4 specificity (e.g. HLA B5, 13, 17, 27). KIR3DL2 has been shown to recognize HLA-A3 and A11

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_23, © Springer Science + Business Media, LLC 2003, 2010

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Table 23-1.  Main natural killer cell surface receptors and their ligands. Inhibitory receptors

Ligands

Inhibitory KIR

HLA-A/B/C

KIR2DL1

HLA C group 2 (e.g. C2,4,5,6)

KIR2DL2/3

HLA C group 1 (e.g. C1,3,7,8)

KIR3DL1

HLA Bw4 (e.g. B5, 13, 17, 27)

KIR3DL2

HLA A3/A11

NKG2A

HLA-E

Activating receptors

Ligands

Natural cytotoxicity receptors (NKp30, NKp44, NKp46)

Unknown

Activating KIR

Unknown (HLA class I ?)

NKG2D

MIC-A/B, ULBPs

DNAM-1

PVR, Nectin-2

CD16

IgG

2B4

CD48

KIR killer cell immunoglobulin-like receptors, HLA human leukocyte antigen, NKG2A/D natural killer cell group 2A/D, MIC major histocompatibility complex class I chain-related, ULBP UL16 binding proteins, DNAM-1 DNAX accessory molecule 1, PVR polio virus receptor, IgG immunoglobulin G

expressed on target cells in  vitro depending on the peptide presented; its significance in  vivo remains unclear [9]. KIR2DL4 recognizes HLA-G, an atypical class I antigen expressed on decidual cells, and is implicated in maintaining the tolerance against the fetal-derived placental tissue [10]. KIR3DL3 and KIR2DL5 are still orphan receptors [11]. Approximately 30% of a Caucasian population carries the gene for a single activating KIR (KIR2DS4), whereas the rest carry genes for between one and six activating KIRs. Ligands for activating KIRs have not been defined. Extensive homology exists in the extracellular domains of activating and inhibitory KIRs suggesting that they might share ligands; functional studies have, however, shown only very weak affinities between activating KIRs and HLA-class I antigens, indicating that HLA class I antigens may not be the true ligand for activating KIR [12]. While activating KIR appears to be implicated in host defense in immunosuppressed patients [13], their possible role in allorecognition remains controversial. Phenotypically, NK cells are defined by an expression of CD56 and lack of the T-cell receptor-associated antigen CD3. NK cells respond to cytokines, their in vitro killing activity can be greatly enhanced by culture in IL-2, and some studies suggest that adoptive transfer of NK cell subsets in an activated state (i.e. after stimulation with IL-2, IL-12, IL-15, or combinations thereof), or in vivo activation (e.g. by administration of IL-2 after infusion of NK cells) may be required for optimal efficacy.

Chapter 23  Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation 

1.1. Transplants from Haploidentical Donors Haploidentical stem cell transplantation (HSCT) from mismatched family donors is a treatment option for patients lacking an HLA-identical sibling, a matched unrelated donor, and a suitable cord blood. As HLA antigens are inherited as two haplotypes, most patients lacking an HLA-identical sibling will have a haploidentical donor, i.e. a sibling or parent, or other relative sharing at least one HLA-haplotype. Haploidentical HSCT remains difficult, even though progress has been made using large doses of highly purified stem cells. Graft-versus-host disease is effectively prevented by extensive T-cell depletion, however, immune reconstitution is slow with a high incidence of infectious complications. Because of T-cell depletion, T-lymphocyte mediated graft-versus-leukemia reactions are limited and many patients relapse. Lymphocyte, especially CD4 counts, remain suppressed for many months after haploidentical HSCT, the first lymphoid population to recover are NK cells. Recent data have shown that these early reconstituting NK cells are immature with impaired cytotoxicity [14]. Such patients are, therefore, candidates for adoptive immunotherapy to enhance immune reconstitution and graft versus leukemia effects. 1.2. NK Cell Alloreactivity As inhibitory signals from self HLA-receptors usually override signals from activating receptors, early trials using autologous NK cells in the 1980s were largely unsuccessful [15], and the focus of NK therapy shifted to the use of allogeneic NK cells. NK cell alloreactivity could be broadly defined as any NK cell effect against cells involving some form of allorecognition. Based on the HLA class I typing of recipient and donor, NK cell alloreactivity, i.e. a lack of inhibition of donor NK cells and hence killing activity can be expected if functional donor NK cells expressing a given KIR encounter recipient cells that lack the corresponding KIR ligand (i.e. HLA class I molecule). Of the relevant inhibitory KIRs with defined ligand specificity, either KIR2DL2 or KIR2DL3 is expressed in all donors, and both KIR2DL1 and KIR3DL1 are found in >90% of donors [16, 17]. Therefore, assessing only KIR ligands will provide a reasonable approximation of potential KIR/KIR-ligand mismatches in most cases; KIR genotyping as well as flowcytometric studies may be performed to document the presence of a KIR in the donor and estimate the magnitude of the potentially alloreactive NK cell subset. NK cells may exert alloreactivity either in the graft versus host/tumor or in the host versus graft direction. NK cell alloreactivity in the host versus graft direction was first described as the phenomenon of “hybrid resistance” in a mouse transplantation model in the 1960s. In the clinical setting of HSCT after myeloablative conditioning, it rarely has any measurable effects due to the intense nature of the conditioning regimen effectively ablating host NK cells before transplantation. NK cell alloreactivity in the graft versus host direction is of specific interest as NK cells may mediate graft versus leukemia effects. There is evidence in animal studies of a multitude of potentially beneficial effects including NK versus leukemia activity, reducing relapse risks; NK-versus residual host T-cell activity, reducing graft rejection risks; and NK versus host antigen presenting cell activity [18], potentially associated with reduced GVHD risks

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as host antigen presenting cells have been implicated in the initiation of acute graft versus host disease [19]. In the setting of human haploidentical HSCT, NK cells take 2–3 weeks to mature from transplanted CD34+ cells; graftderived NK cells may, therefore, emerge too late to have a significant impact on the risk of rejection or GVHD, providing a rationale for infusion of mature donor NK cells along with the graft. Importantly, due to apparent restriction of NK cell alloreactivity to hematopoetic cells, large doses of NK cells may be infused without causing GVHD. In human studies, a positive outcome of KIR/HLA disparity has been demonstrated in the haploidentical HSCT setting [20]. A lower than expected rate of leukemia relapse was noted in patients with AML when the haploidentical donor possessed inhibitory KIRs with their corresponding ligand missing in the recipient (KIR-ligand mismatch). Alloreactive NK clones that killed recipient hematopoeitic cells including leukemic blasts in vitro were isolated from recipients following HSCT [21]. While this translated into a reduction of relapse risk in patients transplanted for an acute myeloid leukemia, no beneficial effect concerning relapse was noted in patients transplanted in chemoresistant relapse (perhaps due to unfavorable effector:target cell ratios) [22] or for adult patients transplanted for B-cell acute lymphoblastic leukemia [18]. Clinical results correlated with in vitro alloreactivity: acute myeloid leukemia blast cells were almost universally killed by alloreactive NK cells, whereas in adult acute lymphoblastic leukemia blast cells were in vitro resistant to NK alloreactivity due to lack of expression of activating NK ligands [23]. In contrast, both in vitro studies [24] and transplant outcome of pediatric patients [25] provide evidence that in pediatric ALL, blast cells are sensitive to NK alloreactivity. 1.3. Adoptive Immunotherapy/Donor Lymphocyte Infusion Adoptive immunotherapy using donor lymphocyte infusion (DLI) has become a standard practice in patients relapsing after HSCT, since the initial description in patients with CML in 1990 [26]. DLI may be administered in bulk doses or in a graded incremental fashion which may be beneficial because of less GVHD [27]. DLI appears to be highly effective in slowly progressing diseases such as CML and less so in diseases with rapid proliferation. This is likely due to the fact that graft versus leukemia activity of unfractionated DLI is mainly exerted by T-lymphocytes: as frequencies of alloreactive T-cells in an HLA matched setting are minute and as antigen-driven expansion requires time, responses take weeks and months to develop. The major risk of DLI is GVHD. Unfractionated DLI have been rarely used in recipients of haploidentical HSCT, mainly because of GVHD risks. Some investigators have been using very small doses of DLI to stabilize the graft and to promote the immune reconstitution; however, GVHD remains a significant problem [28]. DLI with highly selected NK cells in recipients of haploidentical HSCT provide a model to study the effects of NK cells and to elucidate mechanisms of NK cell alloreactivity without carrying a risk for GVHD.

2.  NK Cell DLI Based on the above, several groups have investigated – in the context of allogeneic HSCT – the preparation and infusion of purified, T-cell depleted, donor

Chapter 23  Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation 

NK lymphocytes (NK DLI) with the aim to (a) consolidate engraftment and (b) to induce graft versus leukemia effects. Most of these studies include small numbers of patients, and have been published as abstracts only.

3. Technical Aspects 3.1. Natural Killer Cell Product The aim of NK cell engineering in haploidentical HSCT is to obtain a product with a high number of functionally active CD56+/CD3− NK cells, depleted of CD3+ T lymphocytes. The choice of the target cell number for adoptive immunotherapy is based on the capacity to harvest NK cells, and on the experience with T-cell collection for DLI, where usually between 1.0 × 10×6/ kg and 1.0 × 108/kg CD3+ cells are infused. Therefore, the target NK cell dose for NK-DLI products is often fixed at ³1.0 × 107/kg body weight. However, there are no experimental or clinical data to help define an adequate cell dose. The prerequisite for an NK cell product in the haploidentical setting is T-cell depletion with a target CD3+ T-cell contamination of less than 0.5–1.0 × 105/ kg, the threshold dose which can be administered without risk of causing graftversus-host disease. 3.2. Harvesting of NK Cell Product The efficacy of NK cell collection from a healthy donor is related to the number of NK cells in the peripheral blood at the time of harvest, and the blood volume processed during leukapheresis. The pre-leukapheresis peripheral blood values correlate with the yields, therefore efficacy of NK cell collection can be predicted from the peripheral cell counts of the donor [29]. In our experience of haploidentical donors (leukapheresis volume 10–12  L), the number of collected NK cells ranged between 1.7 and 30 × 108, with contaminating T-cells between 28 and 155 × 108 [30, 31]. The Memphis group reported on 12 leukapheresis products after processing twice the blood volume in healthy adult volunteers. Products contained a median of 65 × 108 mononuclear cells (range, 20–137 × 108), with a median number of NK cell count of 4.4 × 108 (range, 0.58–22.3 × 108) [32]. Finally, the Minneapolis group reported on 70 leukaphereses of 15  L each: mean nucleated cell count after apheresis was 197 × 108, with a 10.7% fraction of NK cells [33].

4. NK Cell Product Engineering The aim of mononuclear cell engineering is to obtain a highly purified NK cell fraction with minimal T-cell contamination and conserved natural cytotoxicity. The NK purification steps should lead to an optimal NK cell yield with minimal loss of the target CD3−/CD56+ cell population. For clinical application, most commonly a large-scale purification method allowing automated, efficient, and relatively rapid isolation of human NK cells is used. This NK selection is based on a two-step immunomagnetic method, with first a CD3+ cell depletion followed by a CD56+ cell enrichment. Using this purification method, NK purity of more than 90% is obtained, with an efficient T-cell depletion of 3–5 logs, allowing an infusion of less than 0.5–1.0 × 105/kg

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CD3+ cells. The high NK cell purity and an extensive T-cell depletion are at the expense of a considerable loss of NK cells during engineering. The final recovery of CD3−/CD56+ NK cells ranges between 30% and 70%, with an inverse correlation between NK recovery and NK purity. Natural cytotoxicity of the purified cells is increased by approximately five-fold as compared to the unpurified mononuclear cells, and may be increased further by stimulation with different cytokines in vitro. The overall processing time is about 8–10 h. Preliminary data using a single-step positive selection of NK cells using antibodies directed against the NK cell-specific NKp46 antigen are promising and this procedure may allow a more rapid and cost effective isolation of NK cells in the future [34]. Enriched NK cells can be infused without any additional manipulation, after overnight incubation in high dose IL-2 or after cytokine driven in vitro expansion. Expansion has two aims: to activate the freshly selected CD56+ cells and to increase the total number of NK cells. Using CD69 as an activation marker, activation of NK cells occurs within 24 h of incubation with IL-2 [35]. When enriched CD56+ NK cells are cultured with either IL-2 alone or IL-2 combined with IL-15, a significant expansion can be observed. However, there is a lag of 1 week before NK cells start to proliferate. During the second week, the expansion occurs, leading to a five- to 20-fold increase of CD56+ NK cells at the end of two weeks of culture [36]. With a protocol that enables the generation of NK cells on a clinical scale using a closed system that allows good manufacturing practice (GMP) conformity, the expanded NK cells are highly cytotoxic against different malignant target cells [35]. As infused cell number appears to be critical, alternative expansion protocols are currently being developed with the aim to augment NK expansion such as co-culture with cytokine producing feeder cells [37] or expansion of NK cells derived from umbilical cord blood units [38, 39]. Clinical scale collection, enrichment, activation, and expansion of purified NK cells are feasible. Most of the technical aspects for adoptive NK cell therapy are mature and ready for clinical application. However, the laboratory procedures involved are time consuming and expensive, need specific skills, and must be performed according to a GMP-compliant protocol. 4.1. NK Cell Infusion Several groups have worked on pilot projects investigating feasibility and effects of adoptive immunotherapy using NK cells. Different approaches have been tested (summarized in Table 23-2). Fifteen recipients of haploidentical HSCT with AML (N = 7) or ALL (N = 5), or other hematological malignancy (N = 3) were selected for the Basel/ Frankfurt pilot protocol [31]. Donors were parents (N = 12) or mismatched siblings (N = 3). NK-DLI were given as an outpatient procedure, several weeks or months after the transplant, either immediately after processing or as cryopreserved NK-units, thawed at the bedside and infused rapidly. During infusion of fresh or thawed NK-products, no immediate adverse reactions were observed. NK cell collection and ex vivo purification was successful in all donors. One patient in each center developed severe (grade III/grade IV) GVHD. These two patients had received the highest T-cell dose. The remaining patients tolerated NK-DLI and did not develop GVHD. Eight patients were alive during the last follow-up [40].

Preemptive on day+2

Preemptive with transplant

DLI for relapse or imminent relapse

Part of conditioning regimen

17

3

8

3

AML 3

AL 3, MDS 2, NHL 2, HD 1

AML 1, ALL 2

AML 10, ALL 3, other 3

AML 7, ALL 5, other 3

Disease

UCB

Haploidentical 3, unrelated 1, sibling 4

Haploidentical

Haploidentical

Haploidentical

Donor

CD3 depleted/IL-2 activated UCB

IL-2 expanded, then CD56 selected

CD3 depleted, CD56 selected and IL-2 expanded

Waste of CD34 selection after CD3 depletion/CD56 selection. Overnight IL-2 activation in 7 patients.

CD3 depletion, CD56 selected

Selection/activation

0

0

0

Grade ³ II a GVHD 7/10 (without IL-2); and 1/7 (with IL-2)

1 Grade III, 1 grade IV

GVHD after NK cells

44

43

30

42

31

References

This table includes all reported data known to the authors of the clinical use of adoptive immunotherapy with purified NK-cells in HSCT recipients AML acute myeloid leukemia, AL acute leukemia, ALL acute lymphoblastic leukemia, MDS myelodysplastic syndrome, NHL non-Hodgkin lymphoma, HD Hodgkin disease, IL-2 interleukin 2, UCB umbilical cord blood

Decreasing chimerism/ incipient relapse/ preemptive

Indication for NK-cell infusion

15

N patients

Table 23-2.  Clinical application of purified NK cells in patients in the context of HSCT.

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Uharek et al. reported on 17 recipients of haploidentical CD34+ cell grafts receiving an add-back of 0.8 × 107 CD56+CD3− NK cells/kg at day +2 after transplant. Ten patients received unstimulated NK products, in the remaining seven patients NK cells were incubated in high-dose IL-2 for 16 h. No severe acute toxicity attributable to NK cell infusion was observed in both groups of patients. Whereas only one patient developed GVHD ³grade II after treatment with IL-2 activated NK cells, seven out of 10 patients showed GVHD ³grade II after transfer of non-activated NK cells. In a parallel study, 18 patients received CD3/CD19 depleted grafts (i.e. grafts depleted of B- and T-cells but containing large numbers of NK cells). Compared to the patients receiving NK-DLI, recovery of NK counts was faster and more sustained in patients receiving CD3/CD19 depleted grafts [41, 42]. Slavin et  al. used IL-2 activated NK cells (CD 56+ selected) following transplantation from haploidentical (3) sibling (4) or unrelated (1) donors. Patients with hematological malignancies (including acute leukemia and lymphoid malignancies), age 4–63 (median 25) years, had relapsed or were at very high risk. Donor lymphocytes were incubated for 4 days with IL-2 and then positively selected for CD56+. Purity of CD56+ was 39 (30–71)% and CD3+ was 3 (2–21)%. The number of CD56+ cells was 120 (10–600) × 106 cells. Cell infusion was uneventful, and no GVHD was observed. One patient with relapsed ALL and a patient with MDS transplanted from a KIR ligand matched mother achieved CR. Four patients are alive; one with disease; three with no evidence of disease at 9–22 months post HSCT [43]. Koehl et al. reported on three pediatric patients with multiply relapsed ALL (2) and AML (1) treated with repeated transfusions of IL-2 activated NK cells post haploidentical HSCT from parental donors (single dose: 3–34 × 106 CD56+CD3−/kg). Blast persistence (37–97%) was demonstrated in pretransplant bone marrow in all three patients. KIR ligand mismatches in GVH direction were demonstrated in all donor:recipient pairs. All patients achieved complete remission 4 weeks post HSCT, which was accompanied by complete donor chimerism. NK cell infusion was well tolerated, two patients died of transplant-related complications, while one patient died of relapse [30]. Miller et  al. reported on three patients with refractory AML treated with a triple umbilical cord blood (UCB) transplantation strategy. UCB unit 1 was immunomagnetically depleted of T-cells, ex vivo treated with high-dose interleukin-2, and infused in patients on day 12 after completion of myeloablative conditioning. NK cells were then expanded in  vivo by administration of subcutaneous interleukin-2 until day 0, when UCB units 2 and 3 were transplanted for hematopoeitic rescue. Unexpectedly, two patients showed neutrophil engraftment on days +3 and +7 from UCB unit 1. NK infusion was tolerated without toxicity and all patients were leukemia-free at the time of engraftment [44]. Other investigators have presented data on CD3/CD19 depletion for haploidentical HSCT which corresponds to selecting CD34+ cells along with, what they call: CD34-progenitors, natural killer, graft-facilitating, and dendritic cells. A recent report describes 29 patients receiving a peripheral stem cell graft containing 7.2 × 107/kg CD56+ cells after a conditioning of reduced intensity with engraftment in all patients and survival in 9/29 patients [45]. Cumulative incidence of grade II–IV acute GVHD was considerable (48%), however, infused T-cell doses with this approach are higher than those achieved

Chapter 23  Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation 

by CD34+ selection (median, 0.44 × 10e5/kg). A retrospective comparison of patients undergoing haploidentical HSCT using CD34+ selection versus CD3+ CD19+ depletion showed faster engraftment and faster NK cell reconstitution with the latter method [46]. Patients were, however, not entirely comparable and there were also differences in the conditioning regimens used. Data available so far show that ex vivo purification of donor NK cells from leukapheresis products is technically feasible, and large numbers of CD56positive, highly CD3-depleted cells can be obtained using the CliniMACS® two-step procedure of T-cell depletion and NK cell enrichment. NK cells are infused without immediate adverse events and possibly without inducing GVHD. Several cases of GVHD occurring after NK cell infusion have been described. In some instances, this has been associated with a less efficient T-cell depletion. Whether GVHD is attributable to contamination by T-cells or is due to the effects of NK cells cannot be determined based on this clinical data. The fact that at least in some cases the T-cell content was highest in patients developing GVHD is in favor of a T-cell effect. Clinical data on efficacy are very limited at this point in time. In addition, it is difficult to separate NK cell effects from effects of small number of residual T-cells in the product. Another question to be resolved is the importance of cytokine activation. While infusion of haploidentical donor NK in a non-transplant setting has only led to in vivo expansion of infused NK cells if patients received concomitant treatment with interleukin-2 [47], most investigators have refrained from treating patients with cytokines after NK infusion, due to concerns that this might trigger graft-versus-host disease. Infusion of ex vivo cytokine activated NK cells may combine the benefits of infusing highly cytotoxic NK cells without activating potentially alloreactive T-lymphocytes in vivo. Finally, the question arises whether allogeneic transplantation is required as a prerequisite for successful therapy with allogeneic NK cells. Interesting data from the Minneapolis group have shown that infusion of a purified NK cell product into patients treated with cyclophosphamide, fludarabine, and IL-2 may lead to transient engraftment of transfused NK cells and induction of remission in refractory AML patients [47]. NK cell chimerism was detectable for up to several months after infusion, and ultimately all patients rejected transferred NK cells and relapsed. However, the data shows the powerful effects of NK cells in vivo and may argue for a combination of (haploidentical) allogeneic transplantation and NK DLI, as a transplant preceding NK infusion will prevent rejection of NK cells and ultimately serve as a source of alloreactive NK cells on its own.

5.  Outlook Future studies will have to determine the usefulness of NK cells as an adoptive immunotherapy in recipients of HSCT from haploidentical and other donors. Open issues include NK cell doses, timing, and appropriate selection of donor and recipients amongst others (see Table  23-3). Whether NK DLI should be used preemptively or as a salvage treatment is unknown. Timing may be crucial. NK cells infused simultaneously with the transplant have the benefit of being administered at a time of minimal tumor load. Conditioning regimen induced aplasia and also upregulation of growth factors important for NK cell survival.

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Table  23-3.  Issues in harvesting, purification, and administration of natural killer (NK) cells. Production issues Mobilizing NK cells

Increased NK-cell numbers and lower adhesion molecule expression with epinephrine and exercise

Positive NK-cell selection

CD3 depletion followed by CD56+ selection

Negative “NK”-cell selection

CD3 (T-cell) depletion and CD19 (B-cell) depletion, may result in similar numbers but different purity of NK cells

In vitro activation of NK cells

IL-2 short- or median-time culture, other cytokines (e.g. IL-12, IL-15); is known to increase NK-cell killing in vitro

In-vitro expansion

Activation of NK cells and expansion of their number without increasing the number of T-cells

Infusion issues Infusion at the time of transplant

Purification from waste after CD34+ selection, potential impact of G-CSF mobilization on NK cells; this will not provide information on safety or on NK-cell effects

Infusion during post-transplant course but pre-emptive

Information on safety, but no information on NK-cell effects to be expected

Infusion with relapse or with falling chimerism

Information on safety and on NK-cell effects to be expected

Patient selection issues AML versus other diseases

NK-alloreactivity has been shown to be strongest in myeloid leukemia and possibly absent in other diseases, but this has not been studied prospectively and not using adoptive immunotherapy protocols

Donor selection issues Any suitable haploidentical donor versus donor with known NK-alloreactivity

The skeptical approach of not restricting inclusion for adoptive immunotherapy protocols to donors with known NK-alloreactivity will allow for testing of NK-cell effects in donor/ recipient pairs with or without predicted or measured NK-cell alloreactivity

Other allogeneic donors or autologous NK cells

There is little or no information available

IL interleukin, G-CSF granulocyte colony stimulating factor, AML acute myelogenous leukemia

However, effects will be much harder to measure than if NK cells were administered later in the course. Some groups have used NK cells purified from the stem cell product, and the impact of mobilization on NK cells needs to be studied. Other groups have reported the enrichment of NK cells by depletion of T- and B-cells, which is attractive in both financial and practical aspects, as a product containing both progenitor cells and high numbers of NK cells that can be produced in one step.

Chapter 23  Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation 

Whether NK cells will be used without additional manipulation, or whether stimulation and culture either in the short- or medium-term with cytokines such as IL-2 will prove to be more effective awaits further studies. Purging and enrichment technology using magnetic beads for clinical application is technologically fascinating but expensive, in direct costs of antibodies and columns and the time of the laboratory personnel. The NK cell enrichment technology will require some improvement for broad application. Future studies may, therefore, include intervention on the part of NK cells by selecting donors with appropriate NK receptor profiles and possibly activating the cells while promoting ligand expression on the blasts to enhance killing. The burden of proof of principle and of usefulness in clinical practice lies, therefore, with those who want to apply this technology. Multi-institutional phase III trials in recipients of haploidentical HSCT, comparing standard transplant to transplant plus NK-DLI could be instrumental in establishing the clinical role, if any, of adoptive NK-cell therapy.

References 1. Farag SS, Caligiuri MA (2006) Human natural killer cell development and biology. Blood Rev 20:123–137 2. Farag SS, Fehniger TA, Ruggeri L, Velardi A, Caligiuri MA (2002) Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood 100:1935–1947 3. Bottino C, Moretta L, Moretta A (2006) NK cell activating receptors and tumor recognition in humans. Curr Top Microbiol Immunol 298:175–182 4. Biassoni R, Cantoni C, Pende D et al (2001) Human natural killer cell receptors and co-receptors. Immunol Rev 181:203–214 5. Valiante NM, Uhrberg M, Shilling HG et al (1997) Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity 7:739–751 6. Kim S, Poursine-Laurent J, Truscott SM et  al (2005) Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature 436: 709–713 7. Fernandez NC, Treiner E, Vance RE, Jamieson AM, Lemieux S, Raulet DH (2005) A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for self-MHC molecules. Blood 105:4416–4423 8. Anfossi N, Andre P, Guia S et al (2006) Human NK cell education by inhibitory receptors for MHC class I. Immunity 25:331–342 9. Hansasuta P, Dong T, Thananchai H et  al (2004) Recognition of HLA-A3 and HLA-A11 by KIR3DL2 is peptide-specific. Eur J Immunol 34:1673–1679 10. Rajagopalan S, Long EO (1999) A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells. J Exp Med 189:1093–1100 11. Estefania E, Flores R, Gomez-Lozano N, Aguilar H, Lopez-Botet M, Vilches C (2007) Human KIR2DL5 is an inhibitory receptor expressed on the surface of NK and T lymphocyte subsets. J Immunol 178:4402–4410 12. Vales-Gomez M, Reyburn HT, Erskine RA, Strominger J (1998) Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors. Proc Natl Acad Sci USA 95:14326–14331 13. Cook M, Briggs D, Craddock C et al (2006) Donor KIR genotype has a major influence on the rate of cytomegalovirus reactivation following T-cell replete stem cell transplantation. Blood 107:1230–1232

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M. Stern et al. 14. Nguyen S, Dhedin N, Vernant JP et  al (2005) NK-cell reconstitution after ­haploidentical hematopoietic stem-cell transplantations: immaturity of NK cells and inhibitory effect of NKG2A override GvL effect. Blood 105:4135–4142 15. Rosenberg SA, Lotze MT, Muul LM et al (1987) 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 316:889–897 16. Uhrberg M, Valiante NM, Shum BP et  al (1997) Human diversity in killer cell inhibitory receptor genes. Immunity 7:753–763 17. Uhrberg M, Parham P, Wernet P (2002) Definition of gene content for nine common group B haplotypes of the Caucasoid population: KIR haplotypes contain between seven and eleven KIR genes. Immunogenetics 54:221–229 18. Ruggeri L, Capanni M, Urbani E et  al (2002) Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 295: 2097–2100 19. Shlomchik WD, Couzens MS, Tang CB et al (1999) Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 285:412–415 20. Ruggeri L, Mancusi A, Burchielli E et al (2008) NK cell alloreactivity and allogeneic hematopoietic stem cell transplantation. Blood Cells Mol Dis 40:84–90 21. Ruggeri L, Capanni M, Casucci M et  al (1999) Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation. Blood 94:333–339 22. Ruggeri L, Mancusi A, Capanni M et al (2007) Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value. Blood 110:433–440 23. Pende D, Spaggiari GM, Marcenaro S et al (2005) Analysis of the receptor-ligand interactions in the natural killer-mediated lysis of freshly isolated myeloid or lymphoblastic leukemias: evidence for the involvement of the Poliovirus receptor (CD155) and Nectin-2 (CD112). Blood 105:2066–2073 24. Pfeiffer M, Schumm M, Feuchtinger T, Dietz K, Handgretinger R, Lang P (2007) Intensity of HLA class I expression and KIR-mismatch determine NK-cell mediated lysis of leukaemic blasts from children with acute lymphatic leukaemia. Br J Haematol 138:97–100 25. Leung W, Iyengar R, Turner V et al (2004) Determinants of antileukemia effects of allogeneic NK cells. J Immunol 172:644–650 26. Kolb HJ, Mittermuller J, Clemm C et  al (1990) Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 76:2462–2465 27. Dazzi F, Szydlo RM, Craddock C et  al (2000) Comparison of single-dose and escalating-dose regimens of donor lymphocyte infusion for relapse after allografting for chronic myeloid leukemia. Blood 95:67–71 28. Lewalle P, Triffet A, Delforge A et  al (2003) Donor lymphocyte infusions in adult haploidentical transplant: a dose finding study. Bone Marrow Transplant 31:39–44 29. Wolf CE, Meyer M, Riggert J (2005) Leukapheresis for the extraction of monocytes and various lymphocyte subpopulations from peripheral blood: product quality and prediction of the yield using different harvest procedures. Vox Sang 88:249–255 30. Koehl U, Sorensen J, Esser R et al (2004) IL-2 activated NK cell immunotherapy of three children after haploidentical stem cell transplantation. Blood Cells Mol Dis 33:261–266 31. Passweg JR, Tichelli A, Meyer-Monard S et al (2004) Purified donor NK-lymphocyte infusion to consolidate engraftment after haploidentical stem cell transplantation. Leukemia 18:1835–1838 32. Iyengar R, Handgretinger R, Babarin-Dorner A et al (2003) Purification of human natural killer cells using a clinical-scale immunomagnetic method. Cytotherapy 5:479–484

Chapter 23  Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation  33. McKenna DH Jr, Sumstad D, Bostrom N et al (2007) Good manufacturing practices production of natural killer cells for immunotherapy: a six-year single-institution experience. Transfusion 47:520–528 34. Bondzio I, Schmitz J, Huppert V (2007) CliniMACS cell enrichment using NKp46. A large scale, single-step NK cell isolation method. Blood 110:abstract 3877 35. Koehl U, Esser R, Zimmermann S et al (2005) Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children. Klin Padiatr 217:345–350 36. Klingemann HG, Martinson J (2004) Ex vivo expansion of natural killer cells for clinical applications. Cytotherapy 6:15–22 37. Fujisaki H, Kakuda H, Lockey T, Eldridge PW, Leung W, Campana D (2007) Expanded natural killer cells for cellular therapy of acute myeloid leukemia. Blood 110:abstract 2743 38. Xing D, Fang W, Decker WK, et al (2007) Ex vivo expansion of cord blood NK cell have in  vivo efficacy against leukemia. ASH Annu Meet Abstr 110(11): abstract 2741 39. Ayello J, Nemiroff J, Satwani P, et al (2006) Enhanced NK cell activation, cytotoxicity and ex-vivo expansion (EvE) of cryopreserved cord blood (CB) natural killer (NK) cells: potential role for CB NK cells in adoptive cellular immunotherapy (ACI). ASH Annu Meet Abstr 108:726 40. Passweg JR, Koehl U, Stern M, et  al (2006) Preemptive immunotherapy with highly purified CD56+/CD3− natural killer cells after haploidentical stem cell transplantation. A prospective phase II study in 2 centers. ASH Annu Meet Abstr 108(11):abstract 411 41. Gentilini C, Haegele M, Muessig A, et al. (2007) NK-Cell recovery and immune reconstitution after haploidentical hematopoietic cell transplantation using either CD34 selected grafts and adoptive NK-Cell transfer or CD3/CD19 depleted grafts: comparison of two strategies for NK cell based immunotherapy. ASH Annu Meet Abstr 110(11):abstract 2988 42. Gentilini C, Hilbers U, Huppert V, et al (2007) Patients Receiving IL-2 Activated Donor NK Cells Show Lower Incidence of Severe GvHD after Haploidentical SCT. ASH Annu Meet Abstr 110(11):abstract #354 43. Slavin S, Morecki S, Shapira M, Samuel S, Ackerstein A, Gelfand Y (2004) Immunotherapy using rIL-2 activated mismatched donor lymphocytes positively selected for the treatment of resistand haematologic malignancies after stem cell transplantation. Bone Marrow Transplant 37(S1) 44. Miller JS, Brunstein CG, Cooley S, et al (2006) A novel triple umbilical cord blood transplant (UCBT) Strategy to promote NK cell immunotherapy (Unit 1) with a fully ablative preparative regimen followed by a double UCBT in patients with refractory AML. ASH Annu Meet Abstr 108:abstract 3111 45. Bethge WA, Faul C, Bornhauser M et al (2008) Haploidentical allogeneic hematopoietic cell transplantation in adults using CD3/CD19 depletion and reduced intensity conditioning: an update. Blood Cells Mol Dis 40:13–19 46. Bethge WA, Haegele M, Faul C et al (2006) Haploidentical allogeneic hematopoietic cell transplantation in adults with reduced-intensity conditioning and CD3/ CD19 depletion: fast engraftment and low toxicity. Exp Hematol 34:1746–1752 47. Miller JS, Soignier Y, Panoskaltsis-Mortari A et  al (2005) Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 105:3051–3057

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Chapter 24 Cryopreservation of Allogeneic Stem Cell Products Noelle V. Frey and Steven C. Goldstein

1. Introduction Donor stem cells for allogeneic transplant are traditionally collected and transfused “fresh” into the recipient on the day of transplant. Alternatively, donor stem cells can be collected in advance and cryopreserved until needed. Due to historical momentum and concerns that the cryopreservation and thawing process may damage the graft and worsen clinical outcomes, most institutions favor the former approach. The use of cryopreserved grafts has, therefore, traditionally been reserved for extreme circumstances of questionable donor reliability or availability. This trend is, however, slowly changing as some individual centers are favoring the use of frozen grafts in their related donor transplants due to increased ease of transplant coordination. Similarly, The National Marrow Donor Program (NMDP), which authorizes the collection of all cryopreserved unrelated grafts, has noticed an increasing trend in the use of frozen stem cell products. The total of cryopreserved stem cell grafts, however, still represents less than 2% of all unrelated products (R King; NMDP, personal communication). The paramount question when considering using a fresh vs. a frozen stem cell allograft is whether the cryopreservation and thawing processes alter the viability or activity of individual mononuclear cell (MNC) subsets in the graft. The next question is whether these alterations in stem cell graft content correlate with clinically meaningful disparate outcomes between cryopreserved graft recipients and fresh graft recipients. Of specific concern is the impact of cryopreservation on T-cell subsets which are important mediators of engraftment, graft vs. tumor (GVT), and graft vs. host disease (GVHD). This data cannot be extrapolated from the autologous literature where cryopreserved products are the mainstays of therapy. In this chapter, we will review the sparse clinical data which evaluates the effects of the cryopreservation and thawing processes on the allogeneic stem cell graft and transplant clinical outcomes. We will also examine the potential advantages and disadvantages of using a cryopreserved allograft [1].

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_24, © Springer Science + Business Media, LLC 2003, 2010

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2. Cryopreservation 2.1. Methodology Hematopoietic stem cells are progressively lost during storage at room ­temperature or 4°C. Peripheral blood (PB) and bone marrow (BM) stem cell grafts are, therefore, cryopreserved if storage for longer than 3–4 days is anticipated. In addition, cryopreservation serves as the primary method of storage for cord blood grafts [2]. There are several aspects of the freeze-thaw process that are designed to minimize the damage to stem cells and other MNCs. To avoid mechanical and osmotic damage to cells from ice crystal formation during the freeze process, colligative cryoprotectants are added to the product prior to freezing. Dimethylsulfoxide (DMSO) at a final concentration of 10% is a commonly used cryoprotectant and its breakdown into dimethylsulfide (DMS) accounts for the characteristic sulfur smell originating from patients after re-infusion. For further optimization of cell survival, the cells are subsequently suspended in a solution containing various protein and solute concentrations, and the graft is frozen at a slow rate (commonly 1–2°C/ min) and ultimately stored at a temperature colder than −80°C. Thawing can safely occur more quickly, often with use of a water bath at the bedside [3]. It should be noted that individual center’s freeze-thaw techniques are variable, which complicates the interpretation and applicability of single center data reporting outcomes of cryopreserved grafts. It is also important to emphasize that in the unrelated setting, stem cell products are collected at the donor center and subsequently transported at 4°C until they are cryopreserved at the recipient center. Transport time has been shown to affect the outcomes in unrelated transplants and is therefore another factor which could influence the cryopreservation outcomes in the unrelated setting [4]. 2.2. Effect of Cryopreservation on Graft content It is important to recognize that a donor graft is composed of several different MNC subtypes including CD34+ stem cells, dendritic cells, T-cells, and NK cells which are variably important for effecting certain transplant outcomes such as engraftment, GVT, and GVHD. These MNC subtypes are also differentially affected by the freeze-thaw process and have different optimal conditions for survival. It is, therefore, reasonable to suspect that cryopreservation may alter a donor graft to such a degree that transplant outcomes are altered. For example in the allogeneic setting, it is known that CD34+ cells, T-cells, and NK cells are important for engraftment and that GVHD is T-cell dose dependent [5–8]. The cryopreservation process could potentially affect the engraftment and GVHD outcomes if any of these subsets were significantly damaged by the freeze-thaw process. The idea that differences in graft content can alter transplant outcomes is best illustrated by reports comparing recipients of PB or BM allografts. Recipients of PB grafts, which have a higher T-cell content, have shortened engraftment times and increased incidence of GVHD compared to recipients of BM grafts [9, 10]. The impact of cryopreservation on graft content and function is best described for CD34+ stem cells, burst forming units-erythroid (BFU-E), and colony forming units granulocyte-macrophage (CFU-GM). As cryopreservation is the mainstay of autologous graft storage, most studies have been

Chapter 24  Cryopreservation of Allogeneic Stem Cell Products 

performed on autologous grafts and have shown a strong association with CD34+ number and time to platelet and neutrophil reconstitution [11–14]. Of interest however, engraftment outcomes in these studies are often correlated with pre-frozen CD34+ numbers with no post-thaw comparison. An interesting study compared pre- and post-thaw MNC, CD34+ cells, and CFU-GM in 83 PB and 43 BM autografts. Pre- and post-thaw CD34+ cells were well correlated with each other while pre- and post-thaw MNC and CFU-GM were less well correlated. In this study, the total CD34+ cells infused were found to be the only factor predictive of engraftment outcomes [14]. A recent large report describing outcomes for 105 recipients of cryopreserved PB allografts reported CFU-GM, BFU-E, and colony forming unit megakaryocyte (CFUMEG) in donor grafts before and after cryopreservation. In this study, the viability of MNCs after cryopreservation was globally assessed by tryptan blue exclusion which showed a median recovery rate of 71%. CFU-GM, BFU-E, and CFU-MEG growth rates were reduced by 25%, 30.5%, and 61%, respectively (see Fig. 2-1). These findings, as discussed in more detail below, did not translate into statistically significant reduction in time to engraftment when compared to recipients of fresh PB allografts [15]. The reported experience regarding the impact of cryopreservation on the viability and functionality of other MNCs is quite limited. Early studies using crude estimates of T-cell number and function suggested no significant reduction in T-cell percentage or function after the cryopreservation and thaw process [16, 17]. A recent investigation incorporating flow cytometry and more sophisticated functional analysis has showed a small but statistically significant reduction in the percentage of CD3+, CD4+, and CD8+ cells after cryopreservation and 3 months of storage [18]. The full impact of donor derived antigen presenting dendritic cells (DCs) in effecting GVT and GVHD responses is yet to be determined [19]. Several studies have suggested that in G-CSF mobilized blood, the freeze-thaw process does not affect the phenotype, viability, or biological activity of immature and mature DCs [20, 21].

Fig. 24-1.  Frequency of clonogenic hematopoietic progenitors in PB allograft before and after cryopreservation (reprinted from [15])

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The assessment of functionality of mononuclear cell subsets after cryopreservation deserves further investigation. Methods to optimize the viability of MNCs in cryopreserved cord blood products may contribute to optimizing the freeze-thaw processes in PB and BM allografts [22]. 2.3.  Effect of Cryopreservation on Transfusion Reactions In addition to potentially altering the graft content, the cryopreservation and thawing processes result in an increased risk of transfusion reactions due to the presence of DMSO as a cryoprotectant. These symptoms are for the most part transient, well tolerated, and are generally not associated with more clinically significant adverse outcomes. One study compared transfusion reactions between 134 recipients of autologous cryopreserved BM and 71 recipients of fresh allogeneic BM [23]. A statistically significant increase in the incidence of nausea (45 vs. 14%; p  20)

13

13

Days to Plt engraftment

a

NR

57.5%

20%

NR

75%

60%

61%

81.2 (p = 0.113)

78.2

aGVHD ³gr II

50%

61%

70%

NR

55%

72%

82%

No difference in OS

Day 100 survival

a Statistically significant difference. NR not reported, HSC hematopoietic stem cell source, ANC absolute neutrophil count, Plt platelet, aGVHD acute Graft-vs.-host disease, OS overall survival

[28]

[27]

[31]

40

Frozen

106

Fresh

[29]

105

Frozen

[15]

N

Storage

Reference

Table 24-1.  Summary of engraftment and outcome data for selected reports of allogeneic stem cell transplantation using cryopreserved grafts.

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the authors compare outcomes of 105 consecutive related cryopreserved PB allograft recipients with 106 historical recipients of related fresh PB allografts. The groups were well matched with regard to the recipient age, donor age, disease type, conditioning regimens, and GVHD prophylaxis. Donor grafts were cryopreserved using 10% DMSO, stored at −86°C and thawed at the bedside in a 40°C water bath. The transfusions were overall well tolerated and no grafts had bacterial contamination. Grafts were stored for a median of 15 days (range 5–238 days) [15]. In multivariate analyses, no statistically significant differences were found between fresh and frozen allograft recipients in terms of times to platelet and neutrophil engraftment, incidence of acute and chronic GVHD, relapse rates, or overall survival. Due to concerns that T-cells may be more deleteriously affected by the cryopreservation process, the authors also compared lymphocyte recovery over time between the two groups and found no difference. In the recipients of cryopreserved allografts, the median time to neutrophil (ANC >0.5 × 109/L for 2 consecutive days) and platelet engraftment (platelets >20 × 109/L for 3 consecutive days) were 17 and 21 days, respectively. Similar engraftment kinetics were also observed in recipients of fresh allografts. Only one subject who received a cryopreserved allograft failed to engraft, compared to five recipients of fresh allografts. The incidence of Grade II–IV acute GVHD was 78.2% in the cryopreserved graft recipients and 81.2% in the fresh graft recipients (p = 0.113) (see Fig. 24-2). Chronic GVHD affected 83.8% of cryopreserved graft recipients and 90.1% of fresh graft recipients (p = 0.673). While no statistically significant difference in acute GVHD was found between fresh and cryopreserved recipients, a trend towards less GVHD from frozen allografts is noted [15]. While well designed, this study is underpowered to detect a 5 or 10% difference in GVHD. As discussed earlier, viability studies on graft content showed a 25–30% reduction of CFU-GM, BFU-E, and MNCs. The authors interestingly noted a significant 61% reduction in CFU-MEG which did not translate into slower platelet engraftment times in recipient’s cryopreserved products. To further evaluate the impact of CFU-MEG on platelet engraftment kinetics, the authors divided patients into quartiles based on their infused CFU-MEG and found a delayed time to platelet recovery (29 vs. 18 days) in the bottom quartile of the cryopreserved graft recipients [15]. The above study by Kim and colleagues is limited to related PB allograft recipients. In an earlier study, Stockshlader and colleagues retrospectively compared 40 patients who received cryopreserved related BM allografts with 40 patients matched for age, disease, and disease stage who received fresh BM allografts [29]. Information on donors was not reported. Indications for cryopreservation appeared to be independent of disease status and included concerns regarding donor age, reliability, scheduling, and operating room availability. The cryoprotectant was DMSO (final concentration 10%) and the median time of graft storage was 17.5 days (range 3–455 days). All patients with two exceptions received the same GVHD prophylaxis with cyclosporine (CSA) and methotrexate (MTX). Conditioning regimens were similar. No statistically significant differences were found in time to neutrophil and platelet engraftment, day 100 survival, or incidence of GVHD between the two groups [29]. This group compared the total number of BFU and CFU-GM infused into recipients of fresh vs. frozen allografts and found no statistically significant difference. Prior published results from a subset of 19 patients who received a

Chapter 24  Cryopreservation of Allogeneic Stem Cell Products 

Fig. 24-2.  Incidence of GVHD in cryopreserved and fresh allograft recipients (reprinted from [15])

cryopreserved product directly measured the impact of cryopreservation and thawing on graft content by comparing CFU-GM and MNC numbers before and after cryopreservation. This analysis revealed no statistically significant difference in the pre-frozen and post-thaw numbers of CFU-GM and MNC [30]. Stockshlader and colleagues also reported their limited experience in using cryopreserved BM for 10 patients undergoing unrelated allogeneic transplantation [31]. Outcomes for these patients were not directly compared to matched or historical controls, and are reported in Table 24-1. Two other small series from the early 1990s also showed no significant reduction in time to platelet or neutrophil engraftment and overall survival in cryopreserved allograft recipients when compared to institutional or historic controls [27, 28].

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One of these studies, however, reported a statistically significant reduction in the incidence of GVHD for 10 recipients of cryopreserved BM compared with 33 unmatched institutional controls [27]. In this study, recipients of cryopreserved products had a 20% incidence of acute GVHD compared to a 56% incidence in the control group. There were, however, significant differences between the two recipient groups with regards to age, disease type, disease stage, and type of GVHD prophylaxis. Attempts to account for these differences with a multivariate analysis failed to identify cryopreservation as an independent predictor of GVHD [27].

4. Donor Lymphocyte Infusion Donor lymphocyte infusion (DLI) is increasingly being used after both myeloablative and non-myeloablative stem cell transplantation to treat and prevent relapse, to establish full donor chimerism, and to treat and prevent infections. It is feasible to collect and cryopreserve DLI products at the time of original donor stem cell collection [33, 34]. The potential benefits of having a readily available DLI product (especially in the unrelated donor setting) needs to be weighed against the potential deleterious effects of cryopreservation and storage of this lymphocyte-rich product as well as the potential cost of long-term storage of DLI products that may never be utilized. It is important to note that due to differences in cell content and timing of administration between stem cell grafts and DLI products, cryopreservation may differentially affect clinical outcomes in recipients. Unfortunately, the data describing outcomes of recipients of cryopreserved DLI products is very limited. Sohn et  al. reported their experience with 17 patients at high risk for relapse whose donors underwent donor lymphocyte collection with cryopreservation at the time of original harvest [34]. DLI was given to transplant recipients without GVHD at pre-specified time points with the goal of preventing recurrent disease (i.e., prophylactic DLI). The incidence of GVHD after DLI was ~60%, a finding consistent with other reports of GVHD incidence after DLI [35, 36]. While this study was not designed to assess the efficacy of a cryopreserved vs. a fresh DLI product, it suggests that cryopreservation of donor lymphocytes at the time of collection for subsequent re-infusion in high risk patients may be a reasonable strategy to avoid delay of DLI in the event of relapse [34]. Lane et al. reported the outcomes of 19 subjects who received cryopreserved DLI for either relapsed disease or low donor chimerism after non-myeloablative transplantation. The infusions were well tolerated. Three of 15 patients who were treated for relapse developed a complete response and donor chimerism improved by a mean of 16% (range 0–50%). GVHD outcomes were not reported [33]. Further investigation of the impact of cryopreservation on DLI outcomes is warranted.

5. Logistics It is logistically challenging to coordinate a patient’s conditioning regimen with donor stem cell collection when a fresh product is used. Securing operating rooms, apheresis time, and total body irradiation (TBI) slots are sometimes difficult. The donor, who must be available on day 0, is often asked to be available at

Chapter 24  Cryopreservation of Allogeneic Stem Cell Products 

times which may not be convenient. These coordinating challenges are intensified in the unrelated setting in which the NMDP helps to organize these events between two different centers. The use of a cryopreserved product introduces a greater amount of flexibility into the system and would inherently be more convenient for the donor and the hospital. Streamlining the donor experience could also help relieve potential recipient guilt over causing inconvenience to family members. An increased cost would be incurred with cryopreservation and storage of stem cell products but these may be balanced by potential “hidden” savings of streamlining bed utilization, and optimal scheduling of stem cell laboratory and radiation oncology personnel. While a rare event, there is always a possibility that a donor will become unavailable for unforeseen circumstances after the conditioning regimen has been initiated. There is also a 2–5% inherent risk that a donor undergoing peripheral blood stem cell collection will be a poor-mobilizer, a disconcerting finding if discovered on day 0 of transplant [37, 38]. In fact, in the report by Kim and colleagues 14% of PB donors required collection over two or more days [15]. While this issue may become less important with the mobilization success rates of AMD-3100 (a CXCR-4 antagonist), the collection of certain donors at high risk for mobilization-failure ahead of time may be appropriate [39–42].

6. Ethical Concerns The increased use of cryopreserved products raises the possibility of an increase of collected, but not utilized grafts. This raises ethical concerns about subjecting some donors to unnecessary, time-consuming, and potentially harmful harvesting procedures. If collection of stem cells were undertaken closer in time to the transplant (e.g., within 30 days), the incidence of unnecessary harvesting procedures would be minimized while maintaining many of the benefits mentioned above. The practicality of this approach is supported by the reported experience of Kim and Stockschlader who noted a median time for graft storage of 15 and 17.5 days, respectively [15, 29]. Of note, the manuscripts discussed above do not comment on how many grafts were collected by the individual centers and never utilized.

7. Summary and Conclusions We have reviewed the potential advantages and disadvantages of using cryopreserved grafts in the allogeneic stem cell transplant setting (Table  24.2). Cryopreserved grafts are associated with a higher incidence of transfusion reactions and bacterial contamination but these are rarely associated with significant morbidity. The limited literature on this subject to date shows no significant difference in overall survival, relapse rates, GVHD, or time to engraftment between recipients of fresh or frozen stem cell products. However, one should be cautious in making firm conclusions based on the available literature as it is quite limited. The studies published to date are retrospective and insufficiently powered to assess for 5–10% differences in GVHD or relapse rates. In fact, two studies reported thus far suggest that there may be a trend towards less GVHD in recipients of cryopreserved products [15, 27].

435

Comments The limited published experience shows no significant delay in time to platelet or neutrophil engraftment. Further, more appropriately powered studies with laboratory correlates are needed Cryopreserved products are associated with more transfusion related nausea, vomiting, and fever but not necessarily more serious events such as hemodynamic instability or pulmonary compromise Higher rates of bacterial contamination do not translate into significantly higher rates of bacteremia and sepsis in recipients. Peripheral blood grafts correlate with a lower incidence of contamination Timing of collection within 1–3 weeks of transplant would minimize this inevitable outcome Comments Coordinating donor collection on Day 0 of transplant is challenging. Cryopreservation introduces greater flexibility into the system Greater flexibility for donor to schedule collection in setting of other obligations Encouraging mobilization results of AMD-3100 may decrease current 2–5% poor mobilization rates among healthy peripheral blood stem cell donors While rare, cryopreservation would allay this commonly reported fear among recipients Most studies to date do not support this hypothesis. Adequately powered clinical studies with laboratory correlates are needed Limited data on effect of cryopreservation on DLI. Prophylactic collection of DLI could increase the number of products that are not infused (concern for costeffectiveness and donor safety)

Potential disadvantages

Concern over delay of neutrophil and platelet engraftment due to damage of the graft during cryopreservation

Increased incidence of transfusion reactions due to the presence of DMSO as a cryoprotectant

Increased incidence of bacterial contamination of the graft due to increased handling in the freeze-thaw process

Increased incidence of collecting grafts which are never utilized, putting the donor through an unnecessary harvesting procedure

Potential Advantages

Decreased stress on the healthcare system

Decreased stress on the donor

Identification of donors who are poor mobilizers before day 0 of transplant

Ensured availability of donor graft in the event of donor death or unavailability

Decreased incidence of GVHD due to preferential destruction of T-cell subsets in the cryopreservation process

Collection and cryopreservation of DLI at the time of original stem cell procedure allows for readily available DLI

Table 24-2.  Potential advantages and disadvantages of using cryopreserved stem cell products over fresh stem cell products for allogeneic stem cell transplantation.

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Chapter 24  Cryopreservation of Allogeneic Stem Cell Products 

There is also a paucity of data on the impact of cryopreservation on outcomes with unrelated donor transplants and DLI, both products which logistically would benefit greatly from cryopreservation. We, in conjunction with the NMDP, are performing a retrospective cohort study comparing outcomes of over 300 recipients of unrelated cryopreserved grafts with matched controls to further shed light on this issue. If further, more appropriately powered studies are performed yielding no significant difference between recipients of cryopreserved and fresh allografts, it may be that the noted trend by individual centers to use cryopreserved products is appropriate. The increased ease of scheduling collections and transplants in conjunction with ensured availability of a suitable donor graft on day 0 may financially and logistically justify the increased cost of cryopreservation. It is important to continue to monitor this practice with an eye on donor safety to ensure that an unacceptable number of unnecessary harvests are not performed.

References 1. Frey NV, Lazarus HM, Goldstein SC (2006) Has allogeneic stem cell cryopreservation been given the ‘cold shoulder’? An analysis of the pros and cons of using frozen versus fresh stem cell products in allogeneic stem cell transplantation. Bone Marrow Transplant 38(6):399–405 2. Harris DT, Schumacher MJ, Rychlik S et  al (1994) Collection, separation and cryopreservation of umbilical cord blood for use in transplantation. Bone Marrow Transplant 13(2):135–143 3. Rowley S (2004) Cryopreservation of hematopoietic Cells. In: Blum K (ed) Thomas’ hematopoitic cell transplantation. Malden, MA, Blackwell, pp 599–612 4. Lazarus HM, Kan F, Tarima S et  al (2007) Rapid Transport and Infusion of Hematopoietic Stem Cells Can Improve Outcome after Unrelated Donor Transplant. In 2007, p. 3063 5. Manilay JO, Sykes M (1998) Natural killer cells and their role in graft rejection. Curr Opin Immunol 10(5):532–538 6. Martin PJ (1993) Donor CD8 cells prevent allogeneic marrow graft rejection in mice: Potential implications for marrow transplantation in humans. J Exp Med 178(2):703–712 7. Marmont AM, Horowitz MM, Gale RP et  al (1991) T-cell depletion of HLAidentical transplants in leukemia. Blood 78(8):2120–2130 8. Kernan NA, Collins NH, Juliano L et  al (1986) Clonable T lymphocytes in T cell-depleted bone marrow transplants correlate with development of graft-v-host disease. Blood 68(3):770–773 9. Group SCTC (2005) Allogeneic peripheral blood stem-cell compared with bone marrow transplantation in the management of hematologic malignancies: An individual patient data meta-analysis of nine randomized trials. J Clin Oncol 23(22):5074–5087 10. Schmitz N, Eapen M, Horowitz MM et al (2006) Long-term outcome of patients given transplants of mobilized blood or bone marrow: A report from the international bone marrow transplant registry and the european group for blood and marrow transplantation. Blood 108(13):4288–4290 11. Robinson SN, Freedman AS, Neuberg DS et  al (2000) Loss of marrow reserve from dose-intensified chemotherapy results in impaired hematopoietic reconstitution after autologous transplantation: CD34(+), CD34(+)38(−), and week-6 CAFC assays predict poor engraftment. Exp Hematol 28(12):1325–1333 12. Tricot G, Jagannath S, Vesole D et al (1995) Peripheral blood stem cell transplants for multiple myeloma: Identification of favorable variables for rapid engraftment in 225 patients. Blood 85(2):588–596

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N.V. Frey and S.C. Goldstein 13. Weaver CH, Hazelton B, Birch R et al (1995) An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 86(10):3961–3969 14. Feugier P, Bensoussan D, Girard F et al (2003) Hematologic recovery after autologous PBPC transplantation: Importance of the number of postthaw CD34+ cells. Transfusion 43(7):878–884 15. Kim DH, Jamal N, Saragosa R et  al (2007) Similar outcomes of cryopreserved allogeneic peripheral stem cell transplants (PBSCT) compared to fresh allografts. Biol Blood Marrow Transplant 13(10):1233–1243 16. Ludgate ME, Dryden PR, Weetman AP, McGregor AM (1983) T-cell subset analysis of cryopreserved human peripheral blood mononuclear cells. Immunol Lett 7(3):119–122 17. Jones HP, Hughes P, Kirk P, Hoy T (1986) T-cell subsets: Effects of cryopreservation, paraformaldehyde fixation, incubation regime and choice of fluoresceinconjugated anti-mouse IgG on the percentage positive cells stained with monoclonal antibodies. J Immunol Methods 92(2):195–200 18. Tollerud DJ, Brown LM, Clark JW et  al (1991) Cryopreservation and long-term liquid nitrogen storage of peripheral blood mononuclear cells for flow cytometry analysis: Effects on cell subset proportions and fluorescence intensity. J Clin Lab Anal 5(4):255–261 19. Shlomchik WD (2007) Graft-versus-host disease. Nat Rev Immunol 7(5):340–352 20. Celluzzi CM, Welbon C (2003) A simple cryopreservation method for dendritic cells and cells used in their derivation and functional assessment. Transfusion 43(4):488–494 21. Hori S, Heike Y, Takei M et al (2004) Freeze-thawing procedures have no influence on the phenotypic and functional development of dendritic cells generated from peripheral blood CD14+ monocytes. J Immunother 27(1):27–35 22. Woods EJ, Liu J, Derrow CW et al (2000) Osmometric and permeability characteristics of human placental/umbilical cord blood CD34+ cells and their application to cryopreservation. J Hematother Stem Cell Res 9(2):161–173 23. Stroncek DF, Fautsch SK, Lasky LC et  al (1991) Adverse reactions in patients transfused with cryopreserved marrow. Transfusion 31(6):521–526 24. Lazarus HM, Magalhaes-Silverman M, Fox RM et al (1991) Contamination during in vitro processing of bone marrow for transplantation: Clinical significance. Bone Marrow Transplant 7(3):241–246 25. Rowley SD, Davis J, Dick J et  al (1988) Bacterial contamination of bone marrow grafts intended for autologous and allogeneic bone marrow transplantation. Incidence and clinical significance. Transfusion 28(2):109–112 26. Padley D, Koontz F, Trigg ME et al (1996) Bacterial contamination rates following processing of bone marrow and peripheral blood progenitor cell preparations. Transfusion 36(1):53–56 27. Eckardt JR, Roodman GD, Boldt DH et al (1993) Comparison of engraftment and acute GVHD in patients undergoing cryopreserved or fresh allogeneic BMT. Bone Marrow Transplant 11(2):125–131 28. Lasky LC, Van Buren N, Weisdorf DJ et al (1989) Successful allogeneic cryopreserved marrow transplantation. Transfusion 29(2):182–184 29. Stockschlader M, Hassan HT, Krog C et al (1997) Long-term follow-up of leukaemia patients after related cryopreserved allogeneic bone marrow transplantation. Br J Haematol 96(2):382–386 30. Stockschlader M, Kruger W, Kroschke G et al (1995) Use of cryopreserved bone marrow in allogeneic bone marrow transplantation. Bone Marrow Transplant 15(4):569–572 31. Stockschlader M, Kruger W, tom Dieck A et al (1996) Use of cryopreserved bone marrow in unrelated allogeneic transplantation. Bone Marrow Transplant 17(2):197–199

Chapter 24  Cryopreservation of Allogeneic Stem Cell Products  32. Shinkoda Y, Ijichi O, Tanabe T et  al (2004) Identical reconstitution after bone marrow transplantation in twins who received fresh and cryopreserved grafts harvested at the same time from their older brother. Clin Transplant 18(6):743–747 33. Lane TA, Medina B, Bashey A et  al (2007) Clinical efficacy of cryopreserved donor lymphocytes for infusion (DLI). Biol Blood Marrow Transplant 13:352 34. Sohn SK, Jung JT, Kim DH et al (2002) Prophylactic growth factor-primed donor lymphocyte infusion using cells reserved at the time of transplantation after allogeneic peripheral blood stem cell transplantation in patients with high-risk hematologic malignancies. Cancer 94(1):18–24 35. Collins RH Jr, Shpilberg O, Drobyski WR et al (1997) Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 15(2):433–444 36. Kolb HJ, Schattenberg A, Goldman JM et al (1995) Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 86(5): 2041–2050 37. Suzuya H, Watanabe T, Nakagawa R et al (2005) Factors associated with granulocyte colony-stimulating factor-induced peripheral blood stem cell yield in healthy donors. Vox Sang 89(4):229–235 38. Anderlini P, Donato M, Chan KW et  al (1999) Allogeneic blood progenitor cell collection in normal donors after mobilization with filgrastim: The M.D. Anderson Cancer Center experience. Transfusion 39(6):555–560 39. Devine SM, Andritsos L, Todt L et al (2005) A pilot study evaluating the safety and efficacy of AMD3100 for the mobilization and transplantation of HLA-matched sibling donor hematopoietic stem cells in patients with advanced hematological malignancies. ASH Annu Meet Abstr 106(11):299 40. Devine SM, Flomenberg N, Vesole DH et al (2004) Rapid mobilization of CD34+ cells following administration of the CXCR4 antagonist AMD3100 to patients with multiple myeloma and non-Hodgkin’s lymphoma. J Clin Oncol 22(6):1095–1102 41. Flomenberg N, Devine SM, Dipersio JF et al (2005) The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone. Blood 106(5):1867–1874 42. Liles WC, Rodger E, Broxmeyer HE et  al (2005) Augmented mobilization and collection of CD34+ hematopoietic cells from normal human volunteers stimulated with granulocyte-colony-stimulating factor by single-dose administration of AMD3100, a CXCR4 antagonist. Transfusion 45(3):295–300

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sdfsdf

Chapter 25 Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic Stem Cell Transplantation Steven C. Goldstein and Selina Luger

The number of reduced-intensity conditioning (RIC)/nonmyeloablative transplants (NMT) has risen steadily over the last 10 years, now comprising approximately 30% of all allogeneic transplants performed annually [1]. Despite the rapid rise in its application, we have much to learn in terms of optimizing conditioning regimens, GvHD prophylaxis, identifying appropriate patient cohorts, and disease states, thus balancing the critical endpoints of chimerism, GvHD, relapse, and toxicity for the optimal utilization of this strategy. Building on the landmark work by Storb et al. [2, 3] in the canine model, the paradigm requiring myeloablation of the host immunohematopoietic system for successful long-term donor hematopoietic engraftment, has been replaced by the view that nonmyeloablative allogeneic transplantation is at its essence, the truest form of cellular immunotherapy. At its inception approximately a decade ago, the initial goal was to offer potentially curative treatment to patients previously excluded from consideration for standard allotransplantation secondary to age and/or other comorbid conditions. Early papers in RIC/ NMT focused on the critical goals of establishing donor hematopoiesis with low early treatment-related mortality; notably absent was the demonstration of long-term disease control [4]. Whether NMT/RIC can improve on the disease outcomes of standard transplantation as opposed to simply broadening the pool of potential candidates for allotransplantation remains an area of active investigation.

1. Defining Dose Intensity Although definitions of truly nonmyeloablative (e.g., TBI 200, fludarabine/ Cyclophosphamide as “immunosuppressive only”) regimens achieve wide consensus among BMT physicians, there remains a gray area for regimens in

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_25, © Springer Science + Business Media, LLC 2003, 2010

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Table 25-1.  Characteristics of NMT versus RIC conditioning. NMT

TBI 200 cGy +/− Fludarabine

RIC

Fludarabine plus

£ 500 cGy TBI £9 mg/kg total busulfan dose £140 mg/m2 total melphalan dose £10 mg/kg total thiotepa dose

NMT nonmyeloablative transplantation, RIC reduced-intensity conditioning, TBI total-body irradiation

which the dosages of fully ablative regimens have been attenuated to decrease early treatment-related morbidity and mortality, but still likely to require hematopoietic progenitor cell support (i.e., reduced intensity conditioning, RIC). Although scientifically interesting, studies to define the exact dosages at which stem cell support is required are not feasible and remain hypothetical in practice. Therefore, there is a spectrum of regimens which are deemed as RIC, but may or may not require stem cell rescue. The distinction between myeloablative, RIC, and NMT strategies goes beyond mere stratification by dose; the underlying principles of balancing treatment-related morbidity and mortality, graft-versus-host disease, and relapse are critical in defining the optimal strategy for an individual patient. In RIC, cytotoxic and immunosuppressive conditioning is combined to provide disease control and suppression of the host-versus-graft reaction, anticipating the several month interim required for the establishment of a graft-versus-tumor (GvT) effect by donor immunohematopoiesis; whereas the noncytotoxic, “immunosuppressive-only” conditioning employed in NMT via aggressive post-grafting immunosuppression is designed to minimize early toxicity with the goal of establishing adequate immunosurveillance and GvT with inherently less emphasis on early disease control. Although often used interchangeably, consensus definitions have been adopted to allow for more consistent characterization of transplant outcomes across standard, RIC, and NMT, as outlined in Table 25-1.

2. Does Dose Intensity Matter? 2.1. Dose Intensity in Standard Transplantation Although the rationale of high dose, myeloablative therapy in optimizing disease control for patients undergoing standard conditioning with allogeneic or autologous stem cell rescue has long been accepted as an important attribute in their curative potential, data demonstrating a significant disease-free or overall survival benefit of increasing dose intensity between myeloablative regimens is lacking. Although a meta-analysis by Hartman et al. [5] suggested a possible trend toward improved survival for patients receiving TBI-containing regimens compared to busulfan/cyclophosphamide, a subsequent study by Socie et al. [6] retrospectively compared the long-term outcomes for patients with myeloid leukemia (CML and AML) receiving busulfan/cyclophosphamide (BuCy2) vs. the more intensive cyclophosphamide/total-body irradiation (Cy/ TBI) across four randomized trials [7–10]; no statistically significant difference in survival was identified. In the report from the IBMTR, summarizing

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic 

the outcomes of AML patients in CR1 who underwent matched sibling bone marrow transplantation after BuCy2 (n = 318) or Cy/TBI (n = 200) between 1988 and 1996, Litzow et  al. [11] also noted no significant differences in treatment-related mortality, disease-free survivalor overall survival. Studies such as these require large number of patients and many years to complete; thus their extrapolation to current practice is potentially confounded by the subsequent implementation of busulfan dose-targeting and use of alternate stem cell sources. As noted above, despite their inherent limitations, most series published to date demonstrate a similar survival after myeloablative conditioning regardless of dose intensity. Recent investigations into novel, myeloablative regimens modified to decrease dose-related toxicity of conditioning (i.e., often referred to as RIC) have been associated with lower TRM without significant increase in relapse or decrease in overall survival, even among patients traditionally excluded by standard transplantation based on age or comorbidities [12–15] (see Table  25-2). These efforts warrant further investigation as they pursue many of the same goals of truly nonmyeloablative transplantation, specifically to expand the potential cohort of patients that might be treated with curative intent without undue toxicity. 2.2. Dose Intensity within RIC/NMT One must be cautious in extrapolating the lack of dose/conditioning effect in myeloablative conditioning to the realm of NMT/RIC where the kinetics of disease control must take into account the time it takes to establish donor immunosurveillance and withdrawal of immunosuppression to maximize GVL responses. In essence, the “intensity” of the various RIC/NMT regimens that may be critical for success is of a different quality; the intensity of immunosuppression, rather than cytotoxicity or myelosuppression. Whereas efforts to maximize dose intensity within standard transplantation have not been definitively proven to improve the outcome, variance between NMT/RIC conditioning regimens are much more likely to be relevant not only to overall outcomes, but will likely be closely linked in terms of tumor burden, disease activity, pace of disease progression, and inherent disease immunogenicity (i.e., susceptibility to GvT effect) at the time of transplantation. 2.2.1. Relevance of Tumor Burden/Disease Activity in NMT In addition to the two most common indications for pursuing NMT over standard transplantation, age and comorbidity, several studies have explored the impact of tumor burden, disease status, and rate of disease progression prior to NMT on outcomes after NMT and their relevance to the decision on whether to pursue NMT and/or choice of NMT regimen. A consistent message that can be drawn from both registry and single-center data is that active disease at the time of NMT/RIC, particularly in patients with AML and aggressive NHL, predicts for a significantly worse outcome as compared to patients transplanted in a minimal residual disease state [16–18]. Of note, however, is that even among leukemic patients with active disease (i.e., >5–10% blasts in marrow or periphery) there is a small subset of long-term disease free survivors after RIC (as opposed to strictly immunosuppressive therapy in NMT), suggesting that dose intensity is relevant for this high-risk cohort [19]. In their report of 102 AML patients receiving reduced intensity conditioning with

443

Bu 1 mg/kg × 10, flu 150/m2

Bu 130 mg/m2, then targeted to AUC 4500–5600; flu 160 mg/m2

Bu 1.0 mg/kg × 16 doses, flu 120 mg/ m2

Bu 130 mg/m /d × 4, flu 160/m2

10 mg/kg po

16 mg/kg po

520 mg/m IV

2

19

NR

54

25

43

NR

49

55

65 ± 11%

65% PFS

OS NR

51% EFS (1 year)

61% OS (1 year)

35% DFS (1.5 years)

42% OS (1.5 years)

52% EFS (1 year)

65% OS (1 year)

non-AML 26 ± 11%

74 ± 8%

4%

24%

0

8%

2%

AML

D100 High risk

Low risk

High risk

DFS % (2 years)

38

12.8 mg/kg IV

8

NRRM

GvHD% Total BU dose Acute Chronic

5% 1 year

17%

1 year

NR

19%

5%

2 years

URD

rel

Flu fludarabine, Bu busulfan, REL related donor, URD unrelated donor, ATG anti-thymocyte globulin, GvHD graft-versus-host disease, OS overall survival, NR not reported, EFS event-free survival, PFS progression-free survival, NRRM non-relapse-related mortality

N = 37 (sib)

Martino [75]

[33 rel, 36 URD]

N = 69

Field et al. [15]

[16 rel, 26 URD]

N = 42

Bornhauser et al. [14]

[60 rel, 36 URD]

N = 96

deLima et al. [13]

[49 rel, 21 URD]

2

Bu 3.2 mg/kg/D × 4 IV, flu 250 mg/m2, ATG

Russell et al. [12]

N = 70

Dose/schedule

N

Reference

Table 25-2.  Novel myeloablative regimens (selected references).

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Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic 

fludarabine/busulfan, Sayer et al. described an EFS of 49% for patients with less than 5% blasts, 24% for patients with 5–20% blasts, and 14% for patients with >20% blasts at 12 months of median follow-up [18]. It should be noted that the correlation between tumor burden and adverse outcome after transplant is not unique to NMT/RIC strategies, a similar finding has been noted for myeloablative approaches for acute leukemia as well [20]. In contrast to Sayer et al. [18], in which a small subset of patients with active disease could be salvaged via RIC, Shimoni et al. concluded that only patients undergoing myeloablative conditioning could be salvaged if they had active disease at the time of transplant [21]. 2.3. Myeloablative Conditioning vs. NMT/RIC The growing list of reports demonstrating a similar overall outcome for patients with acute leukemia and non-Hodgkin lymphoma who have undergone standard, myeloablative conditioning vs. RIC/NMT must be interpreted cautiously in light of the selection bias of the two approaches. These cohorts are inherently unbalanced, as in almost all circumstances patients undergoing RIC/NMT were deemed ineligible to undergo standard allotransplantation. Of course, achieving a similar outcome in a much higher-risk cohort of patients is a critical first step in the broader application of this strategy but, to date, there has been no conclusive data demonstrating a benefit of RIC/NMT in patients eligible to receive standard conditioning. As regimens evolve that are myeloablative, but clearly reduced in toxicity, the debate as to whether to maximize dose with the least toxicity vs. minimizing dose with the most immunotherapeutic potential, and in which circumstance, will continue unabated. Indeed, prospective trials comparing myeloablative to nonmyeloablative conditioning in the same cohort of patients are underway or in development in the United States and Europe. Until these studies are completed, we have only retrospective series with their inherent inadequacies on which to base our decision-making. Despite (or because of) the vagaries in comparing cohorts using different strategies over time, the results of most of the retrospective reports are remarkably similar; each has demonstrated that the overall survival outcomes after fully ablative vs. nonmyleloablative/RIC are nearly identical [21–29] (see Table 25-3).

3.  Does Dose Intensity Impact on Graft-Versus-Host Disease? 3.1. Acute GvHD after RIC/NMT Extrapolating from the initial GvHD paradigm [30] relating the cytokine storm triggered by tissue damage from conditioning regimens as a critical component of the acute pathophysiology of acute GvHD, one would predict that the incidence and severity of acute GvHD after RIC/NMT should be lower than that seen after standard conditioning, a trend often alluded to in small case series [31–33]. However, retrospective studies must be interpreted carefully before drawing this conclusion. Initial reports were in patients receiving TBI-based conditioning. There is remarkably little data available to correlate dose intensity with acute GvHD and whether it is relevant to non-TBI-based conditioning; a

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Table 25-3.  Outcomes after NMT/RIC versus Myeloablative (selected references).

Alyea et al. [28]

71 RIC Flu/bu OS at 1 year, 2 years AML 21, ALL1, CML 9, CLL 13, MDS 15, NHL 9, CMML 3 81 std bucy/cy-tbi

Aoudjhane et al. [22]

Valcarcel et al. [23]

NMT/RIC Myeloablative (%) (%) P-value 51, 39 38, 29 0.06

27, 25

0.24

AML 13, ALL 3, CML 33, CLL2, NRRM cum MDS17, NHL 10

PFS at 1 year, 2 years 40, 36 32

50

0.01

315 RIC

OS at 2 years

47

46

0.43

407 std

LFS at 2 years

40

44

0.8

AML CR1 245, AML CR2 52, AML adv 110

TRM at 2 years

18

32

55 years urd)

25

6.4 mg/kg IV 8 mg/kg po

Dose/schedule

GvHD% OS Total Bu dose/ route Acute Chronic NRRM

N

Reference

Table 25-5.  Fludarabine/busulfan (selected references).

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isolation from the GvHD prophylaxis regimen, stem cell source, and use of DLI. Indeed, each regimen is likely to require its own counterbalance strategy, which takes into account its potential inherent weaknesses. For example, the excellent early protection from acute GvHD provided by alemtuzumab may have a less desirable impact on maintaining full-donor chimerism and/or minimizing risk of relapse thus warranting a planned strategy such as “prophylactic” DLI in order to maximize immune and hematopoietic reconstitution later in the post-transplant course. 4.1. Disease-Specific Regimens? Published prospective, comparative trials across regimens within specific diseases are lacking. Whereas many clinicians may consider one drug or regimen more “anti-lymphoid” (e.g., fludarabine/melphalan or fludarabine/TBI) or “anti-myeloid” (e.g., fludarabine/busulfan), there is no conclusive evidence at present that customizing the regimen based on disease influences outcome (though disease state, i.e., remission vs. MRD vs. active disease, is likely to correlate with outcome based on the “intensity” of the regimen as described above), highlighting the need for disease-specific multi-institutional prospective trials [40]. 4.2. Fludarabine-Based Conditioning Purine analogs, primarily fludarabine, have become the foundation around which most nonmyeloablative and reduced-intensity regimens have been constructed. Indeed, there is also a growing trend in purine analog-based myeloablative conditioning as well [12–15]. Among NMT and RIC regimens, the most common combinations published to date have been fludarabine (125–180 mg/2) with melphalan, busulfan, or TBI with or without the addition of serotherapy in the form of anti-thymocyte globulin (ATG) or alemtuzumab [Tables 25.4, 25.5]. 4.3. Fludarabine-Melphalan vs. Fludarabine-Busulfan In an important retrospective (nonrandomized) study of 151 patients with both myeloid and lymphoid malignancies, comparing 72 patients conditioned with fludarabine/busulfan (flu/bu) vs. 79 patients conditioned with fludarabine/ melphalan (flu/mel), Shimoni et  al. [16] described a lower cumulative incidence of acute GvHD (33 vs. 53%), death from organ toxicity (10 vs. 23%) and graft-versus-host disease (6 vs. 17%) in the flu/bu cohort as compared to flu/mel group, respectively, but did not identify a significant difference in the overall survival between the two regimens. The higher reported incidence of acute GvHD after flu/mel is intriguing in that the flu/mel cohort had significantly fewer patients with unrelated donors and therefore expected to be at a lower risk for acute GvHD as a group. Confounding factors in this observation include the use of ATG in the conditioning of recipients of unrelated grafts, but not sibling grafts, as well as the higher incidence of mucositis among flu/ mel patients as compared to flu/bu (49 vs. 29%), likely not coincidental to the higher observed incidence of gut GvHD in the flu/mel cohort. Although no difference in the overall survival between the two regimens can be identified when patients with active disease are included, a subset analysis of patients

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic 

in remission at the time of transplant suggests a possible overall survival advantage of flu/bu over flu/mel, for patients in remission, likely , due in large part,to a lower rate of NRRM despite a similar rate of relapse. 4.4. Fludarabine/TBI The largest experience in combining low-dose total-body irradiation (200 cGy) with and without fludarabine (90  mg/m2) has been described by the Seattle Consortium [41, 42]. Retrospective cohort analyses of >100 patients with myeloid malignancies (AML, MDS, CML) have demonstrated feasibility with sustained engraftment in recipients of both sibling and unrelated grafts with a 1-year nonrelapse mortality rate of ~15–20% [27, 43]. Of note, though several early patients (with sibling donors) received TBI alone, the relatively high rate of graft failure prompted the addition of fludarabine to the remaining cohort of sibling and all unrelated recipients. This series was one of the first to note the potential for divergent outcome based on donor source, rather than regimen. Specifically, there was a statistically significant decrease in relapse at 2 years among CR1 recipients of unrelated grafts when compared to CR1 recipients of sibling grafts (16 vs. 50%; p = .005) and a trend towards improved 2 years overall survival (63 vs. 44%; p = 0.13), despite a similar incidence of acute and chronic graft-versus-host disease [43]. The potential for an improved GvL effect, putatively attributed to the use of unrelated donors in this cohort, did not translate into a lower relapse rate in patients beyond CR1 however and may have been confounded by differences in the use of fludarabine, prior autologous transplantation, and DLI between the two groups. 4.5. Addition of Alemtuzumab Although the use of serotherapy in the form of pre-transplant conditioning with anti-CD52 monoclonal antibody (alemtuzumab) either in  vivo or ex vivo/“in the bag” has been in clinical practice for more than a decade [44–46], the immunologic mechanism and optimal utilization of this agent remains an area of active investigation. Early reports investigating the addition of alemtuzumab to ablative and nonmyeloablative conditioning regimens have illustrated the double-edged sword of this strategy. While there is a consensus among investigators regarding the reduction of acute graft-versus-host disease among recipients of alemtuzumab, most apparent in the higher-risk unrelated cohort [47], ambiguity persists in the literature in terms of its longerterm impact on donor chimerism, infection and risk of relapse [46, 48–53]. Analyzing these studies in terms of total dose of alemtuzumab, timing of administration, and the specific endpoints being reported provide insights into potential explanations for the disparate conclusions between investigators. For example, depending on the timing and dose of administration of alemtuzumab, the prolonged half-life likely impacts on clinical endpoints via its dual role in both depleting donor T cells, B cells, and APCs in the graft [45] and host lymphoid and antigen-presenting cells in the patient [54, 55]. As the monoclonal antibody may be still in circulation on day 0 at the time of the stem cell infusion, the depleting impact on donor cells may amelioriate GvHD, but may poteniate risk of infecton, relapse, and failure to achieve full-donor chimerism, whereas the impact of alemtuzamab on host lymphoid cells and APCs may facilitate engraftment and decrease GvHD [34]. Although surprisingly little data is

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available in terms of alemtuzumab levels at the time of stem cell infusion and its relevance to in vivo T cell depletion and outcome, recent reports [49, 52, 53, 56] evaluating a range of alemtuzumab dosages provide some important insights. By titrating the alemtuzumab dose to 50 mg from 100 mg given subcutaneously from d-7 to d-3, Khouri et al. [56] may have separated the hostdepletion of lymphoid cells and APCs from the Tcell depletion of the donor graft based on their finding that no detectable alemtuzumab could be identified in circulation via ELISA [57] after only 48 h beyond the last dose. This is in contrast to the findings of Rebello et al. [57] which suggested that the 50-mg dose given over a similar time frame prior to stem cell infusion was associated with peak concentrations of alemtuzumab (2.5 ucg/ml) on day 0 that was still above the level necessary for opsonization of lymphocytes. Indeed, circulating alemtuzumab was still detected up to 11 days after the 50-mg total dose with a terminal half-life of 15–21 days. Other investigators have postulated that “in vitro” alemtuzumab doses, as low as 20 mg, added “to the bag” may provide an alternate strategy, though immune reconstitution remains a concern [49, 53]. While retrospectively reconciling these disparate findings is not possible, one explanation may be that Khouri et al. cohort analysis was limited to patients with B-cell malignancies (i.e., CD-52 positive), thereby serving as a potential “sink” for circulating antibody (as compared to patients undergoing transplantation with nonlymphoid malignancies [57]), and possibly decreasing the levels of detectable antibody [58]. Potentiating functional depletion of host APCs while minimizing T cell depletion of the donor graft may be critical for achieving the long-sought balance between decreasing GvH and maximizing GvT [59]. Optimal titration of both the dose and timing of alemtuzumab [53] (as well as considering the impact of CD-52 positivity of the disease) remains an active area of investigation. 4.6. Extracorporeal Photopheresis-Based Conditioning On the basis of phase I/II results [60, 61] exploring the reduced-intensity combination of 2-deoxycoformycin, TBI (600 rads) and extracorporeal photopheresis (ECP) in patients with hematologic malignancies, a cooperative group Phase II study has been initiated for patients with myelodysplasia. Although the putative immunomodulatory effects of the specific components of the conditioning regimen cannot be distinguished from each other, it is postulated that ECP may (1) attenuate Th1-mediated cytokine secretion by activated T-helper cells, (2) cause a shift in the DC1/DC2 ratio favoring plasmacytoid rather than monocytoid dendritic cell profiles, and (3) decrease antigen responsiveness by dendritic cells [62]. The low rates of GvHD with this regimen appear promising, but the impact on disease control and relapse rates will require longer follow-up. Relating an ECP-mediated increase in Treg’s as a possible mechanism for lowering acute GvHD is supported by the work of Lamioni et  al. who demonstrated an in increase in Treg’s after ECP as therapy for cardiac transplant rejection [63]. 4.7. TLI-Based Conditioning Murine models [64, 65] demonstrating the protective effect of total lymphoid irradiation (TLI) against acute GvHD, (as opposed to total-body irradiation) prompted a phase I trial in humans in which total lymphoid irradiation, rather than TBI was used in conjunction with ATG as protective conditioning against GvHD. This strategy was based on the postulated skewing of the T cell to a

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic 

Th2 phenotype by TLI (but not TBI) and enhanced IL-4 (inhibitory) secretion with “downstream” abrogating effects on the inflammatory cytokine cascade. Another proposed mechanism is the selection of regulatory T cells and NK-T cells by low-dose lymphoid radiation, without compromising the effector CD8+ population, thus allowing for the potential separation of the GvL effect mediated by donor CD8+ T cells from the GvHD-abrogating effect of regulatory T cells and NK-T cells. Thirty-seven patients with lymphoid (n = 24) and myeloid (n = 13) disease received 80  cGy TLI over 11 days with ATG prior to receiving PBSC grafts from matched sibling (n = 23) and unrelated (n = 14) donors, followed by cyclosporine and mycophenolate mofetil [66]. Despite the advanced disease status in the majority of patients entering transplant, the response rates and disease-free survival appear very promising when compared to historical controls, though longer-term follow-up in a larger cohort is required. The incidence of grade II–IV acute GvHD was remarkably low for the entire cohort, 1/37 (3%). Of particular interest is the demonstration of a tenfold increase in NK-T cells after TLI as well as an increase in IL-4 production and decrease in proliferative response in CD4+ cells after TLI when compared to CD4+ cells after TBI-containing regimens [66]. 4.8. Future Directions Despite the rising number of patients undergoing allogeneic transplantation with nonmyeloablative or reduced-intensity conditioning, the proportion of patients achieving cure remains disappointing. Improving overall outcomes will require a two-tiered approach. While smaller, single-center phase I/II studies will continue to explore novel strategies to enhance the graft-versusmalignancy effect while minimizing graft-versus-host disease via incorporation of novel methods of immunosuppression (e.g., TLI [66], targeting of APCs [34, 67]), cellular therapy (e.g., NK infusions [68], preemptive DLI [69–72]), vaccines [73], and gene therapy [74], it remains critical that multicenter, prospective, randomized trials comparing disease-specific regimens in the NMT/RIC and myeloablative setting are prioritized to ultimately define the optimal strategy for the individual patient.

References 1. Pasquini MC (2006) Current use and outcome of hematopoietic stem cell transplantation: part I - CIBMTR Summary Slides, 2005. CIBMTR Newslett 12(1):5–8 2. Storb R et al (1997) Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Blood 89(8):3048–3054 3. Yu C et  al (1995) DLA-identical bone marrow grafts after low-dose total body irradiation: effects of high-dose corticosteroids and cyclosporine on engraftment. Blood 86(11):4376–4381 4. Giralt S et al (1997) Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graft-versus-leukemia without myeloablative therapy. Blood 89(12):4531–4536 5. Hartman AR, Williams S, Dillon JJ (1998) Survival, disease-free survival and adverse effects of conditioning for allogeneic bone marrow transplantation with busulfan/cyclophosphamide vs total body irradiation: a meta-analysis. Bone Marrow Transplant 22:439–443 6. Socie G et al (2001) Busulfan plus cyclophosphamide compared with total-body irradiation plus cyclophosphamide before marrow transplantation for myeloid leukemia: long-term follow-up of 4 randomized studies. Blood 98(13):3569–3574

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S.C. Goldstein and S. Luger 7. Blaise D, Maraninchi D, Archimbaud E (1992) Allogeneic bone marrow transplantation for acute myeloid leukemia in first remission: a randomized trial of a busulfancytoxan versus cytoxan-total body irradiation as preparative regimen: a report from the Groupe d’Etudes de la Greffe de M. Blood 79:2578–2582 8. Ringden O, Ruutu T, Remberger M (1994) A randomized trial of comparing busulfan with total body irradiation as conditioning in allogeneic marrow transplant recipients with leukemia-a report from the Nordic Bone Marrow Transplantation Group. Blood 83:2723–2730 9. Clift RA et  al (1994) Marrow transplantation for chronic myeloid leukemia: a randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide. [comment]. Blood 84(6):2036–2043 10. Devergie A, Blaise D, Attal M (1995) Allogeneic bone marrow transplantation for chronic myeloid leukemia in first chronic phase: a randomized trial of busulfancytoxan versus cytoxan-total body irradiation as preparative regimen: a report from the French Society of Bone Marrow. Blood 85:2263–2268 11. Litzow MR et al (2002) Comparison of outcome following allogeneic bone marrow transplantation with cyclophosphamide-total body irradiation versus busulphancyclophosphamide conditioning regimens for acute myelogenous leukaemia in first remission. Br J Haematol 119(4):1115–1124 12. Russell J et al (2002) Once-daily intravenous busulfan given with fludarabine as conditioning for allogeneic stem cell transplantation: study of pharmacokinetics and early clinical outcomes. Biol Blood Marrow Transplant 8:468–476 13. deLima M, Couriel D, Thall PF (2004) Once-daily intravenous busulfan and fludarabine: clinical and pharmacokinetic results of a myeloablative, reducedtoxicity conditioning regimen for allogeneic stem cell transplantation in AML and MDS. Blood 104:857–864 14. Bornhauser M et al (2003) Conditioning with fludarabine and targeted busulfan for transplantation of allogeneic hematopoietic stem cells. Blood 102:820–826 15. Field T et al (2006) Busulfan area-under-the-curve finding study within a busulfan/ fludarabine (BuFlu) conditioning regimen before allogeneic hematopoietic cell transplantation. Blood 108:832a 16. Shimoni A et  al (2007) Comparison between two fludarabine-based reducedintensity conditioning regimens before allogeneic hematopoietic stem-cell transplantation: fludarabine/melphalan is associated with higher incidence of acute graft-versus-host disease and non-relapse mortality and lower incidence of relapse than fludarabine/busulfan. Leukemia 21(10):2109–2116 17. van Besien K et al (2005) Fludarabine, melphalan, and alemtuzumab conditioning in adults with standard-risk advanced acute myeloid leukemia and myelodysplastic syndrome. J Clin Oncol 23(24):5728–5738 18. Sayer HG et al (2003) Reduced intensity conditioning for allogeneic hematopoietic stem cell transplantation in patients with acute myeloid leukemia: disease status by marrow blasts is the strongest prognostic factor. Bone Marrow Transplant 31(12):1089–1095 19. de Lima M et  al (2004) Nonablative versus reduced-intensity conditioning regimens in the treatment of acute myeloid leukemia and high-risk myelodysplastic syndrome: dose is relevant for long-term disease control after allogeneic hematopoietic stem cell transplant. Blood 104(3):865–872 20. Kebriaei P et  al (2005) Impact of disease burden at time of allogeneic stem cell transplantation in adults with acute myeloid leukemia and myelodysplastic syndromes. Bone Marrow Transplant 35(10):965–970 21. Shimoni A et al (2006) Allogeneic hematopoietic stem-cell transplantation in AML and MDS using myeloablative versus reduced-intensity conditioning: the role of dose intensity. Leukemia 20(2):322–328 22. Aoudjhane M et al (2005) Comparative outcome of reduced intensity and myeloablative conditioning regimen in HLA identical sibling allogeneic haematopoietic

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic  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 19(12):2304–2312 23. Valcarcel D et al (2005) Conventional versus reduced-intensity conditioning regimen for allogeneic stem cell transplantation in patients with hematological malignancies. Eur J Haematol 74(2):144–151 24. Massenkeil G et  al (2005) Survival after reduced-intensity conditioning is not inferior to standard high-dose conditioning before allogeneic haematopoietic cell transplantation in acute leukaemias. Bone Marrow Transplant 36(8):683–689 25. Martino R et al (2006) Retrospective comparison of reduced-intensity conditioning and conventional high-dose conditioning for allogeneic hematopoietic stem cell transplantation using HLA-identical sibling donors in myelodysplastic syndromes. Blood 108(3):836–846 26. Maruyama D et  al (2007) Comparable antileukemia/lymphoma effects in nonremission patients undergoing allogeneic hematopoietic cell transplantation with a conventional cytoreductive or reduced-intensity regimen. Biol Blood Marrow Transplant 13(8):932–941 27. Scott BL et al (2006) Myeloablative vs nonmyeloablative allogeneic transplantation for patients with myelodysplastic syndrome or acute myelogenous leukemia with multilineage dysplasia: a retrospective analysis. Leukemia 20(1):128–135 28. Alyea EP et al (2005) Comparative outcome of nonmyeloablative and myeloablative allogeneic hematopoietic cell transplantation for patients older than 50 years of age. Blood 105(4):1810–1814 29. Hari P et al (2008) Allogeneic transplants in follicular lymphoma: higher risk of disease progression after reduced-intensity compared to myeloablative conditioning. Biol Blood Marrow Transplant 14(2):236–245 30. Ferrara J, Deeg H (1991) Graft-versus-host disease. N Engl J Med 324:667–674 31. Mielcarek M et al (2003) Graft-versus-host disease after nonmyeloablative versus conventional hematopoietic stem cell transplantation. Blood 102(2):756–762 32. Sorror M et  al (2005) Lessened severe graft-versus-host after “minitransplantations”. Blood 105(6):2614 33. Couriel DR et al (2004) Acute and chronic graft-versus-host disease after ablative and nonmyeloablative conditioning for allogeneic hematopoietic transplantation. Biol Blood Marrow Transplant 10(3):178–185 34. Shlomchik W et al (1999) Prevention of graft-versus-host disease by inactivation of host antigen-presenting cells. Science 285:412–415 35. Mielcarek M, Storb R (2005) Graft-vs-host disease after non-myeloablative hematopoietic cell transplantation. Leuk Lymphoma 46(9):1251–1260 36. Mielcarek M et al (2005) Prognostic relevance of “early-onset” graft-versus-host disease following non-myeloablative haematopoietic cell transplantation. Br J Haematol 129(3):381–391 37. Levine J et al (2003) Lowered-intensity preparative regimen for allogeneic stem cell transplantation delays acute graft-versus-host disease but does not improve outcome for advanced hematologic malignancy. Biol Blood Marrow Transplant 9:189–197 38. Couriel D, Giralt S (2005) Graft vs Host Disease in Nonmyeloablative Transplant. In: Ferrara JL, Cooke KR, Deeg HJ (eds) Graft vs Host Disease. Marcel Dekker, New York 39. Loren A et  al (2005) Intensive graft-versus-host disease prophylaxis is required after unrelated donor non-myeloablative stem cell transplantation. Bone Marrow Transplant 35:921–926 40. Deeg HJ et al (2006) Optimization of allogeneic transplant conditioning: not the time for dogma. Leukemia 20(10):1701–1705 41. McSweeney PA et  al (2001) Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graftversus-tumor effects. Blood 97(11):3390–3400

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S.C. Goldstein and S. Luger 42. Maris MB et al (2003) HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with hematologic malignancies. Blood 102(6):2021–2030 43. Hegenbart U et al (2006) Treatment for acute myelogenous leukemia by low-dose, total-body, irradiation-based conditioning and hematopoietic cell transplantation from related and unrelated donors. J Clin Oncol 24(3):444–453 44. Hale G et  al (1998) Improving the outcome of bone marrow transplantation by using CD52 monoclonal antibodies to prevent graft-versus-host disease and graft rejection. Blood 92(12):4581–4590 45. Hale G, Cobbold S, Waldmann H (1988) T cell depletion with CAMPATH-1 in allogeneic bone marrow transplantation. Transplant 45(4):753–759 46. Kottaridis PD et al (2000) In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation. Blood 96(7):2419–2425 47. Loren AW et al (2005) Intensive graft-versus-host disease prophylaxis is required after unrelated-donor nonmyeloablative stem cell transplantation. Bone Marrow Transplant 35(9):921–926 48. Perez-Simon JA et  al (2002) Nonmyeloablative transplantation with or without alemtuzumab: comparison between 2 prospective studies in patients with lymphoproliferative disorders. Blood 100(9):3121–3127 49. Morris EC et  al (2003) Pharmacokinetics of alemtuzumab used for in  vivo and in vitro T-cell depletion in allogeneic transplantations: relevance for early adoptive immunotherapy and infectious complications. Blood 102(1):404–406 50. Morris E et  al (2004) Outcomes after alemtuzumab-containing reduced-intensity allogeneic transplantation regimen for relapsed and refractory non-Hodgkin lymphoma. Blood 104(13):3865–3871 51. Michaelis L et al (2007) Chimerism does not predict for outcome after alemtuzumab based conditioning. Bone Marrow Transplant 40(2):181 52. Juliusson G et  al (2006) Subcutaneous alemtuzumab vs ATG in adjusted conditioning for allogeneic transplantation: influence of Campath dose on lymphoid recovery, mixed chimerism and survival. Bone Marrow Transplant 37(5):503–510 53. Hale G et  al (2001) CAMPATH-1 antibodies in stem-cell transplantation. Cytotherapy 3(3):145–164 54. Klangsinsirikul P et  al (2002) Campath-1G causes rapid depletion of circulating host dendritic cells (DCs) before allogeneic transplantation but does not delay donor DC reconstitution. Blood 99(7):2586–2591 55. Ratzinger G et  al (2003) Differential CD52 expression by distinct myeloid dendritic cell subsets: implications for alemtuzumab activity at the level of antigen presentation in allogeneic graft-host interactions in transplantation. Blood 101(4):1422–1429 [erratum appears in Blood. 2005 Apr 15;105(8):3018 Note: dosage error in text] 56. Khouri IF et al (2004) Low-dose alemtuzumab (Campath) in myeloablative allogeneic stem cell transplantation for CD52-positive malignancies: decreased incidence of acute graft-versus-host-disease with unique pharmacokinetics. [see comment]. Bone Marrow Transplant 33(8):833–837 57. Rebello P et  al (2001) Pharmacokinetics of CAMPATH-1H in BMT patients. Cytotherapy 3(4):261–267 58. Hale G et  al (2004) Blood concentrations of alemtuzumab and antiglobulin responses in patients with chronic lymphocytic leukemia following intravenous or subcutaneous routes of administration. Blood 104(4):948–955 59. Russell NH, Byrne JL (2004) In vivo Campath for the prevention of GvHD following allogeneic HSCT: effects of dose, schedule and antibody type. [comment]. Bone Marrow Transplant 34(12):1101–1102 60. Chan GW et al (2003) Reduced-intensity transplantation for patients with myelodysplastic syndrome achieves durable remission with less graft-versus-host disease. [see comment]. Biol Blood Marrow Transplant 9(12):753–759

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic  61. Miller KB et  al (2004) A novel reduced intensity regimen for allogeneic hematopoietic stem cell transplantation associated with a reduced incidence of graft-versus-host disease. Bone Marrow Transplant 33(9):881–889 62. Foss FM, Gorgun G, Miller KB (2002) Extracorporeal photopheresis in chronic graft-versus-host disease. Bone Marrow Transplant 29(9):719–725 63. Lamioni A et al (2005) The immunological effects of extracorporeal photopheresis unraveled: induction of tolerogenic dendritic cells in vitro and regulatory T cells in vivo. Transplantation 79(7):846–850 64. Lan F et al (2003) Host conditioning with total lymphoid irradiation and antithymocyte globulin prevents graft-versus-host disease: the role of CD1-reactive natural killer T cells. Biol Blood Marrow Transplant 9(6):355–363 65. Lan F et  al (2001) Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: “natural suppressor” cells. J Immunol 167(4):2087–2096 66. Lowsky R et al (2005) Protective conditioning for acute graft-versus-host disease. [see comment]. N Engl J Med 353(13):1321–1331 [erratum appears in N Engl J Med. 2006 Feb 23;354(8):884] 67. Reddy P et  al (2005) A crucial role for antigen-presenting cells and alloantigen expression in graft-versus-leukemia responses. Nat Med 11(11):1244–1249 68. Miller JS et al (2005) Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 105(8):3051–3057 69. Baron F, Beguin Y (2002) Preemptive cellular immunotherapy after T-cell-depleted allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 8:351–359 70. Barrett AJ et al (1998) T cell-depleted bone marrow transplantation and delayed T cell add-back to control acute GVHD and conserve a graft-versus-leukemia effect. Bone Marrow Transplant 21(6):543–551 71. Massenkeil G et  al (2003) Reduced intensity conditioning and prophylactic DLI can cure patients with high-risk acute leukaemias if complete donor chimerism can be achieved. Bone Marrow Transplant 31(5):339–345 72. Montero A et al (2006) T-cell depleted peripheral blood stem cell allotransplantation with T-cell add-back for patients with hematological malignancies: effect of chronic GVHD on outcome. Biol Blood Marrow Transplant 12(12):1318–1325 73. Barrett AJ, Rezvani K (2007) Translational mini-review series on vaccines: Peptide vaccines for myeloid leukaemias. Clin Exp Immunol 148(2):189–198 74. Rossig C, Brenner MK (2004) Genetic modification of T lymphocytes for adoptive immunotherapy. Mol Ther 10(1):5–18 75. Martino R et  al (2002) 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 100(6):2243–2245 76. Giralt S et al (2001) Melphalan and purine analog-containing preparative regimens: reduced-intensity conditioning for patients with hematologic malignancies undergoing allogeneic progenitor cell transplantation. Blood 97(3):631–637 77. Nakamura R et al (2007) Reduced-intensity conditioning for allogeneic hematopoietic stem cell transplantation with fludarabine and melphalan is associated with durable disease control in myelodysplastic syndrome. Bone Marrow Transplant 40(9):843–850 78. Chakraverty R et  al (2002) Limiting transplantation-related mortality following unrelated donor stem cell transplantation by using a nonmyeloablative conditioning regimen. Blood 99(3):1071–1078 79. Tauro S et al (2005) Allogeneic stem-cell transplantation using a reduced-intensity conditioning regimen has the capacity to produce durable remissions and long-term disease-free survival in patients with high-risk acute myeloid leukemia and myelodysplasia. J Clin Oncol 23(36):9387–9393

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S.C. Goldstein and S. Luger 80. Shimoni A et  al (2005) Hematopoietic stem-cell transplantation from unrelated donors in elderly patients (age >55 years) with hematologic malignancies: older age is no longer a contraindication when using reduced intensity conditioning. [see comment]. Leukemia 19(1):7–12 81. Slavin S et al (1998) Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 91:756–763 82. Schetelig J et al (2004) Reduced-intensity conditioning with busulfan and fludarabine with or without antithymocyte globulin in HLA-identical sibling transplantation–a retrospective analysis. Bone Marrow Transplant 33(5):483–490 83. Hamaki T et al (2004) Reduced-intensity stem cell transplantation from an HLAidentical sibling donor in patients with myeloid malignancies. Bone Marrow Transplant 33(9):891–900 84. Bornhauser M et al (2000) Dose-reduced conditioning for allogeneic blood stem cell transplantation: durable engraftment without antithymocyte globulin. Bone Marrow Transplant 26(2):119–125 85. Blaise DP et al (2005) Reduced intensity conditioning prior to allogeneic stem cell transplantation for patients with acute myeloblastic leukemia as a first-line treatment. Cancer 104(9):1931–1938

Chapter 26 Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer Cell Alloreactivity Franco Aversa and Andrea Velardi

1. Introduction Despite advances in chemotherapy, most adults with acute lymphoblastic leukemia (ALL) or Acute Myeloid Leukemia (AML) relapse and few survive when they have unfavorable cytogenetics at diagnosis, when they do not achieve complete remission (CR) after the first induction cycle and when they are in second or later remission [1–3]. Under these circumstances, an allogeneic hematopoietic stem cell transplant (HSCT) is preferred as post-remission therapy [4–6]. As only 30% of patients have a matched sibling donor, the only option is transplantation from an alternative donor. Phenotypically matched unrelated donors are the most widely sought-after for allogeneic transplant but have two major limitations. Molecular analysis ensures more accurate close matching, which lowers the risk of GvHD, but reduces the chance of finding a suitable matched donor [7–9]. The time-lapse from registration to donor identification can lead to disease progression in patients who urgently need transplantation. Unrelated umbilical cord blood transplantation (UCBT) has emerged as a viable option, at least in children. It offers the advantages of immediate availability of cryopreserved samples, easy procurement with no risk to the donor, and acceptance of minor mismatching (2/6 antigens). For adults UCBT is seldom considered because the divergence between body weight and the number of hematopoietic cells in a cord blood unit, particularly if associated with a two-antigen mismatch, increases the risk of graft failure and delays hematopoietic reconstitution [10–13]. Another source of stem cells is the family donor with whom the patient shares only one HLA haplotype for HLA-A, B, C and DR. These donors offer several advantages: (a) immediate availability for all transplant candidates; (b) selection of the best of many relatives on the basis of age, infectious disease status and natural killer (NK) cell alloreactivity (see below) [14–16]; (c) option to change donor if a poor stem cell mobiliser or if optimal graft

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_26, © Springer Science + Business Media, LLC 2003, 2010

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Table 26-1.  HSC Transplantation from alternative donors. MUD

UCB

HAPLO

Candidate donors with HLA-A + B + DRB1 typing 16–56%

~80%

100%

Median search time

3–6 months

90 of the ALL cases. In ALL, IgH and TcR rearrangements are not lineage-restricted and this is referred to as lineage infidelity or cross lineage rearrangements. Thus, clonal rearrangements of TcR genes are seen in a large proportion of B-ALL and a smaller proportion of IgH rearrangements are found in T-ALL [12]. Sensitivity. The detection limit of PCR analysis of junctional regions generally varies between 10−4 and 10−6. Advantages and disadvantages. The main advantages of this method are its high sensitivity and applicability in virtually all ALL patients. The need to sequence junctional regions and to develop probes and primers for each ALL case is time-consuming and a limiting factor of the method. The main disadvantage of using Ig and TcR rearrangements as MRD targets is that continuing rearrangements can occur during the disease course [13–15]. Such changes in rearrangement patterns will lead to false negative PCR results. It is now generally accepted that at least two Ig/TcR gene targets should be used for reliable and sensitive MRD detection in ALL patients. Some studies have made methodological comparisons between flow cytometry and rearrangement analysis for MRD detection [16–18]. High concordance was found between both methods. Discrepant results were usually due to low sample cellularity or the presence of PCR inhibitors [16]. 2.3. Fusion Gene Transcript Analysis Chromosomal abnormalities are present in 70–80% of the patients with AML and ALL and in >95% of the patients with CML [19]. Some of these abnormalities, especially the chromosome translocations, are recurrent and have been associated with leukemogenesis [20]. Chromosome abnormalities can be used as leukemia specific targets for MRD analysis and with the polymerase chain reaction (PCR) technique, the RNA transcripts of fusion genes can be detected with high sensitivity. Applicability. The Philadelphia (Ph) - chromosome was the first specific chromosome abnormality described in leukemia [21]. The product of this translocation, the fusion gene BCR-ABL, can be found in almost all CML patients and also in a fraction of patients with ALL (10–30%) and AML (1%) [22–26]. In AML and ALL, there is no specific translocation associated with disease. There are several numbers of translocations, which occur in 1–30% of the cases, with larger frequencies in specific leukemia subtypes. Sensitivity. Because chromosome abnormalities are highly disease specific, PCR amplification of the fusion gene transcripts can usually detect one leukemic cell among 104–106 normal cells. Advantages and disadvantages. One of the major advantages of the PCR technique is the high sensitivity. In addition, these translocations are leukemia-specific and stable during the disease course. The high sensitivity can be a problem if cross-contamination of PCR products occurs, leading to

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false-positive results. RNA degradation and variations in efficiency of cDNA synthesis may also affect sensitivity of the method. One important methodological aspect regarding the analysis of fusion gene transcripts is the use of an internal control gene [27]. Internal control genes (housekeeping genes, reference genes) are constitutively expressed genes, which are used for quality control of the patient samples. Usually, the number of fusion gene transcripts (BCR-ABL) is normalized to the number of transcripts of a control gene in order to compensate for variations that can occur between samples. The ABL gene is considered to be a suitable control gene in different diseases [28, 29]. 2.4. Chimerism Analysis After SCT, a state of chimerism develops when donor cells in the graft reconstitute the hematological and immunological system [30]. However, in some cases, host cells of hematopoietic origin survive the conditioning treatment and co-exist with donor cells. This state, which is termed mixed chimerism, may be stable or transient. There are some terms describing the chimeric status after SCT [31]. – Donor chimerism (full chimerism, complete chimerism) means that all the circulating hematopoietic cell populations are of donor origin. – Mixed chimerism means that there is a mixture of donor and host cells in peripheral blood (PB) or BM. The percentage of host cells varies between 5 and 95. – Split chimerism describes the situation when one cell lineage is of host origin and another cell lineage is of donor origin – e.g., B-cells are host and T-cells are donor. Mixed chimerism and split chimerism can be difficult to distinguish if chimerism analysis is performed using whole blood cells without prior cell separation. – Microchimerism is a term usually used after organ transplantation where 300 days between the first MRD positive sample and hematological relapse in some patients [47]. These results need of course to be supported by larger studies but it is possible that more effort should be aimed to control the pre-transplant MRD levels. This is especially important in patients who relapse early after transplant, 0.02% in three consecutive samples [28]. DLI treatment at the time of molecular relapse is associated with higher response rates as compared to DLI given at the time of hematological relapse [75–77]. The definition of molecular relapse is also important to avoid unnecessary treatment of patients who show very low MRD levels after SCT without having an increased risk of relapse [78].

Chapter 37  Minimal Residual Disease 

In addition to the importance of serial quantitative analysis, some studies have also shown that BCR-ABL quantification at single time points early after SCT can have a predictive value for relapse. Patients with high BCRABL transcript levels at 3–5 months post SCT have much higher probability of relapse, ~80%, as compared to low level/ MRD negative patients,  7 Gy/mCi. A Phase II study using the same regimen was given to 18 patients with advanced AML or high risk MDS followed by allogeneic HSCT from matched related or unrelated donors [27]. Seven of 18 treated patients (39%) were alive and disease-free after periods up to 3.5 years. A recently published Phase I/II trial utilized 131I-BC8 antibody combined with targeted BU (area-under-curve, 600–900  ng/ml) and CY (120  mg/kg) as a preparative regimen for acute myeloid leukemia patients prior to allogeneic HSCT [28]. The estimated 3-year non-relapse mortality of 21% and disease-free survival of 61% compared favorably to matched historical control patients from the Bone Marrow Transplant Registry who underwent busulfan/ cyclophosphamide alone. The mortality risk was reduced by 35% in antibodytreated patients compared to the registry patients with a p-value approaching significance (p = 0.09). Based on the encouraging outcomes in young patients, 45 elderly patients (median age 62) with advanced AML or high-risk MDS received 131I-BC8 followed by fludarabine, 2 Gy TBI and matched related or unrelated donor allografting. Of the 45 patients treated on this protocol, 13 are alive and 11 remain disease free up to 33 months after HSCT (J. Pagel et al. unpublished data). These favorable results support the use of RIT with antiCD45 antibody in future randomized control trials. Radiolabeled anti-CD66 antibody has been used during conditioning for Philadelphia chromosome positive ALL, advanced CML, and high risk AML/MDS [29, 30]. CD66 is highly expressed on maturing hematopoietic cells but not on leukemic blasts; therefore crossfire effect is responsible for killing neighboring leukemic blasts. The update of a trial utilizing Rhenium-188 labeled antiCD66 antibody for the treatment of Philadelphia chromosome positive ALL or advanced CML showed a 4-year survival of 29% [29]. 188Re-labeled anti-CD66 antibody as part of a conditioning regimen for high-risk AML/MDS showed a 64% disease free survival at a median follow-up of 24 months [30]. A more recent study also treating AML/MDS patients with either 188Re- or 90Y-labeled anti-CD66 antibody showed an estimated 2-year survival of 52% [31]. Burke and others reported the use of 131I anti-CD33 antibody with Bu/Cy conditioning to reduce the burden of the disease prior to allogeneic transplant in leukemic patients [32]. CD33 is a sialic acid adhesion protein highly expressed on myeloid leukemia blasts but not on hematopoietic stem cells. In summary, RIT shows great promise in reducing the toxicities associated with TBI; however, further randomized trials are required to establish its role in allogeneic transplantation.

4. Post-transplant Consolidation Persistence of minimal residual disease after transplant is associated with a higher risk of relapse [33, 34]. Rituximab therapy, after engraftment in patients treated for CD20+ lymphoid malignanies, may eradicate minimal residual disease post-transplant and augment graft versus lymphoma effect by stabilizing the remaining disease. Horwitz et al. reported in a phase II trial the feasibility of rituximab as a consolidation treatment after autologous transplantation for B-cell lymphoma

Chapter 41  Monoclonal Antibodies in Allogeneic Hematopoietic Stem Cell  

with stem cell grafts purged in  vitro with rituximab [35]. All patients were treated with weekly rituximab for 4 weeks beginning day 42 after transplant. A further 14 patients received an additional 4 week course at 6 months post transplant. Immune reconstitution with B-cell recovery was delayed; however, no serious infections were reported. Two-year OS was 80%. Other trials involving autologous transplantation have also shown elimination of minimal residual disease [36–38]. Few published studies describe monoclonal antibody therapy as consolidation after allogeneic transplantation. Shimoni et  al. studied the safety and efficacy of rituximab treatment after autologous (16 patients) or allogeneic (12 patients) transplantation [39]. Nine patients not achieving complete response (CR) converted to CR after treatment with rituximab. The estimated 2-year OS and disease-free survival of patients treated with rituximab were 95% and 64% respectively. Seven patients had recurrent neutropenia and severe hypogammaglobulinemia treated with intravenous immunoglobulin. None of the ten allogeneic recipients treated had severe GVHD. A pilot study investigated the feasibility of treatment with gemtuzumab ozogomicin (anti-CD33 monoclonal antibody) after reduced-intensity allogeneic HSCT in eight children with CD33+ AML [40]. Results showed that two doses of gemtuzumab ozogomicin (first dose between days +60 and +180) were well-tolerated with myeloid suppression being the most commonly observed adverse effect. No episodes of sinusoidal obstructive syndrome occurred. In summary, although there is good data to support the use of rituximab after autologous transplant, there is currently no convincing data for its routine use as maintenance or consolidation therapy after allogeneic transplantation. Use of other monoclonal antibodies for consolidation after transplant similarly remains investigational.

5. Antibody Prophylaxis for Graft Versus Host Disease GVHD remains a serious complication of allogeneic HSCT and accounts for about 15% of deaths after allogeneic transplants [41]. GVHD is a T-lymphocyte mediated inflammatory disease in which donor T-cells attack host cells. Tissue injury during conditioning may contribute partly to development of GVHD. GVHD is associated with reduced risk of relapse presumably due to donor lymphocyte recognition and eradication of recipient tumor cells surviving the preparative regimen. Acute GVHD occurs in 38–60% of transplant recipients while chronic GVHD occurs in 46–67% [42]. Peripheral blood stem cells have increasingly become the source for allogeneic transplantation due to rapid hematopoietic reconstitution after transplant and the relative ease of procurement when compared to bone marrow sources. However, peripheral blood stem cell harvests contain more donor T-cells than marrow harvests. This leads to an increased incidence of acute and chronic GVHD with peripheral blood stem cell transplantation compared with bone marrow transplantation [43]. Depletion of donor T-cells reduces the frequency and/or severity of GVHD; however, this carries an increased risk of graft rejection, viral infection and relapse compared with T-cell replete grafts [44]. In vivo depletion of T-cells with antithymocyte globulin (ATG) has been used in the past. Monoclonal antibodies directed against T-cells are increasingly being used.

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The Campath family of antibodies directed against the human CD52 antigen was first used in the mid-1980s for the purpose of reducing the incidence of acute GVHD [45]. CD52 is highly expressed on both normal and malignant B- and T-lymphocytes. Anti-CD52 antibodies have been used both in vitro and in vivo for purging. Several early studies supported the use of anti-CD52 antibodies in the allogeneic setting to reduce acute GVHD [46, 47]. The most widely used anti-CD52 monoclonal antibody is alemtuzumab (CAMPATH-1H) which is a humanized monoclonal antibody derived from an IgG rat antibody. Several groups have used alemtuzumab as part of conditioning regimens with fludarabine and melphalan prior to allogeneic HSCT. Alemtuzumab appears to permit a reduced intensity of conditioning with no adverse effect on survival in older patients. A recent study showed that older patients tolerated reduced intensity myeloablative conditioning and T cell depleted grafts as well as younger patients with a similar rate of hematopoietic recovery, treatment related mortality and survival [48]. GVHD occurred in only 13% of recipients. However, T-cell depletion of allogeneic grafts has not reliably translated into improved disease-free survival because the rates of graft rejection and relapse are often increased as a consequence. In one randomized phase II–III trial, the risk of CML relapse was significantly increased in patients receiving T-cell depleted grafts [49]. Other studies have also shown that while T-cell depletion reduces GVHD incidence, the risks of relapse, rejection and infection are also significantly increased [44]. In at least one study, reduced GVHD rate as a result of T-cell depletion came at the expense of reduced graft versus myeloma effect [50]. Reduction of the total dose of Campath-1H in one trial was necessary due to loss of graft in five of the six patients [51]. A study comparing the efficacy of alemtuzumab versus methotrexate for GVHD prophylaxis illustrated the pros and cons of anti-CD52 mediated lymphocyte depletion [52]. One hundred twenty-nine patients undergoing nonmyeloablative HLA-matched sibling HSCT for chronic lymphoproliferative disorders enrolled in two prospective studies were treated with melphalan and fludarabine as a conditioning regimen. GVHD prophylaxis consisted of cyclosporin A combined with either alemtuzumab or methotrexate. The alemtuzumab group had a significantly lower rate of both acute (21.7% versus 41.5%, P = 0.006) and chronic GVHD (5% versus 66.7%, P  or = 60 years old with relapsed or refractory B-cell lymphoma. J Clin Oncol 25:1396–1402 24. Fietz T, Uharek L, Gentilini C et al (2006) Allogeneic hematopoietic cell transplantation following conditioning with 90Y-ibritumomab-tiuxetan. Leuk Lymphoma 47:59–63 25. Gopal AK, Rajendran JG, Pagel JM et al (2006) A phase II trial of 90Y-ibritumomab tiuxetan-based reduced intensity allogeneic peripheral blood stem cell (PBSC) transplantation for relapsed CD20+ B-cell non-Hodgkins lymphoma (NHL). Blood 108:316a 26. Matthews DC, Appelbaum FR, Eary JF et  al (1995) Development of a marrow transplant regimen for acute leukemia using targeted hematopoietic irradiation delivered by 131I-labeled anti-CD45 antibody, combined with cyclophosphamide and total body irradiation. Blood 85:1122–1131 27. Matthews DC, Appelbaum FR, Eary JF et  al (1999) Phase I study of (131) I-anti-CD45 antibody plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood 94:1237–1247 28. Pagel JM, Appelbaum FR, Eary JF et  al (2006) 131I-anti-CD45 antibody plus busulfan and cyclophosphamide before allogeneic hematopoietic cell transplantation for treatment of acute myeloid leukemia in first remission. Blood 107: 2184–2191 29. Zenz T, Glatting G, Schlenk RF et  al (2006) Targeted marrow irradiation with radioactively labeled anti-CD66 monoclonal antibody prior to allogeneic stem cell transplantation for patients with leukemia: results of a phase I-II study. Haematologica 91:285–286 30. Bunjes D (2002) 188Re-labeled anti-CD66 monoclonal antibody in stem cell transplantation for patients with high-risk acute myeloid leukemia. Leuk Lymphoma 43:2125–2131 31. Ringhoffer M, Blumstein N, Neumaier B et al (2005) 188Re or 90Y-labelled antiCD66 antibody as part of a dose-reduced conditioning regimen for patients with acute leukaemia or myelodysplastic syndrome over the age of 55: results of a phase I-II study. Br J Haematol 130:604–613 32. Burke JM, Caron PC, Papadopoulos EB et  al (2003) Cytoreduction with iodine131-anti-CD33 antibodies before bone marrow transplantation for advanced myeloid leukemias. Bone Marrow Transplant 32:549–556 33. Gribben JG, Neuberg D, Freedman AS et al (1993) Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma. Blood 81:3449–3457 34. Zwicky CS, Maddocks AB, Andersen N, Gribben JG (1996) Eradication of polymerase chain reaction detectable immunoglobulin gene rearrangement in nonHodgkin’s lymphoma is associated with decreased relapse after autologous bone marrow transplantation. Blood 88:3314–3322 35. Horwitz SM, Negrin RS, Blume KG et al (2004) Rituximab as adjuvant to highdose therapy and autologous hematopoietic cell transplantation for aggressive nonHodgkin lymphoma. Blood 103:777–783 36. Brugger W, Hirsch J, Grunebach F et al (2004) Rituximab consolidation after highdose chemotherapy and autologous blood stem cell transplantation in follicular and mantle cell lymphoma: a prospective, multicenter phase II study. Ann Oncol 15:1691–1698

Chapter 41  Monoclonal Antibodies in Allogeneic Hematopoietic Stem Cell   37. Neumann F, Harmsen S, Martin S et al (2006) Rituximab long-term maintenance therapy after autologous stem cell transplantation in patients with B-cell nonHodgkin’s lymphoma. Ann Hematol 85:530–534 38. Mangel J, Leitch HA, Connors JM et al (2004) Intensive chemotherapy and autologous stem-cell transplantation plus rituximab is superior to conventional chemotherapy for newly diagnosed advanced stage mantle-cell lymphoma: a matched pair analysis. Ann Oncol 15:283–290 39. Shimoni A, Hardan I, Avigdor A et al (2003) Rituximab reduces relapse risk after allogeneic and autologous stem cell transplantation in patients with high-risk aggressive non-Hodgkin’s lymphoma. Br J Haematol 122:457–464 40. Roman E, Cooney E, Harrison L et al (2005) Preliminary results of the safety of immunotherapy with gemtuzumab ozogamicin following reduced intensity allogeneic stem cell transplant in children with CD33+ acute myeloid leukemia. Clin Cancer Res 11:7164s–7170s 41. Welniak LA, Blazar BR, Murphy WJ (2007) Immunobiology of allogeneic hematopoietic stem cell transplantation. Annu Rev Immunol 25:139–170 42. Giralt S (2006) The role of alemtuzumab in nonmyeloablative hematopoietic transplantation. Semin Oncol 33:S36–S43 43. Cutler C, Giri S, Jeyapalan S, Paniagua D, Viswanathan A, Antin JH (2001) Acute and chronic graft-versus-host disease after allogeneic peripheral-blood stem-cell and bone marrow transplantation: a meta-analysis. J Clin Oncol 19:3685–3691 44. Marmont AM, Horowitz MM, Gale RP et  al (1991) T-cell depletion of HLAidentical transplants in leukemia. Blood 78:2120–2130 45. Wasil T, Rai KR, Mehrotra B (2004) The role of monoclonal antibodies in stem cell transplantation. Semin Oncol 31:83–89 46. Cull GM, Haynes AP, Byrne JL et  al (2000) Preliminary experience of allogeneic stem cell transplantation for lymphoproliferative disorders using BEAMCAMPATH conditioning: an effective regimen with low procedure-related toxicity. Br J Haematol 108:754–760 47. Lush RJ, Haynes AP, Byrne J et al (2001) Allogeneic stem-cell transplantation for lymphoproliferative disorders using BEAM-CAMPATH (+/- fludarabine) conditioning combined with post-transplant donor-lymphocyte infusion. Cytotherapy 3:203–210 48. Novitzky N, Thomas V, Hale G, Waldmann H (2005) Myeloablative conditioning is well tolerated by older patients receiving T-cell-depleted grafts. Bone Marrow Transplant 36:675–682 49. Wagner JE, Thompson JS, Carter SL, Kernan NA (2005) Effect of graft-versus-host disease prophylaxis on 3-year disease-free survival in recipients of unrelated donor bone marrow (T-cell Depletion Trial): a multi-centre, randomised phase II-III trial. Lancet 366:733–741 50. D’Sa S, Peggs K, Pizzey A et al (2003) T- and B-cell immune reconstitution and clinical outcome in patients with multiple myeloma receiving T-cell-depleted, reduced-intensity allogeneic stem cell transplantation with an alemtuzumab-containing conditioning regimen followed by escalated donor lymphocyte infusions. Br J Haematol 123:309–322 51. Shore T, Harpel J, Schuster MW et al (2006) A study of a reduced-intensity conditioning regimen followed by allogeneic stem cell transplantation for patients with hematologic malignancies using Campath-1H as part of a graft-versus-host disease strategy. Biol Blood Marrow Transplant 12:868–875 52. Perez-Simon JA, Kottaridis PD, Martino R et  al (2002) Nonmyeloablative transplantation with or without alemtuzumab: comparison between 2 prospective studies in patients with lymphoproliferative disorders. Blood 100:3121–3127 53. Novitzky N, Davison GM, Hale G, Waldmann H (2002) Immune reconstitution at 6 months following T-cell depleted hematopoietic stem cell transplantation is predictive for treatment outcome. Transplantation 74:1551–1559

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M.C. Palanca-Wessels and O. Press 54. Davison GM, Novitzky N, Kline A et  al (2000) Immune reconstitution after allogeneic bone marrow transplantation depleted of T cells. Transplantation 69: 1341–1347 55. Morris EC, Rebello P, Thomson KJ et  al (2003) Pharmacokinetics of alemtuzumab used for in  vivo and in  vitro T-cell depletion in allogeneic transplantations: relevance for early adoptive immunotherapy and infectious complications. Blood 102:404–406 56. Dodero A, Carrabba M, Milani R et  al (2005) Reduced-intensity conditioning containing low-dose alemtuzumab before allogeneic peripheral blood stem cell transplantation: graft-versus-host disease is decreased but T-cell reconstitution is delayed. Exp Hematol 33:920–927 57. Chakrabarti S, MacDonald D, Hale G et  al (2003) T-cell depletion with Campath-1H “in the bag” for matched related allogeneic peripheral blood stem cell transplantation is associated with reduced graft-versus-host disease, rapid immune constitution and improved survival. Br J Haematol 121:109–118 58. Avivi I, Chakrabarti S, Milligan DW et al (2004) Incidence and outcome of adenovirus disease in transplant recipients after reduced-intensity conditioning with alemtuzumab. Biol Blood Marrow Transplant 10:186–194 59. Martin SI, Marty FM, Fiumara K, Treon SP, Gribben JG, Baden LR (2006) Infectious complications associated with alemtuzumab use for lymphoproliferative disorders. Clin Infect Dis 43:16–24 60. Chakrabarti S, Milligan DW, Pillay D et al (2003) Reconstitution of the EpsteinBarr virus-specific cytotoxic T-lymphocyte response following T-cell-depleted myeloablative and nonmyeloablative allogeneic stem cell transplantation. Blood 102:839–842 61. Avivi I, Chakrabarti S, Kottaridis P et al (2004) Neurological complications following alemtuzumab-based reduced-intensity allogeneic transplantation. Bone Marrow Transplant 34:137–142 62. Barge RM, Starrenburg CW, Falkenburg JH, Fibbe WE, Marijt EW, Willemze R (2006) Long-term follow-up of myeloablative allogeneic stem cell transplantation using Campath “in the bag” as T-cell depletion: the Leiden experience. Bone Marrow Transplant 37:1129–1134 63. Meyer RG, Britten CM, Wehler D et  al (2007) Prophylactic transfer of CD8depleted donor lymphocytes after T-cell-depleted reduced-intensity transplantation. Blood 109:374–382 64. Khouri IF, Saliba RM, Admirand J et al (2007) Graft-versus-leukaemia effect after non-myeloablative haematopoietic transplantation can overcome the unfavourable expression of ZAP-70 in refractory chronic lymphocytic leukaemia. Br J Haematol 137:355–363 65. Kebriaei P, Saliba RM, Ma C et al (2006) Allogeneic hematopoietic stem cell transplantation after rituximab-containing myeloablative preparative regimen for acute lymphoblastic leukemia. Bone Marrow Transplant 38:203–209 66. Escalon MP, Champlin RE, Saliba RM et al (2004) Nonmyeloablative allogeneic hematopoietic transplantation: a promising salvage therapy for patients with nonHodgkin’s lymphoma whose disease has failed a prior autologous transplantation. J Clin Oncol 22:2419–2423 67. Ho AY, Devereux S, Mufti GJ, Pagliuca A (2003) Reduced-intensity rituximabBEAM-CAMPATH allogeneic haematopoietic stem cell transplantation for follicular lymphoma is feasible and induces durable molecular remissions. Bone Marrow Transplant 31:551–557 68. Khouri IF, Lee MS, Saliba RM et  al (2003) Nonablative allogeneic stem-cell transplantation for advanced/recurrent mantle-cell lymphoma. J Clin Oncol 21: 4407–4412

Chapter 42 Treatment of Acute Graft-vs-Host Disease Steven C. Goldstein, Sophie D. Stein, and David L. Porter

1. Introduction Over the last two decades, advances in our understanding of the pathophysiology of acute GVHD (aGVHD) [1, 2] have not yet translated into significant changes in upfront management. Corticosteroids have remained the cornerstone of aGVHD therapy for the last several decades, and still, failure to respond to steroids remains the primary predictor of poor overall outcome. Several issues limit the usefulness of most retrospective reports of therapeutic intervention for aGVHD. First, we must acknowledge that the majority of series using the “temporal onset” definition of acute and chronic GvHD actually included a mix of patients with either late onset acute or early chronic GvHD, confounding their interpretation and extrapolation to recent efforts to define these syndromes on the basis of clinical features regardless of timing of onset. Second is the lack of prognostic tools to identify cohorts who may need more (vs less) aggressive induction. Third, we also must acknowledge the significant variability in clinical practice between transplant physicians, often well-founded in their efforts to individualize therapy on the basis of specific organ involvement, severity of symptoms, etc. Rather than surrendering to the potentially insurmountable task of establishing a single and perhaps suboptimal standard of care for the upfront treatment of aGVHD, advances in the field and improved patient outcomes will require modern and well-designed clinical trials supported throughout the transplant community. 1.1. Indications for Intervention for AGVHD Although the definitions of acute and chronic GvHD have been recently revised to characterize the respective syndromes on the basis of their clinical manifestations rather than timing of onset [3], the indication for intervention among most transplant physicians has remained rather constant, taking into account the level of immunosuppression and severity of symptoms.

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_42, © Springer Science + Business Media, LLC 2003, 2010

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Grade A (aka grade I aGVHD) is typically observed without intervention, though there is the occasional patient in whom a rash may be extremely symptomatic on topical steroids despite its limited body surface area involvement of less than 25%. It may be relevant for the clinician to consider the recent evolution in staging systems for aGVHD when deciding to start steroid therapy as the literature for intervention is primarily on the basis of the traditional grade I–IV staging system [4], typically defining grade II or greater as an indication for the initiation of steroid therapy (see Tables 42-1–42-3). The recent integration of the IBMTR severity index [5] was designed to enhance the predictive value of patterns of organ involvement on outcome on the basis of more objective organ-specific measurements, but may obfuscate traditional criteria for initiation of therapy in certain circumstances. For example, while Index grade A aGVHD may overlap entirely with grade I aGVHD (both typically requiring no intervention), index grade B represents a spectrum of patients with both traditional Glucksberg grade I disease (skin only, less than 50% BSA or S = 2, G = 0, L = 0) and Glucksberg grade II disease (S 50% of body surface area of generalized erythroderma

Bilirubin >6–15

>1500 cc diarrhea/day

4

Generalized erythroderma with bullous formation and desquamation

Bilirubin >15

>1500 cc diarrhea/day plus severe abdominal pain with or without ileus

Source: Przepiorka et al. [95]

Table 42-2.  Acute graft-vs-host disease: IBMTR severity index. Index

Skin (max), or

Liver (max), or

Intestine (max)

A

1 (1500 cc)

D

4 (bullae)

4 (>15)

4 (severe pain and ileus)

Assign index based on maximum involvement in an individual organ system Source: Rowlings et al. [5]

Chapter 42  Treatment of Acute Graft-vs-Host Disease 

Table 42-3.  Glucksberg (modified) criteria. Grade

Skin

Liver

Intestine

I

1–2 (20  mg/kg/d has been reported [8, 9] with few inroads toward improved efficacy, but clearly with higher toxicity at higher doses, predominantly from fungal infection [8, 9] (it is important to note that poor outcomes with high dose steroids were published in an era without effective anti-fungal prophylaxis.). Although the goal of achieving clarity in this area is clouded by decades of empiricism and a lack of outcome data controlled for by starting dose [9], 2 mg/kg of solumedrol is reasonable for the majority of patients. Oral beclomethasone has been reported to benefit a significant number of patients with primarily GI symptoms [10].

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1.4. Duration of Therapy In contrast to the therapy for chronic GvHD in which steroid therapy is prolonged over many months, (reviewed in a later chapter), the goals of therapy for aGVHD focus on rapid suppression of the effector phase of organ damage with rapid taper to minimize the sequelae of both short and long term steroid exposure. The duration of “full dose” therapy and length of taper remain highly individualized between patients and physicians, but some general trends in practice have been established. A typical patient with ³ grade II aGVHD [4] may be treated 14 days at their starting dose before initiating a taper schedule every 4–7 days over approximately 2–3 months with close monitoring for flare. Studies comparing the length of taper demonstrate no advantage of a prolonged taper beyond 2–3 months in duration [11]. In patients on cyclosporine or tacrolimus, tapering of calcineurin inhibitors is usually deferred until after successful steroid taper. Critical early milestones of response and possible triggers for additional salvage therapy often include: progression by day 3, no change by day 5–7, and/or incomplete resolution by day 14. Unfortunately, objective early response data are confounded by the extremely subjective nature of assessing many patients with aGVHD. 1.5. Outcome The response rates to primary corticosteroid therapy for aGVHD have been unfortunately quite consistent over the last three decades. Typically 25–35% of patients achieve complete resolution with an additional 15–20% achieving partial responses [6, 7, 12]; effective for some, yet inadequate for many. The need for improved first line therapy is evident in light of the dismal prognosis for patients failing to respond to prednisone: only 5–30% long term survival was observed among those with steroid resistant GvHD as compared to the 50–60% of patients with stable responses to first line treatment who achieve long term survival. 1.6. Combination Therapy as First-line Treatment These sobering results have prompted efforts to both combine corticosteroids with newer agents in first-line treatment strategies (vs attempts to add second/ third line agents in patients deemed steroid-refractory, see below) and to target more organ-specific modalities. Depending on the timing of onset of aGVHD post-transplant, most physicians will increase cyclosporine/tacrolimus to therapeutic levels if the syndrome develops during the taper phase, and empirically resume a calcineurin inhibitor if they have completed their taper. Although a common practice, no randomized trials assessing corticosteroids ± CSA or tacrolimus as primary treatment for aGVHD have been performed. Rapid progress in the evolution of cell-targeted monoclonal antibody therapy and cytokine inhibition/blockade has coincided with attempts at achieving synergy in this area, with mixed results. Up-front combination strategies of targeting T-cells [ATG [13, 14], anti-CD5 mAb [15], anti-IL2R mAb [16, 17]], cytokines [TNF [18], IL1RA [19]], and T-cell signaling [MMF [20], beclomethasone [10]] have been reported in small Phase II studies with similar themes; initial enthusiasm for improved efficacy, but little long term improvement

Chapter 42  Treatment of Acute Graft-vs-Host Disease 

in overall outcomes when compared to historical controls. Unfortunately, many of these studies lack adequate statistical power and include heterogeneous and small populations making interpretation difficult. However, the case of daclizumab, in particular, speaks to the critical importance of well-designed clinical trials before presuming efficacy of more aggressive therapy; a prospective, randomized, multi-center study of methylprednisone ± Daclizumab as up-front treatment of aGVHD in 102 patients demonstrated statistically significant inferior 100 day survival in the combination arm, in large part due to death from disease and GVHD, prompting early closure of the trial [17]. Although daunting, meeting the challenge of improving outcomes for the primary treatment of aGVHD (as well as improving prophylaxis) is essential for the field to advance. One must account for (1) the limitations of small phase II studies, (2) the poor outcome of patients once they are deemed steroid refractory, (3) the increasing numbers of high-risk patients undergoing allogeneic transplantation, and (4) the plethora of novel immunomodulatory agents now available. Toward this end, well designed studies of newer agents are desperately needed. 1.7. Does Treatment of aGVHD Impact on GvL? A continued concern is whether inhibition of T-cell function by corticosteroids for GvHD also inhibits the graft-vs-leukemia potential. Data are limited and only indirect inferences can be drawn; a retrospective analysis of 197 patients treated for  ³ grade II aGVHD at a single center found no correlation between risk of relapse and prior immunosuppressive therapy for GvHD [12]. In contrast, Kataoka et al. [21] drew a different conclusion from their observation in AML and CML patients that grade I aGVHD (i.e., untreated) was associated with lower relapse rates and improved disease-free survival than grade II or greater GvHD, implying that the primary factor was suppression of moderate/ severe aGVHD rather than potential differences in underlying path physiologic mechanisms and effector pathways between mild and moderate aGVHD and their relevance to the GvL response. Additional indirect evidence for the suppression of GvL via aggressive immunosuppression of GvHD may be inferred from the higher relapse rate and death from disease found in the daclizumab arm of the study of Lee et al. [17]. 1.8. Supportive Care As patients who develop aGVHD may experience a generalized decline secon­ dary to the disease itself as well as from the effects of steroids, it is important to anticipate potential complications and optimize supportive care early in the course of disease. For patients with gastrointestinal symptoms of nausea, vomiting, and diarrhea, bowel rest, hyperalimentation, and pain control are critical ancillary interventions. A trial of octreotide may be of benefit with significant secretory diarrhea [22]. Aggressive prophylaxis and early intervention for opportunistic infections such as HSV, VZV, PCP, and fungus are essential. Many patients will develop hyperglycemia secondary to corticosteroids that requires intervention. Attention to bone mineral retention and ongoing physical therapy is important to minimize the debilitation associated with steroidinduced osteoporosis and myopathy.

751

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S.C. Goldstein et al.

2.  Second Line Therapy Only approximately half of patients treated with corticosteroids for new onset aGVHD will obtain a significant and durable response. Tacrolimus or cyclosporine initiated at the time of transplant is usually continued when steroids are added. The significant morbidity and mortality associated with steroidrefractory aGVHD have led to intense interest in alternative treatments. 2.1. Broad Anti-T Cell Agents/Antibodies 2.1.1. Anti-Thymocyte Globulin Until recently, anti-thymocyte globulin (equine ATG, ATGAM) has been the most common therapy for steroid-refractory GvHD. Unfortunately, outcomes after ATG have been disappointing. Khoury et  al. [23] used three different schedules of equine ATG in 58 steroid-resistant aGVHD patients. Twenty-one days after ATG treatment, only 8% (4/52 subjects) had achieved a complete response. Furthermore, 90% (52/58 subjects) had died at a median of 40 days from the initiation of ATG (see Fig. 42-1). Macmillan et al. [24] and Arai et al. [25] reported similar dismal outcomes with a complete response rate of 20% (N = 79; evaluation at day 28) and 14% (N = 69), respectively. Patients with GVHD involving the skin showed the most significant responses in all studies [23–26] (Table 42-4). Although the more recent introduction of ATG prepared from rabbits (thymoglobulin) has been associated with less infusion related toxicity, there have been no published studies evaluating whether it has any advantages over the equine preparation in the treatment of steroid refractory GvHD [27]. Published studies of thymoglobulin have focused primarily on its potential benefits in the prevention, rather than treatment of GvHD [28–30].

Fig.  42-1.  Kaplan-Meier survival estimate for 58 patients with SR aGVHD treated with ATG. Day 0 represents the first day ATG was initiated. Censored observations are indicated with a plus. Khoury et al. [23]

Chapter 42  Treatment of Acute Graft-vs-Host Disease 

753

Table 42-4.  ATG/thymoglobulin for GvHD therapy-selected trials. Reference

Formulation

N

Response rate

Comments

Khoury et al. [23]

Equine

N = 58

8% CR79% PR/CR in patients with skin gvh

52 of 58 pts expired by d40 ATG

Macmillan et al. [24]

Equine

N = 79

20% CR61% PR/CR in patients with skin gvh

Arai et al. [25]

Equine

N = 69

14% CR

Multiorgan involvement and age >35 predicted no response to ATG

Graziani et al [96]

Rabbit

N = 28

38–74% PR/CR

Steroid refractory not clearly defined

McCaul et al [27]

Rabbit

N = 36

38% CR21% PR

High incidence (25%) of PTLD

2.1.2. Alemtuzumab (Campath; Genzyme) Alemtuzumab is a humanized monoclonal antibody directed against CD52, an antigen known to be expressed on T cells. Unlike the T-cell specificity of ATG, alemtuzumab targets a broader population of cells expressing CD52, such as T-cells, B-cells, and some APCs. A potential advantage of Campath may therefore be a lower incidence of post-transplant lymphoproliferative disease than seen after ATG therapy; a disadvantage would likely be the higher incidence of opportunistic infection as a complication of such broad immunodepletion. There are several studies supporting the use of alemtuzumab to prevent GVHD [31–38] but its role in treating aGVHD has been limited to isolated case reports [39–42], each suggesting possible efficacy in the refractory patient. Although several of the published reports involve patients with hepatic involvement, the numbers are too small to confirm organ specificity. 2.1.3. Visilizumab Another humanized monoclonal antibody, HuM291 (visilizumab, PDL) [43], directed against the invariant CD3 epsilon chain of the T-cell receptor remains under investigation. In a multicenter Phase II study, Carpenter et al. [44] reported an overall response of 32% (14% complete) in a cohort of 44 high-risk patients with predominantly grade III–IV steroid refractory aGVHD. Although response rates may appear to be lower than reported for other agents, this more likely reflects the advanced disease of participants in this study (86% with grades III–IV aGVHD) as compared to other studies enrolling patients with grade I–II aGVHD, rather than lack of efficacy. The relatively high incidence of patients (19/44) with elevation of EBV titers [no patient developed post-transplant lymphoproliferative disease (PTLD), 17 of 19 pre-emptively treated with Rituxan] highlights the importance of following EBV titers in future studies as well as the role for pre-emptive Rituxan in this setting to minimize the risk of PTLD. 2.2. Broad Anti-T Cell Agents/Immunomodulatory Agents 2.2.1. Mycophenolate Mofetil/MMF (CellCept; Roche) In addition to anti-lymphocyte monoclonal antibodies, immunomodulatory drugs targeting signal transduction pathways may have significant activity in

754 

S.C. Goldstein et al.

GVHD [45]. The ability of MMF to selectively inhibit lymphocyte proliferation is on the basis of the blockade of de  novo purine synthesis by MPA, the active metabolite of MMF. Unlike other cell types, lymphocytes do not have a salvage pathway, allowing for a rather selective inhibition of B and T-cell proliferation [45]. Although the availability of MMF in both oral and IV formulations provides the clinician more dosing flexibility, the optimal frequency for dosing and relevance of MPA levels in the plasma remain an active area of investigation. In solid organ transplant recipients, recommendations for specific target levels of MPA AUC have been established [46]. Among stem cell recipients, however, a consensus regarding the utility of drug level monitoring has not been reached, although it will likely be a focus of any prospective study with this agent. Although the role of MMF in the prophylaxis of GvHD has increased over the last several years, particularly in non-myeloablative strategies, data regarding its efficacy once GvHD is established remain rather limited [45]. Most series include both acute and chronic GvHD patients and are divided in terms of whether MMF has similar efficacy in both settings. Kim et  al. [47] evaluated 26 patients with GvHD, split evenly between refractory acute and chronic, and concluded that MMF was more efficacious in patients with chronic GvHD than aGVHD (with response rates of 54 vs. 33%, respectively), although it does not appear that the study carried adequate statistical power to compare outcomes. In contrast, in their retrospective analysis of 21 patients with refractory GvHD (10 acute, 11 chronic), Krejci et  al. [48] reported a similar response rate of ~60% in both cohorts. Basara et  al. [49] reported a response rate of 72% (26 of 36) among patients who developed grade I–IV aGVHD on a prednisone-containing prophylaxis regimen. Interestingly, MMF was given on a QID schedule in this study, potentially providing higher plasma levels when enterohepatic circulation and drug interactions are taken into account. A smaller series by Takami et al. [50] prospectively studied 11 patients with refractory GvHD (6 acute, 5 chronic) and again demonstrated a remarkably similar response rate of 67% among patients with aGVHD. A common theme among these reports was the steroid-sparing effect allowed by MMF for the responding patients (Table 42-5). Table 42-5.  MMF for GvHD-selected trials. Reference

N

Response rate

Kim et al. [46]

N = 26N = 13 with aGVHD

30.8% overall response in agvh 30.8% response in skin agvh 44.4% response in liver agvh 22.9% response in gut agvh

Krejci et al. [47]

N = 21N = 10 with aGVHD

6/10 (60%) with AGVHD 7/11 (64%) with Chronic GvHD

Basara et al. [48]

N = 48N = 36 with AGVHD

72% overall response 86% response in skin agvh 75% response in liver agvh 50% response in gut agvh

Takami et al. [49]

N = 11N = 6 with AGVHD

67% response rate in patients with aGVHD

Chapter 42  Treatment of Acute Graft-vs-Host Disease 

2.2.2. Deoxycoformycin (Pentostatin, Nipent) Deoxycoformycin is a nucleoside analog that inhibits adenosine deaminase (ADA). Lymphocytes contain one of the two isoforms of ADA which metabolizes 2¢-deoxyadenosine to 5¢-triphosphate (dATP). Without the production of dATP, lymphocyte growth is inhibited and apoptosis results. A phase I dose-finding study conducted by Bolaños-Meade et  al. [51] investigated the use of pentostatin in steroid refractory or steroid unresponsive GVHD. The MTD was determined to be 1.5 mg/m2/d × 3 days and careful dose adjustment for renal insufficiency is warranted. In 22 assessable patients, the complete and partial response rates were 64 and 14%, respectively and 1 year overall survival was 26%. Dose limiting toxicity was determined to be infections occurring greater than 3 weeks from the time of enrollment. 2.2.3. Sirolimus (Rapamycin; Wyeth–Ayerst) The IL-2 pathway can be targeted using Sirolimus. Sirolimus binds to FK-binding protein (FKBP12) in the cytosol of cells. The complex then inhibits the mammalian target of Rapamycin (m-tor) and blocks the response to IL-2. In the presence of sirolimus, B- and T-cells cannot become activated. In a pilot study of 21 patients conducted by Benito et al. [52], the overall response rate to sirolimus was 57% (24% complete response and 33% partial response). However, myelosuppression and hypertriglyceridemia were significant dose limiting toxicities. Even more concerning were two cases of hemolytic uremic syndrome reported, although the two study participants were also on cyclosporine. 2.2.4. Extracorporeal Photopheresis Although experience with extracorporeal photopheresis (ECP) has been primarily in the treatment of chronic cutaneous GVHD [53], recent studies suggest a potential benefit for patients with SR aGVHD as well [54]. The exact mechanism by which ECP works is not well understood, but proposed hypotheses focus on the apoptosis of 8-methoxypsoralen-exposed leukocytes induced by UVA irradiation. Upon return to the patient, the apoptic cells are taken up by antigen-presenting cells and ultimately lead to inhibition of T-cell function, cytokine release, and induction of regulatory T-cells [55]. Greinix et  al. [56] reported a series of 21 patients with aGVHD treated with ECP. After 3 months of treatment (median response time was 4 months), the complete response rate was 60%. Nine of the 12 patients who achieved a complete response had grade II aGVHD and the best responses were seen in patients with liver and/or skin involvement. 2.3. Narrow Anti-T Cell Agents/Receptor and Cytokine Targets 2.3.1. Daclizumab (Zenepax; Hoffman-La Roche) Daclizumab is a humanized monoclonal antibody directed against the IL-2 receptor, specifically the alpha chain (CD 25), found on activated immune cells, including T-cells. It competitively inhibits IL-2 binding to CD 25, thereby downregulating the T-cell immune response. Response rates to daclizumab have ranged from 20 to 69% (Table 42-6) with the best responses noted for cutaneous GVHD. Interestingly, when daclizumab was added to corticosteroids as first line therapy for aGVHD, outcomes were worse than that of patients receiving steroids alone [17]. The higher mortality in the daclizumab

755

756 

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Table 42-6.  Trials of daclizumab in GvHD-selected trials. Reference

Patients

Response/survival

Lee et al. [17]

N = 49 steroids +  placeboN = 53 steroids +  daclizumab

OR:53% (steroid + placebo)

Comments

Survival was worse in combination group due to higher rate 51% (steroid and daclizumab) of relapse and GVHD-related P = 0.85 mortality 100 day survival: 94% (steroid + placebo) 77% (steroid + daclizumab) P = 0.02

Anasetti et al. [97]

N = 19 SteroidCR = 20%PR = 20% refractory Skin GVHD = 56% response GVHDN = 1 primary treatment 100 day survival: 40%; median 76 days

Przepioka et al. [98]

N = 39 steroid CR = 37%PR = 14% refractoryN = 4 Skin GVHD = 54% response primary treatment 120 day survival: 40%; median 77 days

Bordigoni et al. [99]

N = 62 steroid refractory

CR = 68.8%PR = 21.3% Skin GVHD

Patients could receive ATG on study as a salvage regimen but were considered nonrespondersAll 4 patients receiving daclizumab for untreated visceral GVHD failed to respond 64% of study participants were under the age of 18

72.7% CR (stage I–II) 33.3% CR (stage III–IV) P = 0.018 Srinivasan et al. [100]

N = 12 steroid refractory

CR = 100%

Willenbacher et al. [101]

N = 16 steroid refractory

CR = 8%PR = 58%

Majority of subjects were transplanted for solid malignancy;Subjects could receive daclizumab alone (N = 6) or w/atg or infliximab

treated patients was due in part to a higher rate of death from disease and GVHD. As noted above, this study highlights the need to balance effective immunosuppression with GVL activity, infection risks, and other morbidities, and emphasizes the continued need for randomized trials for GVHD therapy. 2.3.2. Denileukin Diftitox (Ontak; Ligand Pharmaceutical Inc.) Denileukin diftitox is another therapy directed against CD25. It is a recombinant fusion protein consisting of an active portion of diphtheria toxin bound to human IL-2, and thus it selectively targets the IL-2 receptor. The toxin inhibits protein synthesis and induces T cell apoptosis once it has gained entry into the cell via the IL-2 receptor. Ho et al. [57] found a 33% (8/24 patients) CR rate and 38% (9/24 patients) PR rate at 29 days after the first denileukin diftitox treatment. Four patients who had a PR converted to a CR after day 29. However, survival data were still quite poor with a median survival of only 7.2 months in the 30 original study

Chapter 42  Treatment of Acute Graft-vs-Host Disease 

subjects. Encouragingly, responses were not limited to patients with grade I–II GVHD. Shaughnessey et al. [58] reported their experience with Ontak in 22 subjects and showed a similar 41% complete response rate on day 36 from initiation of Ontak, but again median survival in the cohort was only 121 days. In these two phase II trials, transaminitis was the dose limiting toxicity, as seen in studies of denilukin diftitox in lymphoma. 2.3.3. ABX-CBL/Anti CD147 This antibody was tested in patients with SR aGVHD with reasonable response rates leading to a phase III trial comparing ABX-CBL to ATG. Unfortunately, as seen repeatedly in other trials, there was no advantage in terms of response or survival using this novel antibody [59]. 2.4. TNF Inhibition 2.4.1. Infliximab (Remicade; Centocor) A number of recent trials have focused on targeting the cytokine cascade involved in the initiation and propagation of SR GVHD [60]. Tumor necrosis factor-alpha (TNFa) has been implicated as central to this process [61]. Infliximab is a humanized TNFa receptor that binds solubleTNFa, causing its neutralization, and the membrane-bound precursor of TNFa, causing cytotoxicity via the complement cascade and antibody-mediated cellular apoptosis. Couriel et al. [62] investigated the use of infliximab in the treatment of aGVHD and reported a 62% (13/21 patients) complete response rate on day 7, the most of which was in subjects who had gastrointestinal involvement. Of note, all of the surviving patients who had a complete response developed chronic GVHD. Over half of the patients in the original group of 21 patients had gastrointestinal GVHD and this may reflect the treatment bias of attending physicians, which is inherent in retrospective analysis. Patriarca et al. [63] conducted another retrospective study of infliximab in 32 patients with steroid refractory aGVHD and found only a 19% complete response rate on day 7. The low response rate in this study may have been impacted by the large percentage of patients (60%) in the group with grade IV GVHD (Table 42-7). Table 42-7.  Categories of agents used in GvHD therapy. Broad

Antibody

Signal transduction

Agent

Target

ATG

CD3

Thymogloblulin

CD3

Alemtuzumab

CD52

Mycophenolate mofetil Sirolimus

M-TOR

Deoxycoformycin Narrow

Cytokine inhibition

?

ECP

?Treg’s

Monoclonal antibody

Denileukin diftitox

IL2-R

Daclizumab

IL2-R

Visilizumab

TCR

Infliximab

TNF

Etanercept

TNF

Monoclonal antibody

757

758 

S.C. Goldstein et al.

2.4.2. Etanercept (Enbrel; Amgen and Wyeth) In contrast to the neutralizing effect of Infliximab, etanercept is a recombinant human soluble tumor necrosis factor receptor fusion protein that inhibits TNF-a. Busca et al. [64] reported the outcomes of 21 patients with refractory GvHD (13 with SR aGVHD, eight with chronic GvHD) treated with etanercept, in addition to other salvage agents. Six of the 13 patients with aGVHD responded (4 CR, 2 PR) and five of the eight patients with chronic GvHD responded for an overall response rate of 52%. Interestingly, 55% of patients with gastrointestinal involvement responded. In a pre-emptive strategy, Uberti et  al. [18] reported a 75% CR rate in patients treated with up-front etanercept and prednisone in patients with new onset aGVHD. Although greater than 50% of patients demonstrated a response prior to the addition of etanercept and patients with grade IV disease were excluded (potentially exaggerating the response rate to etanercept), the results provide enthusiasm for ongoing efforts to modulate TNF function. 2.4.3. IL1-RA IL1 is another inflammatory cytokine implicated in the pathogenesis of GVHD [60]. Antin and colleagues [19] treated 17 patients with SR AGVHD with an IL-1 receptor antagonist (IL-1RA) in a phase I/II manner and found high levels of response; 63% of patients had improvement of GVHD by at least 1 grade, though this therapy has not yet been pursued further.

3. Related Questions and Future Directions 3.1. Non-Myeloablative Transplantation and GvHD The evolution of non-myeloablative transplantation in patients already at higher risk for the development of aGVHD on the basis of the increasing age of recipient and donor [65–67] has allowed a critical test of “proof of principle” for the established paradigm linking tissue damage and cytokine cascade triggered by myeloablative conditioning to the development of aGVHD [68, 69]. In theory, the low-intensity conditioning in NMT would be associated with a lower incidence and potentially different clinical presentation of acute and chronic GvHD than with classic myeloablative conditioning. Although there have been no large-scale, systematic studies to adequately address this issue, recently published outcomes from single centers comparing standard to non-myeloablative conditioning provide some intriguing insights. Despite the potentially confounding impact of traditional definitions and grading systems on more recently recognized syndromes of “late-onset aGVHD” and mixed manifestations of acute and chronic GvHD in the same patient, several retrospective studies [70–73] allow for some generalizations regarding the timing, severity, and response to therapy of aGVHD after NMT: (1) Initial observations that NMT may be associated with less AGvHD have to be tempered on the basis of studies with longer follow-up. Although the incidence of AGvHD may be lower in the early post-transplant period (6 kg) can be performed by using apheresis machines used for collection of peripheral blood stem cell grafts in children, by administering granulocyte colony stimulating factor (rhG-CSF,100 mg/kg) [54–57] in combination with stem cell factor (SCF) as a mobilizing agent to stimulate bone marrow cells [6, 13]. For optimal peripheral blood stem cell collection, the animal is first treated with SCF alone at 25 mg/kg/day subcutaneously for 3 days beginning on day 0. On day 3, a dose of 100 mg/kg of G-CSF is combined with the 25 mg/kg/day dose of SCF on a daily basis. SCF alone resulted in minimal CD34+ cell mobilization; the addition of G-CSF to SCF led to a remarkable egress of CD34+ cells into the peripheral blood. On day 7, the mobilized peripheral blood stem cells can be collected by a single leukapheresis [12, 58]. The CD34+ cell contents

Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies 

of grafts isolated from seven baboons mobilized with G-CSF alone or G-CSF and SCF are shown in Table 43-2. These data indicate that there is some animal to animal variation but the combination of SCF and G-CSF is more effective in stem cell mobilization than G-CSF alone. A mobilization strategy using SCF is not useful in humans because of the severe adverse effects associated with its use. Human umbilical cord blood grafts have been established as an important alternative source of transplantable HSC grafts [14, 15]. Baboon cord blood grafts can be isolated following full term delivery by cesarean section. It is important to note that veterinary support is critical in this model for several reasons: Nonhuman primates deliver spontaneously at night and tend to consume their own placenta following delivery, thereby negating the opportunity of obtaining cord blood stem cells. Therefore, it is essential to preemptively schedule a cesarean section to deliver the live neonate and obtain the placenta and cord blood. Hematopoietic progenitor cells from the placenta and umbilical cord blood can be collected by using standard methods following aseptic techniques [16]. Several cord blood units have been collected from pregnant baboons at the University of Illinois using such techniques and have been utilized for experimental transplantation or other preclinical studies. The volume of cord blood collected following such deliveries ranged between 10 and 75 ml and the total nucleated cell number ranged between 1 and 20 × 107 cells. Low density mononuclear cells can be separated from baboon cord blood using similar techniques utilized to separate bone marrow cells from red blood cells, by incorporating density gradient separation methods. CD34+ cells can be enriched by immunomagnetic beads conjugated to monoclonal antibodies specific for CD34. These cells can be cryopreserved and thawed successfully for various experimental purposes including in vitro functional studies, such as transplantation of cryopreserved HSC grafts and reconstitution of all blood cell lineages. One of the major limitations of using such a nonhuman primate model is the retrieval and cost of maintaining the pregnant primate, and, if performing an autologous transplant, the cost of maintaining the offspring until they can undergo conditioning and transplantation (approximately 24 months). Achieving statistical power can be quite challenging, because of the

Table  43-2.  Growth factor mobilized peripheral blood stem cell product in baboon. Animal

Cytokine used

CD34 + Cells/kg 106

1

G-CSF

4.40

2

G-CSF

3.06

3

G-CSF

13.33

4

G-CSF, SCF

9.49

5

G-CSF, SCF

9.86

6

G-CSF, SCF

61.13

7

G-CSF, SCF

24.38

a

The calculations are based on an animal weighing 8 kg as a recipient G-CSF granulocyte colony stimulating factor, SCF stem cell factor CD34 cell number indicates product of single day leukapheresis

773

774 

E. Szilagyi et al.

costs involved. This model may be best suited to demonstrate safety of the proposed therapy rather than superiority of treatment.

4. Conditioning Regimens 4.1.  Radiation Based Regimens Radiation studies involving nonhuman primates have provided early and important insights on hematopoiesis, demonstrating hematopoietic deficiencies in response to radiation injury [4, 5]. This model is particularly useful for investigating in vivo kinetics of blood cells following radiation or chemotherapy. For example, exposure to sub-lethal (250  cGy) total body irradiation, in which endogenous recovery occurs, can be used to study the cellular and molecular responses involved in the kinetics of blood cell recovery and immune system in  vivo. A myeloablative conditioning regimen has been utilized by several investigators to perform allogeneic HSC transplantation in baboons using bone marrow or growth factor mobilized peripheral blood stem cells grafts [5, 59]. Successful protocols have been established to support animals during periods of prolonged pancytopenia (~60 days) [10]. Beginning 4 days prior to transplant, the animals are given two daily doses of 125 cGy total body irradiation from a linear accelerator over 4 days for a total dose of 1,000  cGy. The delivery of a fractionated dose of radiation has been shown to be lethal and myeloablative but results in relatively low nonhematologic toxicities [10]. The baboons that have received such myeloablative regimens have had tolerable gastrointestinal toxicities and almost no detectable pulmonary toxicity [8]. Support for these animals following the delivery of the conditioning regimen requires aggressive supportive care including prophylactic antimicrobial drugs, IV fluid and drug administration, blood component transfusions, and nutritional support via gavage. Conservative myeloablative conditioning regimens prior to clinical HSC transplantation promote hematopoietic cell engraftment and permit transplantation across limited histocompatability barriers (Table  43-3). In allogeneic transplants a combination of cyclophosphamide with busulfan or total body irradiation is commonly employed. The fully myeloablative approach is associated with higher risks of infectious complications when compared to novel nonmyeloablative regimens. Nonmyeloablative combined regimens are largely utilized in primate studies using combined approaches with lower doses of chemotherapy and radiation [60]. Major aims of these studies [60–66] are to induce stabile mixed chimerism for transplantation tolerance or gene therapy for hematologic or metabolic genetic disorders. One approach has been to include nonlethal total body irradiation (TBI), local thymic irradiation (TLI), and T-cell depletion with monoclonal antibodies like antithymocyte globulin (ATG) or antiCD2. Nonlethal low dose TBI ranging from 1.5 to 5 Gy is principally used in NHP HSC transplant studies [56, 60] with or without supplemental local thymic radiation with higher 7 Gy doses [67]. ATG 50 mg/kg has been generally employed for T-cell depletion [68], while adjunctive splenectomy has been used to further complete the reduction of donor-reactive lymphocyte population. Such regimens as reported by Kawai and others have led to stabile chimerism for a transient period of time, but this has been sufficient for the induction of tolerance to renal allografts

Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies 

775

Table 43-3.  Conditioning regimens in allogeneic transplantation. Conditioning regimen T-cell depletion/ suppression

Drugs

2 × 150 rad 700 rad

ATG

CsA

+ (multiline- 196, 198, 150, >40 Kawai [64, 67] age)

1.5 Gy

7 Gy

ATG, splenectomy,

CsA

+11/13

>3,478, >2,569, 834, 771, 405, 260, 198, 196, 137, 72, 44, 40, 37

Kawai [61]

1.5 Gy

7 Gy

ATG, anti-CD154

CsA

+8/8

>1,710, >1,167, 837i, 755, 401, 373, 206, 58

Kawai [61]

–

–

Thymoglobulin, fludarabine

Melphalan

8/8

180, 110, 100

Bartholomew [71]

–

–

Anti-IL2, betalecept Busulfan, + (ave 119 Not tested (CD28block), sirolimus days, max H106 196 days) (antiCD154)

TBI

TLI

Stabile chimerism

In vivo donor specific tolerance

[63, 69]. This approach has been successfully translated into clinical practice, using HLA mismatched donor and recipient pairs [70]. 4.2. Non-radiation Based Regimens In other studies, radiation can be entirely eliminated for the induction of mixed chimerism [66], with thymoglobulin, fludarabine, melphalan, or busulfan, the anti-IL2-receptor antibody basiliximab, blockade of CD28 signaling with the belatacept fusion protein, CD154 blockade with the H106 monoclonal antibody, and mTOR inhibitor sirolimus.

5. Models of Stem Cell Transplantation 5.1. Autologous Autologous HSC transplants can successfully engraft in rhesus [54, 56, 72], cynomolgus [73] and baboon [57] models and have been employed in the study of gene therapy, gene marking, mobilizing cytokines [74], and hematopoiesis. In addition to the mobilization strategy described above with recombinant human G-CSF [54–57], new strategies using SDF [54], myelopoetin [75], or other chemotactic factors [74] have also been studied. HSCs have been administered either intravenously or, more recently, directly intramarrow [57] HSC to deliver genetically modified grafts for transplant. 5.2. Autotransplantation for Studies in Gene Therapy HSCs are ideal targets for genetic manipulation in the treatment of several congenital and acquired disorders affecting the hematopoietic compartment. Large animals such as rhesus monkeys, baboons, cats, and dogs have similar

Reference

Kean [66]

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stem cell dynamics, cytokine responsiveness, and retroviral receptor properties as humans. Murine retroviruses based on the Moloney murine leukemia virus (Mo-MLV) were the first and still are the most widely used vectors in gene transfer studies [76]. They are one of the vector systems that have been used to transduce human hematopoietic stem or progenitor cells in a clinical trial. They stably integrate into the genome of the marked cell allowing the transduced cell to be tracked for its entire life span and for the lifespan of its progeny cells. Low level expression of the transgene in vivo, the potentially limiting level of specific cell surface receptors on certain target cells, their requirement for cell cycling, and safety issues including insertional mutagenesis from replication-competent retroviruses have resulted in an intense search for alternative viral and nonviral vectors. To overcome low level of cell surface receptors limiting transduction efficiency, pseudotyping with envelope proteins from different viruses like gibbon ape leukaemia virus (GALV) 10A1, or RD114, a feline endogenous retrovirus with higher receptor densities on target cells was developed [77, 78]. In the baboon model, direct comparisons between GALV and amphotropic vectors favor the GALV pseudotype to be studied [79]. Lentiviral vectors, based on disabled HIV-1 or HIV-2 genomes, are promising transducers without prolonged ex vivo stimulation of haematopoietic stem cells [80]. However, there is evidence that cytokine stimulation may be necessary for optimal transduction of haematopoietic cells using lentiviral vectors [81]. In the macaque, early generation vectors demonstrated low-level long-term in  vivo marking, even with cells cultured in vitro for short time periods without multiple stimulatory cytokines [82, 83]. In vivo gene transfer levels of 5–10% or higher have been achieved [84]. The use of G-CSF and stem cell factor (SCF)-mobilized peripheral blood (PB) CD34+ cells provides significantly higher in  vivo marking levels compared with that of G-CSF alone or that of G-CSF + Flt3-L-mobilized cells in the rhesus macaque competitive repopulation model [54]. Using the autologous HSC transplant model, several studies have illustrated the feasibility of tranduced HSCs to successfully and stably engraft [85–87]. To date, the efficiency of transduction may still be insufficient to attain therapeutic expression of gene products; however, the nonhuman primate model remains the best model to further refine this approach prior to moving to clinical trials. Using this preclinical model has additional benefits in testing the safety of this approach. Five years after receiving replication-defective retroviral transfected autograft a rhesus macaque developed a fatal myeloid sarcoma [88]. This observation highlights the significance of the nonhuman primate as a necessary preclinical stepping-stone. 5.3. Allogeneic Allogeneic HSCT in NHP models can reproduce the immunologic sequelae following fully mismatched allogeneic HSC transplant between nonhaploidentical individuals [71]. With access to breeding colonies, haploidentical transplants can also be performed either between parent and offspring or between siblings. HSCs derived from bone marrow and leukopheresis products both were found to be successful in inducing high-level hematopoietic mixed chimerism in a NHP model [66]. Stabile mixed chimerism is in the

Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies 

focus of organ transplant tolerance research as well [61]. Various promising conditioning regimens may provide mixed chimerism [61, 66] but its duration can be transient, and GVHD and infectious or malignancy complications due to immunosuppressive state still pose as fertile areas of study. 5.4. GVHD One of the most threatening complications of allogeneic bone marrow transplant is the graft-versus-host disease (GVHD). Experimental research on GVHD with laboratory animals has been performed with rodents, rhesus monkeys, and dogs. The basic immunological mechanisms operative in GVHD are largely similar in these three species and in human patients, although the patterns of GVHD in the three animal species show differences. The predictive value for clinical GVHD of the results obtained in the different animal species is different in histocompatibility, T cell numbers in the graft, and the intestinal microflora. Rhesus monkeys score highest as regards clinical relevance for the first two variables [89]. 5.5. MHC Typing In humans, apes, and Old World monkeys, the class II loci (DR) are highly conserved; however, there are species specific differences [90–94]. Like humans, chimpanzees, gorillas, and rhesus macaques have variable numbers of MHC-DRB loci per haplotype [95, 96]. Rhesus macaques (macaca mulatta, hence the MHC designation “Mamu” after the first two letters of macacca and mulatta, respectively), consangineously bred to achieve homozygosity at their MHC region and revealed that the number of Mamu-DRB loci per haplotype varies from two to six with up to three -DRB genes expressed [97]. Recently, Prasad et al. [98] identified also marmoset major MHC Class II DRB genes (Caja-DRB*W1201, Caja-DRB1*03, Caja-DRB*W16) using sequence-based typing techniques. They investigated whether matching at MHC Class II DRB loci alone could predict alloreactivity, as assessed in vitro by two-way mixed lymphocyte reactions. Fully mismatched and partially mismatched animal pairs exhibited significant in  vitro T-cell proliferation above single cell controls. Using DRB genotyping, suitable alloreactive donor-recipient pairs may therefore be rapidly and accurately identified for use in NHP studies of cellular and solid organ transplantation. Lymphohematopoietic chimerism in the primates was investigated in MHCmismatched allogeneic bone marrow transplantation (BMT) in the rhesus monkey detecting restriction fragment length polymorphism. A human MHC (HLA) class II DR beta gene cDNA probe was tested on rhesus peripheral blood mononuclear cell DNA digested with any of three restriction enzymes. The human DR beta probe hybridized to as many as 15 restriction fragments per rhesus DNA sample, suggesting cross-hybridization at more than one locus of rhesus MHC class II beta genes; restriction fragment length polymorphisms were common among outbred monkeys as a result of class II beta gene polymorphisms and would be sufficient for chimerism detection in the majority of random pairs of outbred monkeys utilizing only a single restriction enzyme. Sensitivity (5–10% chimerism) was comparable to that reported for restriction fragment length polymorphism assays utilizing nonMHC probes in

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clinical HLA-identical BMT. [99] Efforts are underway to provide MHC typed rhesus for haploidentical transplants using molecular characterization of the MHC (personal communication, Leslie Kean, Emory). These efforts will be used to direct breeding of rhesus monkeys to obtain specific haplo-identical pairs for further study. 5.6. Combined Stem Cell and Whole Organ Transplants Inducing whole organ tolerance through mixed chimerism after HSC transplant is a prominent area of investigation using NHP hematopoietic transplantation models. Kawai et  al. [60] developed a nonmyeloablative preparative regimen that can produce mixed chimerism and renal allograft tolerance between MHC-disparate nonhuman primates. The induction of transient mixed hematopoietic chimerism led also to long-term heart allograft survival in MHC disparate monkeys without chronic immunosuppression. However, unlike kidney allografts, full tolerance to cardiac allografts was not achieved. Moreover, chronic cellular and humoral immune responses were elicited by cardiac allografts [67]. Significantly, modifications of these early studies were used to define a successful clinical regimen which has recently demonstrated renal transplant tolerance [70]. In this regimen, the same investigators have observed human subjects off all immunosuppression for greater than 6 years (personal communication David Sachs, Harvard). 5.7. Fetal and In Utero Transplants BMT is a promising treatment to reconstitute defective hematopoietic cell lines in children with congenital defects but is limited by donor availability, graft rejection, and GVHD. These problems can be limited by transplanting normal preimmune fetal HSCs or adult HSCs into an unrelated preimmune fetal recipient. In clinical cases only fetuses with immunological defects were transplanted with adult HSC with limited outcomes [100]. Fetal transplantation addressing the composition and cell number of graft, and recipient age has been studied systematically in sheep models. The few NHP studies did provide some valuable insights that can be directly transferred to human clinical situations. Initial studies [33, 101] showed no chimerism after adult bone marrow transplant more than 0.44 (baboons) and 0.42 (cynomolgus) gestation time of the recipient. Zanjani et al. in their studies demonstrated first that the injections of allogeneic fetal stem cells into preimmune fetal monkeys result in long-term stable hematopoietic chimerism. HSCs harvested from the livers of preimmune fetal monkeys when injected into the peritoneal cavity of young unrelated fetal monkey recipients lead to stable, long-term postnatal multilineage hematopoietic chimerism. Donor cell engraftment was achieved without the use of cytoablative procedures and without the development of GVHD [102]. Shields et  al. investigated in baboon and macaques models the level of chimerism reached after in utero transplantation of purified, haploidentical CD34+ allogeneic bone marrow cells and the influence of T-cell number on engraftment [36, 103, 104]. Donor HSCs grafts containing different doses of donor T-cells were administered two or three times into the abdominal cavity of the fetuses between 0.38 and 0.42 gestation time. Chimerism was detected in cord blood or bone marrow cells in 87% of the recipients during the first month of life. Successful postnatal engraftment appeared to correlate with

Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies 

the total CD34+ cell dose as well as donor T-cells number. Interestingly, the amount of long term peripheral blood chimerism did not appear to be of sufficient levels to potentially induce therapeutic effect in most diseases; however this requires further study. 5.8. Xenotransplantation The use of xenotransplantation has been used in immunodeficient mouse models to study human hematopoiesis. Nonhuman primate models have also been used to study xenotransplantation, however this has been directed toward the study of organ transplants, typically between pigs and nonhuman primates. This topic has spanned several volumes in its own right and is not covered herein; however, it is important to note that such studies have investigated the ability to induce mixed chimerism through xenogeneic engraftment of porcine HSCs in nonhuman primates. The overarching hypothesis is on the basis of the premise that the induction of stable mixed chimerism will lead to the induction of immunologic tolerance. HSC xenotransplantation is challenged by problems of natural and elicited anti-pig antibodies, recipient platelet adhesion to pig hematopoietic progenitor cells resulting disseminated intravascular coagulation, and the rapid removal of pig HSC by the host macrophage-phagocytic system. Initial bone marrow xenotransplant studies of Sablinski et al. [105] investigated bone marrow transplants in the pig-to-cynomolgus monkey model. Similar to the conditioning regimen described for allotransplants, the recipient underwent pre-transplant splenectomy (day-6), total body irradiation in two fractions of 150  cGy (on days-6 and -5), and thymic irradiation (TI) of 700  cGy (on day-1). Preformed antibodies directed to Gal[alpha]1,3Gal (Gal) determinants were depleted by ex vivo adsorption through a pig liver (on day 0). Cyclosporine (15 mg/kg per day) was administered for 4 weeks, and 15-deoxyspergualin (6  mg/kg per day) for 2 weeks. In one of the two recipients, pig-specific interleukin-3 (IL-3, 10  mg/kg per day) and stem cell factor (SCF, 10 mg/kg per day, which is not pig-specific) were administered for 14 days. BM cells from MHC-defined miniature swine were transplanted into each recipient at doses of 2.3 and 2.5 × 108 cells/kg, respectively, on day 0. This regimen was not sufficient to induce engraftment, with porcine DNA only detectable by PCR on days 180 and 302 after BM Tx in one animal treated with porcine growth factors. In these and subsequent studies, porcine stem cells were rapidly removed from the circulation, suggesting macrophagephagocytic activity [106, 107] with some detectable DNA in animals treated with porcine IL-3 and SCF [108]. These studies highlight the difficulty of hematopoietic engraftment across disparate species and serve as an important base for greater investigation. Platelet Aggregation and anti Gal antibody production remain the major concerns in xenotransplant models. The infusion of pig HSCs alone or following the nonmyeloablative regimen led to a marked and sustained fall in platelet count and a rise in LDH with hemorrhagic events and even fatal outcome [109]. Alwayn et al. [110] demonstrated that porcine PBPC directly mediate aggregation of baboon platelets and that this process likely contributes to the thrombotic microangiopathy observed after porcine HSC transplant in the pig-to-baboon model. A combination of heparin and eptifibatide appeared to be the most beneficial in preventing a thrombotic disorder while maintaining

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adequate hemostatic responses [111]. Other studies showed that combination of eptifibatide-heparin-methylprednisolone could also prevent thrombotic events [109, 112, 113]. A significant biotechnical advance was the use of [alpha]1,3-galactosyltransferase gene-knock out pigs as donors of HSCs. Such grafts were infused (11 × 108 cells/kg) to three baboons [114]. Although the platelet count fell because of the conditioning regimen and following the infusion of the BM cells, the profound loss of platelets seen previously did not occur. Pig cell chimerism was detected by flow cytometry during the second posttransplant week, suggesting transient pig cell engraftment in baboon BM. With the elimination of this significant obstacle, attention is now directed toward engineering the bone marrow microenvironment by grafting pig spleen [115] with the HSC transplant. Such dramatic advances could never be accomplished nor insights gained in smaller animal models. Nonhuman primate models provide a critical stepping stone for sorting viable and nonviable directions for clinical development. 5.9.  The Hematopoietic Bone Marrow Microenvironment and Mesenchymal Stem Cells The bone marrow microenvironment plays a pivotal role in HSC engraftment. Osteoblasts of the endosteal niche and endothelial cells of the vascular niche can regulate stem cell quiescence and proliferation, respectively [116]. Mesenchymal stem cells (MSC), also active within the bone marrow microenvironment, give rise to osteoblasts, have been implicated in control of hematopoiesis, and are now being used therapeutically to improve hematopoietic engraftment [117–120]. MSC serve another function, providing important immunoregulatory signals that can control both adaptive and innate immune responses. The nonhuman primate model was pivotal in illustrating the powerful effect of a single infusion of MSC on the allogeneic immune response, revealing that such a treatment was capable of prolonging skin graft survival in baboons [12]. Extensions of these studies have been undertaken in humans, using MSC for the successful control of GVHD following BMT [121–123]. Additional pre-clinical studies undertaken in the nonhuman primate have included MSC tracking studies to demonstrate their ability to take up residence in a variety of tissues [11, 124] and gene transfection studies illustrating the feasibility of using such cells for gene therapy [125] and as vehicles in regeneration, promoting their administration to facilitate healing following radiation injury in clinical practice [126, 127].

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Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies  46. Gengozian N, Batson JS, Eide P (1964) Hematologic and cytogenetic evidence for hematopoietic chimerism in the marmoset, tamarinus nigricollis. Cytogenetics 10:384–393 47. Haig D (1999) What is a marmoset? Am J Primatol 49:285–296 48. Watkins DI, Chen ZW, Hughes AL, Hodi FS, Letvin NL (1990) Genetically distinct cell populations in naturally occurring bone marrow-chimeric primates express similar MHC class I gene products. J Immunol 144:3726–3735 49. Crawford DH, Janossy G, Hetherington CM et al (1981) Immunological characterization of hemopoietic cells in the common marmoset, rhesus monkey, and man. In search of a model for human marrow transplantation. Transplantation 31:245–250 50. Michalevicz R, Hetherington CM, Hann I, Blacklock HA (1985) Studies of subhuman primate (marmoset) pluripotent hemopoietic stem cells (CFU-GEMM) in vitro. J Med Primatol 14:245–253 51. Hibino H, Tani K, Ikebuchi K et  al (1999) The common marmoset as a target preclinical primate model for cytokine and gene therapy studies. Blood 93: 2839–2848 52. Izawa K, Tani K, Nakazaki Y et al (2004) Hematopoietic activity of common marmoset CD34 cells isolated by a novel monoclonal antibody MA24. Exp Hematol 32:843–851 53. Ryffel B, Car BD, Woerly G et al (1994) Long-term interleukin-6 administration stimulates sustained thrombopoiesis and acute-phase protein synthesis in a small primate-the marmoset. Blood 83:2093–2102 54. Hematti P, Tuchman S, Larochelle A, Metzger ME, Donahue RE, Tisdale JF (2004) Comparison of retroviral transduction efficiency in CD34+ cells derived from bone marrow versus G-CSF-mobilized or G-CSF plus stem cell factor-mobilized peripheral blood in nonhuman primates. Stem Cells 22:1062–1069 55. Rosenzweig M, MacVittie TJ, Harper D et  al (1999) Efficient and durable gene marking of hematopoietic progenitor cells in nonhuman primates after nonablative conditioning. Blood 94:2271–2286 56. Wagemaker G, Neelis KJ, Hartong SC et  al (1998) The efficacy of recombinant thrombopoietin in murine and nonhuman primate models for radiation-induced myelosuppression and stem cell transplantation. Stem Cells 16:375–386 57. Jung CW, Beard BC, Morris JC et al (2007) Hematopoietic stem cell engraftment: A direct comparison between intramarrow and intravenous injection in nonhuman primates. Exp Hematol 35:1132–1139 58. Ageyama N, Kimikawa M, Eguchi K et al (2003) Modification of the leukapheresis procedure for use in rhesus monkeys (Macaca mulata). J Clin Apher 18:26–31 59. Basser RL, To LB, Begley CG et  al (1998) Rapid hematopoietic recovery after multicycle high-dose chemotherapy: Enhancement of filgrastim-induced progenitor-cell mobilization by recombinant human stem-cell factor. J Clin Oncol 16:1899–1908 60. Kawai T, Cosimi AB, Colvin RB et  al (1995) Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation 59:256–262 61. Kawai T, Sogawa H, Boskovic S et  al (2004) CD154 blockade for induction of mixed chimerism and prolonged renal allograft survival in nonhuman primates. Am J Transplant 4:1391–1398 62. Kimikawa M, Sachs DH, Colvin RB, Bartholomew A, Kawai T, Cosimi AB (1997) Modifications of the conditioning regimen for achieving mixed chimerism and donor-specific tolerance in cynomolgus monkeys. Transplantation 64:709–716 63. Kawai T, Abrahamian G, Sogawa H et  al (2001) Costimulatory blockade for induction of mixed chimerism and renal allograft tolerance in nonhuman primates. Transplant Proc 33:221–222 64. Kawai T, Poncelet A, Sachs DH et al (1999) Long-term outcome and alloantibody production in a non-myeloablative regimen for induction of renal allograft tolerance. Transplantation 68:1767–1775

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E. Szilagyi et al. 65. Kawai T, Sogawa H, Koulmanda M et al (2001) Long-term islet allograft function in the absence of chronic immunosuppression: A case report of a nonhuman primate previously made tolerant to a renal allograft from the same donor. Transplantation 72:351–354 66. Kean LS, Adams AB, Strobert E et  al (2007) Induction of chimerism in rhesus macaques through stem cell transplant and costimulation blockade-based immunosuppression. Am J Transplant 7:320–335 67. Kawai T, Cosimi AB, Wee SL et  al (2002) Effect of mixed hematopoietic chimerism on cardiac allograft survival in cynomolgus monkeys. Transplantation 73:1757–1764 68. Preville X, Flacher M, LeMauff B et  al (2001) Mechanisms involved in antithymocyte globulin immunosuppressive activity in a nonhuman primate model. Transplantation 71:460–468 69. Kawai T, Cosimi AB, Colvin RB et  al (1995) Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation 59:256 70. Kawai T, Cosimi AB, Spitzer TR et al (2008) HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med 358:353–361 71. Bartholomew A, Sturgeon C, Siatskas M et al (2000) A non-radiation based regimen results in mixed chimerism in MHC-mismatched monkeys. Blood 96 72. Huhn RD, Tisdale JF, Agricola B, Metzger ME, Donahue RE, Dunbar CE (1999) Retroviral marking and transplantation of rhesus hematopoietic cells by nonmyeloablative conditioning. Hum Gene Ther 10:1783–1790 73. Hanazono Y, Terao K, Shibata H et al (2002) Introduction of the green fluorescent protein gene into hematopoietic stem cells results in prolonged discrepancy of in vivo transduction levels between bone marrow progenitors and peripheral blood cells in nonhuman primates. J Gene Med 4:470–477 74. Larochelle A, Krouse A, Metzger M et al (2006) AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates. Blood 107:3772–3778 75. MacVittie TJ, Farese AM, Davis TA, Lind LB, McKearn JP (1999) Myelopoietin, a chimeric agonist of human interleukin 3 and granulocyte colony-stimulating factor receptors, mobilizes CD34+ cells that rapidly engraft lethally X-irradiated nonhuman primates. Exp Hematol 27:1557–1568 76. Miller AR, Skotzko MJ, Rhoades K et al (1992) Simultaneous use of two retroviral vectors in human gene marking trials: Feasibility and potential applications. Hum Gene Ther 3:619–624 77. Barrette S, Douglas J, Orlic D et al (2000) Superior transduction of mouse hematopoietic stem cells with 10A1 and VSV-G pseudotyped retrovirus vectors. Mol Ther 1:330–338 78. Barrette S, Douglas JL, Seidel NE, Bodine DM (2000) Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to moloney murine leukemia virus-based vectors. Blood 96:3385–3391 79. Kiem HP, Heyward S, Winkler A et al (1997) Gene transfer into marrow repopulating cells: Comparison between amphotropic and gibbon ape leukemia virus pseudotyped retroviral vectors in a competitive repopulation assay in baboons. Blood 90:4638–4645 80. Case SS, Price MA, Jordan CT et  al (1999) Stable transduction of quiescent CD34(+)CD38(−) human hematopoietic cells by HIV-1-based lentiviral vectors. Proc Natl Acad Sci USA 96:2988–2993 81. Sutton RE, Reitsma MJ, Uchida N, Brown PO (1999) Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent. J Virol 73:3649–3660 82. An DS, Kung SK, Bonifacino A et  al (2001) Lentivirus vector-mediated hematopoietic stem cell gene transfer of common gamma-chain cytokine receptor in rhesus macaques. J Virol 75:3547–3555

Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies  83. An DS, Wersto RP, Agricola BA et al (2000) Marking and gene expression by a lentivirus vector in transplanted human and nonhuman primate CD34(+) cells. J Virol 74:1286–1295 84. Kiem HP, Andrews RG, Morris J et al (1998) 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 92:1878–1886 85. Van Beusechem VW, Bart-Baumeister JA, Bakx TA, Kaptein LC, Levinsky RJ, Valerio D (1994) Gene transfer into nonhuman primate CD34 + CD11b-bone marrow progenitor cells capable of repopulating lymphoid and myeloid lineages. Hum Gene Ther 5:295–305 86. Hanawa H, Hematti P, Keyvanfar K et al (2004) Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virusbased lentiviral vector system. Blood 103:4062–4069 87. Kiem HP, Sellers S, Thomasson B et al (2004) Long-term clinical and molecular follow-up of large animals receiving retrovirally transduced stem and progenitor cells: No progression to clonal hematopoiesis or leukemia. Mol Ther 9:389–395 88. Seggewiss R, Pittaluga S, Adler RL et al (2006) Acute myeloid leukemia is associated with retroviral gene transfer to hematopoietic progenitor cells in a rhesus macaque. Blood 107:3865–3867 89. van Bekkum DW (1994) Biology of acute and chronic graft-versus-host reactions: Predictive value of studies in experimental animals. Bone Marrow Transplant 14(Suppl 4):S51–S55 90. Gyllensten U, Sundvall M, Ezcurra I, Erlich HA (1991) Genetic diversity at class II DRB loci of the primate MHC. J Immunol 146:4368–4376 91. Otting N, Bontrop RE (1995) Evolution of the major histocompatibility complex DPA1 locus in primates. Hum Immunol 42:184–187 92. Thiel C, Bontrop RE, Lanchbury JS (1995) Structure and diversity of the T-cell receptor alpha chain in rhesus macaque and chimpanzee. Hum Immunol 43: 85–94 93. Heise ER, Cook DJ, Schepart BS et al (1987) The major histocompatibility complex of primates. Genetica 73:53–68 94. Otting N, de Vos-Rouweler AJ, Heijmans CM, de Groot NG, Doxiadis GG, Bontrop RE (2007) MHC class I A region diversity and polymorphism in macaque species. Immunogenetics 59:367–375 95. Kenter M, Otting N, de Weers M et al (1993) Mhc-DRB and -DQA1 nucleotide sequences of three lowland gorillas. Implications for the evolution of primate Mhc class II haplotypes. Hum Immunol 36:205–218 96. Schonbach C, Vincek V, Mayer WE, Golubic M, O’HUigin C, Klein J (1993) Multiplication of Mhc-DRB5 loci in the orangutan: Implications for the evolution of DRB haplotypes. Mamm Genome 4:159–170 97. Balner H (1980) The DR system of rhesus monkeys: A brief review of serology, genetics, and relevance to transplantation. Transplant Proc 12:502–508 98. Prasad S, Humphreys I, Kireta S et al (2007) The common marmoset as a novel preclinical transplant model: Identification of new MHC class II DRB alleles and prediction of in vitro alloreactivity. Tissue Antigens 69(Suppl 1):72–75 99. Moses RD, Beschorner WE, Singer D et  al (1989) Restriction fragment length polymorphism analysis with a cross-reactive HLA class II DR-beta gene probe for the detection of engraftment of MHC-mismatched marrow in the rhesus monkey. Bone Marrow Transplant 4:475–481 100. Flake AW, Roncarolo MG, Puck JM et  al (1996) Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow. N Engl J Med 335:1806–1810 101. Brent L, Linch DC, Rodeck CH et al (1989) On the feasibility of inducing tolerance in man: A study in the cynomolgus monkey. Immunol Lett 21:55–61

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E. Szilagyi et al. 102. Zanjani ED, Mackintosh FR, Harrison MR (1991) Hematopoietic chimerism in sheep and nonhuman primates by in utero transplantation of fetal hematopoietic stem cells. Blood Cells 17:349–363 discussion 364–346 103. Shields LE, Gaur L, Delio P, Potter J, Sieverkropp A, Andrews RG (2004) Fetal immune suppression as adjunctive therapy for in utero hematopoietic stem cell transplantation in nonhuman primates. Stem Cells 22:759–769 104. Shields LE, Gaur L, Delio P et al (2005) The use of CD 34(+) mobilized peripheral blood as a donor cell source does not improve chimerism after in utero hematopoietic stem cell transplantation in non-human primates. J Med Primatol 34:201–208 105. Sablinski T, Emery DW, Monroy R et al (1999) Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcinespecific growth factors. Transplantation 67:972–977 106. Kozlowski T, Ierino FL, Lambrigts D et al (1998) Depletion of anti-Gal(alpha)13Gal antibody in baboons by specific alpha-Gal immunoaffinity columns. Xenotransplantation 5:122–131 107. Kozlowski T, Monroy R, Xu Y et  al (1998) Anti-Gal(alpha)1-3Gal antibody response to porcine bone marrow in unmodified baboons and baboons conditioned for tolerance induction. Transplantation 66:176–182 108. Kozlowski T, Monroy R, Giovino M et al (1999) Effect of pig-specific cytokines on mobilization of hematopoietic progenitor cells in pigs and on pig bone marrow engraftment in baboons. Xenotransplantation 6:17–27 109. Buhler L, Awwad M, Treter S et al (2002) Pig hematopoietic cell chimerism in baboons conditioned with a nonmyeloablative regimen and CD154 blockade. Transplantation 73:12–22 110. Alwayn IP, Buhler L, Appel JZ III et al (2001) Mechanisms of thrombotic microangiopathy following xenogeneic hematopoietic progenitor cell transplantation. Transplantation 71:1601–1609 111. Alwayn IP, Appel JZ, Goepfert C, Buhler L, Cooper DK, Robson SC (2000) Inhibition of platelet aggregation in baboons: Therapeutic implications for xenotransplantation. Xenotransplantation 7:247–257 112. Appel JZ III, Alwayn IP, Correa LE, Cooper DK, Robson SC (2001) Modulation of platelet aggregation in baboons: Implications for mixed chimerism in xenotransplantation. II. The effects of cyclophosphamide on pig peripheral blood progenitor cell-induced aggregation. Transplantation 72:1306–1310 113. Appel JZ III, Alwayn IP, Buhler L, DeAngelis HA, Robson SC, Cooper DK (2001) Modulation of platelet aggregation in baboons: Implications for mixed chimerism in xenotransplantation. I. The roles of individual components of a transplantation conditioning regimen and of pig peripheral blood progenitor cells. Transplantation 72:1299–1305 114. Tseng YL, Dor FJ, Kuwaki K et al (2004) Bone marrow transplantation from alpha1, 3-galactosyltransferase gene-knockout pigs in baboons. Xenotransplantation 11:361–370 115. Dor FJ, Tseng YL, Kuwaki K, Ko DS, Cooper DK (2004) Pig spleen transplantation induces transient hematopoietic cell chimerism in baboons. Xenotransplantation 11:298–300 116. Shiozawa Y, Havens AM, Pienta KJ, Taichman RS (2008) The bone marrow niche: Habitat to hematopoietic and mesenchymal stem cells, and unwitting host to molecular parasites. Leukemia 22:941–950 117. Koc ON, Gerson SL, Cooper BW et  al (2000) Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 18:307 118. Angelopoulou M, Novelli E, Grove JE et al (2003) Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice. Exp Hematol 31:413–420

Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies  119. Fibbe WE, Noort WA (2003) Mesenchymal stem cells and hematopoietic stem cell transplantation. Ann NY Acad Sci 996:235–244 120. Le Blanc K, Samuelsson H, Gustafsson B et al (2007) Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia 21:1733–1738 121. Le Blanc K, Rasmusson I, Sundberg B et  al (2004) Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 363:1439–1441 122. Lazarus HM, Koc ON, Devine SM et  al (2005) Cotransplantation of HLAidentical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant 11:389–398 123. Le Blanc K, Frassoni F, Ball L et  al (2008) Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: A phase II study. Lancet 371:1579–1586 124. Devine SM, Cobbs C, Jennings M, Bartholomew A, Hoffman R (2003) Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates. Blood 101:2999–3001 125. Bartholomew A, Patil S, Mackay A et al (2001) Baboon mesenchymal stem cells can be genetically modified to secrete human erythropoietin in vivo. Hum Gene Ther 12:1527–1541 126. Chapel A, Bertho JM, Bensidhoum M et al (2003) Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiationinduced multi-organ failure syndrome. J Gene Med 5:1028–1038 127. Lataillade JJ, Doucet C, Bey E et al (2007) New approach to radiation burn treatment by dosimetry-guided surgery combined with autologous mesenchymal stem cell therapy. Regen Med 2:785–794

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Chapter 44 In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation Lisbeth A. Welniak and William J. Murphy

1. Introduction Animal models have been vital to the development of allogeneic hematopoietic stem cell transplantation (AlloHSCT) as well as our understanding of its biology. Rodent models led the way in the demonstration of the whole body radiotherapy for effective anti-tumor responses [1] and the ability to rescue mice from high dose radiation with a transfusion of bone marrow cells [2, 3] in the early 1950s. However, the ability to promote prolonged tumor-free survival in mice using allogeneic bone marrow transplantation after myeloablative doses of radiation was offset by the recognition that allogeneic bone marrow transplant (BMT) could result in a lethal “secondary” disease of wasting, diarrhea and skin lesions [4] now known as graft-versus host disease. Interestingly, graft-versus-tumor (GVT) activity was also recognized in studies during this time period. [5, 6].

2. Preclinical Models for the Study of Allogeneic HSCT After the initial studies described above, much of the development of protocols for alloHSCT as well as the prevention and treatment of GVHD were performed in dogs [7]. Donnall Thomas, in collaboration with other investigators at Fred Hutchinson Cancer Center, pioneered the field of alloHSCT with his work in beagles [8]. Other animal models that have been used include the miniature swine model for work in allogeneic and xenogeneic hematopoietic cell and/or organ transplantation [9, 10] and the fetal sheep model for the study of human stem cells in a tolerant xenogeneic transplant [11]. Rodent models and in particular mouse models are the principle animal species used for the study of hematopoiesis and immunology. Mouse models remain an essential tool to understanding the biology of alloHSCT due to the availability of resources, the advantage of inbred strains,

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_44, © Springer Science + Business Media, LLC 2003, 2010

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the availability of genetically engineered strains and the ability of researchers to perform studies with sufficient numbers of animals for statistical power. Genetic and instrumentation technologies have evolved around the mouse model, which take advantage of the small body size of the mouse and aid in our understanding of alloHSCT. One of the most used technologies is in vivo bioluminescent-based imaging (BLI), which is based, on the observation that light can pass through tissues. Cells or mice of interest can be modified to express a bioluminescent marker such as the luciferase gene or green fluorescence protein (GFP). These cells can then be infused and tracked in unlabeled recipient mice using low light sensitive cameras. Trafficking and proliferation of labeled tumor cells, donor-derived bone marrow or specific cell populations such CD4+ and CD8+ T cells in mice have been examined using this technique and provide powerful real-time physical orientation and temporal information in engraftment, GVHD and GVT/GVL studies [12–14]. Other advantages of the technique are that it is non-invasive, provides quantitative information, and requires the use of far fewer numbers of animals than necropsy followed by analysis of multiple tissues per animal. Work in the mouse model is the foundation for our knowledge of the hematologic and the immunological mechanisms related to alloHSCT. However, there are many differences too, between man and mouse that can complicate the interpretation of findings. Some of the differences are obvious such as variation between species but other factors can be less apparent but can exert just equally important influences on the outcome and interpretation of results. The following are some of the important considerations that need to be taken into account when evaluating the results of animal studies. 2.1. Species Differences Evolution has conserved many aspects of the immune system in mammalian development where it has allowed for the development of functional similarities in molecularly disparate systems (i.e., Ly49 and KIR in mouse and human NK cells, respectively). However, other differences can result in discrepancies between pre-clinical mouse studies and clinical findings. The physical structure of lymphoid tissue in mouse and man are very similar but many differences that affect the innate as well as the adaptive immune systems exist between these two species and are detailed in depth in the 2004 review by Mestas and Hughes [15]. In addition, other physiological differences between the two species exist that can alter the action and or toxicity of drugs that are commonly used in alloHSCT. Finally, many biological targets are not sufficiently conserved between mouse and man. The use of species-specific biological reagents such as cytokines or antibodies can have unexpected differences in the specificity or strength of reactions. For example, the small variation between non-human primate and human CD28 may have been responsible for the differences in the disastrous outcome in the preclinical and clinical trial with agonist anti-CD28 [16]. 2.2. Conditioning Regimens Typically in mouse alloHSCT studies, unfractionated or split dose myeloablative irradiation is used to prepare the recipient but treatments can range from no conditioning for the adoptive transfer of allogeneic T cells in GVHD

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation 

studies to myeloablative doses of whole body irradiation followed by HSC rescue. Despite the increasing use of reduced intensity conditioning in clinical practice, most mouse studies have failed to reflect this change although some studies have investigated these therapies in mice [17, 18]. Part of the problem arises from the differences between species. Fludarabine is a common component in reduced intensity conditioning regimens. However, the metabolism and pharmokinetics of this drug differs in mice and man, due to differences in the activity of the enzyme that converts the prodrug into its active form [19], thus hindering its use in mouse models. Work in the mouse model has demonstrated the influence of conditioning on various outcome parameters after alloHSCT. Conditioning not only induces hematopoietic suppression, including lympho-depletion to facilitate engraftment of donor hematopoietic stem cells, but can also induce tissue damage and the translocation of lipopolysaccharide (LPS) from the intestine resulting in immune-stimulation and increased acute GVHD mortality and morbidity [20], as well as increased anti-tumor responses [21]. The intensity and composition of the conditioning regimen may also affect not only the incidence and intensity of GVHD but also the physiopathology as demonstrated in studies by Claman et al. [22] and studies by Xun and Widmer [23]. In addition, mouse models have shown that under lower intensity conditioning regimens, residual host T cells can provide a veto effect on the alloreactive donor T cells [24] that can result not only in reduced rates of GVHD [25] but also result in a loss of the beneficial activity of GVL [26]. These studies demonstrate that the use of different conditioning regimens can result in varied outcomes in the induction, intensity and clinical presentation of GVHD, GVL as well as affecting donor chimerism. In addition, species differences between mouse and man may alter the influence of various conditioning regimens on outcomes in alloHSCT. 2.3. Mouse Strains and Immunologic Disparity Several different combinations of major and/or minor histocompatible mismatches between donor and host are commonly transplanted in humans. However, the histocompatibility differences between mouse strains commonly used in experimental models do not always reflect the common clinical situations. This may be influenced in part by the maintenance of animals in specific-pathogen free housing. The availability of inbred strains, MHC congenic strains and semi-allogeneic (parent into F1) strains also allows for experimental designs to address or control for particular histocompatibility disparities between donor and host. Thus, lethal acute GVHD, which is mediated primarily by CD4+ cells, CD8+ cells or both, can be investigated using the appropriate combination of mouse strains [27]. Issues of host T cell mediated rejection of donor T cells can be eliminated by using parent into F1 models of alloHSCT. Selection of donor-host combinations is usually dependent on the amount of MHC and MiHA mismatched desired, the availability of reagents, histocompatible tumor cell lines, and the background genes of specialize mice such as transgenics and mice with targeted mutations. While genetically engineered mice are invaluable to the study of immunology, they do have limitations that are not always apparent, as the genetic manipulation may result in

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unexpected and/or subtle differences that may confound the interpretation of the experiment. Unexpected influences on neighboring genes, antigenicity of the inserted gene product in transplanted cells and developmental changes in these animals are all complications that have arisen in different experimental settings. Additionally, the time and expense of backcrossing genetically modified mice (gene knockout or transgenics) onto different genetic backgrounds can also limit the choice of strain combinations for alloHSCT. 2.4. Tissue Source and Cellular Composition of the Graft While peripheral blood and/or bone marrow or umbilical cord blood are the primary source of T cells in the grafts of human HSCTs, spleen cells and/ or lymph node cells are added to the bone marrow graft to provide sufficient numbers of T cells for the induction of GVHD in mice. The number of T cells required for GVHD is dependent on the frequency of allo-reactive precursors and therefore is dependent on the strain combination. Until very recently it was less clear as to the importance of expression of homing receptors on T cells taken from different tissues. CD62L, CCR7 and MAdCAM (a4b7 integrin) are differentially expressed on cells found in the blood or tissues such as the skin, liver and intestine and in lymphoid tissues. The homing receptors CD62L and CCR7 have been shown to play a critical role in the development of GVHD but expression of these receptors also partially define the maturation status of the T cell. CD62L and CCR7 are expressed on naïve T cells, which comprise the alloreactive T cell population in unsensitized donor mice [28, 29]. Forced expression of CD62L on mouse T memory cells has shown that it is not required for the induction of GVHD [30]. However, expression of the mucosal addressin molecule, MAdCAM, on donor T cells does appear to be important in liver and intestinal GVHD [31, 32]. In support of these findings, a study from Beilhack et al. [33] has shown that while homing of T cells to lymph nodes or spleen is required for the induction of GVHD, organ associated lymphoid tissue is not required. Thus, mesenteric lymph nodes and Peyer’s Patches are not required for the induction of intestinal GVHD. The caveat to these observations are discussed in Sect. 2.2, as it has been shown that in the absence of conditioning, intestine associated lymphoid tissue is critical to the development of GVHD [34, 35]. In addition to T cells, other cell populations in the graft such as NKT, NK and myeloid cells can affect GVHD and GVT. The role of NKT and NK cells in alloHSCT are described in greater detail in Sect. 4. 2.5. Endogenous Microflora and Opportunistic Pathogens Mice are maintained in specific-pathogen-free facilities which can alter the immune responses in marrow rejection [36] and GVHD. This practice can result in the use of larger numbers of T cells or greater amounts of conditioning than would be required if animals were maintained in conventional housing. The use of non-absorbable antibiotics can influence the outcome in both murine studies and clinical studies [37]. Gut flora is an especially important factor for the induction of intestinal lesions of acute GVHD in mice, since bacterial flora may up-regulate minor histocompatibility antigens in the recipient animals and result in the activation of toll-like receptors following translocation of microorganisms resulting in the induction of cytokine production and

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation 

increased immune responses including GVHD [20, 38, 39] and anti-tumor responses [21]. 2.6. Age and Sex of the Donors and Recipients The age of donor and recipient can be important variables for the immune reconstitution and for the development and severity of GVHD. However, most animal studies use young adults of a single sex to minimize the variables in the study and improve reproducibility. A few murine studies have examined the influence of age on GVHD development by using middle aged or old mice as either donors or recipients [40–43].

3. Immunobiology of Allogeneic HSCT Animal models have been instrumental in understanding the biology of alloHSCT. The roles of conditioning, immunodepletion, alloreactive and immunoregulatory cells on the various clinically relevant facets of a hematopoietic cell transplants as illustrated in Fig. 44-1 are described in the remainder of this chapter. 3.1. Graft Rejection Successful allogeneic transplants require that the immunocompetent host not reject the graft. Allogeneic HSC graft rejection can be mediated by NK cells [44, 45],

Fig. 44-1.  Sequelae of alloHSCT and factors influencing their development. Donor T cells recognition of host antigens are responsible for both GVHD and GVT while donor NK cells may provide GVT effects. Host NK and/or T cell recognition of the donor graft can result in graft rejection. Other parameters affecting a successful outcome after alloHSCT include immunodepletion or immunosuppression of the donor or host

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NKT cells [46] and/or T cells [47–50] that recognize histocompatibility determinants on the donor cells. The mechanisms of graft rejection have been extensively studied in animal models. In fact, NK cells were originally described based on their ability to reject bone marrow grafts in a MHC independent manner [44, 45]. To this day, the graft rejection model remains one of the few functional assays for NK activity in mice. Mouse models have also been used to demonstrate that increasing the intensity of host conditioning decreases the number of immunocompetent cells in the recipient to overcome rejection of T-cell depleted grafts [49]. It was also through the use of mouse models that the strategy of transplanting “megadoses” of T-depleted or CD34+ selected HSC partially matched (haploidentical) grafts from related donors was developed and shown that these large doses may tolerize the recipient to the engrafting cells [51, 52]. A great deal of our understanding of the effector pathways used by recipient T cells to reject hematopoietic grafts comes from work with mice. It has been shown that in naïve, unsensitized recipient mice, CD8+ T cells can mediate rejection through the use of the effector molecules, perforin, granzyme B and fas/fasL [36, 53, 54]. CD4+ T cells can also mediate MHC mismatched bone marrow destruction [55] and this activity is dependent on donor CD4+ T cell derived interferon-gamma [56]. Still to be determined are all of the effector pathways that can mediate rejection of bone marrow since CD8+ T cells in recipients pre-sensitized to alloantigen can reject the bone marrow by an unknown mechanism [57]. In addition, as in solid organ transplants, antibodies can induce rapid bone marrow graft rejection in presensitized recipients [58]. These animal studies have been used to demonstrate the types of cells and the effector mechanisms that are involved in graft rejection. 3.2. Immune Reconstitution A major hurdle limiting the efficacy of alloHSCT is prolonged immune suppression in patients due to factors including cytoreductive conditioning, the immunosuppressive drugs to prevent GVHD and the small proportion of transplanted T cells compared to size of the T cell compartment in an immunocompetent person. In addition, lymphoid hypoplasia, resulting from suppression of both thymic dependent and independent expansion of lymphocytes, is associated with acute GVHD [59–63]. These factors leave the patient susceptible to a number of opportunistic infections. Unfortunately, very few pre-clinical models that have been developed to study these opportunistic infections in the allogeneic transplant setting and the complicating effects of GVHD. Studies in rodent models have been employed to investigate therapies to enhance immune reconstitution in young and more importantly mature mice, as it has been demonstrated that conditioning protocols for alloHSCT can damage the cells that support lymphohematopoiesis [64–66]. A variety of experimental approaches have been examined in mice as a means to enhance immune reconstitution following alloHSCT. These approaches include the administration of keratinocyte growth factor (KGF) prior to transplant to protect the thymus resulting in increased immune reconstitution post-transplant [67–69], the administration of the T cell growth factor, interleukin-7, to increase thymopoiesis [70–72] although it also enhances peripheral T cell expansion [72–74], and sex steroid hormone blockade to remove negative regulators of thymopoiesis [75–77].

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation 

The mouse model has also allowed investigators the opportunity to explore the consequences of improved T cell reconstitution post-transplant. Responses to infectious diseases following alloHSCT and the promotion or aggravation of GVHD can be tested to examine the function of the immune system following these experimental manipulations. However, it has been shown that administration of growth factors after alloHSCT can promote peripheral expansion of T cells that may exacerbate GVHD [78–80]. 3.3. Acute GVHD Graft-versus-host disease (GVHD) is a major complication of alloHSCT. Shortly after the development of experimental bone marrow transplantation as a cure for radiation sickness, it was recognized that the animals developed a wasting syndrome with tissue destruction to the gut, liver and skin. Based on the work performed in animal models, Billingham put for a set of principles necessary for the development of GVHD [81]. These principles are (1) GVHD requires that the host must be incapable of adequately rejecting the graft; (2) the graft must contain immunocompetent cells and (3) there must be incompatibilities in transplantation antigens between the host and donor such that the host tissues express antigens not present on the donor cells [81]. In general these principles have been upheld through vigorous investigation into the biology of GVHD. However, it has been shown that GVHD can develop after syngeneic or autologous transplants due to a loss of tolerance in the reconstituted immune system [82]. Mouse models have been instrumental in understanding the role of effector mechanisms in acute GVHD. Evaluation of clinical trials has provided insight into the roles of cell populations and their products on alloHSCT outcome, however there is a limit to the definitive cause and effect that can be derived. Mouse models provide a means for in-depth evaluation which may lead to better and more specific targeting of therapeutic strategies in the clinic. For example, work in mouse models of GVHD have not only been able demonstrate the critical role of cytokines, Tumor Necrosis Factor-a (TNFa), and interferon-g (IFNg) in acute GVHD and GVT, but have led to the knowledge that only the production of these cytokines by specific T cell subpopulations are responsible for potentiating GVHD and GVT, respectively. Thus this provides a greater understanding of how TNFa blockade modulates GVHD disease [83–86] and why neutralization of IFNg [87, 88] can lead to deleterious effects in both GVHD and GVT. The commonly used mouse models for acute GVHD have been well characterized for the requirement of strain specific doses of irradiation for myeloablation, the extent of major and minor histocompatibility differences, the involvement of CD4+ and/or CD8+ T cells in pathophysiology, severity of disease and prevalence of organ-specific disease. A few of the most frequently utilized strain combinations for GVHD are listed in Table 44-1. 3.4. Chronic GVHD Mouse models of acute GVHD provide a wide range of clinically relevant BMT models that allow for the investigation of either CD4+ and/or CD8+ T cell mediated acute GVHD, as well as the selection of histocompatibility antigen mismatch. In addition, the spectrum of clinical features of acute GVHD

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Table 44-1.  Mouse models of GVHD. Donor

Host

BALB/c (H2d)

C57BL/6 (H2b)

b

d

Histocompatibility differences

Disease manifestations

Primary T cell dependence

MHC I, II, mHAs

Acute GVHD

CD4+

C57BL/6 (H2 )

BALB/c (H2 )

MHC I, II, mHAs

Acute GVHD

CD4+

B10.BR (H2d)

C57BL/6 (H2b)

MHC I, II

Acute GVHD

CD4+

b

b

C3H.SW (H2 )

C57BL/6 (H2 )

mHAs

Acute GVHD

CD8+

Bm1 (H2bm1)

C57BL/6 (H2b)

MHC I

Acute GVHD

CD8+

C57BL/6 (H2 )

MHC II

Acute GVHD

CD4+

BALB/c (H2d)

MHC I, II, mHAs

Bm12 (H2

bm12

FvB (H2q) d

)

b

d

CD4+ or CD8+

B10.D2 (H2 )

BALB/c (H2 )

mHAs

Chronic GVHD-scleroderma

CD4+

LP/J (H2b)

C57BL/6 (H2b)

mHAs

Chronic GVHD-scleroderma

CD4+

Chronic GVHD-SLE

CD4+

d

DBA (H2 )

(DBA x C57BL/6) MHC I, II, mHAs F1 (H2d/b)

MHC Major histocompatibility complex; mHA minor histocompatibility antigen; SLE systemic lupus erythematosus

in mice resembles the spectrum seen in humans. Unfortunately, the situation is not the same for chronic GVHD. The five most common murine models of chronic GVHD are all dependent on CD4+ T cells and only two models use conditioning and bone marrow transplant. Of these two models, the B10. D2 donor cells into BALB/c recipient, after sub lethal or lethal irradiation, is probably the most commonly utilized strain combination. Both the B10.D2 → BALB/c and the second transplant model, LP/J → C56BL/6, model the scleroderma features of chronic GVHD that is observed clinically in patients [89, 90]. 3.5. Graft Versus Tumor Graft versus Leukemia (GVL) or the broader term Graft versus Tumor (GVT) refers to the anti-tumor response that is associated with alloHSCT. Since the earliest rodent studies it has been recognized that GVL/GVT is associated with the occurrence GVHD [5, 6]. While the antigenic targets for GVL/GVT are not always clear in MHC-matched HSCT (it is speculated to be minor HAs and/or tumor associated or tumor-specific antigens), alloantigens in MHC-mismatched transplants may elicit potent anti-tumor responses. Mouse tumor models allow for study of GVL/GVT in both MHC-matched and MHCmismatched models. However, a caveat to all tumor studies in mice is the use of tumor cell lines that are immunogeneic to some degree in syngeneic strains, as many of these tumors were initiated by viruses, and because the hosts are not tolerized/tolerant to the tumor-associated antigens. In addition, many but not all of the tumor cell lines commonly used in GVL/GVT studies do not express MHC II and thus are not recognized by CD4+ T cells and not susceptible to CD4+ T cell mediated killing.

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation 

4.  Insights from Mouse Studies into the Role of Non-T Cell Lymphoid Populations on Recovery After Allogeneic HSCT Murine models have been instrumental in delineating the role of non-T cell lymphoid populations, present in both the recipients and in the donor graft, which can affect the outcome of graft rejection, induction of tolerance, GVHD and GVT after alloHSCT. 4.1. T Regulatory Cells CD4+ T regulatory cells are defined by the expression the IL-2 receptor alpha chain (CD25) and the restricted expression of the transcription factor, Foxp3. These cells have a potent immunoregulatory suppressor activity that has been shown to have profound effects in mouse models. Depletion of T regulatory cells in the mouse can be accomplished by the administration of the PC61, a monoclonal antibody that recognizes CD25. Reducing the number of CD25+ cells from the graft or in the recipient immediately after alloHSCT promotes GVHD in several mouse models [91–94]. Conversely, adding more CD4+CD25+ T cells to the graft can reduce GVHD in both acute and chronic murine models [92–94]. Interestingly, GVT does not appear to be diminished by the addition of Treg cells [95]. In addition to reducing GVHD, donor-derived Treg cells have been shown to increase engraftment and tolerance of MHC disparate allografts after sub lethal conditioning [96, 97]. Expression of CD62L is critical for the protective effects of infused donor-derived Treg cells, suggesting that homing of these cells to secondary lymphoid tissues and inhibition of alloreactive T cell priming is the mode of action of this cell population during GVHD [96, 98]. The adoptive cell transfer studies with Treg cells usually used ex vivo activated and expanded populations due to the low frequency of this population in mice, as well as man. This expansion protocol provides evidence of feasibility and potential efficacy in human trials but it is worth noting that freshly isolated Treg cells are also capable of inhibiting GVHD lethality [99] which demonstrates that passenger Tregs in the graft or host-derived Tregs may play an important function in suppressing GVHD, which could be manipulated to improve outcome. Immunosuppressive drugs given to prevent or control GVHD have also been shown to affect Treg cell expansion and function. In animal studies, cyclosporine A can lead to a reduction in donor Treg cell proliferation and function resulting in increased GVHD severity [100], while rapamycin can expand functional murine Treg cells in ex vivo culture [101]. The hematopoietic graft is not the only source of Treg cells. CD4+CD25+ Treg cells are less radiosensitive than conventional T cells and host-derived Treg cells are found at a higher frequency in recipient animals then other T cell populations [92]. The presence of this cell population in conditioned hosts can also be advantageous to the outcome of alloHSCT. Recipient-derived CD4+CD25+ Treg cells also can reduce acute and chronic GVHD in murine models [92, 102], inhibit NK cell-mediated BM graft rejection [103] as well as improve immune reconstitution and GVT [102].

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4.2. Gamma/Delta (gd) TCR T Cells gd TCR T cells comprise a small proportion of the T cell population (0.5–3% in the PB and spleen of mice). In experimental murine models, recipient gd T cells can reject allogeneic hematopoietic cell grafts [104, 105] while donortype gd TCR T cells can promote engraftment in mice [105, 106] and can promote GVHD [107]. Unlike recipient ab TCR T cells that may reduce GVHD by rejecting donor T cells, it has been proposed that recipient gd TCR T cells promote GVHD by activating stimulating host APCs even in heavily irradiated hosts [108] however, another investigator found no role for recipient gd TCR T cells in GVHD [92]. 4.3. Natural Killer Cells Unlike B and T cells, natural killer (NK) cells are primitive immune cells that provide one of the first lines of defense during an immune response. NK cells lack the T-cell markers CD3 and TCR and express NK specific markers (NK1.1 and DX5 in mouse, CD56 in humans). Although early mouse studies suggested host NK cells could reject donor BM in a non-MHC restricted manner [44, 45] it is now recognized that NK cells express inhibitory and activating receptors on their cell surface, that are directed to MHC and other cellular determinants on target cells that are critical for target identification and subsequent NK cell mediated killing (reviewed in [109]). Murine models have also been used to demonstrate that adoptive transfer of activated NK cells early after transplant inhibit GVHD and promote GVT [110] although, administration of activated NK cells later in the course of GVHD could exacerbate the disease [110]. Additional studies in animals are needed to determine how one can best exploit the potential benefit of NK cells in alloHSCT. 4.4. NKT Cells Natural Killer T (NKT) cells are characterized by the expression NK cell markers and a TCR. NKT cells can be stimulated through the TCR by recognition of glycolipid antigens and peptides presented in the non-classical MHC I molecule CD1d. Upon stimulation these cells can produce IFNg or IL-4 and can enhance or suppress responses in a wide range of immunological models. Murine studies have demonstrated that both donor and host NKT cells can attenuate GVHD and that this protection is dependent on the production of IL-4 [46, 111, 112] of invariant TCR type NKT or IFNg [113] by CD8+ NKT cells. While NKT cells suppressed GVHD in these experimental models, NKT cells can also provide anti-tumor activity to the graft [113, 114] and promote graft rejection by the recipient [46].

5. Concluding Remarks Animal models have been instrumental in the development of allogeneic as a medical therapy for the treatment of a number of hematologic, immunologic and oncology related disorders in humans. Animal models remain an important tool in pre-clinical and translational medicine and mouse models, in particular, continue to provide insight into the mechanisms underlying cellular therapy and greater understanding of the basic workings of the immune and hematopoietic systems.

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation 

Acknowledgments.  The work from the authors’ laboratories was supported by NIH R01 CA93527, R01 HL089905, R01 CA102282 and R01 AG022661.

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L.A. Welniak and W.J. Murphy 92. Anderson BE, McNiff JM, Matte C, Athanasiadis I, Shlomchik WD, Shlomchik MJ (2004) Recipient CD4+ T cells that survive irradiation regulate chronic graftversus-host disease. Blood 104(5):1565–1573 93. Cohen JL, Trenado A, Vasey D, Klatzmann D, Salomon BL (2002) CD4(+) CD25(+) immunoregulatory T Cells: new therapeutics for graft-versus-host disease. J Exp Med 196(3):401–406 94. Hoffmann P, Ermann J, Edinger M, Fathman CG, Strober S (2002) Donor-type CD4(+)CD25(+) regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J Exp Med 196(3):389–399 95. Edinger M, Hoffmann P, Ermann J et al (2003) CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med 9(9):1144–1150 96. Taylor PA, Panoskaltsis-Mortari A, Swedin JM et al (2004) L-Selectin(hi) but not the L-selectin(lo) CD4+25+ T-regulatory cells are potent inhibitors of GVHD and BM graft rejection. Blood 104(12):3804–3812 97. Hanash AM, Levy RB (2005) Donor CD4+CD25+ T cells promote engraftment and tolerance following MHC-mismatched hematopoietic cell transplantation. Blood 105(4):1828–1836 98. Ermann J, Hoffmann P, Edinger M et  al (2005) Only the CD62L+ subpopulation of CD4+CD25+ regulatory T cells protects from lethal acute GVHD. Blood 105(5):2220–2226 99. Taylor PA, Noelle RJ, Blazar BR (2001) CD4(+)CD25(+) immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade. J Exp Med 193(11):1311–1318 100. Zeiser RS, Nguyen VH, Beilhack A et al (2006) Inhibition of CD4+CD25+ regulatory T cell function by calcineurin dependent interleukin-2 production. Blood 108(1):390–399 101. Battaglia M, Stabilini A, Roncarolo MG (2005) Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood 105(12):4743–4748 102. Trenado A, Charlotte F, Fisson S et al (2003) Recipient-type specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia. J Clin Invest 112(11):1688–1696 103. Barao I, Hanash AM, Hallett W et  al (2006) Suppression of natural killer cellmediated bone marrow cell rejection by CD4+CD25+ regulatory T cells. Proc Natl Acad Sci USA 103(14):5460–5465 104. Xu H, Exner BG, Cramer DE, Tanner MK, Mueller YM, Ildstad ST (2002) CD8(+), alphabeta-TCR(+), and gammadelta-TCR(+) cells in the recipient hematopoietic environment mediate resistance to engraftment of allogeneic donor bone marrow. J Immunol 168(4):1636–1643 105. Blazar BR, Taylor PA, Bluestone JA, Vallera DA (1996) Murine gamma/deltaexpressing T cells affect alloengraftment via the recognition of nonclassical major histocompatibility complex class Ib antigens. Blood 87(10):4463–4472 106. Drobyski WR, Majewski D (1997) Donor gamma delta T lymphocytes promote allogeneic engraftment across the major histocompatibility barrier in mice. Blood 89(3):1100–1109 107. Blazar BR, Taylor PA, Panoskaltsis-Mortari A, Barrett TA, Bluestone JA, Vallera DA (1996) Lethal murine graft-versus-host disease induced by donor gamma/ delta expressing T cells with specificity for host nonclassical major histocompatibility complex class Ib antigens. Blood 87(2):827–837 108. Maeda Y, Reddy P, Lowler KP, Liu C, Bishop DK, Ferrara JL (2005) Critical role of host gammadelta T cells in experimental acute graft-versus-host disease. Blood 106(2):749–755 109. Barao I, Murphy WJ (2003) The immunobiology of natural killer cells and bone marrow allograft rejection. Biol Blood Marrow Transplant 9(12):727–741

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation  110. Asai O, Longo DL, Tian ZG et al (1998) Suppression of graft-versus-host disease and amplification of graft-versus-tumor effects by activated natural killer cells after allogeneic bone marrow transplantation. J Clin Invest 101(9):1835–1842 111. Zeng D, Lewis D, Dejbakhsh-Jones S et  al (1999) Bone marrow NK1.1(-) and NK1.1(+) T cells reciprocally regulate acute graft versus host disease. J Exp Med 189(7):1073–1081 112. Lan F, Zeng D, Higuchi M, Huie P, Higgins JP, Strober S (2001) Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: “natural suppressor” cells. J Immunol 167(4):2087–2096 113. Baker J, Verneris MR, Ito M, Shizuru JA, Negrin RS (2001) Expansion of cytolytic CD8(+) natural killer T cells with limited capacity for graft-versus-host disease induction due to interferon gamma production. Blood 97(10):2923–2931 114. Morris ES, MacDonald KP, Rowe V et al (2005) NKT cell-dependent leukemia eradication following stem cell mobilization with potent G-CSF analogs. J Clin Invest 115(11):3093–3103

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Chapter 45 Dendritic Cells Jacalyn Rosenblatt and David Avigan

1. Introduction Dendritic cells (DCs) represent a complex network of antigen-presenting cells that play a crucial role in the initiation of primary immunity, as well as maintaining the balance between immune tolerance and reactivity [1]. The modern field of DC biology was initiated in 1973 by Steinman and Cohn, who identified a subpopulation of murine splenocytes that had distinctive morphologic and phenotypic characteristics and powerfully stimulated T cell responses [2]. DCs have subsequently been described as the most potent antigen-presenting cells, which demonstrate the unique capacity to induce primary immune responses. Stimulation of naive T cells requires antigen presentation in the context of co-stimulatory and adhesion molecules, which serve as secondary signals needed for the activation of primary immunity. Antigen-presenting cells, such as B cells and macrophages, are effective in maintaining immune responses, but are incapable of initiating primary responses to novel antigens. In contrast, DC richly express MHC class I, II, co-stimulatory and adhesion molecules and are uniquely potent in initiating cellular immunity (Fig. 45-1) [3–6]. DCs also mediate humoral responses through the activation of helper T cells and direct effects on B cells [7]. DC activation of innate immunity has been demonstrated through their effects on NK cells and NKT cells [8, 9]. DCs have emerged as an area of intense interest in the fields of tumor immunotherapy and transplant biology. Although DCs represent only a small fraction of circulating mononuclear cells, large number of DCs can be generated from precursor populations derived from blood, marrow, and cord blood, allowing for the potential clinical use for immunotherapy [10–12]. A major focus of tumor immunotherapy has been the use of DCs to reverse tumor-induced anergy by the presentation of antigen in the context of co-stimulatory molecules [13]. Tumor cells evade host immunity through a variety of mechanisms including the presentation antigen in the absence of co-stimulation, secretion of inhibitory cytokines that suppress T cell function and DC maturation, and

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_45, © Springer Science + Business Media, LLC 2003, 2010

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Fig. 45-1.  Antigen presentation

the associated increase in inhibitory cells such as regulatory T cells. In contrast, DCs with a normal phenotype may be generated ex vivo from patients with malignancy. DCs, manipulated to express tumor antigens, have been shown to induce tumor-specific immunity in preclinical animal and human studies, and are now being studied in clinical trials [14]. DCs are also essential for maintaining tolerance toward self antigens and may mediate rejection or tolerance towards allogeneic tissue [15–18]. Distinct DC populations participate in the process of clonal deletion of autoreactive T cell clones and the establishment of peripheral tolerance by preventing expansion and activation of those clones that escaped central deletion in the thymus [19]. As such, DCs are thought to play a crucial role following allogeneic hematopoietic stem cell transplantation with the capacity to promote or inhibit graft versus host disease dependent on the phenotypic characteristics of the DC population [20]. Recent studies have sought to exploit this issue by manipulating DCs to minimize the risk of GVHD while maintaining the potent graft versus disease effect. Understanding the pattern DC reconstitution post-transplant and the balance between donor and recipient cells is crucial for this endeavor [21]. This review will focus on hematopoietic development of DC populations and the intimate link between the circumstances of DC development and the nature of its impact on cellular immunity. The use of ex vivo generated DC populations for tumor immunotherapy will be discussed including attempts to translate these finding into the clinical setting. The role of DCs in eliciting tolerance will be reviewed. Studies examining the role of DCs in allogeneic transplantation and the risk of graft versus host disease will be discussed.

Chapter 45  Dendritic Cells 

2. DC Subsets DC arise from precursor populations that differentiate along distinct pathways of maturation [22, 23]. In mice, CD11c+ DCs are found in the lymph nodes, spleen, and thymus and are subdivided by the expression of CD4 and CD8, the latter of which is expressed by thymic DCs responsible for deletion of autoreactive lymphocytes [24]. Other subsets include skin-derived DCs (Langerhans Cells) and tissue interstitial DCs which manifest an immature phenotype and migrate to draining lymph nodes upon activation. In humans, two primary pathways of DC development include the generation of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) [25]. mDCs are found at sites of antigen capture in the peripheral tissues, in the secondary lymph nodes at sites of T cell interaction, and in the circulation. In the skin, mDCs are differentiated into classic Langerhans cells (LCs) and interstitial DCs found in the dermis [26]. These two subsets express different cytokines and respond to unique chemokine signaling. Dermal DCs promote the differentiation of B cells into plasma cells, stimulate CD4 mediated help of immunoglobulin class switching, and migrate to the area of the LN adjacent to the B cell follicles. In contrast, LCs are far more potent in inducing primary CTL responses and migrate to T cell areas of the draining lymph node. Blood-derived mDCs are characterized by DR+/lineage-/CD11c+ expression. These cells are likely a reservoir for tissuebased DCs and are often referred to as DC1 because they characteristically induce expression of TH1 cytokines by reactive T cell populations. pDCs are characterized by DR+/lineage-/CD123+ expression, further divided into CD2+/ and CD2− subsets and are potent secretors of type I IFN in response to viral pathogens [27, 28]. In contrast to mDCs, pDCs do not prominently express IL-12 and may polarize T cells towards a TH2 phenotype. However, depending on the nature of the milieu present during their development and T cell interaction, these subsets may be associated with the expansion of activated or suppressor T cells [20, 29, 30].

3. Phenotypic Characterization of Immature DCs DCs pass through a complex life cycle, in which their phenotypic characteristics evolve with maturation (Fig. 45-2) [1, 3, 23, 31, 32]. DCs originate from marrow progenitors and subsequently migrate to sites of exposure to foreign antigens [31]. In murine studies, DC’s have been shown to differentiate from myeloid and lymphoid precursor populations [33]. Lymphoid-derived DCs express CD8a and share a common precursor with T cells, B cells and natural killer (NK) cells [34, 35]. Myeloid DC progenitor are defined by the absence of CD8a, expression and exquisite sensitivity to granulocyte–macrophage colony stimulating factor (GM-CSF). In humans, DCs differentiate from marrow-derived CD34+ precursors and migrate to the sites of antigen uptake [10, 20, 36]. DCs are found in the epithelial surface of the skin, gastrointestinal tract, and lung, as well as the interstium of all organs with the exception of the immunoprivileged sites of the brain, testis, and eye [37]. The precise mechanisms that are responsible for the recruitment and localization of DCs in tissue has not been fully elucidated, but appears to be related to intrinsic features of the progenitor cells, as well as the release of cytokine and inflammatory signals [38]. For example, the presence

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Fig. 45-2.  Dendritic cell life cycle

of a “skin-homing receptor” on a subset of CD34+ cells is associated with their subsequent migration to the epidermis and their acquisition of phenotype of a Langerhans cell (LC) [39]. Expression of E-cadherin by LCs facilitates the binding of these cells into the epidermal layer. The migration of DCs into the brochoepithelium is induced by the presence of aerosolized lipopolysaccharide (LPS), soluble protein, bacterial or viral products, or the release of GM-CSF secondary to local inflammation or by pulmonary tumors. In a murine model, administration of FLt3L resulted in the accumulation of immature DCs in the bone marrow, gastrointestinal lymphoid tissue, peripheral blood, peritoneal cavity, liver, lymph nodes, lung, spleen, thymus, and dermis which strongly express class II, CD 11c, DEC205, and CD86 [40–42]. LCs represent a well-characterized immature DC population found in the skin, which express CD1a, Lag antigen, E-cadherin, and contains cytoplasmic inclusion bodies known as Birbeck granules (Fig. 45-3) [3, 31]. DCs residing in other tissues do not share all of these morphologic characteristics, but demonstrate similar properties with regard to antigen processing and presentation. The morphology of immature DCs is characterized by a highly organized cytoskelatin, slow motility, and the absence of prominent dendrites. They express low levels of co-stimulatory molecules and are poor stimulators of allogeneic T cell proliferation. Immature DCs demonstrate potent capacity to internalize exogenous antigens [38, 43]. Studies of freshly isolated LCs and bone marrow-derived immature DCs demonstrate the ability to internalize protein latex microspheres, bacille Calmette-Guerin (BCG), colloidal gold, apoptotic and necrotic cell fragments, heat shock proteins, viral and bacterial products, as well as whole bacteria. Phagocytic properties of DCs are distinct from that seen with macrophages [3, 38, 43, 44]. Macrophages are responsible for the scavenging and clearance of foreign material, which are transferred to cytoplasmic lysosomal compartments for degradation. DCs are more selective, demonstrating the uptake of smaller quantities of antigen, which are incorporated into MHC class Il compartments for subsequent antigen presentation. DC-mediated antigen uptake

Chapter 45  Dendritic Cells 

Fig. 45-3.  Phenotypic properties of immature and mature dendritic cells

Fig. 45-4.  Functional characteristics of immature and mature dendritic cells

occurs via both receptor-mediated endocytosis as well as macropinocytosis. Immature DCs express Fc receptors, complement, and mannose receptors thought to mediate internalization of exogenous proteins [45, 46]. In contrast to macrophages, DCs express the avB5 integrin, which is crucial for the uptake of apoptotic bodies and the subsequent presentation of antigen along the class l pathway [47]. An essential component facilitating endocytosis is the presence of DEC205, a receptor homologous to the macrophage mannose receptor [45, 48]. Antigen-bound DEC 205 is transferred via coated pits into endosomal compartments for subsequent processing and presentation in the context of MHC class II molecules. Endocytosis mediated by this pathway has been demonstrated to be 100-fold more effective than bulk macropinocytosis [49]. Mannosylyation of the ingested antigen markedly increases in immunogenicity with an increase in the levels of T cell responsiveness by 200- to 10,000fold (Fig. 45-4).

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4. Phenotypic Characteristics of Mature DC Upon maturation, DCs migrate as veiled cells via the afferent lymphatics to regional lymph nodes and designated areas of the spleen, which serve as the site of antigen recognition and T cell activation [1, 3, 31]. During this process, DCs undergo fundamental changes in their phenotypic and functional characteristics. DCs do not recirculate in the efferent lymphatics, and it is thought that those cells that do not present antigen undergo T cell-mediated apoptosis. A variety of factors have been demonstrated to induce the maturation and migration of DCs. Receptors for GM-CSF are prominently expressed by immature DCs and GM-CSF has been shown to support the differentiation, viability, and long-term survival of DC [6, 12, 36, 50–53]. Innate immunity against infectious pathogens are mediated through signaling pathways associated with the toll-like receptors (TLRs) and other receptor families, and is intimately involved in the maturation and development of distinct DC subsets [54]. Response to microbial products, such as lipids (TLR 1, 2, 4, and 6) and nucleic acids (TLR 3, 7, 8, and 9), are mediated by individual TLR receptors. For example, exposure to oligodeoxynucleotides containing CpG motifs signals via TLR9 and augments DCs maturation as manifested by a transient increase in antigen processing followed by loss of capacity to internalize and process exogenous protein antigens [55]. These signaling pathways may act synergistically to activate DCs and promote maturation and the stimulation of inflammatory cytokines such as IL-12 [56]. Myeloid DCs express TLR 1-6 and 8 while plasmacytoid DCs express TLR 7 and 9 [54]. Cell populations of the innate system secrete cytokines that dictate the nature of DC polarization and maturation [22]. For example, IFNg expression by NK cells or plasmacytoid DCs support DC activation, IL-12 production, and the stimulation of TH1 responses. Similarly, mature DC activate innate immune cells such as NKT cells to express IFNg. In this manner, the innate and adaptive immune system have developed a complex interaction that modulate host responses [20]. Material from dying cells has also been shown to induce DC maturation either directly or by stimulation of other accessory cells such as macrophages. Heat shock proteins (HSP), high mobility group box 1 (HMGB1), b-defensin, and uric acid derivatives have been shown to activate DCs via TLR mediated signaling and other pathways [57–59]. Maturation signals also impact properties of cell adhesion promoting the migration of maturing DCs. For example, loss of E-cadherin by LCs is associated with their capacity to migrate from skin epithelium and travel toward lymphocyte-rich areas [60, 61]. Maturing DCs downregulate CCR6 and lose sensitivity towardmacrophage-inhibiting protein (MIP)-3 [3, 62, 63]. Conversely, maturing DCs upregulate expression of CCR7 and demonstrate increased sensitivity towards chemokines MIP-3a and 6Ckine [64]. Expression of these chemokines is found in lymphatic vessels and the T cell-rich paracortical areas of the draining lymph nodes and mediates migration of DCs through the afferent lymphatics [53, 65]. Mature DCs release MIP-3a and 6Ckine, further amplifying the effect, as well as attracting naive T cells to the site of antigen presentation [66]. Absence of MIP-3a and 6Ckine expression is associated with deficient homing of DCs and T cells to lymphatic issue [67, 68]. Adenoviral transfection of tumor cells with MIP-3o resulted in the migration of DCs into the tumor bed and inhibition of tumor growth [69].

Chapter 45  Dendritic Cells 

With the onset of maturation, there is a transient increase in the production of cytoplasmic class II molecules and antigen loaded during this period is particularly immunogenic [70]. Terminal maturation associated with a decrease in the synthesis of MHC molecules, and MHC class II are thrust onto the cell surface resulting in stable presentation of the incorporated antigen. Localization of co-stimulatory molecules and peptide–MHC complexes in cytoplasmic compartments is subsequently translated to clustering of these molecules on the membrane surface for antigen presentation. In contrast, exposure to IL-10 inhibits the translocation of antigen expressing class II molecules onto the plasma membrane [71]. Mature DCs are distinguished by the prominent expression of MHC class I, II, adhesion and co-stimulatory molecules [4, 5, 7, 72, 73]. Ligation of corresponding molecules on T cells, most notably CD28, provides the essential secondary signals for the initiation of primary immune responses. Interference with this crucial dialog through antibody blockade abrogates DC-mediated T cell stimulation. Although signaling occurs via the entire network of adhesion and co-stimulatory molecules, disruption of CD86 binding appears paramount, which results in the reduction in T cell stimulation. Mature DCs are distinguished morphologically by the presence of prominent dendrites that facilitate motility and provide a large surface area for the simultaneous interaction with multiple T cells [1]. Morphologic changes are mediated by the actin-bundling protein p55 fascin. Fascin expression is augmented by cytokines that induce DC maturation and has been associated with increased capacity to stimulate T cell proliferation [74]. Mature DCs are distinguished by low buoyant density, lack of adherent properties, absence of expression of lineage-specific surface markers characteristic of T, B, and NK cells, macrophages, and the presence of CD83 in some populations [3, 75]. Expression of Fc receptors and nonspecific esterase is downregulated and Birbeck granules are no longer detected. Mature DCs lack phagocytic capacity and are incapable of processing and presenting exogenous protein. Mature cells are far more effective than macrophages and B cells in stimulating mitogen or allogeneic T cell proliferation and induce significantly higher levels of IL-2 secretion [67, 68, 76, 77]. Unlike other antigen-presenting cells, DCs are uniquely capable of inducing CD8 proliferative and CTL responses in the absence of CD4 helper cells [78]. Mature DCs potently stimulate T cell responses induce TH1 responses following repetitive stimulation of T cells. In contrast, co-culture of T cells with immature DCs resulted in upregulation of the inhibitory molecule CTLA-4, lack of proliferation, and an inability for the T cells to subsequently respond to stimulation with mature DCs [79].

5. DC:T Cell Interactions Endogenously generated antigens are characteristically presented along the class I pathway to CD8+ T cells while exogenous proteins are processed and presented to helper T cells via the class II pathway. DCs may also perform cross presentation in which exogenous protein antigens are presented along the class I pathway [80–83]. DCs initially aggregate with T cells in an antigen-independent manner in an effort to survey the repertoire for T cells with the capacity to recognize the presented antigen. IL-I5 induces the release of chemokines which mediate the migration of T cells to the site of

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antigen presentation, while IL-14 facilitates T cell clustering around the DC [84]. Adhesion molecules such as CD1a, CD54, and CD58 are responsible for DC–T cell binding, which is strengthened by the presence of antigen recognition [85]. T cell activation is significantly impaired by antibody blockade of these interactions or genetic defects in which animals lack the full complement of adhesion molecules. Danger signals expressed at sites of inflammation induce DC maturation [86, 87]. The activation of T cells is mediated through the ligation of co-stimulatory molecules and the subsequent release of a complex network of cytokines (Fig. 45-5). CD40L and IL-12 play an important role in DC-mediated stimulation of T cells [88, 89]. Upon binding to DCs, T cell expression of CD40L is upregulated. This results in increased expression of MHC class II adhesion and co-stimulatory molecules and prolonged DC survival [6, 90–95]. Ligation of RANK, a member of the TNF receptor family, and release of IFNg in response to DC–T cell binding also results in the secretion of IL-12 [96]. IL-12 release is also stimulated by the antigenspecific activation of T cells and exposure of the antigen-presenting cells to inflammatory factors such as TNF, segmental allergen, challenge (SAC), and LPS [97]. IL-12 augments T and NK cell cytotoxicity and biases T cell development towards the TH1 phenotype that is associated with the release of IFNg [98, 99] IL-12 has been shown to be far more potent than IL-2 in amplifying antigen-specific responses mediated by DCs activation of T cells [73]. Exogenous IL-12 has been demonstrated to replace the need for helper T cells in generating effective immune responses directed against tumor lines of poor immunogenicity [100]. Maturation along the DC1 or DC2 pathways is determined by exposures occurring during differentiation such as presence of bacterial or helminth products, respectively [101]. TLRs are expressed by DCs and bind bacterial and viral products and stimulate DC maturation and expression of stimulatory cytokines such as IL-12. TLRmediated signaling operates in concert with cytokines via the TNF receptor family to stimulate DC activation and proliferation [86]. In several animal models, exposure to necrotic cells stimulates DC maturation and activation [102]. Intracellular uric acid that is released as a part of tissue necrosis has been shown to contribute to the DC response. This response may be a critical step in promoting pathways associated with graft versus host disease and transplant rejection.

Fig. 45-5.  DC-T cell interaction

Chapter 45  Dendritic Cells 

IL-12 enhances T cell proliferation following stimulation with DCs pulsed with tumor peptide, and transfection of murine DCs with IL-12 gene markedly upregulates their capacity to induce tumor-specific CTL responses. DC-mediated signaling though OX40 and 4-1BB on T cells provides proliferative stimuli to reactive T cell populations [103]. Another approach to enhance DC mediated stimulation is through the suppression of inhibitory signaling mediated by SOCS1 using siRNA [104]. Stimulatory signals such as CD40 deliver antiapoptotic effects that facilitate DC survival and promote DC capacity to stimulate T cells [105]. In contrast, a variety of factors such as IL-10 inhibit DC maturation resulting in the downregulation of co-stimulatory molecule expression, and the suppression of the release of inflammatory cytokines such as IL-1, IL-6, IL-8, TNF, and GM-CSF [106]. DCs generated in the presence of IL-10 induce anergy in potentially reactive T cell populations that is not reversed upon exposure to IL-10 naive DCs. In contrast, DCs that mature in the absence of IL-10 are subsequently resistant to its inhibitory effects. IL-10 secretion has been demonstrated by a variety of malignancies including melanoma, renal cell, and colon cancer, and may play an important role in the tumor evasion of host immunity. DC subsets also impart unique homing characteristics on the responding T cell population [92].

6. Ex Vivo Generation of DCs DCs have been generated from CD34+ or progenitor populations that undergo differentiation through in  vitro exposure to cytokines [36, 51, 93, 94]. In murine models, exposure of bone marrow mononuclear cells to GM-CSF induces the presence of cells with a DC phenotype as characterized by the strong expression of co-stimulatory molecules with potent immunostimulatory capacity. In humans, CD34+ cells cultured with GM-CSF give rise to mixed myeloid colonies of DCs and macrophages [36]. Stem cell factor (SCF) and FLt3L promotes the recruitment and proliferation of early progenitors resulting in an increase in the number and size of the colonies [95]. TNFa acts in concert with GM-CSF in promoting the differentiation of DCs giving rise to pure DCs as well as mixed myeloid colonies. Large yields of DCs may be generated from CD34+ cells isolated from cord blood, bone marrow, and mobilized peripheral blood stem cells that are cultured in the presence of GMCSF and TNF a [10, 21, 22, 51, 135] IL-4 suppresses monocyte maturation and improves the purity of DCs in the resultant population [107]. SCF and Flt3L promote the expansion of early progenitor populations increasing the overall cell yields in suspension cultures. Exposure to Flt3L also facilitates the generation of CDla+ and CDla-immature DC from CD34+ progenitors [108, 109]. DCs derived from CD34+ progenitors that pass through intermediate stages of differentiation with the capacity to mature into DCs or macrophages dependent on the nature of cytokine exposure [110]. Mature myeloid and plasmacytoid DCs can be directly isolated from peripheral blood using CMRF-44 or other antigens [111, 112]. Myeloid DCs (CD11c+) may also be generated in significant numbers in vitro from partially differentiated monocyte precursors in peripheral blood [113]. Plastic adherent PBMC cultured with GM-CSF, TNFx, and IL-4 generate cell populations that are potent in allogeneic mixed leukocyte reaction (MLR) and express CD83

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and CDla [84]. Alternatively, DC precursors may be isolated by magnetic bead isolation of CD14+ cells that are then differentiated into DCs [114]. Monocyte precursors cultured with GM-CSF and IL-4 yield DCs with an immature phenotype that maintain the capacity to internalize and present exogenous protein [115]. Blood precursor populations may also differentiate into an LC phenotype under the influence of TGFb or IL-15 [99, 116] Plasmacytoid DCs are derived from CD11c− cells and their growth and maturation is supported by GM-CSF, IL-3, and TNFa [117]. Some studies have demonstrated that CPG ODN promote plasmacytoid DCs to adopt an immunosuppressive phenotype characterized by an increased expression of IL-10, TGFb, and IL-6 and their capacity to stimulate the expansion of regulatory T cells [29]. Alternatively, CPG has also been shown to increase expression of IFNa and the capacity to stimulate B cell activation [118]. A variety of agents promote terminal maturation of myeloid DCs in vitro with the concomitant loss of phagocytic capacity, increased expression of the co-stimulatory markers CD80, CD86, and CD40, increase in the maturation marker, CD83, and enhanced ability to stimulate T cell responses. These include TNFa, LPS, IL-3, CD40L, LPS, and PG-E2. Exposure to monocyteconditioned media potently induces terminal DC maturation and activation which persists following withdrawal of cytokines. Its effects may be recapitulated by the addition of combination of IL-1b, IL-6, TNFa, and PG-E2 [119, 120]. The effects of PG-E2 on DC development are complex with some studies demonstrating its ability to promote polarization of DCs towards an inhibitory phenotype characterized by IL-10 expression, stimulation of TH2 responses and the expansion of regulatory T cells [121]. Activation of signaling pathways associated with innate immunity also induce DC maturation. Ligation of the TLRs by LPS (TLR4), polyI:C (TLR3), Imiquomod (TLR 7/8), and CpG ODN (TLR9) promote the activation of immature DCs and has been shown to be more potent than stimulation with cytokines [14, 56, 122]. Another strategy for DC maturation involves the combined use of TNF, IL-1, POLY I:C, IFNa and IFNg which results in cells that potently express IL-12 in response to CD40-mediated stimulation [123]. DC maturation may also be induced by relatively brief exposure of monocytes (2 days) to IFNg and LPS or GM-CSF and type I IFN which generates a population of antigen presenting cells that secrete high levels of IL-12 [124]. Vaccination with these rapidly activated DCs pulsed her2neu peptide resulted in anti-tumor immune and clinical responses in patients with breast cancer [125]. Maturation of immature DCs be induced in vivo by exposure to adjuvants such as imiquomod that also facilitate migration to draining lymph nodes [126]. The ideal strategy for DC generation is dependent on the nature of the vaccine design and the method of antigen loading. Immature Dcs have been associated with a tolerogenic phenotype. In one study, vaccination with immature DCs with influenza peptide resulted in flu specific anergy [127]. In contrast, peptide-loaded mature DCs effectively stimulated anti-influenza responses [128]. Several studies have compared the immunologic efficacy of mature vs. immature DCs loaded with tumor antigens [129]. In one study, vaccination of melanoma patients with peptide-pulsed mature DCs resulted in DTH and clinical responses while none were seen following vaccination with immature DCs [130]. Similar findings were observed in comparing the efficacy immature and mature DCs in patients with glioma [131]. Another strategy involves the use

Chapter 45  Dendritic Cells 

of immature DCs that undergo antigen-loading techniques that directly induce maturation. Loading of DCs with necrotic tumor cells or DC–tumor fusion has been shown to activate and mature immature DCs [132]. Similarly, vaccination with immature DCs in conjunction with adjuvant has been shown to induce maturation in  vivo [126]. Use of TH1-polarized DCs for immunization was associated with increased IL-12 expression and enhanced responses [133]. In one study, vaccination with Langerhans cells was more potent than monocytederived DCs [134]. Despite these findings, there is considerable complexity regarding the balance of DC-mediated stimulation and inhibition of T cells and the precise strategy for DC generation for vaccine therapy remains to be elucidated. Mature DCs may also be tolerogenic and promote the expansion of regulatory T cell populations. Mature DCs have been shown to express IDO or inhibitory cytokines resulting in an inhibitory phenotype [135]. Therefore, the phenotypic characteristics of DC populations are the product of multiple factors that include maturation state and the nature of the culture stimuli [108]. Another concern regarding optimizing DC vaccination involves identifying the population with the capacity to migrate to sites of T cell traffic. In a study of patients with melanoma, mature DCs were far more capable to reach the draining lymph nodes as compared to immature DCs [109]. A correlated question is “what is the optimal mode of administration to facilitate migration of vaccine cells and immunologic potency?”. In a study of patients with prostate cancer undergoing vaccination with DCs pulsed with tumor antigen, T cell expression of IFNg was most pronounced following intradermal or intralymphatic injection while antibody responses occurred most commonly following intravenous administration [110]. In a study of patients with melanoma, intranodal as compared to intravenous or intradermal administration was associated with greatest DTH responses and T cell cytokine expression to antigen-bearing targets [136]. However, the superiority of the intranodal route has not been uniformly observed. In another study, subcutaneous as compared to intravenous administration was associated with increase in tumor-reactive memory cells in the lymph nodes with protective effects against skin based disease [113].

7. The Role of DCs in Establishing Tolerance The T cell repertoire is generated by random DNA rearrangements of the T cell receptor genes resulting in a diverse population of T cells, many of which with the capacity to recognize self antigens. Tolerance towards self antigens and protection from autoimmunity is provided by thymic deletion of autoreactive T cell clonal populations and peripheral mechanisms by which potentially noxious T cells are anergized [137]. DCs play a crucial role in the maintenance of both central peripheral tolerance and establishing the balance between immune activation of suppression [17, 138]. Autoreactive T cells are eliminated in the thymic medulla via interactions mediated by thymic epithelial cells and mature DCs. Tolerogenic DCs are thought to be essential for suppressing potentially autoreactive T cells that escape thymic deletion. This DC population is most commonly characterized by an immature phenotype, relatively low expression of co-stimulatory molecules, impaired ability to secrete stimulatory cytokines such as IL-12, and strong expression of ­inhibitory

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cytokines [15]. A greater understanding of DC-mediated immune tolerance is crucial for establishing strategies to immunize against shared self and tumor-associated antigens. In addition, the balance of DC-mediated immune activation and tolerance is thought to play a critical role in the development of alloreactive rejection and GVHD and is a potential focus for therapeutic manipulation. The nature of DC development determines the nature of the signaling pattern to reactive T cells and subsequent polarization towards an activated or inhibitory phenotype [1, 3, 16]. Immature DCs promote immune tolerance by processing and presenting self antigens at tissue sites [115]. Presentation of antigen in the absence of co-stimulatory molecules by immature DCs and their expression of cytokines such as IL-10 results in the delivery of an inhibitory signal to reactive T cell populations. Immature DCs also express receptors for apoptotic cells promoting tolerance toward these antigens [116]. Immature DCs may express high level of Indoleamine 2,3-dioxygenase (IDO), an enzyme responsible for tryptophan degradation [139]. Tryptophan is thought to be an essential amino acid for T cell survival and its absence may induce T cell death. Animal models suggest that DC capture and presentation of apoptotic bodies polarizes DCs towards a tolerogenic phenotype which is thought to be responsible, in part, for the therapeutic efficacy of photopheresis in the treatment of graft versus host disease. In contrast, defects in apoptotic mechanisms are thought to contribute in the disruption of immune tolerance in patients with systemic lupus erythematosus [140]. Presence of cytokines such as IL-10, TGFb, and VEGF expressed in the tumor bed prevents DC differentiation and polarizes DCs towards an inhibitory phenotype. Other inhibitory signaling pathways thought to be important in DC–T interactions include the expression of the programmed death ligand-1 (PDL1/B7-H1) by DCs that binds PD-1 on T cells and provides an inhibitory signal to T cell development [141]. Of note, B7-H1 expression is also found on tumor cells and abrogation of expression is associated with T cell activation and autoimmunity. A variety of agents have been identified that inhibit DC maturation and function in  vitro resulting in an inhibitory phenotype. These include IL-10, TGFb, inducers of cyclic AMP such as prostaglandin E2, and immunosuppressive drugs such as rapamycin and corticosteroids [15]. For example, rapamycin-treated DCs produce low levels of IL-12p70, do not respond to TLR-mediated signaling and markedly expand regulatory T cell populations with the capacity to enhance graft survival in an allogeneic transplant model [142]. The activated form of vitamin D3, in concert with other immunosuppressive agents, has also been shown to polarize DCs towards a tolerogenic phenotype. Other agents associated with the development of tolerogenic DCs include G-CSF, M-CSF and IL-4, thrombopoietin, and IFNl [143–147]. DC mediate signaling via CTLA-4. and PD-1 pathways induces tolerance in CD8+ cells [148]. Regulatory T cells represent a thymic-derived population of immunosuppressive T cells and play a vital role in maintaining peripheral tolerance [149–151]. Regulatory T cells co-express CD4 and high levels of CD25, minimally proliferate in response to mitogenic stimuli, and inhibit T cell responsiveness through the expression of inhibitory ligands and cytokines such as TGFb. Distinctive phenotypic characteristics include co-expression of the glucocorticoid-induced tumor-necrosis factor receptor (GITR),

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cytotoxic T lymphocyte antigen-4 (CTLA-4), and, most importantly, the forkhead–winged helix transcription factor, Foxp3 [152]. Mature DCs facilitate the expansion of regulatory T cells in the thymus. Regulatory T cells consist of 5–10% of circulating lymphocytes and their adoptive transfer has been associated with mitigation of autoimmunity and GVHD [153–155]. Regulatory T cells express inhibitory cytokines such as IL-10 and TGFb but also demonstrate the capacity to suppress T cell activation by direct cell contact via the CD95 signaling pathway. T regulatory 1 cells (Tr1) are expanded from peripheral CD4 T cells and suppress T cell activation by IL-10 and TGFb expression in a nonantigen-specific manner, similar to thymic derived regulatory cells [156]. DCs play a crucial role in the maintenance and expansion of regulatory T cell populations in vivo and in vitro [15, 19, 138, 155]. In an animal model, immature DCs loaded with ovalbumin were shown to induce the expansion of T cells which demonstrated specific inhibition of ovalbumin-directed immunity [157]. In a murine model, DCs that have been modified to express co-stimulatory molecules with low levels of co-stimulatory molecules were highly effective in stimulating regulatory T cell responses [158]. Expansion of regulatory T cells may also be facilitated by TGFb expression by DCs (72). However, mature DCs have been shown to the most effective antigen presenting cell with regard to the expansion of regulatory T cells in an antigen-dependent fashion [159, [160]. Of note, the suppressive function of DC-expanded regulatory cells is greater than cells directly isolated from the circulation. Once activated in the presence of a specific antigen, regulatory T cells have the capacity to inhibit T cell activation generally towards other antigens. Mature DCs induce the expansion of both activated effector and regulatory T cell populations [161]. Co-stimulatory molecule-mediated signaling appears to potently stimulate both populations suggesting that regulatory T cells may represent a homeostatic mechanism that ultimately blunts response to DC-mediated stimulation. In concert with these findings is the observation that response to DC-based vaccination is augmented by regulatory T cell depletion [162]. The interaction between DCs and regulatory T cells is bidirectional. Regulatory T cells may interfere with DC maturation and activation further augmenting their inhibitory potential [163]. Regulatory T cells inhibit the upregulation of co-stimulatory molecules on DCs and the interaction of DCs with effector cells [159]. One potential mechanism is CTLA-4-mediated signaling resulting in increased IDO expression in DCs which in turn result in suppressive effects on reactive T cell populations. Tolerogenic DCs may also inhibit DC populations with an immunostimulatory phenotype. Plasmacytoid DCs may direct T cells towards a TH2 phenotype, upregulate IDO expression and induce the differentiation of regulatory T cells [164]. Allograft tolerance may be induced by inhibitory donor-derived DCs or recipient DCs that have been loaded with donor allopeptides [165]. The effect of plasmacytoid DCs in vivo may be determined by their site of tissue migration. In a cardiac allograft model, plasmacytoid DCs homing to the lymph nodes were found to expand regulatory T cells and promote allograft tolerance while those migrating to the spleen-induced rejection [18]. Plasmacytoid DC precursors have been shown to promote hematopoietic stem cell engraftment in the absence of GVHD and are an important subpopulation of transplantfacilitating cells [166]. Regulatory T cells expanded by allogeneic DCs dem-

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onstrate the capacity to suppress rejection specific to the allogeneic stimulus [159]. Similar findings were observed in an in vivo GVHD model in which mice that received donor regulatory T cells expanded by recipient allogeneic DCs were better protected than those receiving regulatory cells expanded by third party DCs. Tumor cells utilize a variety of mechanisms to inhibit DC maturation, activation, and antigen presentation resulting in the polarization of T cell responses towards tolerance [167]. Tumor cells constitutively express STAT3 which inhibits the production of inflammatory cytokines and promotes the release of factors that inhibit DC function. Tumor cells secrete VEGF, IL-10, and IL-6 which have been shown to prevent DC maturation towards an activated phenotype. This results in the localization of tolerizing immature DCs in the tumor bed with mature cell largely confined to the periphery [168]. In contrast, inhibitors of VEGF have restored normal DC differentiation but not necessarily their stimulatory capacity [169]. Tumor-associated glycoproteins such as MUC1 have been shown to disrupt antigen processing, inhibit DC expression of IL-12, and promote the development of TH2 responses [170]. Of note, in a breast cancer model, tumor growth is supported by IL-13, a TH2 associated cytokine [171]. In a human myeloma model, DCs have been shown to support the growth of myeloma cells [172].

8.  The Role of DCs in Immune Reconstitution, Graft Versus Disease and Graft Versus Host Disease Following Hematopoietic Stem Cell Transplantation Mobilization of DC precursors has been studied as a part of stem cell collections for autologous and allogeneic transplantation. Use of GM-CSF as a part of the cytokine regimen has been associated with increased presence of DCs in the mobilized product [173]. Another study demonstrated that mobilization with GM-CSF and G-CSF as compared to G-CSF alone resulted in a decrease in the DC2 subset in the graft suggesting that these cells may be less tolerogenic [174]. Following allogeneic transplantation, DCs are thought to play an important role in the pathogenesis of GVHD [175]. DCs may be activated in the setting of cytokine storm that prevails following transplant conditioning resulting in the presentation of alloantigens to donor T cell populations [176]. DC-mediated signaling of CD4 T cells via OX40 has been shown to be an important pathway for the induction of GVHD in animal models [177]. DCs were found to be predominantly of donor origin in the early post-transplant period [178]. However, DCs are nonproliferating cells and may transiently survive ablative conditioning regimens [20]. Host-derived DCs may persist for a long-time post-transplant particularly after reduced intensity conditioning regimens [179]. In one animal model, as compared to activated B cells, recipient DCs were shown to be uniquely capable of stimulating alloreactive CD4 and CD8 T cells resulting in GVHD [180]. In contrast, in reduced intensity murine transplant model, high autoantibody levels predictive of cGVHD were seen in the setting of mixed chimerism and associated with the persistence of host B cells rather than DCs [181].

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The importance of host-derived DCs was emphasized in a study in which host skin Langerhans cells persist following T cell-depleted transplantation despite the conversion to full donor chimerism in other tissue sites [182]. GVHD responses in the skin following infusion of donor lymphocytes were seen exclusively in animals with residual host Langerhans cells. Donor T cells were shown to be crucial for the eradication of recipient Langerhans cells and the recruitment of donor Langerhans cells to the site. While host DCs are sufficient to initiate GVHD, it is further intensified by the presence of donor antigen-presenting cells which cross prime alloreactive CD8 cells [183]. Of note, donor-derived antigen-presenting cells were not thought to be responsible for stimulating graft versus leukemia responses. A major research focus has been the separation of GVHD from graft versus disease effects. In a murine transplant model, host DCs stimulated T cell subsets differentiated by CD44 expression in which the CD44low/CD8+ fraction was responsible for GVHD and depletion of these cells was protective without impacting graft versus leukemia effects of the graft [184]. The nature of DCs reconstitution following allogeneic transplantation is likely to have profound implications for the development of donor/host tolerance with clinical implications for the incidence of rejection, graft-vs-host disease (GVHD), infection, and disease relapse [185]. The nature of the recovering DCs subpopulations and their functional characteristics is strongly associated with levels of alloreactivity and tolerance. Rapid establishment of donor chimerism for DC populations has been noted in the early posttransplant period [179]. In animal models of solid-organ transplants, treatment with immature DCs, lymphoid-derived DCs, or DCs following blockade of the CD40 pathway resulted in prolonged survival of the allograft tissue [186, 187]. In an effort to manipulate the kinetics of DC reconstitution, investigators have examined the in vivo effect of CAMPATH-1G, an antibody directed against CD52 expressed on T cells and certain DC subsets [188, 189]. In one study, the use of CAMPATH-1G as a part of transplant conditioning resulted in the depletion of host DCs but did not impact recovery of donor DC subsets post-transplant. Following transplantation with G-CSF-mobilized allogeneic stem cells, increased numbers of circulating DC2 cells are found that may mediate tolerance [147]. This finding was thought to potentially explain the lack of increase in acute GVHD associated with allogeneic peripheral blood stem cell grafts, despite the increased numbers of T cells as compared to bone marrow. In patients undergoing allogeneic peripheral blood stem cell transplantation, increased number of DC2 in the stem cell graft was associated with decreased incidence of GVHD and increased risk of relapse [190]. In a pediatric study, decreased circulating levels of monocytoid and plasmacytoid DCs early post-transplant was associated with the subsequent development of acute but not chronic GVHD [191]. In a study of 31 adult patients undergoing allogeneic peripheral blood stem cell transplantation, both myeloid and plasmacytoid DC subsets were suppressed in patients with grade II–IV aGVHD [192]. Similar to these findings, a study of 50 patients undergoing transplant demonstrated that lower levels of circulating DCs was associated with a higher risk of relapse, death, and aGVHD [185]. Analysis of DC1 and DC2 subsets demonstrated a trend towards similar effect as that seen with the total DC population. In another study, an increase in myeloid

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and ­plasmacytoid DCs was observed at the onset of GVHD, the latter of which was promptly suppressed with steroid therapy [193]. Administration of G-CSF was associated with higher initial levels of myeloid DCs but ­subsequently resulted in lower levels of myeloid and plasmacytoid DCs as well as IL-12-producing cells. Persistence of host-derived DCs was associated with graft versus leukemia effects following donor lymphocyte infusions in patients with CML [194]. Umbilical cord blood is an effective source of hematopoietic stem cells used to support allogeneic transplantation. Cord blood transplantation is associated with a greater degree of tolerance as compared to adult bone marrow such that that a greater degree of HLA mismatch results in similar levels of GVHD. Of note, DCs isolated from cord blood demonstrated a more immature phenotype than those from peripheral blood [195]. Cord blood-derived DCs were associated with lower levels of TNFa and IFNg secretion and potently induced the expansion of regulatory T cells. There has been strong interest in developing strategies to expand tolerogenic DC as a means of inhibiting the GVHD response following allogeneic transplantation. In animal models, infusion of tolerogenic DCs was found to inhibit GHVD while preserving graft versus tumor responses [196]. Similarly, DCs differentiated in the presence of vasoactive intestinal peptide induce the expansion of regulatory T cells and blunt GVHD following allogeneic transplantation [197]. In contrast, these cells do not inhibit the capacity of alloreactive CD8+ T cells to lyse leukemia targets. Co-transplantation of TGFb treated DCs resulted in the prolongation of the survival of animals undergoing MHC-disparate allogeneic transplantation [198]. DCs generated in  vitro with GM-CSF, IL-10 and TGFb prevent the development of lethal GVHD but not anti-leukemia responses in a murine allogeneic transplant model [196]. In contrast, transplantation of mature DCs intensified the resulting course of lethal GVHD. Extracorporeal photochemotherapy has become established as a therapeutic strategy for patients with chronic GVHD. A proposed mechanism based on animal models is that apoptotic cells resulting from this procedure are ingested by native DC populations polarizing them towards a tolerogenic phenotype [199, 200]. DCs generated from patients treated with FK506 following allogeneic transplantation demonstrated decreased functional capacity. Exposure to rapamycin or corticosteroids results in inhibited maturation and expression of co-stimulatory molecules [142, 165, 201].

9. DC-Based Immunotherapy for Cancer Tumor cells express unique antigens that serve as potential targets for cancer immunotherapy. Tumor-associated antigens have been identified that are aberrantly expressed by malignant cells allowing for their differentiation from normal tissue [202–204]. T cells with the capacity to recognize tumor antigens have been found in the immune repertoire of patients with malignancy. However, tumor cells evade recognition leading to immunologic tolerance that supports the growth and dissemination of malignant disease [205]. Tumor cells present antigens in the absence of co=stimulatory molecules necessary for the initiation of primary immune responses [206]. As outlined above, tumor cells secrete factors that disrupt function and maturation of native antigen-

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p­ resenting cells [207, 208]. In addition, tumor cells blunt the functional potency of effector cell populations by a variety of mechanisms including the increased presence of regulatory T cells that suppress T cell-mediated response. Tumor cells may exist in an equilibrium with host immunity until progressive tumor-mediated immune suppression and emergence of poorly immunogenic clones result in the promotion of tumor growth [205]. In contrast, DCs potently richly express co-stimulatory molecules and cytokines necessary for immune activation. As such, there has been strong interest in the manipulation of DCs to process and present tumor-associated antigens to generate productive anti-tumor immunity. Strategies to introduce tumor antigens into DCs have been pursued in an effort to induce tumor-specific CTL responses. One approach has been the in vivo loading of tumor antigens by DCs recruited to the site of malignancy. Introduction of DCs into the tumor bed has been shown to directly inhibit tumor growth [209, 210]. Tumor cells genetically engineered to express GM-CSF or co-administered with GM-CSF-secreting cells induce tumorspecific immunity through the recruitment of DCs to the site of inoculation with subsequent internalization and presentation of tumor antigens [211, 212]. Administration of immature DCs in conjunction with the TLR agonist, imiquimod, enhanced the capacity of the DC vaccine to the draining lymph node and the induction of anti-tumor immunity [126]. Systemic administration of Flt3L results in the tissue accumulation of DCs and the potential internalization, processing, and presentation of tumor antigens at the site of malignant disease [41]. Therapy with Flt3L has been shown to induce tumor regression in animal models [213]. Tumor-specific immunity was transferred by CD8+ splenocytes isolated from mFlt3L-treated animals. In another murine model, administration of Flt3L was protective against an otherwise lethal challenge of myeloid leukemia cells and induced anti-leukemia CTL responses but did not generate long-term memory responses and was ineffective for treating established disease [214]. In another study, animals treated with a combination of radiation and FLT3L experienced a decrease in pulmonary metastases and improved survival as compared to those treated with FLI3L alone [215]. The investigators postulated that radiation facilitated the loading of tumor antigens onto infiltrating DCs. DCs isolated from patients with malignancy demonstrate functional deficiencies [207, 216, 217]. As such, the use of native DC populations for cancer immunotherapy is potentially problematic. Alternatively, DCs generated ex vivo from progenitor populations have been shown to be functionally competent [173, 218–220]. As such, manipulation of these population have been pursued to design tumor vaccines. A variety of in vitro strategies to introduce tumor antigens into DCs have also been examined in animal models. (Fig. 45-6) Exogenous loading of DCs with tumor peptides allows for the use of DCs with a mature phenotype. Response to individual peptides is governed by their affinity to MHC binding and their capacity to induce responses against various epitopes [221]. In murine models and pre-clinical human studies, DC pulsed with tumor-associated peptides effectively induce antigen-specific CTL responses resulting in protection from tumor challenge [155–160, 204, 222–224]. In one study, vaccination with DCs pulsed with Her2neu peptide, which was altered to augment binding to the MHC complex resulted in higher levels of CTL activity [225]. Of note, weekly immunization resulted in decreased

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Fig. 45-6.  Strategies to load tumor antigens onto dendritic cells

levels of response, while animals vaccinated every 3 weeks did not experience diminution in CTL immunity. In the hematological malignancy setting, peptides derived from the BCR/ABL fusion region have also been shown to be immunogenic when presented by antigen-loaded DCs [226]. CD4-mediated responses were generated that lysed CML cells containing the associated breakpoint region. T cells stimulated with DCs pulsed with a bcr–abl peptide lyse patient-derived chronic myeloid leukemia (CML) cells containing the same breakpoint, but not autologous monocytes [227]. DCs loaded with peptides eluted from AML cells have also be used to stimulate tumor-specific responses [228]. Peptide-pulsed DC-derived exosomes have also been shown to be potent anti-tumor immunogens resulting in eradication of established disease in murine models [178]. Although effective in animal models, the efficacy of peptide-pulsed DCs in generating tumor-specific immunity is limited. The immunogenicity of identified antigens is variable, the stability of antigen presentation following exogenous pulsing is uncertain, and the clinical efficacy of an immune response directed against a single epitope may be muted. Of note, patientderived CTL induced by DCs pulsed with p53-derived peptides were unable to lyse autologous squamous cell carcinoma cells due to the downregulation of expression of this epitope [229]. In addition, there is a lack of defined tumorspecific peptides in many malignancies, and treatment is limited to patients of a particular HLA genotype. Another approach to designing DC-based tumor vaccines is through the exogenous loading of whole proteins [230]. In this way, multiple epitopes may be presented in the appropriate HLA context. As an example, vaccination with DCs pulsed with lymphoma-derived idiotype protein stimulates antigen-specific T cell response and protection from challenge with idiotypeexpressing tumor cells [231]. The efficacy of protein loading may also be limited in that it is dependent on the use of DCs with the capacity to internalize

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and process antigen, and the ability of T cell repertoire to recognize presented epitopes is less easily defined. The processing of exogenous proteins also results in their presentation along a class II pathway, producing a primary helper as compared to cytotoxic T cell response. However, cross-presentation of internalized antigens to cytotoxic T cells has been documented as an important mechanism of DC-mediated immune responses. One strategy that has been used to use whole proteins as a source of peptide epitopes is the generation of overlapping long peptides that cover the coding sequence of the identified tumor antigen [232]. Another approach to introducing tumor antigens into DCs has been the use of viral vectors to insert genes encoding for tumor-specific proteins. ln this way, tumor antigens are processed through endogenous mechanisms and presented along a class I pathway to reactive CD8+ T cells. Viral transduction may induce DC activation enhancing immune responses. Another potential advantage is that insertion of tumor-associated genes potentially provides an ongoing source of antigens for presentation. In contrast, peptide or protein antigens may be cycled off the cell surface by the time that DCs arrive at sites of T cell interaction [14]. Transduction of DCs with recombinant pox viruses expressing the co-timulatory and adhesion molecules (TriCOM complex) markedly augments their capacity to stimulate antigen-specific responses [233]. DCs infected with vaccinia virus bearing melanoma-derived gp 100 stimulated CTL responses that lysed HLA-matched targets that had been pulsed with a variety of gp100-derived peptides [234]. Transduction of DCs with retroviral or lentiviral vectors has also been investigated [235, 236]. Investigators have explored the feasibility of the retroviral insertion of tumor genes into CD34+ cells that are subsequently differentiated into DCs in the presence of cytokines. Using this approach, stable expression of the MUC-1 tumor antigen was generated in DCs derived from retrovirally transfected precursor cells [237]. Similarly, the MART1 gene was expressed in approx 25% of DC following its retroviral insertion into CD34+ cells that were then cultured with SCF, TNFcr, and GM-CSF [238]. Vaccination with DCs transduced with an adenoviral vector bearing the MAGE-1 gene resulted in suppression of tumor growth in a subcutaneous melanoma model, with 10% of animals experiencing long-term survival [239]. In contrast, vaccination with tumor cells expressing IL-12, GM-CSF, or CD40L were unable to contain tumor progression. Paradoxically, levels of MAGE-I expression were significantly higher in tumor cells as compared to DCs. A potential concern regarding the use of viral vectors is their generation of potent immunologic responses directed against viral proteins. These antigens are potentially far more immunogenic and may overwhelm the response against the designated tumor antigens and prevent repetitive dosing from being effective. Another potential limitation of this approach is the demonstration that viral infection may be associated with decreased DC function [240]. Transfer of tumor-specific genes into DCs has also been accomplished through the use of tumor-derived RNA [241]. This strategy is facilitated by established methods to isolate and amplify RNA from biopsy specimens, allowing for its potential general applicability in the clinical setting. As an example, DCs pulsed with CEA-mRNA stimulate tumor-specific CD8+ CTL [242]. Similarly, DCs pulsed with RNA encoding for prostate-specific antigen (PSA) induced CTL responses against cells expressing PSA but not kallikrein,

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a self antigen that shares homology with PSA [243]. DCs transfected with RNA encoding for the MUC-I tumor antigen induced tumor-specific responses in immunized animals resulting in protection from tumor challenge and regression of established disease [244]. Vaccination with DCs cotransfected with MUC-I RNA and IL-12 resulted in MUC-1-specific responses in a transgenic murine model. Similarly, DCs pulsed with mRNA encoding for human telomerase reverse transcriptase are highly effective in stimulating responses against diverse malignancies for this effective target antigen [245]. One potential concern is that mRNA-based strategies will induce a cytotoxic T cell response in the absence of a helper response. Approaches to concomitantly stimulate CD4 T cells include the insertion of RNA encoding for a lysosomal targeting fusion signal [246]. All of the above mentioned-strategies involve the targeting of known tumor-associated antigens. The use of single gene products for DC-based immune strategies limits one to a small group of potential antigens of uncertain immunogenicity. Immunotherapeutic approaches that rely on induction of immunity against a particular antigen are also potentially subject to tumor cell resistance mediated by the downregulation of expression of that single gene product. One approach to circumvent this limitation is the pulsing of DCs with antigens extracted from whole tumor cells or whole tumor-derived RNA [241, 247]. DCs generated from cord blood CD34+ cells have been successfully transduced with RNA derived from a leukemia cell line [248]. DCs loaded with lysate generated by freeze–thawing of an Epstein–Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) line stimulated tumorspecific CD4+ and CD8+ responses with TH1 phenotype [249]. Loading of DCs with lysate prior to terminal maturation with TNFa, IL-1b, and PG-E2 was the most effective approach in generating tumor immunity. Another study examined the capacity of monocyte-derived immature DCs to process and present tumor antigens from tumor cells that had undergone lethal l irradiation or exposure to anti-Fas antibody [250]. DCs loaded with leukemia lysates generate tumor-specific CTL responses [251]. As a measure of their capacity to stimulate class I responses via cross-priming, DCs pulsed with breast cancer lysate-induced CD8 mediated MUC1 specific responses associated with the production of TH1 cytokines [252]. Another strategy for generating tumor immunity involves the use of apoptotic bodies as a means of introducing tumor antigens into DCs with subsequent cross-presentation along the class I pathway [81, 253]. DCs were found to express a unique receptor which facilitates phagocytosis of apoptotic bodies and is downregulated upon maturation [47]. Mactophages ingest apoptotic bodies more readily, but lack the capacity to cross-present antigenic material from the apoptotic bodies. In one study, DCs demonstrated the capacity to internalize necrotic as well as apoptotic tumor bodies, but only the latter induced DCs maturation and resulted in potent CD8+ tumor-specific responses [81]. ln another study, DCs pulsed with melanoma cells that underwent apoptosis were more effective in generating tumor responses than those loaded with live or necrotic cells [254]. One strategy that is currently being explored for the generation of DC-based immunotherapy for leukemia has been the differentiation in vitro of leukemic clones into DCs. In this manner, tumor antigens retained from the malignant clone can be endogenously processed and presented by a functionally active

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antigen-presenting cell. CML cells cultured with GM-CSF andlL-4 developed phenotypic characteristics of DCs that contained the bcr–abl translocation [255]. Stimulation of autologous T cells resulted in cytotoxic activity against CML cells and bcr–abl-expressing targets as well as the inhibition of growth in CML clonogenic precursors in colony-forming assays in vitro. Retroviral transduction with the gene encoding for IL-7 further increased the potency of bcr–abl-expressing DCs generated from CML patients [256]. Investigators have also demonstrated that functionally active DCs can be generated from acute myeloid and leukemia cells [257, 258]. Leukemic blasts were cultured in cytokines and subsequently found to express co-stimulatory molecules such as CD80, CD86, and CD40, DC-specific markers such as CD83, and retained the chromosomal abnormalities of the original leukemic clone. DCs stimulated autologous T cell-lysed leukemic targets. Of note, immature CD34+/CD38− leukemia progenitors are resistant to differentiating toward DCs [259]. A potent strategy for designing DC-based tumor vaccines involves the fusion of DCs with tumor cells. In this approach, multiple tumor antigens, including those yet unidentified, are presented in the context of DC-mediated co-stimulation. DC/tumor fusions stimulate CD4- and CD8-mediated immunity resulting in greater potential durability of the anti-tumor response. In diverse animal models, vaccination with DC/tumor fusions has been shown to potently induce tumor-specific CTL responses, is protective from an otherwise lethal challenge of tumor cells and may eradicate established metastatic disease [260–263]. DC/tumor fusions were also found to break immunologic tolerance toward the MUCl tumor antigen in transgenic mouse models [264]. In another study, vaccination with fusion cells resulted in protection from tumor challenge as well as efficacy as therapy for metastatic disease in melanoma and lung carcinoma models [265]. Subsequent studies have demonstrated that DC/tumor fusions are potent stimulators of tumor-specific immunity in preclinical human studies in multiple myeloma, breast and ovarian cancer [266–269]. Fusion cells were generated from patient-derived tumor cells and autologous DCs, and were found to co-express tumor antigens and DC-derived co-stimulatory molecules. Fusion cells induced prominent tumor-specific CTL responses in vitro following a single stimulation. CTLs did not lyse autologous monocytes and were inhibited by incubation with anti-class I antibody. Similar findings were demonstrated with DCs fused with AML cells in which fusion cells potently stimulated CTL responses directed against leukemia cells including those with core binding factor mutations [270–272]. Fusion cells were also shown to prevent the spontaneous development of mammary tumors [273].

10. DC Immunotherapy for Cancer: Clinical Studies DC-based tumor immunotherapy is now being pursued in the clinical setting. One strategy involves the attempt to stimulate in  vivo generation and mobilization of native DC populations that then undergo antigen uptake and presentation at the tumor site [274]. In one study, patients with colon cancer were treated with Flt3L prior to resection of metastatic lesions in the lung or liver [275]. Increased number of CD11c/CD14− DC were noted in the peripheral blood-as well as at the tumor margins. Patients receiving Flt3L following autologous stem cell transplant experienced an increase in monocyte and plasmacytoid DCs that could be further matured ex vivo with CPG ODN [276].

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Clinical responses have also been demonstrated in patients with melanoma and breast cancer undergoing injection with autologous DCs directly into the tumor bed [205]. This was further facilitated by radiation therapy into the tumor bed in patients with hepatoma [277]. Another approach is the use of IFNa as an adjuvant to melanoma peptides vaccine to enhance native DC processing and presentation [278]. Exposure of DCs to alpha-GalCer enhances their capacity to stimulate NK T cells as anti-tumor effector cells in lung cancer [279]. Another approach is the use of the leukemia clone to generate DCs expressing leukemia-associated antigens. In patients with CML – vaccination in patients with incomplete response to imatinib or interferon therapy – 4/10 patients had potential evidence of further response to vaccination which was associated with the emergence of tumor-reactive T cells in three patients [280]. In a study of 22 patients with AML, patients underwent vaccination with leukemia derived DCs following completion of chemotherapy. Although T cell responses were documented in a subset of patients, only two patients remained in remission for more than 12 months [281]. In a small study of patients with AML undergoing vaccination with DCs generated from leukemia cells, immune responses were noted against the leukemia-associated antigen, PRAME [282]. Vaccination of patients with DCs pulsed with tumor peptides has been studied in clinical trials particularly in patients with melanoma. In an early trial, 16 patients with melanoma were treated with DCs pulsed with melanoma peptides or lysate as well as KLH to induce helper responses [283]. Following vaccination, 11 out of 16 patients developed DTH responses at the vaccine site and associated tumor-specific CTL responses were noted. Six out of 16 patients showed evidence of clinical response. In a similar study of patients undergoing vaccination with peptide-pulsed DCs activated with IFNa, significant clinical or immunologic responses were not observed [284]. Vaccination with DCs pulsed with MAGE peptide demonstrated that clinical responses were associated with a 20- to 400-fold increase in antigen-specific CTL which demonstrated a polyclonal profile [285]. In another study, 18 patients with metastatic melanoma underwent vaccination with CD34+-derived DCs pulsed with several melanoma peptides [286]. Sixteen out of 18 patients demonstrated evidence of T cell response to the control antigen, influenza, and KLH, and at least one of the melanoma peptides. Of note, clinical response was associated with immunologic response to at least two melanoma peptides as manifested by an increased percentage of T cells expressing IFNg in response to ex vivo exposure to the peptide. In a follow-up report, four patients remained alive 5 years after completion of the study, with survival correlating with tumor peptide-specific immunity [287]. Based on the encouraging pre-clinical data, a study was performed in which 15 patients with melanoma were treated with DC-derived exosomes pulsed with MAGE peptides [288]. Only one patient demonstrated evidence of a partial response and no evidence of circulating antigen-reactive T cells was observed. Several studies have examined the efficacy of vaccination with DCs pulsed with peptides in other malignancies. In one study, 17 previously untreated patients with prostate cancer underwent monthly intravenous infusions with DCs pulsed with prostate-specific membrane-antigen (PSMA) peptides [207, 289] Three partial responders and one complete response were noted. No significant treatment related toxicity was reported. Immunological assessment

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revealed that clinical response was associated with skin test response to recall antigens, T cell responsiveness to cytokines, and, in some patients, cytotoxicity against the immunizing peptides [290]. In other studies of patients with prostate cancer, vaccination with DCs pulsed with tumor-associated peptides has resulted in antigen-specific T cell responses and disease stabilization in a subset of patients [212, 291, 292]. A phase I trial was conducted in which ten patients with breast and ovarian cancer underwent vaccination with DCs pulsed with Her2neu- or MUC1-derived peptides [293]. In five out of ten patients, peptide-specific CTL responses were noted with immunodominance of two particular epitopes. DCs pulsed with Her2neu peptide and activated with IFNg and LPS were administered intranodally to patients with DCIS results in T cell responses, decreased hers2neu expression in the resected tumor tissue and regression in areas of disease [125]. Patients undergoing vaccination with DC pulsed with peptides eluted from CNS tumors demonstrated cellular immune response and intratumoral T cell infiltration [294]. Response to vaccination of patients with glioblastoma with DCs pulsed with tumoreluted peptides was inversely related to expression of TGFb in the tumor bed [295]. Vaccination of patients with hepatocellular carcinoma with DC pulsed with alpha fetoprotein resulted in the development of T cell immunity against this tumor-associated antigen [296]. In a study of 13 patients with colon cancer undergoing vaccination with DCs matured with IFNg and Klebsiella wall fraction, CEA-specific responses were noted in a minority of patients but no clinical responses were noted [297]. In another study, vaccination of patients with colon cancer resulted in immunologic responses against CEA and disease stabilization in a subset of patients [298]. DC-based vaccines using telomerase peptide has also been successfully employed for generating tumor-specific immunity [299, 300]. Pre-clinical models have demonstrated that peptides altered to enhance binding to the MHC complex may be associated with enhanced immunogenicity. Vaccination with DCs pulsed with a mutant CEA or p53 peptide was associated with responses against the wild-type antigen and disease response [301, 302]. Another strategy that is examined in the clinical setting has been the vaccination of patients with DCs pulsed with tumor-associated proteins. In one study, patients with low-grade lymphoma underwent vaccination with DCs pulsed with idiotype protein. In an initial report, all the patients showed evidence of idiotype-specific cellular immunity, while humoral responses were absent [303]. Eight out of ten patients demonstrated idiotype-specific cellular immunity. Disease response was seen in four patients, including two patients who experienced a complete response. In a subsequent report, of patients undergoing vaccination following completion of chemotherapy, 70% remained without evidence of progression with a median follow up of 43 months [304]. In a study of 11 patients with multiple myeloma, who were treated with CD34derived DCs pulsed with idiotype protein, four out of ten patients developed evidence of increased idiotype-specific cellular immunity as determined by ELIspot analysis, and three patients showed evidence of anti-idiotype tumoral response [305]. One patient demonstrated evidence of marrow regression of plasma cells. Another study examined the impact of the infusion of DCs pulsed with idiotype protein following high-dose chemotherapy with stem cell rescue in patients with multiple myeloma [306]. Four out of 26 patients demonstrated evidence of idiotype-specific T cell proliferative responses in the

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post-transplant period. In another study of patients with myeloma, subcutaneous as compared to intravenous vaccination with DCs pulsed with the idiotype protein resulted in T cell anti-tumor immunity [307]. DCs loaded with tumor-associated RNA has been shown to stimulate antitumor immune responses in patients with prostate cancer [308]. Vaccination was associated with reduction of the log slope in PSA and molecular evidence of circulating tumor cells in a subset of patients. Vaccination with DCs pulsed with renal carcinoma-derived RNA-induced immune responses against a broad array of tumor-specific antigens including G250 and oncofetal antigen [309]. In a similar study, immunologic responses were not improved following intranodal as compared to intradermal injection [310]. In another study, vaccination with DCs pulsed with telomerase reverse transcriptase RNA and lysosomal-associated membrane protein 1 (LAMP1) induced antigen-specific CD4 and CD8 T cell responses and was associated with a decrease in levels of disease as measured by surrogate markers [246]. Insertion of tumor-associated antigens into DCs for vaccination has also been examined using viral vectors. Vaccination with DCs transduced ex vivo with fowl pox virus engineered to express CEA resulted in a minor response or transient disease stabilization in 14 patients with advanced colon or lung cancer. T cell responses to CEA were higher in those patients with evidence of clinical benefit [311]. Another strategy that has been explored in clinical trials involves the use of whole tumor cells as a source of antigen for DC-based vaccines. Vaccination with DC pulsed with autologous tumor lysate has been pursued by multiple investigators [312, 313]. In a study of patients with melanoma, DCs pulsed with autologous lysate appeared more immunologically potent than those pulsed with peptides in stimulating T cell-mediated IFNg responses [314]. In a trial of patients with glioma, vaccination resulted in disease response and stabilization in a subset of patients with longer survival seen in patients with undergoing both intratumoral and intradermal vaccination [131]. In a study of nine patients with renal carcinoma undergoing vaccination with DCs pulsed with renal carcinoma lysate, five patients experienced stable disease and one achieved a partial response and these patients demonstrated treater antigenspecific proliferative responses ex vivo than those with progressive disease [315]. Vaccination with DCs pulsed with killed allogeneic melanoma cells induced disease regression and tumor antigen-specific immune responses in a subset of patients with advanced disease [316]. Of note, some patients experienced extended overall survival raising the possibility that anti-tumor immunity may demonstrate clinical benefit even in those patients without overt disease regression. Similarly, vaccination of patients with non-small lung cancer with DCs pulsed with necrotic malignant cells obtained from pleural effusions resulted in the induction of tumor-specific T cells in a minority of patients who experience disease stabilization [317]. Alternatively, patients have undergone vaccination with apoptotic cells derived from a non-small cell carcinoma cell line that overexpressed the tumor antigens, survivin, Her2/neu, CEA, MUC1, and WT1 [318]. Another promising strategy for DC-based immunization involves the use of DCs fused with patient-derived tumor cells. In one study, 23 patients with advanced breast and renal carcinoma, who underwent vaccination with autologous DC/tumor fusions, were considered [319]. Vaccination was well tolerated and not associated with the development of autoimmunity. 10/18 evaluable

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patients demonstrated evidence of anti-tumor immunity as manifested by an increase in the percent of circulating T cells that expressed IFNg following ex vivo exposure to autologous tumor lysate. Two patients demonstrated evidence of disease regression and an additional six patients had stabilization of the disease. In a study of 15 patients with glioma, vaccination with autologous DC/ tumor fusions in conjunction with IL-12 resulted in >50% regression in four patients [320]. Vaccination of 17 patients with melanoma with patient-derived tumor cells fused with allogeneic DCs resulted in one complete remission, one partial response and six disease stabilization. Fourteen patients demonstrated evidence of immunologic response as manifested by T cell response to tumor antigens. Progression was associated with loss of antigen expression and presentation [321]. In another study, 24 patients with metastatic renal carcinoma underwent vaccination with autologous tumor cells fused with DCs generated from normal donors. Ten patients demonstrated evidence of immunologic response against antigens in autologous tumor lysate. Ten out of 20 evaluable patients experienced either disease regression or stabilization. A statistically significant association between immunologic and clinical response was noted [322].

11. DC Immunotherapy: Potential Limiting Factors While DC-based immunotherapy has emerged as a promising therapeutic strategy, its clinical role has not been defined. In a randomized study of patients with melanoma, DC vaccines were not shown to improve outcomes as compared to standard DTIC therapy [323]. The nature of antigen loading, DC generation, vaccine administration, and the schedule of priming and boosting are all likely to be essential in determining the effectiveness of vaccination [14, 324]. Tumor cells generate an immunosuppressive environment that disrupts the function of host antigen presenting and effector cells and allows for their escape from host immunosurveillance. Tumor-mediated immunosuppression may prevent response to DC-based vaccination. Tumor cells secrete factors such as VEGF, TGFb, IL-6, IL-10, and M-CSF which inhibit DC maturation [207, 325]. Tumor cells secrete high levels of MIP-3a which fosters the migration of immature DCs into the tumor bed and exert a tolerizing influence on host immunity. In contrast, the more functionally active mature DCs are characteristically found in the peritumoral areas [168]. The tumor-associated antigens, MUC-1 and HER-2/neu, are internalized by DCs, but are not consistently transported to late MHC class II endosomes, thus abrogating their ability to undergo appropriate processing and presentation [326]. DCs isolated from peripheral blood and tumor-draining lymph nodes were studied in 93 patients with breast, head and neck, and lung cancer [327]. Decreased numbers of circulating mature DCs was noted and impaired function was seen in DCs derived from both lymph nodes and blood, suggesting a systemic effect of the tumor. Partial reversal of these findings was noted after tumor resection. In contrast, functionally active DCs derived from patients with malignancy can be generated from precursor populations cultured in vitro with cytokines. DCs generated from patients with multiple myeloma, breast cancer, lymphoma, and renal cancer have been shown to prominently express co-stimulatory molecules and stimulate autologous and allogeneic T cell responses [218, 223–225, 328, 329]

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Tumor cells suppress T cell function through a variety of mechanism that limit the capacity of patients with malignant disease to respond to vaccination. T cell dysfunction has correlated with disease bulk. Activation of inhibitory pathways such as those mediated by PDL1 inhibits cytotoxic responses. Migration of tumor-reactive T cells to the tumor bed may also be a target of tumor-mediated immune suppression. In one study, functionally potent Melan-A-specific T cells were detected in the circulation but T cells isolated from the tumor bed demonstrated a suppressive phenotype [330]. Regulatory T cells play a central role in tumor-mediated tolerance, predict for clinical outcomes and may inhibit responses to DC based tumor vaccines [331, 332]. Regulatory T cells are increased in the tumor bed, draining lymph nodes and circulation of patients with malignancy [333]. Circulating levels of regulatory cells may be paradoxically increased following DC-based vaccination. In animal models, depletion of regulatory cells enhances vaccine efficacy and the capacity to induce tumor rejection [103, 162]. In a clinical study, vaccination in conjunction with anti-CD25 linked to diphtheria toxin (ONTAK) resulted in the transient depletion of regulatory cells and a corresponding increase in tumor-reactive T cell responses [103]. The use of chemotherapy prior to vaccination has also been shown to deplete circulating regulatory T cells that may potentially provide an improved platform for DC immunization. Of note, animal models have demonstrated that a transient increase capacity to respond to tumor vaccines is noted following high-dose chemotherapy with stem cell rescue. It is thought that tumor-mediated tolerance is disrupted during the early lymphopoietic reconstitution period post-transplant, potentially due to the elimination of regulatory T cell populations [334]. In an animal model, vaccination with lysate-pulsed DCs following autologous transplantation was associated with heightened and more durable responses [335].

12. Conclusion DCs are potent antigen-presenting cells that play a crucial role in the initiation of cellular immunity and maintenance of the delicate balance between tolerance and immune recognition. DC biology plays an important role in posttransplant immune reconstitution and the development of GVHD. The use of DC-based immunotherapy has emerged as a major field of investigation and has yielded promising preliminary findings. Its integration into hematopoietic stem cell transplantation offers a potential avenue to modulate tumor-specific immunity and improve outcomes: Ongoing efforts in this arena will hopefully bear fruit in the struggle to generate clinically meaningful immunotherapeutic strategies.

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Chapter 45  Dendritic Cells  MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells. Proc Natl Acad Sci USA 95:6279–6283 265. Celluzzi CM, Falo LD Jr (1998) Physical interaction between dendritic cells and tumor cells results in an immunogen that induces protective and therapeutic tumor rejection. J Immunol 160:3081–3085 266. Raje N, Hideshima T, Davies FE, Chauhan D, Treon SP, Young G, Tai YT, Avigan D, Gong J, Schlossman RL, Richardson P, Kufe DW, Anderson KC (2004) Tumour cell/dendritic cell fusions as a vaccination strategy for multiple myeloma. Br J Haematol 125:343–352 267. Gong J, Avigan D, Chen D, Wu Z, Koido S, Kashiwaba M, Kufe D (2000) Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells. Proc Natl Acad Sci USA 97:2715–2718 268. Gong J, Koido S, Chen D, Tanaka Y, Huang L, Avigan D, Anderson K, Ohno T, Kufe D (2002) Immunization against murine multiple myeloma with fusions of dendritic and plasmacytoma cells is potentiated by interleukin 12. Blood 99: 2512–2517 269. Gong J, Nikrui N, Chen D, Koido S, Wu Z, Tanaka Y, Cannistra S, Avigan D, Kufe D (2000) Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce antitumor immunity. J Immunol 165:1705–1711 270. Banat GA, Usluoglu N, Hoeck M, Ihlow K, Hoppmann S, Pralle H (2004) Dendritic cells fused with core binding factor-beta positive acute myeloid leukaemia blast cells induce activation of cytotoxic lymphocytes. Br J Haematol 126:593–601 271. Gong J, Koido S, Kato Y, Tanaka Y, Chen D, Jonas A, Galinsky I, DeAngelo D, Avigan D, Kufe D, Stone R (2004) Induction of anti-leukemic cytotoxic T lymphocytes by fusion of patient-derived dendritic cells with autologous myeloblasts. Leuk Res 28:1303–1312 272. Klammer M, Waterfall M, Samuel K, Turner ML, Roddie PH (2005) Fusion hybrids of dendritic cells and autologous myeloid blasts as a potential cellular vaccine for acute myeloid leukaemia. Br J Haematol 129:340–349 273. Chen D, Xia J, Tanaka Y, Chen H, Koido S, Wernet O, Mukherjee P, Gendler SJ, Kufe D, Gong J (2003) Immunotherapy of spontaneous mammary carcinoma with fusions of dendritic cells and mucin 1-positive carcinoma cells. Immunology 109:300–307 274. Higano CS, Vogelzang NJ, Sosman JA, Feng A, Caron D, Small EJ (2004) Safety and biological activity of repeated doses of recombinant human Flt3 ligand in patients with bone scan-negative hormone-refractory prostate cancer. Clin Cancer Res 10:1219–1225 275. Morse MA, Nair S, Fernandez-Casal M, Deng Y, St Peter M, Williams R, Hobeika A, Mosca P, Clay T, Cumming RI, Fisher E, Clavien P, Proia AD, Niedzwiecki D, Caron D, Lyerly HK (2000) Preoperative mobilization of circulating dendritic cells by Flt3 ligand administration to patients with metastatic colon cancer. J Clin Oncol 18:3883–3893 276. Chen W, Chan AS, Dawson AJ, Liang X, Blazar BR, Miller JS (2005) FLT3 ligand administration after hematopoietic cell transplantation increases circulating dendritic cell precursors that can be activated by CpG oligodeoxynucleotides to enhance T-cell and natural killer cell function. Biol Blood Marrow Transplant 11:23–34 277. Chi KH, Liu SJ, Li CP, Kuo HP, Wang YS, Chao Y, Hsieh SL (2005) Combination of conformal radiotherapy and intratumoral injection of adoptive dendritic cell immunotherapy in refractory hepatoma. J Immunother 28:129–135 278. Di Pucchio T, Pilla L, Capone I, Ferrantini M, Montefiore E, Urbani F, Patuzzo R, Pennacchioli E, Santinami M, Cova A, Sovena G, Arienti F, Lombardo C, Lombardi A, Caporaso P, D’Atri S, Marchetti P, Bonmassar E, Parmiani G,

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Chapter 46 Augmentation of Hematopoietic Stem Cell Transplantation with Anti-cancer Vaccines Edward D. Ball and Peter R. Holman

1. Introduction Hematopoietic stem cell transplantation (HSCT) is a curative therapy for a variety of hematological malignancies including acute and chronic leukemia, non-Hodgkin lymphoma, and Hodgkin lymphoma [1]. In addition, better disease control and improved survival can be achieved in patients with multiple myeloma (MM), though it is presently unclear whether patients can be cured with HSCT [2]. However, relapse of the underlying malignant disease is still a significant clinical problem. After autologous HSCT, the relapse rate is as high as 60% while after allogeneic HSCT up to 30% of patients relapse [3]. If relapse occurs, the prognosis is generally poor. Thus, new and more effective treatments of relapse and/or means of preventing relapse are urgently needed. It is widely believed that much of the success of allogeneic HSCT for patients with leukemia and lymphoma is due to the graft-versus-leukemia (GVL) effect [4]. This belief is based on the observations that GVHD correlates with superior disease control [4] and that clinical responses result from maneuvers such as infusions of donor lymphocytes and withdrawing immunosuppression [5]. The target antigens recognized by the donor immune system are not wellcharacterized and certainly are not identified in routine clinical practice [6]. Many tumor-associated antigens (TAA) have been described (see Table 461). For example, leukemia cells from patients with acute myeloid leukemia (AML) express antigens such as WT-1, PR1, and others [7]. The most tumor-specific antigen known is the idiotype of the surface immunoglobulin, expressed by non-Hodgkin lymphoma cells [8]. This unique region of the immunoglobulin molecule allows selective targeting of essentially each malignant lymphoma cell without cross-reaction with normal cells. The general purpose of vaccines in the context of stem cell transplantation is to amplify the immune response to tumor antigens at a time of tumor

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_46, © Springer Science + Business Media, LLC 2003, 2010

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Table 46-1.  Potential leukemia-associated antigenic targets. Antigen

Disease

Reference

WT-1

AML

[6]

Proteinase 3

AML, CML

[7]

HAGE

CML

[6]

Survivan

CLL, NHL, CML, MM

[6]

RHAMM

AML, MDS, MM, CML, CLL

[6]

PRAME

CML, CLL, ALL

[6]

HTERT

CML, CLL

[6]

MPP11

CML, CLL

[6]

Ig Idiotype

NHL, MM

[8]

vulnerability. Success is dependent on choosing the right antigen, engaging the immune system effectively, and choosing the optimal clinical setting in which to observe definitive results. This chapter will review this promising new approach to improving outcomes through reduction of relapse following HSCT. There are few published clinical trial results but many interesting trials are in progress or in the planning stage. This chapter will focus on the underlying principles of “vaccine”-based therapeutic approaches in hematological malignancy, a review of clinical trials underway, and of vaccine approaches in the developmental phase of clinical application.

2. Types of Vaccines The term vaccine in the setting of cancer vaccines is somewhat misleading. Traditionally, vaccines have been defined as materials used to prevent disease, such as the polio vaccine, an attenuated strain of virus, that induces an immune response in an unaffected individual that protects if challenged with live wildtype virus. In cancer therapy, “vaccine” has evolved to engender the use of both molecular and cellular products in affected individuals to induce primary immune responses to cancer cells. In a sense, they are attempts to break tolerance to cancer-associated antigens. Vaccines may, therefore, consist of a peptide or whole protein with sequences known to be expressed more or less exclusively on cells of the cancer, or, at least, its specific cellular lineage. Alternatively, cytotoxic T lymphocytes may be generated that recognize relatively tumor-specific antigens and that can be directly infused into the circulation of afflicted patients. Further, such TAA-directed T cells may be targeted further through genetic manipulation (transduction of membrane-bound antibodies: e.g., anti-CD19 or 20). Some of the advantages and disadvantages of these approaches are summarized in Table 46-2.

Chapter 46  Augmentation of Hematopoietic Stem Cell Transplantation 

Table 46-2.  Advantages and disadvantages of various vaccine approaches. Advantages

Disadvantages

Protein-based vaccine (e.g., ID, PR-1)

Ease of administration, inexpensive

All malignant cells may not express target antigen. CTL precursors may be limited

Cell-based vaccine

Generation of polyclonal responses

Cumbersome to generate cells. Requires GMP facility. Potential autoimmunity

Immunomodulation e.g., Ease of administration, Potential autoimmune events. anti-CTLA-4 monogeneration of polyclonal No FDA-approved reagents clonal antibody response at present

3. Clinical Settings 3.1. NHL and Multiple Myeloma 3.1.1. Vaccinations in Transplantation for Lymphoma and Myeloma Non-Hodgkin’s lymphoma (NHL) and Multiple Myeloma (MM) are both attractive disorders to target for the development of new immunotherapeutic approaches. Both these groups of diseases are amenable to immunotherapeutic approaches which have already been successful to varying degrees. Supporting the application of immune therapies to lymphoma is the observation of spontaneous remissions seen in a small number of patients with follicular lymphoma [9], the identification of an immune signature within follicular lymphoma as a good prognostic marker [10], the success of the monoclonal antibody rituximab [11] and the efficacy of allogeneic transplantation [12]. Bone marrow- or blood-derived stem cell transplantation is frequently offered to patients with NHL and MM and this setting offers both challenges and opportunities for the development of further innovative immunotherapeutic strategies. The recognition that a graft versus disease effect is primarily responsible for the curative potential of allogeneic transplantation has resulted in the emergence and widespread application of nonmyeloablative allogeneic transplantation protocols. However, owing to the lack of an available matched donor, this form of active immunotherapy is not applicable to the vast majority of patients. The success of monoclonal antibody therapy, a form of passive immunotherapy, and allogeneic transplantation, a form of active immunotherapy, clearly illustrate the susceptibility of these disorders to immunotherapeutic approaches. In NHL and MM, both clonal B cell disorders, the malignant clone as with normal B lymphocytes undergo rearrangement of the V, D and J genes during development. As a result, all cells of the malignant clone express immunoglobulins with the same unique variable sequence termed the idiotype. The idiotype can function as a tumor-specific antigen and as a result, these B cell-derived malignancies should be particularly well suited to a vaccinebased immunotherapeutic approach. Additional methods of inducing an active immune response are also being explored in these diseases.

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3.1.2. Autologous Transplantation High-dose chemotherapy with autologous stem cell transplant (HDT/ASCT) is widely applied in the management of both lymphoma and myeloma. When used to treat patients with indolent lymphoma, progression-free survival is improved but relapse occurs in virtually all patients [13]. Relapse is most likely due to disease surviving the high-dose therapy and/or lymphoma cells surviving in the graft. Similarly, for patients with relapsed aggressive lymphoma an improved OS for patients receiving high-dose therapy and autologous bone marrow transplantation has been established by Philip et al. [14]. Patients with mantle cell lymphoma also frequently undergo HDT/ASCT with an improvement in progression-free survival (PFS) [15, 16]. In MM, even though the introduction of newer targeted therapies is challenging this approach, HDT/ASCT (single or tandem) is widely accepted as initial standard consolidation therapy for younger patients with stage 2 and 3 disease [17, 18]. In all of these situations, however, with the exception of the aggressive lymphomas, HDT/ASCT is not curative. For this reason, many patients will be offered an allogeneic approach. In the case of the aggressive lymphomas, the relapse rate post-autologous transplant is still significant and a further improvement in outcome is required. 3.1.3. Post-autologous Transplant Immunization Idiotype vaccination has been evaluated in the autologous transplant setting in both MM and NHL. A report from the Stanford group described 26 MM patients who received idiotype vaccination following HDT/ASCT. Initially, in the setting of minimal residual disease post-transplant, patients received two intravenous infusions of dendritic cells pulsed with Id or Id-KLH. Subsequently, they received subcutaneous injections of Id-KLH. 24/26 patients developed a KLH-specific immune proliferative response but only 4 (3 in CR) developed an Id-specific immune response [19]. In another report from Stanford, 12 patients with relapsed or refractory B cell lymphomas receiving idiotype vaccination following HDT/ASCT were evaluated for the development of an immune response. Two different vaccination approaches were utilized, one with Id-KLH + GM-CSF and the other also incorporated the use of dendritic cells. Patients received vaccinations starting at 2–12 months post-transplant and all developed KLH-specific immune responses, supporting the use of active immunotherapy in the post-transplant time period. Ten patients developed an Id-specific humoral or cellular immune response [20]. At UCSD, we have conducted a pilot study of idiotype vaccination following HDC/ASCT. Fifteen patients with mantle cell lymphoma, follicular lymphoma grade 1 or 2 or transformed lymphoma received Id-KLH + GM-CSF vaccination. Starting at 3 months following HDT/ASC,T five vaccinations were administered over a 6-month period. Ten patients developed an antiKLH humoral or cellular immune response after one to four immunizations, and seven developed an anti-Id humoral or cellular response after one to five immunizations. Improvements in the clinical response from the early posttransplant status to the end of vaccination were seen in a number of patients and surprisingly, long-lasting remissions were seen in this heavily pre-treated group of patients [21]. At the time this manuscript was prepared, ten of the 15 patients are surviving at a median follow-up time of 55 (range 44–81) months and 7/10 are in complete remission. Further modifications to this approach

Chapter 46  Augmentation of Hematopoietic Stem Cell Transplantation 

could include pre-transplant immunization and continuing vaccinations through the period of immune recovery post-transplant which may allow the response of all arms of the immune system to the vaccination. 3.1.4. Allogeneic Transplantation Allogeneic transplantation is being increasingly offered to carefully selected patients. The development of the reduced intensity and nonmyeloablative conditioning approaches, based on the therapeutic graft-versus-malignancy effect has been increasingly applied and has resulted in a decrease in nonrelapse peri-transplant mortality. A number of trials, mostly small and single center have reported low transplant-related mortality and a low relapse rate in responding patients. There is, however, a significant risk of chronic graft-versus-host disease and other transplant-associated complications which continue to limit the general applicability of this approach. The incidence of graftversus-host disease has tended to correlate with the graft-versus-lymphoma effect but they may occur independently as has been seen in clinical trials [22]. Attempts to separate these effects are ongoing and progress is being made in the identification of lymphocyte subsets that are involved in each of these processes; reviewed in [23]. An improvement in the outcome following allogeneic transplants would occur if the incidence of graft-versus-host disease could be decreased while specific methods to augment graft-versus-malignancy could be enhanced. Disease relapse remains a considerable problem following allogeneic transplantation and molecular remission status is an important predictor of freedom from relapse. Inducing molecular remission following an allogeneic transplant may be achieved with donor lymphocyte infusions; however, this carries a significant risk of graft-versus-host disease. Vaccine approaches may eventually be a safer means of accomplishing this. Meanwhile, these improvements have extended the opportunity to undergo this form of immunotherapy to older patients and those with comorbidities who would have otherwise been excluded from an ablative allogeneic transplant. 3.1.5. Immunotherapy Including Idiotype Directed Therapy Before applying the concept of active immunotherapy with tumor vaccines to the transplant setting for lymphoma and myeloma, a little background information may be helpful. Passive immunotherapy with antibodies such as rituximab has proven to be a very successful therapy [24]. The mechanism of action of this antibody continues to be debated but is thought to function primarily through antibody-dependant cellular cytotoxicity (ADCC). Additionally, the direct induction of apoptosis and complement-mediated cytotoxicity are felt to be important. Recently, it has been postulated that an additional mechanism for rituximab is a vaccinal effect, whereby the dying cell releases components that are taken up by antigen-presenting cells from where an immune response could be initiated [25]. Whether rituximab does have active immunotherapeutic properties remains to be proven. Unlike passive immunotherapy, active immunotherapy with a vaccine has the potential to induce a polyclonal cellular and humoral immune response with a memory component that may prove to be more durable. The potential of idiotype-directed immunotherapy was first demonstrated in the early 1970s. Lynch et al. demonstrated the immunogenicity of myelomarelated proteins and the ability of the resulting antibodies to suppress growth of the corresponding tumor cells [26]. In 1987, it was shown that mice with

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lower, rather than greater, tumor burdens had a greater ability to develop an idiotype-specific immune response. As is the case with most tumor associated antigens, the idiotype protein is a relatively weak immunogen and subsequent studies demonstrated the utility of adding a more immunogenic carrier protein such as KLH to augment the immunogenicity of the administered proteins [27]. A further advance was the administration of an adjuvant in the form of GM-CSF which acts by recruiting antigen-presenting cells to the region of protein administration [8]. Idiotype vaccination has been evaluated in phase 1 and 2 clinical trials. In a pilot study from Stanford, nine patients received idiotype vaccination in the setting of minimal residual disease or complete response following chemotherapy. Two patients developed an idiotype-specific humoral response, four developed an idiotype-specific cellular immune response and one developed a humoral and cellular immune response [28]. Subsequently, the result of a larger cohort of patients was reported. Id-specific immune responses were demonstrated amongst a cohort of 41 patients with follicular lymphoma, also with either a CR or with minimal residual disease following chemotherapy. Forty-one percent of patients developed an anti-idiotype antibody response and 17% developed an anti-idiotype cellular response. Two patients with residual disease achieved a complete response along with the development of an idiotype-specific immune response [29]. Bendandi et al. reported the results of a further clinical trial of idiotype vaccination in 20 patients with follicular lymphoma. All were in the first CR after chemotherapy and were vaccinated starting 6 months after completing chemotherapy. This delay was included to allow recovery of the immune response prior to idiotype vaccination. All were in a CR at the time of vaccination. All patients developed anti-KLH cellular and humoral responses. Anti-idiotype humoral responses developed in 15 of the 20 patients and anti-idiotype cellular responses were noted in 19 patients. Importantly for establishing clinical efficacy, minimal residual disease as demonstrated by a positive test for the t(14;18) was present in 11 patients following chemotherapy, prior to idiotype vaccination. Eight of these patients became negative following vaccination, suggesting an idiotype-specific clinically relevant benefit. In 19 of the 20 patients, an HLA class 1-restricted idiotype-specific cellular proliferation was identified when post-vaccine peripheral blood mononuclear cells were co-cultured with autologous follicular lymphoma cells. This suggested the importance of a cytotoxic cellular response to the anti-lymphoma activity [30]. A long-term follow-up report of this study has been published and with a median follow-up of 9.2 years, the median disease free survival was 8 years and the overall survival rate was 95% [31]. Other studies have suggested the importance of a humoral cellular response for therapeutic benefit. It is likely that components of both may be important; however, following rituximab administration, cellular immune responses can be identified in the absence of humoral responses. The clinical utility of idiotype vaccination is currently being evaluated in three randomized clinical trials. The method of idiotype preparation varies but in all the three, the final product is idiotype protein complexed to the immunogenic carrier protein KLH and administered along with GM-CSF as an adjuvant. In one trial, hybridoma methodology is used for vaccine preparation. In the other two, recombinant DNA techniques are used. The latter

Chapter 46  Augmentation of Hematopoietic Stem Cell Transplantation 

techniques allow for a shorter preparation time, allowing for a more rapid time to administration. The trial designs also differ. In two of the trials, patients receive chemotherapy prior to vaccination. They are required to have at least a partial response in order to receive vaccination. In the third trial, patients are vaccinated following rituximab given alone. Patients who have a response to rituximab, or who have stable disease can be vaccinated. As the role of the humoral response is unclear, maintenance vaccinations are continued as B cell recovery occurs following rituximab and also beyond until there is evidence of disease progression. Individual patient-specific protein vaccines are cumbersome, being laborintensive and time-consuming. Alternative formulations to induce idiotypespecific immunity are under development. These include DNA vaccines [32]. Additionally, insight from studies evaluating the mechanism of action of rituximab and other antibodies is spurring on the development of more effective passive immunotherapies which may render the more cumbersome patient-specific therapies less useful. The peritransplant period has not been extensively evaluated as an opportunity for therapeutic vaccination. In both autologous and allogeneic transplantation, there is a long-lasting immune deficit that was felt to render attempts at augmenting tumor-specific immunotherapy approaches futile. However, a number of different strategies to augment disease control without increasing graft-versus-host disease have been developed in both myeloma and lymphoma. 3.1.6. Donor Immunization One strategy involves immunizing allogeneic stem cell donors. Kwak et al. and Neelapu et  al. have reported the use of myeloma-associated idiotype conjugated to an immunogenic carrier protein and emulsified in an adjuvant to immunize healthy sibling donors prior to allogeneic bone marrow transplant. They demonstrated the induction of Id- and carrier-specific T cell responses in three of the five evaluable recipients who survived more than 30 days post-transplantation. Two of the three patients remained disease free 7 and 8 years post-transplant. The third patient died from renal failure 5.5 years post-transplant while in complete remission [33, 34]. An alternative strategy involves the ex vivo priming of allogeneic donor T cells with recipient Id-KLH-pulsed dendritic cells [35]. Minor Histocompatibility antigendirected donor T cells are felt to mediate graft-versus-malignancy effects. Augmenting the presentation of such antigens is another strategy to augment graft-versus-malignancy following allogeneic transplantation. HA-1 and HA-2 are two such minor histocompatibility antigens that are restricted to hematopoietic lineages. HA-1- and HA-2-positive patients with leukemia and myeloma have been treated with DLI from HA-1- or HA-2-negative donors with the appearance of HA-1- and HA-2-specific CD8+ T cells 5–7 weeks following DLI, coinciding with the induction of a complete remission [36]. Recently, an additional minor histoincompatibility antigen (LB-ADIR-1F) relevant to a myeloma was reported following the study of a tumor-reactive clone that resulted in a complete remission following donor lymphocyte infusion in a patient with relapsed multiple myeloma [37]. These antigens and others may be useful in the development of immunotherapeutic strategies following allogeneic transplantation.

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3.2. Leukemia 3.2.1. Peptide Immunization Leukemia cells are subject to immune attack through their expression of cell surface antigens that are either unique to the particular leukemia or that are lineage-associated. Several leukemia-associated antigens (LAA) expressed on AML cells have been described [6]. These include a peptide-derived from proteinase 3, known as PR1 [6], the Wilm’s tumor antigen WT-1 [6], and many others [6]. Vaccination strategies using these peptide vaccines are underway [7, 38–40]. The group from M.D. Anderson reported that PR1 vaccination elicited immunological responses after hematopoietic stem cell transplantation in 55% of 20 patients [38]. Immune response was defined as a >twofold increase in PR1-CTL by tetramer assay. There was good correlation between immune and clinical responses with 9/11 immunoresponsive patients having a clinical disease response. 3.2.2. Cell-Based Therapies Another therapeutic approach is the generation of cytotoxic T cells reactive with known or unknown antigens expressed on AML cells. At UCSD, we have generated polyclonal T cell cultures, as well as T cell lines that are cytotoxic to autologous AML cells and that could be used in adoptive immunotherapy after autologous stem cell transplantation. This was accomplished by co-culturing AML and normal T cells from the peripheral blood of patients with active disease in the presence of GM-CSF and IL-4 [41, 42]. In these cultures, AML blasts differentiated into dendritic cells that presented antigen(s) to the autologous T cells. The T cells are then expanded through the addition of IL-2 and OKT3 to the cultures. Large quantities of T cells (1010) that are cytotoxic to autologous AML cells can be generated in a closed bag system. We are currently preparing to initiate a clinical trial of autologous stem cell transplantation for patients with AML in remission followed by infusion of the CTL. Blood will be obtained from the patients at diagnosis and the mononuclear cells cryopreserved. The patients will then undergo chemotherapy according to standard treatment protocols. If a patient is considered to be eligible for autologous stem cell transplantation (no HLA-match sibling donor, intermediate- to good-risk cytogenetics) their cryopreserved cells will be thawed and placed into the culture system designed to generate dendritic cell differentiation followed by expansion of autoreactive T cells. The patient will then undergo an autologous stem cell transplant using Bu/ Cy conditioning. After engraftment, the patients will then receive an infusion of CTLs generated from the cultures that were initiated prior to PBSCT. The study will use graded doses of T cells starting with 0.5 × 108 cells/kg body weight, then 108, and then 2 × 108 cells/kg. End points of the study include safety (absence of significant auto-immune events) and efficacy (with comparisons to our historical control database). Given the known correlation of graft-versus-host disease and control of leukemia after allogeneic stem cell transplantation [4], it is attractive to consider specific methods of amplifying anti-leukemia responses after allogeneic transplantation. Toward this end, we have studied the use of a cytotoxic T cell antigen (CTLA)-4 blocking human monoclonal antibody (mAb) in patients relapsing after allogeneic stem cell transplantation [43, 44]. This phase I/II study treated patients relapsing after allogeneic HSCT who relapsed and who

Chapter 46  Augmentation of Hematopoietic Stem Cell Transplantation 

did not have GVHD with safety as the primary end point. Patients received a single dose of ipilimumab, an IgG1 human monoclonal antibody that blocks the binding of CD80/86 to CTLA-4 expressed on activated T cells. Ipilimumab, therefore, blocks the down-regulatory effect of CTLA-4 ligation and allows T cells stimulated by antigen-presenting cells to continue proliferating and potentially mediate anti-tumor activities. Fortunately, we have not seen graft-versus-host disease in any of the 29 patients, though we did observe a few breakthrough autoimmune events (Immune Adverse Events). We have seen several intriguing clinical responses including partial and complete remissions of nodal masses in patients with non-Hodgkin Lymphoma and Hodgkin Disease, and a molecular remission in a patient with chronic myeloid leukemia. Examination of lymphocyte subsets following ipilimumab infusion revealed that T regulatory (Treg) cells were not affected, while there were increased numbers of activated T cells in many patients [44]. Rousseau et  al. studied eight patients with high-risk acute leukemia with a cellular vaccine of autologous leukemia cells mixed with fibroblasts transduced with CD40 ligand and IL-2 [45]. CD40L generates immune responses in leukemia-bearing mice, an effect that is potentiated by IL-2. They studied the feasibility, safety, and immunologic efficacy of an IL-2- and CD40Lexpressing recipient-derived tumor vaccine consisting of leukemic blasts admixed with skin fibroblasts transduced with adenoviral vectors encoding human IL-2 (hIL-2) and hCD40L. Ten patients (including seven children) with high-risk acute myeloid (n = 4) or lymphoblastic (n = 6) leukemia in cytologic remission (after allogeneic stem cell transplantation [n = 9] or chemotherapy alone [n = 1]) received up to six subcutaneous injections of the IL-2/CD40L vaccine. No severe adverse reactions were noted. Immunization produced a 10- to 890-fold increase in the frequencies of major histocompatibility complex (MHC)-restricted T cells reactive against recipient-derived blasts. These leukemia-reactive T cells included both T-cytotoxic/T-helper 1 (Th1) and Th2 subclasses, as determined from their production of granzyme B, interferongamma, and interleukin-5. Two patients produced systemic IgG antibodies that bound to their blasts. Eight patients remained disease free for 27–62 months after treatment (5-year overall survival, 90%). Thus, even in heavily treated patients, including recipients of allogeneic stem cell transplants, recipient-derived anti-leukemia vaccines can induce immune responses reactive against leukemic blasts.

4. Future Directions Augmentation of anti-tumor immune recognition and control is an attractive and important goal, given that relapse after both autologous and allogeneic HSCT continues to be one of the obstacles to cure using this therapy. Tantalizing hints of efficacy of various means of boosting immunity are reviewed above. Continued research into the nature of tumor-associated antigenic targets, means of enhancing antigen presentation, and methods of immune cell activation, proliferation and long-term survival will hopefully result in more specific and rational immunotherapy in the future.

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Chapter 46  Augmentation of Hematopoietic Stem Cell Transplantation  relapse of malignancy after allogeneic hematopoietic stem, cell transplantation. Blood 110:952a 45. Rousseau RF, Biagi E, Dutour A, Yvon ES, Brown MP, Lin T, Mei Z, Grilley B, Popek E, Heslop HE, Gee AP, Krance RA, Popat U, Carrum G, Margolin JF, Brenner MK (2006) Immunotherapy of high-risk acute leukemia with a recipient (autologous) vaccine expressing transgenic human CD40L and IL-2 after chemotherapy and allogeneic stem cell transplantation. Blood 107:1332–1341

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ERRATUM TO:

Allogeneic Stem Cell Transplantation Second Edition Edited by Hillard M. Lazarus University Hospitals Case Medical Center, Cleveland, OH, USA Mary J. Laughlin Case Western Reserve University, Cleveland, OH, USA

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients Flora Hoodin1, Felicity W.K. Harper 2, and Donna M. Posluszny 3 1

 Department of Psychology, Eastern Michigan University, Ypsilanti, MI, USA [email protected]

2

 Communication and Behavioral Oncology Program, Barbara Ann Karmanos Cancer Institute and Department of Family Medicine and Public Health Sciences, Wayne State University School of Medicine, Detroit, MI, USA

3

 Department of Medicine, University of Pittsburgh School of Medicine and Behavioral Medicine Clinical Service, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

H.M. Lazarus and M.J. Laughlin (Eds.), pp. 619–656, © Springer Science+Business Media, LLC 2003, 2010

DOI 10.1007/978-1-59745-478-0_47 The abstract for Chapter 35 Psychological Care of Adult Allogeneic Transplant Patients was erroneously substituted for the first three paragraphs of the text. The original paragraphs follow. References are cited at the end of the chapter. This chapter is devoted to the recognition, detection, and management of psychological concerns of adult patients who undergo allogeneic hematopoietic cell transplantation (HCT). As HCT is typically indicated for life threatening conditions, or when other treatment avenues are no longer curative, the resulting psychological distress cuts across disease types, sources of stem cells, and types of transplant. We attempt to focus exclusively on outcomes for allogeneic patients; however, given that the psychosocial literature for this subgroup of patients is limited, we also draw on empirical studies of

DOI 10.1007/978-1-59745-478-0_47 The abstract for Chapter 35 Psychological Care of Adult Allogeneic Transplant Patients was erroneously substituted for the first three paragraphs of the text. The original paragraphs follow. References are cited at the end of the chapter. autologous transplant patients. Thus, in this chapter, we use the term “HCT” to encompass both types of transplant, and we note explicitly when mixed patient samples are referenced. The intensity of psychological distress is likely amplified for allogeneic as opposed to autologous patients, for whom undergoing HCT is a tightrope-walk between the life-extending, sometimes curative effects of transplant and the potential of the treatment to hasten their death. For example, the treatment related mortality for allogeneic patients at 1 year is 16–29% in contrast to 2% for autologous HCT patients [1]. Further, psychological distress persists for the approximately 50% [2] to 77% [3] of allogeneic patients who do survive 2 or more years and face lingering or latent long-term effects [3], negatively impacting the quality of their lives. In the words of Macklin Smith, poet and long-term survivor of a matched unrelated allograft, a HCT brings both long term positive aspects such as “being alive, amazement at being alive, enjoying life, appreciation for the small things in life, living a productive life,” and negative aspects “fatigue, forgetfulness, chronic sorrow, general bewilderment, fear of death, indifference to death, confusion about death” [4]. The challenge, therefore, in effectively caring for the psychological needs of allogeneic HCT patients is to recognize when psychological symptoms and distress exceed normative responses to HCT with its risks and long-term effects and to provide appropriate psychological intervention and support as needed.

The online version of the original chapter can be found at http://dx.doi.org/10.1007/978-1-59745-478-0_35

sdfsdf

Index

A Acquired hypercoagulable disorders, 708 Acute graft versus host disease (aGVHD) graft manipulation ex vivo TCD, 567 T cells removal, 565 in vivo negative selection techniques, 566 PBSCT, 286 pharmacologic prevention cyclophosphamide, 571–572 cyclosporine, 567 IBMTR, regimens, 568 methotrexate, 567 mycophenolic acid (MPA), 571 novel calcineurin inhibitors, 567 sirolimus, 569–570 treatment cellular subsets optimization, 759 corticosteroid dose, 749 definition, 747 first-line treatment, combination therapy, 750–751 Glucksberg criteria, 749 IBMTR severity index, 748 impact, GvL, 751 mesenchymal stromal cells (MSCs), 759 non-myeloablative transplantation, 758–759 pathophysiologic mechanisms, 759–760 second line therapy, 752–758 standards of care, 749 steroid therapy, 748 supportive care, 751 therapy duration, 750 Acute leuekemia, 356 Acute lymphoblastic leukaemia (ALL) biological randomization, 30 CNS disease, 198–199 conditioning regimens, 194 CR1/CR2, 32 definitions, 31 donor lymphocyte infusions, 198–199 first remission, sibling allograft, 194–195 haploidentical donors, 36 palifermin, 199 Philadelphia chromosome positive ALL, 37–38

prognostic factors, 29–30 rationale and GvL effect, 193–194 refractory disease, 199 relapsed disease allograft, 198 remission, 37 RIC regime, 36–37 role, RIC, 196–197 sibling allo HSCT, 34–35 sibling donor, 32 T-cell depletion, 36 UD-SCT, 33 UKALL XII/ECOG 2993 study, 32 umbilical cord blood HSCT, 33 Acute myeloid leukemia (AML), 11 myeloablative conditioning regimen Bu-based regimens, 16 CR1, 13 donor lymphocyte infusions (DLI), 20 donor vs. no-donor analyses, 14 optimal conditioning regimen, 16 optimal stem cell source, 17 outcome, disease relapse, 19 reduced intensity allografts, 18 RIC regimen, 18 salvage chemotherapy, 15 T-cell depletion strategies, 16–17 Acute nonlymphoblastic leukemia, 3 Adenoviruses, 514–515 Adoptive cell therapy, TRM reduction, 465 Adult cord blood transplantation double cord blood, 368–369 refractory lymphoma, single cord blood, 368 Aggressive B-cell lymphomas chemosensitivity, 118 DLBCL, 98, 117 RIT, 119 single arm cohort studies, 99 Alemtuzumab, 451–452, 753 ALL. See Acute lymphoblastic leukaemia Allogeneic gene therapy, thalassemia alternative donors transplantation, 498–499 BMT vs. medical treatment, 492 disease eradication, 493–494 engraftment, 494

871

872 

Index

Allogeneic gene therapy, thalassemia (cont.) ex-thalassemic management, 499–500 graft failure/rejection, 497–498 GVHD, 498 mixed chimerism, 497 morbidity and mortality, 494 preparatory regimens, 493 risk classes, 495 transplant outcome, HLA matched related donors, 495–497 adult patients, 496–497 class 1and 2 patients, 495 class 3 patients, 495–496 transplant procedures, 492 AlloSCT favorable-risk AML HOVON/SAKK study, 186 LFS, 184 myeloablative conditioning therapy, 183 OS, 185 Alternative donors, SCT alternative related donors, 498 unrelated bone marrow transplantation, 498–499 unrelated cord blood transplantation, 499 AMD3100, hematopoietic cells mobilization mononuclear cell (MNC) fraction, 394–395 PBSC, 395 phase 3 clinical trials, 395 SCID-repopulating capacity (SRC), 394 AML. See Acute myeloid leukemia Antibody-dependant cellular cytotoxicity (ADCC), 859 Anti-cancer vaccines, HSCT augmentation leukemia cell-based therapies, 862–863 peptide immunization, 862 NHL and MM allogeneic transplantation, 859 autologous transplantation, 858 donor immunization, 861 idiotype directed therapy, 859–861 post-autologous transplantation immunization, 858–859 potential leukemia-associated antigenic targets, 856 vaccines types, 856–857 Anti-thymocyte globulin (ATG), 588, 752–753 Aplastic anemia, allograft, 3 Aspergillus infections Aspergillosis, 536 ELISA assay, 537 galactomannan, 537 symptoms, 536 voriconazole, 538 B B cell immune reconstitution, immunotherapy development, B cell, 547–548 naïve phenotype, 548 post-transplant, 548 BCR. See Breakpoint cluster region

Bioluminescent-based imaging (BLI), 790 Birbeck granules, 810 Blood and marrow transplant clinical trials network (BMT CTN) GVHD, 570 psychological morbidity, 644 tandem autologous transplantation, 148 TA-TMA, 706 TCD, 567 unrelated donor CB, 381 Breakpoint cluster region (BCR), 54 Bronchiolitis obliterans (BO), cGVHD, 583 Burkitt's lymphoma (BL), 102, 225 C CALGB. See Cancer and leukemia group B Cancer and leukemia group B (CALGB), 178 Candida infections beta glucan assay, 534 common clinical syndromes, 535 mucosal injury, 533 symptoms, 534 Catheter thrombosis, 704 Cell infusion, NKcell CD 56+, 420 clinical application, 419 CliniMACS ®, 421 cytokine activation, 421 NK-DLI, 418–420 UCB, 420 Center for International Blood Marrow Transplant Research (CIBMTR) allograft recipients age, 130 CML, 57 follicular non-Hodgkin's lymphoma (FL), 112 peri-TKI era, 62 RIC transplantation, 134 RIT regimens, 114 Centers for Disease Control (CDC) evidence-based rating system, 722 guidelines, infection control, 723, 724 level I evidence, 722–723 level II/III evidence air, 725 fomites, 725 food and water, 726 host, 724–725 human to human, 725 soil, construction and cleaning, 726 Central nervous system (CNS) ALL, 198–199 bleeding intracranial hemorrhage (ICH), 701 subdural hematoma (SDH), 701 tacrolimus (FK 506), 702 Childhood ALL. See Pediatric ALL Chromosomal abnormalities, 669 Chronic graft-versus-host disease (cGVHD) diagnosis of, 578

Index  eyes, 582–583 gastrointestinal tract, 583 genitalia, 583 grades II–IV, probabilities, 286 GVHD prophylaxis, 591 hairs, 582 hematopoietic and immune system, 584 liver, 583 lungs, 583 mouth, 582 musculoskeletal system, 583 nails, 582 NIH consensus criteria, 579 risk factors development of, 589 HLA, 587 KPC, 587 peripheral blood stem cell transplantation (PBSCT), 586 sBAFF and anti-dsDNA, 587 severity and score, 585–586 signs and symptoms, 580 skin, 578, 582 treatment cyclosporine (CSA)/tacrolimus, 591 salvage therapy, 590 Chronic lymphocytic leukemia (CLL) treatment. See Hematopoietic progenitor cell transplantation Chronic myeloid leukemia (CML), 4 CIBMTR. See Center for International Blood Marrow Transplant Research Clinical psychologist role evidence-based HCT psychological treatment psychological intervention literature, 634 psycho-oncology, 634–635 somatic and emotional symptoms, 632–633 pathways, psychosocial care post-hospitalization, 638–639 pre-and peri-hospitalization, 637–638 psychopharmacological intervention antidepressant mirtazapine, 636 CBT, 636 psychotropic medications, 635 social workers normal trajectory, 631 practical issues, 629 suggested instruments, 630 Clonal hematopoietic cell disorder. See Chronic myeloid leukemia Cognitive impairment, 624 Co-morbidity index, 688–689 Complete response (CR) rates, 153, 180, 657 Conditioning regimens, hematopoiesis non-radiation based regimens, 775 radiation based regimens, 774–775 Cord blood banking, 364 Cord blood units selection vs. peripheral blood progenitor cells, 375 principles, 376 unrelated donor CB

cell dose, 377–378 diagnosis effect, 381 double cord blood units, 381–382 factors, 377–378 product, 376–377 Texas Transplant Institute Perspective, 382–383 untreated donor CB HLA matching, 378–379 non-inherited maternal allele matching, 379–380 Core-binding factor (CBF) abnormality, 179 Corticosteroid dose, 749 CR1. See First complete remission Cryopreservation advantages and disadvantages, 436 bacterial contamination, 430 clinical outcomes BM allograft comparison, 432–433 engraftment and outcome data, 431 GVHD incidence, 433 multivariate analyses, 432 PB allograft comparison, 432 unrelated allogeneic transplantation, 433–434 donor lymphocyte infusion, 434 ethical concerns, 435 graft content donor graft alteration, 428 impact, 428–430 MNC subtypes, 428 logistics, 434–435 methodology, 428 transfusion reactions, 430 Cytokine-primed marrow transplantation, 290 Cytomegalovirus (CMV) immune therapy and monitoring, 509 preemptive therapy, 508 prophylaxis, 507–508 risk factors, 507 Cytotoxic T-lymphocyte precursors (CTL-ps), 462 D Daclizumab, 755–756 Dendritic cells (DC) ex vivo generation IL, 816 SCF, 815 T cell population, 817 immune reconstitution, GVHD vs. HSCT CAMPATH, 821 co-transplantation, 822 cytokine regimen, 820 donor T cells, 821 pathogenesis, 820 umbilical cord blood, 822 immunotherapy cancer, 822–827 clinical studies, cancer, 827–831 potential limiting factors, 831–832

873

874 

Index

Dendritic cells (DC) (cont.) phenotypic characterization Birbeck granules, 810 GM-CSF, 809 immature DC, 810 mature DC, 812–813 skin-homing receptor, 810 TLRs, 812 subsets, 809 T cell interactions adhesion molecules, 814 danger signals, 814 IL-12, 814, 815 stimulatory signals, 815 tolerance establishment IDO, 818 programmed death ligand-1, 818 T cells, 817 thymic-derived population, 818 Tr1, 819 in vivo GVHD model, 820 Denileukin diftitox, 756–757 Deoxycoformycin, 755 Diffuse alveolar hemorrhage (DAH), 700–701 Diffuse large B cell lymphoma (DLBCL), 98, 117 DLBCL. See Diffuse large B cell lymphoma Donor leukocyte infusion (DLI), myeloablative and non-myeloablative SCT, 434 therapy, 602 Donor lymphocyte infusions (DLI) GVL response, 20 reduced intensity regimens, 81 salvage therapies, 657 survival rate, 272 Donor selection. See also Alternative donors, SCT CLL, 50 hematopoietic progenitor cell transplantation, 50 HLA-Bw4 mismatches, 470–471 HLA-C group 1 alleles, 469–470 principles allele level typing, 313–315 first-degree relatives, 315 KIR ligand groups, 315 molecular methods, 313 Dose intensity, novel regimens definition, 441–442 GvHD, impact acute GvHD, 445–447 TRM, 447 myeloablative conditioning vs. NMT/RIC, 442–443 RIC/NMT, 443 standard transplantation, 442–443 Double cord blood transplantation, 368–369 Down syndrome (DS), 224 Dural (venous) sinus thrombosis, 708 E EBMT transplantation risk score, 57 Emotional disorders

anticipatory nausea, 622 effects, 634 insomnia, 622 psychological distress, 621 symptoms, 622 Epratuzumab, 122 Epstein-Barr Virus (EBV), 510–511, 598 Etanercept, 758 European group for Blood and Marrow Transplantation (EBMT), 45, 49, 346 Event free survival (EFS), 161, 465 F Favorable-risk AML alloSCT HOVON/SAKK study, 186 LFS, 184 myeloablative conditioning therapy, 183 OS, 185 ASCT, 181–183 CR1, 181 GVHD, 181 ITT analysis, 181 LFS, 182 overall survival, 182 CALGB, 178 cytarabine-/anthracycline based induction chemotherapy, 178 definition, 178 molecular pathogenesis, 179 post-remission therapy, 179–180 refractory AML, 186 relapsed AML, 186–187 RIC SCT, 187 SCT, 180–181 First complete remission (CR1) LFS vs. ASCT, 182 OS vs. ASCT, 182 pediatric ALL chemotherapy vs. transplantation, 231 disease-free survival, 234 eligibility criteria, 228–230 HSCT outcome, 232 transplantation, 230, 234–235 umbilical cord outcomes, 236 very high risk ALL frontline treatment, 227 FLIPI. See Follicular lymphoma international prognostic index Fludarabine, 450–451 Follicular center cell NHL. See also Non-Hodgkin's lymphoma CD20 vs. chemotherapy, 161 chemoimmunotherapy (R-CHOP), 163 CR/PR, 161 EFS, 161 FLIPI, 160 myeloablative therapy allogeneic HSCT, 163–165

Index  relapsed/refractory FL, 163 vs. RIC, 166–167 nonmyeloablative therapy allogeneic HSCT, 165 graft versus tumor/GvL effect, 166 RIC regime, 165 relapsed follicular lymphoma treatment, 162 transformed follicular NHL, 167–168 Follicular lymphoma international prognostic index (FLIPI), 160 Follicular non-Hodgkin's lymphoma (FL), 110 Foundation for the accreditation of cellular therapy (FACT), 349, 717 Full and reduced intensity, AML GVL effects, 12 leukemic stem cells (LSC), 11 myeloablative conditioning regimen Bu-based regimens, 16 CR1, 13 DLI, 20 donor vs. no-donor analyses, 14 optimal conditioning regimen, 16 optimal stem cell source, 17 outcome, disease relapse, 19 reduced intensity allografts, 18 RIC regimen, 18 salvage chemotherapy, 15 T-cell depletion strategies, 16–17 post-transplant cyclosporine (CyA) impact, 12 Fungal infections antifungal agents, 540–541 Aspergillus infections airborne infection, 720 Aspergillosis, 536 Clostridia difficile-associated diarrhea, 720 ELISA assay, 537 galactomannan, 537 symptoms, 536 voriconazole, 538 Candida infections beta glucan assay, 534 common clinical syndromes, 535 mucosal injury, 533 symptoms, 534 evaluation and treatment approaches, 542 Fusarium, 539 Pneumocystis carinii, 539 Zygomycetes infections, 538–539 G Gamma/delta ( g d ) TCR T cells, 798 Gastrointestinal bleeding, 700 Grade 4 mucositis, 199 Graft failure/rejection, 497–498 Graft versus host disease (GVHD) aGVHD cellular subsets optimization, 759 corticosteroid dose, 749 definition, 747

875

first-line treatment, combination therapy, 750–751 Glucksberg criteria, 749 graft manipulation, 565–567 IBMTR severity index, 748 impact, GvL, 751 mesenchymal stromal cells (MSCs), 759 non-myeloablative transplantation, 758–759 pathophysiologic mechanisms, 759–760 PBSCT, 286 pharmacologic prevention, 569–572 second line therapy, 752–758 standards of care, 749 steroid therapy, 748 supportive care, 751 therapy duration, 750 antibody prophylaxis alemtuzumab vs. methotrexate, 740 peripheral blood stem cells, 739 T-cell depletion, 740 T-lymphocyte, 739 cGVHD cyclosporine (CSA)/tacrolimus, 591 diagnosis of, 578 eyes, 582–583 gastrointestinal tract, 583 genitalia, 583 GVHD prophylaxis, impact, 591 hairs, 582 hematopoietic and immune system, 584 HLA, 587 KPC, 587 liver, 583 lungs, 583 mouth, 582 musculoskeletal system, 583 nails, 582 NIH consensus criteria, 579 peripheral blood stem cell transplantation (PBSCT), 586 salvage therapy, 590 sBAFF and anti-dsDNA, 587 severity and score, 585–586 signs and symptoms, 580 skin, 578, 582 treatment HLA mismatch adverse effect, 311 infectious complications CD8-depleted DLI, 741–742 immunity suppression, 741 lymphocyte count, 741 mitigate graft allodepletion, 325 non-inherited maternal antigens, 324–325 replete graft, T Cell, 326–327 T cell depletion and infusion lymphocytes, 325–326 prophylaxis, 352 Graft vs. leukemia (GVL), 796, Graft vs. lymphoma effect (GVLy), 91 Graft vs. tumor (GVT), 796

876 

Index

Granulocyte–macrophage colony stimulating factor (GM-CSF), 809 GROb, 393–394 GVL/GVT, 796 GVLy. See Graft vs. lymphoma effect H Haploidentical transplantation engraftment and GvHD CD34+ and T-cells, 462 transplantation procedure, 462–463 event free survival (EFS), 465 leukemia relapse, 464 NK cell alloreactivity cytokine secretion and cytotoxicity, 467 KIR genetics activation, 466–467 matched unrelated donor transplants, 467–469 transplant related mortality (TRM) adoptive cell therapy, 465 G-CSF impact, 464 immunological recovery, 464 overlapping factors, 464 procedure, 463 HCT specific comorbidity index (HCT–CI), 136 Health and behavior current procedural terminology (H & B CPT) codes, 643 Hematopoiesis bone marrow microenvironment, 780 conditioning regimens non-radiation based regimens, 775 radiation based regimens, 774–775 HSC grafts and growth factors, 772–774 MHC, 767 non-human primate models baboons, 770 macaques, 770–801 marmosets, 771–772 in vivo models, attributes, 769 SCT models allogeneic, 776–777 autologous, 775 gene therapy, autotransplantation, 775–776 GVHD, 777 MHC typing, 777–778 whole organ tolerance induction, 778 xenotransplantation, 779–780 Hematopoietic bone marrow microenvironment, 780 Hematopoietic cells mobilization cytokines, mobilizing donor cells, 402–403 G-CSF effect, normal donor PBSC mobilization, 400 short-term stimulation effect, 402 splenomegaly, 401 toxicity, 401 mechanisms adhesion molecules, 388 CD26, 389 chemokines, 388 cytokines, 388–389 IL-8 activity, 389

mobilizing agents, 403 normal donors cell doses, 398 factors, 399–400 G-CSF, 399 GM-CSF, 399 immunomodulatory effect, 400 novel agents AMD3100, 394–396 CXCR4 peptide, 393 GRO[$$], 393–394 parathyroid hormone (hrPTH), 393 pegfilgrastim, 391–392 recombinant human growth hormone (rhGH), 391 stem cell factor, 391 thrombopoietin, 392 regimen selection aldehyde dehydrogenase (ALDH), 397 AMD3100, 398 CD34+ cells, 397 chemotherapy/HGF, 396–397 mobilization capacity, 396 stem cell mobilization regulation neural signals, 389–390 osteolineage derived cells Hematopoietic progenitor cell transplantation autologous transplantation, 44 genomic high risk CLL, 48 myeloablative conditioning regime, 45–47 non-myeloablative conditioning regime, 47–48 treatment, 44–45 chronic immunosuppression, 49 co-morbidity index, 688–689 donor selection, 50 functional assessment tools, 689–690 high dose therapy (HDT), 687 indications, 49 performance status (PS), 688 prognostic factors, 691 Hematopoietic stem cell, thalassemia allogeneic gene therapy alternative donors transplantation, 498–499 BMT vs. medical treatment, 492 disease eradication, 493–494 engraftment, 494 ex-thalassemic management, 499–500 graft failurerejection, 497–498 graft-versus-host disease, 498 mixed chimerism, 497 morbidity and mortality, 494 preparatory regimens, 493 risk classes, 495 transplant outcome, HLA matched related donors, 495–497 transplant procedures, 492 clinical manifestations, 491 hemoglobin disorder, 491 treatment, 491 Hemorrhagic cystitis, 698–700 Hepatic veno-occlusive disease (VOD) diagnosis, 704–705

Index  gemtuzumab ozogamicin, 705–706 morbidity and mortality, 706 pathogenesis, 705 sinusoidal obstruction syndrome (SOS), 704 Hepatitis B and C viruses, 515 Herpes simplex virus (HSV), 509 High dose therapy (HDT), 687 Histocompatibility, 5 HLA. See Human leukocyte antigens HLA-haploidentical related donors clinical outcomes allodepletion, 323–324 G-CSF primed donors, 319–321 megadose stem cell transplantation, 318–319 nonmyeloablative HLA-haploidentical SCT, 321–323 replete grafts, T cell, 317 T cell depletion effect, 317–318 complications aGvHD, 311–312 graft failure, 309–311 GVHD, 311–312 impaired immune reconstitution and infection, 312–313 complications reducing strategies mitigate GVHD, 324–327 prevent/treat relapse, 327–329 feature, 299 HLA typing and donor selection principles allele level typing, 313–315 first-degree relatives, 315 KIR ligand groups, 315 molecular methods, 313 iKIRs and HLA ligands interactions, 303 immunobiology human T cell responses, 300–301 limitations, 301–302 natural killer cell alloreactions, 302–304 reactivity models, 304–307 T and NK cells interactions, 308 inhibitory KIR types, 304 natural killer cell alloreactivity models, 305 unrelated donor umbilical cord blood cell dose, 330 double unit UBCT, 330–331 grafts typing, 329 leukemia-free survival, 329 Hodgkin’s lymphoma allogeneic bone marrow transplantation fully ablative regimens, 80 high risk patients, 85 intensity preparative regimens, 82 ASCT, 78 autologous bone marrow transplant, 77 FFS/OS rates, 78 high-dose therapy, 77 reduced intensity regimens chemoresistant disease, 83 donor lymphocyte infusions (DLI), 81 fludarabine-melphalan, 83 GVL effect, 84

877

nonrelapse mortality, 84 peripheral blood stem cells (PBSC), 81 risk factors, 78 standard therapy, 76 Homeostatic peripheral expansion (HPE), 549–550 Human Herpes Virus Type 6 (HHV-6), 511–512 Human leukocyte antigens (HLA), 5, 587 Hyperleukocytosis, 223–224 I IBMTR. See International Blood and Marrow Transplant Research Idiotype directed therapy ADCC, 859 anti-idiotype humoral responses, 860 trial designs, 861 vaccination, 858, 860 Immunobiology, HLA-haploidentical related donors human T cell responses, 300–301 allogeneic response strength, 300 cross-reactivity phenomenon, 301 determinant density, 300–301 determinant frequency, 301 minor histocompatibility Ags (minor H Ags), 300 limitations, 301–302 natural killer cell alloreactions DNA damage, 304 inhibitory KIRs (iKIRs), 302–304 licensing, 304 missing self hypothesis, 302 molecular basis, 302 reactivity models gene–gene model, KIR, 307 KIR ligand incompatibility, 305 missing ligand model, 307 receptor-ligand model, 306–307 T and NK cells interactions, 308 Immunotherapy B and T cells, 547 B cell immune reconstitution B cell development, 547–548 post-transplant, 548 cancer fusion cells, 827 immature DC administration, 823 melanoma cells, 826 mRNA, 826 tumor-associated antigens, 823, 826 vaccination, 825 viral transduction, 825 cellular immunotherapy post-transplant, 553–554 clinical studies, cancer CD11c/CD14, 827 CPG ODN, 827 ELIspot analysis, 829 MAGE peptides, 828 prostate-specific membrane-antigen (PSMA) peptides, 828 vaccination, 831 considerations, 555

878 

Index

Immunotherapy (cont.) immune reconstitution, 546 killer immunoglobulin-like receptor (KIR), 545 potential limiting factors chemotherapy, 832 ONTAK, 832 tumor cells secrete factors, 831 T cell reconstitution cellular immunotherapy, thymopoiesis, 552–553 HPE, 549–550 initial post-transplant period and implications, 550–551 thymic dependent, 548–549 Indoleamine 2,3-dioxygenase (IDO), 818 Indolent lymphoma, NHL dose intensive chemotherapy, 91 EBMT/IBMTR review, 93 European CUP trial, 91 GVLy effect, 92 low treatment-related mortality (TRM), 92 single arm cohort study, 93 transplantation outcome, 94 Infliximab, 757 Influenza viruses, 514 Intent-to-treat (ITT) analysis, 181 Interleukin (IL), 549 International Blood and Marrow Transplant Research (IBMTR), 46, 81, 93 International Prognostic Scoring System (IPSS), 206 Invasive fungal infections (IFI). See Fungal infections In vivo models, AlloHSCT immunobiology aGVHD, 795 cGVHD, 795–796 graft rejection, 793 graft vs. tumor (GVT), 796 immune reconstitution, 794–795 non-T cell lymphoid populations gamma/delta ( g d ) TCR T cells, 798 natural killer cells, 798 NKT cells, 798 preclinical models age and sex, 793 BLI, 790 endogenous microflora, 792–793 mouse strains and immunologic disparity, 791–792 species differences, 790 IPSS. See International Prognostic Scoring System Isolation methodology, allografts CDC level I evidence, 722–723 level II/III evidence, 723–726 guidelines, infection control, 723, 724 isolation costs finances, 726–727 patient interactions, 727 necessity benefits, 721–722 infection outbreaks, 719–720

infectious complications, 718–719 vancomycin-Resistant Enterococcus (VRE), 720–721 outpatient care, 728 K Karnofsky performance status (KPS), 587 Killer-cell immunoglobulin-like receptors (KIR) genes, 460, 545 gene–gene model, 307 ligand incompatibility model, 305–306 L Lentiviral vectors, 776 Leukemia-associated antigens (LAA), 862 Leukemia-free survival (LFS), 180, 182 Leukemic cell characteristics pediatric ALL Burkitt-leukemia, 225 cytogenetics, 225 morphology, 224 precursor B-cell, 224 Lymphoblastic lymphoma (LBL), 103 Lymphohematopoietic chimerism, 777 M Major histocompatibility complex (MHC), 5, 767 Mantle cell lymphoma (MCL) nonmyeloablative/reduced intensity transplantation, 116 nonmyeloablative/RIT ergimes, 117 Matched unrelated donor (MUD), 95. See also MUD BMT MDS. See Myelodysplastic syndromes Megadose stem cell transplantation, 318–319 Mesenchymal stem cells bone marrow-derived MSCs, 477–478 clinical autologous and allogeneic MSCs transplantation, 480–482 clinical expansion, 485–486 growth factor supplementation, 482–483 immunologic properties, 479–480 limitations, 482–483 MHC barrier, 480 multipotent adult progenitor cells (MAPCs), 477 multipotentiality, 478 senescence, 479 Mesenchymal stem cells (MSC), 776 Mesenchymal stromal cells (MSC), 759 MHC. See Major histocompatibility complex Minimal residual disease (MRD), 226, 237 antigen receptor rearrangement analysis, 668–669 chimerism analysis, 670–671 clinical significance acute lymphoblastic leukemia, 671–672 acute myeloid leukemia, 672 chimerism results, 673–674 chronic myeloid leukemia, 672–673 reduced intensity conditioning regimen, 674–675

Index  fusion gene transcript analysis, 669–670 immunophenotype analysis, 667–668 methods, 668 Missing ligand model, 307 Mixed hematopoietic chimerism (MC), 497 Monoclonal antibodies, hematologic malignancies advantage, 733 CD20, 734 conditioning regimens radiolabeled antibody, 736–738 unlabeled antibody, 735 GVHD prophylaxis alemtuzumab vs. methotrexate, 740 infectious complications, 741–742 peripheral blood stem cells, 739 T-cell depletion, 740 T-lymphocyte, 739 morbidity and mortality, 734 post-transplant consolidation, 738–739 total body irradiation (TBI), 734 MSC. See Mesenchymal stem cells Mucosal Candida infections, 533 MUD BMT comparative analyses, 101 GVLy effect, MCL, 96 mantle cell lymphoma (MCL), 95 national marrow donor program (NMDP), 95 RICSCT techniques, 95 Multiple myeloma age, 131, 132 applications, 137–138 donor availability, 133 GVHD, 133 RIC regimens CIBMTR data, 135 multiple retrospective studies, 134 SEER incidence rates, 129 single vs. tandem autologous transplant, HSCT maintenance therapy, 151 novel pre-transplant regimens, 153 optimal time, transplant, 148 salvage therapy, 151 specific comorbidity index, 136 treatment allogeneic stem cell source and alternative donors, 267 vs. autologous transplantation, 265–267 DLI and post-transplant management, 273 phase II studies, 268–269 prognostic factors, 268–270 rationale, 263–264 syngeneic transplantation, 267 tandem autologous, 270–273 T-cell depletion, 267–268 TRM and OS, 264–265 trends in, 130–131 Multipotent adult progenitor cells (MAPCs), 477 Murine retroviruses, 776 Mycophenolate mofetil, 571 Mycophenolic acid (MPA), 571

879

Myeloablative allogeneic transplantation allogeneic stem cell source and alternative donors, 267 vs. autologous transplantation, 265–267 prognostic factors, 268 syngeneic transplantation, 267 T-cell depletion, 267–268 TRM and OS, 264–265 Myeloablative conditioning regime, AML Bu-based regimens, 16 donor lymphocyte infusions (DLI), 20 donor vs. no-donor analyses, 14 first complete remission (CR1), 13 optimal conditioning regimen, 16 optimal stem cell source, 17 outcome, disease relapse, 19 reduced intensity allografts, 18 RIC regimen, 18 salvage chemotherapy, 15 T-cell depletion strategies, 16–17 Myelodysplastic syndromes (MDS) age and disease state, impact, 207 bone marrow transplantation, 206 chemotherapy induction, 209 classification, 205 conditioning regimens, 210–211 donor and stem cell source, 209–210 JMML transplantation, 213 optimal timing, transplantation HLA identical sibling donors, 208 IPSS scores, 207 prognostic factors, 204 transplantation, CMML, 211–212 UD HSCT, 354–356 Myeloid DCs (mDCs), 809 N National marrow donor program (NMDP) CML, 57 cord blood banks, 364 CR2 ALL, 243 cryopreserved unrelated grafts, 427 MUD BMT, 95 Natural killer (NK) cell activation process, 460 donor selection HLA-Bw4 mismatches, 470–471 HLA-C group 1 alleles, 469–470 haploidentical transplantation cytokine secretion and cytotoxicity, 467 engraftment and GvHD, 462 event free survival (EFS), 465 KIR genetics activation, 466–467 leukemia relapse, 464 matched unrelated donor transplants, 467–469 transplant related mortality (TRM), 464–465 HLA haplotype, 459–460 HSC transplantation, alternative donor, 460 immune cells, 798 matched unrelated donors, 459

880 

Index

Natural killer (NK) cell (cont.) post-transplant infection, 492 strength and weakness, 472 treatment adoptive immunotherapy/DLI, 416 cell expansion, 418 cell infusion, 418–421 definition, alloreactivity, 415 donor lymphocyte infusion (DLI), 416–417 extracellular domain, 414 graft vs. host direction, 415–416 haploidentical donors transplantation, 415 haploidentical HSCT, 416 harvesting, 417 inhibitory receptor, 413–414 localization, 413 product, 417 production issues, 422 purification, 417–418 umbilical cord blood transplantation (UCBT), 459 NMDP. See National marrow donor program Non-Hodgkin's lymphoma (NHL) advantages and disadvantages, 91 aggressive B-cell lymphomas chemosensitivity, 118 diffuse large B cell lymphoma (DLBCL), 98, 117 single arm cohort studies, 99 allogeneic reduced intensity conditioning regime follicular (FL) NHL, 112 nonmyeloablative/reduced intensity transplantation, 113 treatment-related mortality (TRM), 111 Burkitt's lymphoma (BL), 102 chemosensitivity importance, 100 indolent lymphoma dose intensive chemotherapy, 91 EBMT/IBMTR review, 93 European CUP trial, 91 GVLy effect, 92 low treatment-related mortality (TRM), 92 single arm cohort study, 93 transplantation outcome, 94 lymphoblastic lymphoma (LBL), 103 mantle cell lymphoma (MCL) nonmyeloablative/reduced intensity transplantation, 116 nonmyeloablative/RIT ergimes, 117 MUD BMT comparative analyses, 101 GVLy effect, MCL, 96 mantle cell lymphoma (MCL), 95 national marrow donor program (NMDP), 95 RICSCT techniques, 95 NK and T-cell lymphomas, 102 T-cell lymphomas allogeneic nonmyeloablative/RIT, 120 novel/emerging therapies, 121–122 Nonmyeloablative allogeneic transplantation phase II studies EBMT data, 269 TRM, 268

prognostic factors, 269–270 tandem autologous, 270–273 O Ocular bleeding, 702 Overall survival (OS), 264–265 P Palifermin, 199 Papovaviruses, 516 Partial remission (PR), 161 PBSC. See Peripheral blood stem cells Pediatric ALL conditioning regimens, 248–249 donor choice, 246–247 first complete remission (CR1) chemotherapy vs. transplantation, 231 eligibility criteria, 228–230 HSCT outcome, 232 transplantation, 230, 234–235 umbilical cord outcomes, 236 very high risk ALL frontline treatment, 227 MRD and allogeneic transplantation, 249–250 NMDP retrospective study, 243 oncology, 220 polychemotherapy, 221 prognostic factors age, 222–223 cytogenetics, 225–226 down syndrome (DS), 224 early multidrug response, 226 extramedullary involvement, 224 hyperleukocytosis, 223–224 immunophenotype, 224–225 morpholog, leukemic cell characteristics, 224 MRD after induction therapy, 226 prednisone poor response (PPR), 226 second complete remission (CR2) ALL in advanced phase, 244 eligibility criteria, 236–238 relapsed ALL, 236 transplantation, 238, 240–244 umbilical cord outcomes, 244 stem cell source, 245–246 toxicity and mortality, 249 UCBT vs. BMT, unrelated donors, 247 UCBT vs. haploidentical transplantation, 247–248 very high risk (VHR)/ultra high risk (UHR), 220 Pediatric cord blood transplantation, 365–366 Pegfilgrastim, 391–392 Peripheral blood progenitor cells (PBPC) AMD3100, 282–283 vs. BMT, 285 CD34, 281–282 CD133+ graft, 282 clinical aspects cytokine-primed marrow transplantation, 290 engraftment, 284 graft characteristics, 283–284

Index  GvHD, 284–287 infections, 288 quality of life, 288–289 survival, 288 unrelated peripheral blood transplants, 289 cost, 291 cytokines, 282 donor considerations severe adverse reactions, 291 short-term adverse effect, 290–291 Peripheral blood stem cells (PBSC), 17, 81 PFS. See Progression-free survival rates Philadelphia chromosome positive ALL, 37–38 Plasmacytoid DCs (pDCs), 809 Polyomavirus hominis 1, 698 Post-transplant lymphoproliferative disorder (PTLD) definition, 597 pathophysiology EBV, 599 hepatitis C virus, 601 OKT3, 599 risk factors, 600 umbilical cord blood transplants, 600 photomicrographs, 601 prophylaxis and treatment anti-B-cell antibodies, 608–609 antiviral therapy, 603–607 cellular immunotherapy, 610–611 cytokine therapy, 609 cytotoxic chemotherapy, 609–610 immunosuppression reduction, 607–608 local therapy, 607 surveillance, 602–603 WHO classification, 598 Potential leukemia-associated antigenic targets, 856 Prednisone poor response (PPR), 226 Preparative regimens dose intensity definition, 441–442 GvHD, impact on, 445–447 myeloablative conditioning vs. NMT/RIC, 442–443 NMT/RIC vs. myeloablative, 446 novel myeloablative regimen, 444 RIC/NMT, 443 standard transplantation, 442–443 regimens exploration alemtuzumab, 451–452 extracorporeal photopheresis, 452 fludarabine-melphalan vs. fludarabine-busulfan, 450–451 fludarabine/TBI, 450–451 total lymphoid irradiation (TLI), 452–453 Prognostic factors pediatric ALL age, 222–223 cytogenetics, 225–226 down syndrome (DS), 224 early multidrug response, 226 extramedullary involvement, 224 hyperleukocytosis, 223–224 immunophenotype, 224–225

881

morpholog, leukemic cell characteristics, 224 MRD after induction therapy, 226 prednisone poor response (PPR), 226 Progression-free survival (PFS) rates, 75 Prophylaxis and treatment, PTLD anti-B-cell antibodies, 608–609 antiviral therapy EBV infections, 603 therapeutic options, 604–607 cellular immunotherapy, 610–611 cytokine therapy, 609 cytotoxic chemotherapy, 609–610 immunosuppression reduction, 607–608 local therapy, 607 proposed algorithm, 612 Psychological care assessment issues clinical interview, 626 tools, 626–629 clinically significant psychological problems cognitive dysfunction, 623–626 emotional disorders, 621–623 clinical psychologist role evidence-based HCT psychological treatment, 631–635 pathways, psychosocial care, 636–638 psychopharmacological intervention, 635–636 social workers, 629–631 HCT psychological services, 642–643 mental health parity policy implications health and behavior codes, 643–644 resources, 644 treatment adherence and caregiver role caregiver issues, 640–642 treatment regimen, 639–640 Pulmonary cytolytic thrombi (PCT), 707–708 R Radiation chimaera, 1 Radiolabeled monoclonal antibodies anti-CD66 antibody, 738 leukemia treatment, 737–738 radioimmunoconjugate, 736 90Y-labeled ibritumomab tiuxetan, 737 Receptor-ligand model, 306–307 Recombinant human growth hormone (rhGH), 391 Reduced intensity conditioning (RIC) regime, 36–37, 187 Respiratory syncytial virus (RSV), 513, 720 Respiratory viruses, 512–513 Rituximab, 122, 735 S SCT models, hematopoiesis allogeneic, 776–777 autologous, 775 gene therapy, autotransplantation, 775–776 GVHD, 777 MHC typing, 777–778 whole organ tolerance induction, 778 xenotransplantation, 779–780

882 

Index

Second allogeneic transplantation graft failure back-up autologous stem cells, 664 cyclosporine, 663 immunologic rejection, 662 positive CMV serology, 663 relapsed acute leukemia DLI, 657 GVHD risk, 659–661 prognostic factors, 658 reduced intensity conditioning regimens, 662 TBI vs. non-TBI therapies, 658 Second complete remission (CR2), pediatric ALL ALL in advanced phase, 244 eligibility criteria, 236–238 relapsed ALL, 236 transplantation, 238, 240–244 umbilical cord outcomes, 244 Second line therapy, aGVHD treatment broad anti-T cell agents/antibodies alemtuzumab, 753 anti-thymocyte globulin, 752 visilizumab, 753 broad anti-T cell agents/immunomodulatory agents deoxycoformycin, 755 extracorporeal photopheresis (ECP), 755 mycophenolate mofetil/MMF, 753–754 sirolimus, 755 narrow anti-T cell agents/receptor and cytokine targets ABX-CBL/anti CD147, 757 daclizumab, 755–756 denileukin diftitox, 756–757 TNF inhibition etanercept, 758 IL-1 receptor antagonist (IL1-RA), 758 infliximab, 757 SEER. See Surveillance epidemiology and end results Single vs. tandem autologous transplant, HSCT complete response (CR) rates, 153 HDT/transplant vs. SDT OS benefit, 147, 150 PFS benefit, 146 maintenance therapy, 151 cancer and leukemia group B (CALGB), 152 thalidomide, 151 novel pre-transplant regimens, 153 optimal time, transplant blood and marrow transplant clinical trials network (BMT CTN), 149 bortezomib, 148 lenalidomide maintenance therapy, 150 single vs. double autologous ASCT, 149 PFS benefit, 146 salvage therapy, 151 United States Intergroup Study, S9321, 146 Sirolimus FK binding protein 12 (FKBP12), 569 Streptomyces hygroscopicus, 569 tacrolimus, 570

Soluble BAFF (sBAFF), cGVHD, 587 Somatic side-effects, 632–634 Stem cell factor (SCF), 815 Surveillance epidemiology and end results (SEER), 129 T T cel depletion (TCD), 565 T cell receptor rearrangement excision circles (TREC), 548 T cell reconstitution, immunotherapy cellular immunotherapy, thymopoiesis anti-tumor immunity, 553 diagrammatic representation of, 552 HPE endogenous proliferation, 550 homeostatic proliferation, 550 IL-7/15, 549 TGFß, 550 initial post-transplant period and implications oligoclonal peripheral expansion, 551 thymopoiesis, 550 thymic dependent, 548–549 naïve phenotype, 548 recent thymic emigrants (RTE), 549 TREC bearing cells, 549 Texas transplant institute perspective, 382–383 Thalassemia, allogeneic gene therapy alternative donors transplantation, 498–499 BMT vs. medical treatment, 492 disease eradication, 493–494 engraftment, 494 ex-thalassemic management, 499–500 graft failure/rejection, 497–498 GVHD, 498 mixed chimerism, 497 morbidity and mortality, 494 preparatory regimens, 493 risk classes, 495 transplant outcome, HLA matched related donors, 495–497 adult patients, 496–497 class 1and 2 patients, 495 class 3 patients, 495–496 transplant procedures, 492 Thrombopoietin, 392 Thrombotic and hemostatic complications acquired hypercoagulable disorders, 708 bleeding central nervous system (CNS), 701–702 complications, 699 diffuse alveolar hemorrhage (DAH), 700–701 events, 698 gastrointestinal, 700 hemorrhagic cystitis, 698–700 ocular, 702 catheter-related thrombosis, 704 dural (venous) sinus thrombosis, 708 endothelial cell, 695 etiology, 703

Index  GVHD acute GVHD, 696–697 chronic GVHD, 697–698 mechanisms, 697 hepatic veno-occlusive disease (VOD), 704–706 non-myeloablative (NM) and RIC regimens, 708–710 pulmonary cytolytic thrombi (PCT), 707–708 pulmonary veno-occlusive disease (VOD), 707 thrombosis risk factors, 702–703 transplantation-associated thrombotic microangiopathy (TA-TMA), 706–707 Toll-like receptors (TLRs), 812 Total lymphoid irradiation (TLI), 452–453 Total nucleated cells (TNC), 376–377 Transplantation-associated thrombotic microangiopathy (TA-TMA), 706–707 Transplant related mortality (TRM) adoptive cell therapy, 465 G-CSF impact, 464 immunological recovery, 464 myeloablative transplantation, 263 overlapping factors, 464 procedure, 463 Treatment related morbidity and mortality (TRM), 57 Treatment-related mortality (TRM), 187 T regulatory 1 cells (Tr1), 819 Tumor-associated antigens (TAA), 855 Tyrosine kinase inhibitors (TKI), CML patients BCR-ABL gene, 54 breakpoint cluster region (BCR), 54 EBMT transplantation risk score, 57 imatinib use, 65 novel kinase inhibitors, 67 Philadelphia (PH) chromosome, 54 survival percentage, 57 treatment related morbidity and mortality (TRM), 57 U UD-SCT. See Unrelated donor stem cell transplantation Umbilical cord blood (UCB) transplantation adult cord blood double cord blood, 368–369 reduced intensity regimen, 367 refractory lymphoma, single cord blood, 368 single unit ablative regimen, 367 unrelated cord blood vs.bone marrow/peripheral blood stem cell, 366–367 challenges, 370 cord blood banking, 364 ethical issues, 365 pediatric cord blood, 365–366 pre-clinical characteristics, 363 strategies, 369–370 Unrelated donor cord blood cell dose, 377–378

883

diagnosis effect, 381 double cord blood units, 381–382 factors, 377–378 product, 376–377 Texas Transplant Institute Perspective, 382–383 Unrelated donor stem cell transplantation (UD-SCT), 33 Unrelated donor transplants actuarial disease free survival (DFS), 357 acute leuekemia, 356 aplastic anemia, 357–358 bone marrow donors, 347 clinical outcome, 348–349 conditioning regimens, 352–353 HLA and matching criteria antigenic matching, 347–348 haplotypes matching, 348–349 HLA mismatch effect, 349 HSC donation collection quality, 350–351 safety issue, 349 human leukocyte antigen (HLA) typing technology, 346 myelodysplastic syndromes, 356–357 patient selection and indications, 350–351 prophylaxis acute GvHD, 352 chronic GvHD, 352 stem cell source, 353–354 V Vancomycin-resistant enterococcus (VRE), 720–721 Varicella-Zoster virus (VZV), 509–510 Veno-occlusive disease (VOD), 707 Veto activity, 462 Viral infections adenoviruses, 514–515 cytomegalovirus (CMV) immune monitoring and immune therapy, 509 preemptive therapy, 508 prophylaxis, 507–508 risk factors, 507 diagnostic tests, 506 Epstein-Barr Virus (EBV), 510–511 hepatitis B and C viruses, 515 herpes simplex virus (HSV), 509 human herpes virus type 6 (HHV-6), 511–512 influenza viruses, 514 papovaviruses, 516 respiratory syncytial virus (RSV), 513 respiratory viruses, 512–513 Varicella-Zoster virus (VZV), 509–510 Y Yakoub-Agha, MDS, 209