Chronic Myeloid Neoplasias and Clonal Overlap Syndromes: Epidemiology, Pathophysiology and Treatment Options

Richard Greil Lisa Pleyer Daniel Neureiter Viktoria Faber Editors * * Chronic Myeloid Neoplasias and Clonal Overlap S...

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Richard Greil Lisa Pleyer Daniel Neureiter Viktoria Faber Editors *

*

Chronic Myeloid Neoplasias and Clonal Overlap Syndromes Epidemiology, Pathophysiology and Treatment Options

SpringerWienNewYork

Univ.-Prof. Dr. Richard Greil Dr. Lisa Pleyer Dr. D.I. Viktoria Faber Paracelsus Medizinische Privatuniversit€at, Universit€atsklinik f€ur Innere Medizin III, Salzburg, Austria PD Daniel Neureiter Paracelsus Medizinische Privatuniversit€at, Universit€atsinstitut f€ur Pathologie, Salzburg, Austria

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re use of illustrations, broadcasting, reproduction by photocopying machines or similar means, and storage in data banks. Product Liability: The publisher can give no guarantee for all the information contained in this book. This does also refer to information about drug dosage and application thereof. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. 2010 Springer Verlag/Wien Printed in Austria SpringerWienNewYork is part of Springer Science þ Business Media springer.at Typesetting: Thomson Press (India) Ltd., Chennai Printing: Druckerei Theiss GmbH, 9431 St. Stefan im Lavanttal, Austria

Printed on acid free and chlorine free bleached paper SPIN: 12161454 With 85 (partly colored) Figures

Library of Congress Control Number: 2009939839

ISBN 978 3 211 79891 1 SpringerWienNewYork

Preface

Introduction The understanding of the genetic, epigenetic, immunological and biological causes of myeloproliferative disorders has substantially improved in the last few years. Together with refined tools in pathology, the successful establishment of mouse models mimicking at least some of the myeloproliferative disorders, and murine models allowing to carefully dissect the role of mutations and gene dosage effects of, for example JAK2, this has led to ever increasing numbers of modified classification schemes. It is therefore important for the heamtologist or oncologist to keep up with this rapid change in classification language, the upcoming of new entities or differentiation between, or subclassification of, rare diseases such as CMML in its myeloproliferative and myelodysplastic variant. In addition, it has become clear that similar clinical conditions may be caused by different genetic alterations and pathological signalling pathways. These developments point to the future of individualized cancer medicine. Due to the increasing molecular and pathoethiological knowledge, a more diversified field of diseases is emerging, requiring different and much more diversified tailored approaches. The aim of this book is to summarize the current understanding of myeloproliferative and myelodysplastic disorders as well as overlap syndromes for students, physicians in education for internal medicine or preparing for board certification for hematology, as

well as for practicing hematologists or oncologists. Each chapter follows a similar architecture and leads through epidemiology, genetic and molecular causes, hematological and clinical findings, prognostic factors and current treatment approaches of the diseases. Effort has been made to point out the evolving field of novel drugs in this arena but simultaneously differentiate between standard and experimental treatment approaches. Together with the co-editors and all the authors of the various chapters I hope that the readers of the book will enjoy reading and benefit from the information provided.

Acknowledgements I would like to thank all the physicians and scientists contributing to this book. We are indebted to Prof. Klaus Hergan, chief of the Radiology Department at the Private Medical University Hospital in Salzburg for providing the CT scans and radiological findings depicted in this book. Finally, I would like to dedicate this book to the spirit and enthusiasm of all the members of my department and my daughter Raphaela. Richard Greil

Contents

1

Introduction to Classic Chronic Myeloproliferative Disorders (CMPDs) – Molecular and Cellular Biology ::::::::::::::::::::: 1 Lisa Pleyer and Richard Greil

1.1 Pathogenetic Role of the JAK2V617F Mutation Definition of JAK2V617F+ CMPDs with Common Pathogenesis and Natural Disease Evolution from ET to PV to post ET/PV MF vs. JAK2V617F2 CMPDs ::::: 1.1.1 The Clonal Stem Cell Nature of Classic CMPDs :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 1.1.2 JAK2V617F is an Acquired Somatic Mutation :::::::: 1.1.3 Timing of the JAK2 Mutation Relationship Between its Emergence and Clonal Hematopoiesis: JAK2V617F is an Early, but not the Earliest Event in the Transformation Process :::::::: 1.1.4 JAK2 Mutations in Murine Systems Disease Phenotype and Biologic Consequences ::::::::::::::::: 1.1.5 Gene Dosage and the Role of JAK2 Mutations in the Generation of Different Types of CMPD ::::: 1.1.6 JAK2 Mutations, Signaling Aberrations and Consequences for Cell Biology::::::::::::::::::::::::::::: 1.1.7 Altered Downstream JAK2 Signaling and STAT Phosphorlyation States for the Discrimination Between Classic CMPD Entities::::::::::::::::::::::::::: 1.2 Other Important (Epi)genetic Factors Functionally Equivalent to the JAK2V617F Mutation::::::::::::::::::::::::::::: 1.3 Therapeutic Targeting of the JAK2 STAT Signaling Axis :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

2

2.4 2.5 2.6 2.7 2 2.8 2 4

2.9 2.10 2.11

4 4 5 7

8 8 9

Essential Thrombocythemia (ET)::::::::::::::::: 15 Lisa Pleyer, Victoria Faber, Daniel Neureiter, and Richard Greil

2.1 Epidemiology of ET ::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.2 Course of Disease and Prognosis of ET ::::::::::::::::::::::::: 2.3 Cellular and Biological Abnormalities Observed in ET:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.3.1 Monoclonality Versus Polyclonality in ET :::::::::: 2.3.2 Endogenous Megakaryocytic Colony (EMC) Formation and Endogenous Erythroid Colony (EEC) Formation:::::::::::::::::::::::::::::::::::: 2.3.3 Overexpression of the PRV 1 Gene ::::::::::::::::::::: 2.3.4 Decreased cMPL Expression and Elevated Serum Thrombopoietin (TPO) Levels ::::::::::::::::: 2.3.5 Quantitative and Qualitative Defects in Platelets and Leukocyte Biology in ET (and PV) ::::::::::::::

16 16 16 16

17 17 17 17

2.12

2.3.6 JAK2V617F Mutations and Role of Allelic Burden in ET ::::::::::::::::::::::::::::::::::::::::::::::::::: 2.3.7 Thrombopoietin Receptor Gene (MPL) Mutations in ET ::::::::::::::::::::::::::::::::::::::::::::::: Cytogenetics in ET ::::::::::::::::::::::::::::::::::::::::::::::::::::: Clinical Presentation and Disease Complications of ET:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Diagnosis and Differential Diagnosis of ET ::::::::::::::::: Pathophysiology of Thrombosis and Microcirculatory Disturbances in ET (and PV)::::::::::::::::::::::::::::::::::::::: Pathophysiology of Hemorrhagic Complications in ET (and PV) ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Risk Factors for Thrombotic Events in ET/PV ::::::::::::: Risk Factors for Myeloid Disease Progression to PV, Post ET MF and/or Leukemic Transformation:::::::::::::: Indication for Treatment and Choice of Drugs in Patients with ET ::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.11.1 Acetylic Salicylic Acid (ASA, aspirin):::::::::::: 2.11.2 Platelet Reducing Agents Current State of the Art ::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.11.2.1 Hydroxyurea :::::::::::::::::::::::::::::::::: 2.11.2.2 Anagrelide ::::::::::::::::::::::::::::::::::::: 2.11.2.3 Interferon a (IFN a)::::::::::::::::::::::: 2.11.2.4 Pipobroman :::::::::::::::::::::::::::::::::::: 2.11.2.5 Busulphan :::::::::::::::::::::::::::::::::::::: 2.11.2.6 Radiophosphorus 32P :::::::::::::::::::::: 2.11.3 Treatment of Bleeding Events and Indications for Platelet Apheresis ::::::::::::::::::::::::::::::::::::: 2.11.4 Life Style Modifications and Control of Other Risk Factors ::::::::::::::::::::::::::::::::::::: 2.11.5 Effect of Therapeutic Strategies on Re thrombosis :::::::::::::::::::::::::::::::::::::::::::::::: 2.11.6 Should the Site of Occurrence of the Thrombotic Event Have an Influence on Preventive Treatment Strategy? :::::::::::::::::: 2.11.7 Should JAK2V617F Positivity Influence the Choice of Cytoreductive Therapy? :::::::::::: 2.11.8 Antithrombotic Prophylaxis for Elective Surgery in ET (and PV):::::::::::::::::::::::::::::::::: ET in Pregnancy ::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.12.1 Course of Pregnancies in Women with ET :::::: 2.12.2 Prediction of Pregnancy Outcome ::::::::::::::::::: 2.12.3 Management and Treatment of Pregnant Women with ET ::::::::::::::::::::::::::::::::::::::::::::: 2.12.3.1 General Considerations ::::::::::::::::::: 2.12.3.2 Antiaggregatory Platelet Therapy During Pregnancy::::::::::::::::::::::::::: 2.12.3.3 Cytoreductive Therapy During Pregnancy ::::::::::::::::::::::::::::::::::::::

18 19 20 20 21 25 26 27 28 29 31 32 32 34 35 35 36 36 37 37 37

38 38 38 39 39 39 39 39 40 40

viii

Contents

2.12.3.4

Relevance of Periodic Platelet Apheresis in Pregnancy::::::::::::::::::: 2.12.3.5 Recommendations for Treatment of Pregnant Women with ET :::::::::: 2.13 Childhood ET ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.14 Familial, Hereditary Thrombocytosis ::::::::::::::::::::::::::: 2.15 Rare ET Varients :::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.15.1 Philadelphia Chromosome (Ph) Positive ET::::::::::::::::::::::::::::::::::::::::::::: 2.15.2 Bcr Abl Positive Ph Negative ET :::::::::::::::::::

3

40

4

41 41 42 42 42 43

Polycythemia Vera (PV) ::::::::::::::::::::::::::::::: 51

Lisa Pleyer, Victoria Faber, Daniel Neureiter, and Richard Greil 4.1 4.2 4.3 4.4 4.5 4.6

Lisa Pleyer, Daniel Neureiter, and Richard Greil 3.1 Epidemiology of PV ::::::::::::::::::::::::::::::::::::::::::::::::::: 3.2 Should ET and PV be Considered as the Same Disease? ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 3.3 Pathophysiology and Molecular Biology of PV:::::::::::: 3.3.1 Overview of the Role of JAK2V617F Mutations in PV ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 3.3.2 Overexpression of the PRV 1 Gene in PV ::::::::: 3.3.3 Other Molecular Features Implicated in the Pathogenesis of PV :::::::::::::::::::::::::::::::::::::::::: 3.3.4 Exon 12 Mutations in JAK2V617F Negative PV:::: 3.3.5 Single Nucleotide Polymorphisms (SNPs) in JAK2 and EPO R Contribution of Host Genetic Variation to CMPD Phenotype ::::::::::::: 3.4 Cytogenetics in PV ::::::::::::::::::::::::::::::::::::::::::::::::::::: 3.5 Clinical Features and Symptoms Occurring in PV ::::::::: 3.6 Disease Complications :::::::::::::::::::::::::::::::::::::::::::::::: 3.7 Diagnosis of Polycythemia Vera (PV):::::::::::::::::::::::::: 3.8 Differential Diagnosis of Polycythemia Vera:::::::::::::::: 3.8.1 Absolute Polycythemia/Erythrocytosis :::::::::::::: 3.8.2 Relative and Spurious/Apparent Polyglobulia:::: 3.8.3 Idiopathic Erythrocytosis (IE)::::::::::::::::::::::::::: 3.9 Risk Stratification of Patients with PV::::::::::::::::::::::::: 3.10 Treatment of PV ::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 3.10.1 Phlebotomy:::::::::::::::::::::::::::::::::::::::::::::::::::: 3.10.2 Antiaggregatory Therapy :::::::::::::::::::::::::::::::: 3.10.3 Indications for Treatment and Choice of Cytoreductive Drugs in Patients with PV :::: 3.10.3.1 Hydroxyurea :::::::::::::::::::::::::::::::::: 3.10.3.2 Interferon a:::::::::::::::::::::::::::::::::::: 3.10.3.3 Pipobroman :::::::::::::::::::::::::::::::::::: 3.10.3.4 Other Cytoreductive Agents only Rarely Used Nowadays::::::::::::::::::: 3.10.4 Allogeneic Bone Marrow Transplantation in PV ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 3.10.5 Future Treatment Possibilities JAK2 Inhibitors :::::::::::::::::::::::::::::::::::::::::::::: 3.11 Polycythemia Vera in Pregnancy ::::::::::::::::::::::::::::::::: 3.12 Childhood Polycythemias/Erythrocythosis ::::::::::::::::::: 3.12.1 Primary Familial and Congenital Polycythemia ::::::::::::::::::::::::::::::::::::::::::::::::: 3.12.2 Sporadic Pediatric Non Familial PV ::::::::::::::: 3.12.3 Familial Polycythemia Vera :::::::::::::::::::::::::::: 3.12.4 Congenital Secondary Erythrocytosis :::::::::::::: 3.12.4.1 High Affinity Hemoglobin Variants:::: 3.12.4.2 Congenital 2,3 Bisphosphoglycerate (BPG) Deficiency ::::::::::::::::::::::::::: 3.12.4.3 Polycythemias due to Abnormal Hypoxia Sensing ::::::::::::::::::::::::::::

52 52 52 52 53 53 54

4.7 4.8 4.9 4.10 4.11 4.12 4.13

54 54 56 57 58 63 63 65 66 67 68 68 68 69 70 70 70 70 71 71 71 72 72 72 73 73 73 74 74

Primary Myelofibrosis (PMF) [Previously Chronic Idiopathic Myelofibrosis (CIMF), Myelofibrosis with Myeloid Metaplasia (MMM), Agnogenic Myeloid Metaplasia (AMM)] :::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 81

4.14

Introduction to PMF:::::::::::::::::::::::::::::::::::::::::::::::::::: 82 Epidemiology of PMF::::::::::::::::::::::::::::::::::::::::::::::::: 82 Pathophysiology and Molecular Biology of PMF ::::::::: 84 Cytogenetics in PMF::::::::::::::::::::::::::::::::::::::::::::::::::: 86 Clinical Features of PMF :::::::::::::::::::::::::::::::::::::::::::: 86 Laboratory Findings in PMF ::::::::::::::::::::::::::::::::::::::: 88 4.6.1 Abnormal Laboratory Tests :::::::::::::::::::::::::::::: 88 4.6.2 Blood Cell Anomalies Observed in the Hyperproliferative Phase :::::::::::::::::::::::::::::::::: 88 4.6.3 Blood Cell Anomalies Observed During the Late Stage Osteosclerotic Phase:::::::::::::::::: 89 Cytological Findings in PMF::::::::::::::::::::::::::::::::::::::: 91 Histological Findings of Bone Marrow Biopsy Specimen in PMF ::::::::::::::::::::::::::::::::::::::::::::::::::::::: 91 Imaging in Patients with PMF ::::::::::::::::::::::::::::::::::::: 91 Diagnosis of Primary Myelofibrosis::::::::::::::::::::::::::::: 91 Differential Diagnosis for Primary Myelofibrosis ::::::::: 93 Prognostic Scores and other Prognostic Factors in PMF ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 94 Treatment of Patients with Myelofibrosis::::::::::::::::::::: 96 4.13.1 Curative Treatment Options Allogeneic Stem Cell Transplantation::::::::::::::::::::::::::::::: 99 4.13.2 Treatment of Symptomatic Myeloproliferation as well as Constitutional Symptoms ::::::::::::::: 100 4.13.3 Treatment of Cytopenias in Advanced Stage Myelofibrosis :::::::::::::::::::::::::::::::::::::::::::::::: 100 4.13.3.1 Growth Factors::::::::::::::::::::::::::::: 100 4.13.3.2 Androgens:::::::::::::::::::::::::::::::::::: 100 4.13.3.3 Bisphosphonates ::::::::::::::::::::::::::: 101 4.13.3.4 Cyclosporine A :::::::::::::::::::::::::::: 101 4.13.4 Targeting and Modulating the Bone Marrow Microenvironment in PMF::::::::::::::::::::::::::::: 101 4.13.4.1 Thalidomide ::::::::::::::::::::::::::::::::: 101 4.13.4.2 Thalidomide Analogues :::::::::::::::: 102 4.13.4.3 Targeting TNF a with Etanercept:::: 102 4.13.4.4 Interferons:::::::::::::::::::::::::::::::::::: 102 4.13.4.5 Targeting TGF b::::::::::::::::::::::::::: 103 4.13.5 A Possible Role for Epigenetic Therapy in PMF?:::::::::::::::::::::::::::::::::::::::::::::::::::::::: 103 4.13.6 Tyrosine Kinase Inhibitors in PMF::::::::::::::::: 104 4.13.6.1 Targeting Constitutively Activated JAK2 by Selective Tyrosine Kinase Inhibitors:::::::::::::::::::::::::::::::::::::: 104 4.13.6.2 Imatinib Mesylate (STI571, Gleevec) :::::::::::::::::::::::::::::::::::: 104 4.13.6.3 Farensyltransferase Inhibitors:::::::: 104 4.13.6.4 Other Tyrosine Kinase Inhibitors that have been Used in PMF ::::::::: 104 4.13.7 Indications for Splenectomy in PMF :::::::::::::: 105 4.13.8 Indications for Splenic Irradiation :::::::::::::::::: 106 4.13.9 Treatment of Other Foci of Extramedullary Hematopoiesis and Their Complications :::::::: 106 4.13.9.1 Irradiation of Tumor like Manifestations of Extramedullary Hematopoiesis :::::::::::::::::::::::::::::: 106 Atypical Myelofibrosis Variants:::::::::::::::::::::::::::::::::: 107

Contents

ix

4.14.1

Secondary Myelofibrosis, i.e., Post Polycythemia and Post Essential Thrombocythemia Myelofibrosis :::::::::::::::::::::::::::::::::::::::::::::::: 107 4.14.2 Primary Autoimmune Myelofibrosis (AIMF) ::: 108 4.14.2.1 Treatment of AIMF :::::::::::::::::::::: 108 4.14.3 Familial Myelofibrosis::::::::::::::::::::::::::::::::::: 108 4.14.4 Idiopathic Myelofibrosis in Childhood ::::::::::: 109

5

Chronic Myeloid Leukemia (CML) ::::::::::::: 117 Nikolas von Bubnoff, Lisa Pleyer, Daniel Neureiter, Victoria Faber, and Justus Duyster

Introduction ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Epidemiology :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Course of Disease :::::::::::::::::::::::::::::::::::::::::::::::::::::::: Etiology and Pathogenesis of CML :::::::::::::::::::::::::::::: Classification of CML:::::::::::::::::::::::::::::::::::::::::::::::::: Clinical Features and Disease Complications in CML ::::: Diagnosis of CML ::::::::::::::::::::::::::::::::::::::::::::::::::::::: 5.7.1 Baseline Diagnostics ::::::::::::::::::::::::::::::::::::::::: 5.7.2 Cytology of Peripheral Blood in CML :::::::::::::: 5.7.2.1 Changes in the Myeloid Compartment ::::::::::::::::::::::::::::::::::::: 5.7.2.2 Changes in the Lymphoid Compartment ::::::::::::::::::::::::::::::::::::: 5.7.2.3 Changes in the Platelet Compartment ::::::::::::::::::::::::::::::::::::: 5.7.2.4 Changes in the Erythroid Compartment ::::::::::::::::::::::::::::::::::::: 5.7.3 Bone Marrow Cytology in CML:::::::::::::::::::::::: 5.7.4 Bone Marrow Histology in CML ::::::::::::::::::::::: 5.7.5 Laboratory Findings in CML::::::::::::::::::::::::::::: 5.7.6 Molecular Diagnostics in CML:::::::::::::::::::::::::: 5.7.6.1 Conventional Cytogenetics in CML::::::: 5.7.7 Differential Diagnosis of CML :::::::::::::::::::::::::: 5.8 Treatment of Patients with CML :::::::::::::::::::::::::::::::::: 5.8.1 Treatment in Chronic Phase CML ::::::::::::::::::::: 5.8.1.1 Hydroxyurea, Busulphan and Alpha Interferon ::::::::::::::::::::::::::::::::::::::::::: 5.8.1.2 Imatinib in the Treatment of CML :::::: 5.8.2 Treatment of Accelerated and Blast Phase ::::::::: 5.8.3 Response Criteria in CML::::::::::::::::::::::::::::::::: 5.8.4 Monitoring Response in CML ::::::::::::::::::::::::::: 5.8.5 Resistance to Imatinib in CML:::::::::::::::::::::::::: 5.8.5.1 Definition and Incidence of Suboptimal Response and Treatment Failure ::::::::::::::::::::::::::::::::::::::::::::::: 5.8.5.2 Mechanisms of Resistance to Imatinib in CML:::::::::::::::::::::::::::::::::::::::::::::: 5.8.6 Novel Abl Kinase Inhibitors :::::::::::::::::::::::::::::: 5.8.6.1 Preclinical Data:::::::::::::::::::::::::::::::::: 5.8.6.2 Approved 2nd Generation Kinase Inhibitors in Imatinib Resistant or Intolerant CML :::::::::::::::::::::::::::::: 5.8.7 Outlook Promising Strategies in Current and Future Clinical Trials:::::::::::::::::::::::::::::::::: 5.8.7.1 Novel Compounds in Clinical Trials:::::: 5.8.7.2 Second Generation Abl Kinase Inhibitors for 1st Line Treatment of Chronic Phase CML::::::::::::::::::::::: 5.8.7.3 Can Tyrosine Kinase Inhibitors Cure CML? :::::::::::::::::::::::::::::::::::::::::::::::: 5.8.7.4 Immunotherapy of CML::::::::::::::::::::: 5.8.8 Allogeneic Stem Cell Transplantation :::::::::::::::: 5.8.9 Prognostic Scores in CML:::::::::::::::::::::::::::::::::

5.1 5.2 5.3 5.4 5.5 5.6 5.7

118 118 118 118 120 120 121 122 123 123 123 123 123 123 124 124 124 125 125 125 126 126 126 127 128 128 129

129 129 135 135

135 137 137

140 140 140 140 141

5.9 CML Variants :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 5.9.1 Philadelphia Chromosome Negative CML (Formerly Atypical CML) ::::::::::::::::::::::::::::::::: 5.9.2 CML with an Initial Thrombocythemic Phase, CML with a Polycythemic Prophase, CML with Marrow Fibrosis (Formerly Inappropriately Termed Ph Positive ET, PV or PMF):::::::::::::::::: 5.9.3 Other Ph+ Entities :::::::::::::::::::::::::::::::::::::::::::: 5.9.4 CML with Atypical Breakpoints and an Indolent Clinical Course (Formerly Neutrophilic CML) ::::::::::::::::::::::::::::::::::::::::

6

141 141

141 142

142

Myelodysplastic Syndromes (MDS) ::::::::::: 153 Lisa Pleyer, Daniel Neureiter, Victoria Faber, and Richard Greil

6.1 Introduction ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.2 Epidemiology of MDS ::::::::::::::::::::::::::::::::::::::::::::::::: 6.3 Pathophysiology and Molecular Biology of MDS ::::::::: 6.3.1 Disturbances in Apoptosis ::::::::::::::::::::::::::::::::: 6.3.2 Alterations in T Cell Functions and Cytokines ::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.3.3 Microenvironment in MDS:::::::::::::::::::::::::::::::: 6.3.4 The Role of Tumor Suppressor Genes and Oncogenes in MDS Disease Initiation/ Perpetuation:::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.3.4.1 Somatically Acquired Mutations of the AML 1 Gene in MDS::::::::::::::: 6.3.4.2 Overexpression of EVI 1 in MDS ::::::::: 6.3.4.3 Oncogenic Fusion Products in MDS:::::::::::::::::::::::::::::::::::::::::::::: 6.3.4.4 Mutation of the Ras Protooncogene in MDS:::::::::::::::::::::::::::::::::::::::::::::: 6.3.4.5 The Role of Interferon Regulatory Factor 1 (IRF 1) in MDS:::::::::::::::::::: 6.4 Clinical Features in MDS::::::::::::::::::::::::::::::::::::::::::::: 6.4.1 Infectious Complications in MDS:::::::::::::::::::::: 6.5 Laboratory Features in MDS :::::::::::::::::::::::::::::::::::::::: 6.6 Typical Bone Marrow Findings in MDS ::::::::::::::::::::::: 6.7 Diagnosis and Classification of MDS ::::::::::::::::::::::::::: 6.8 Prognostic and Predictive Parameters in MDS :::::::::::::: 6.8.1 Cytogenetics in MDS :::::::::::::::::::::::::::::::::::::::: 6.8.1.1 Frequency of Cytogenetic Aberrations in MDS ::::::::::::::::::::::::::: 6.8.1.2 Clinical and Prognostic Features of Patients with Particular Cytogenetic Aberrations in MDS ::::::::::::::::::::::::::: 6.8.2 Molecular Factors Associated with Progression of the Disease ::::::::::::::::::::::::::::::::::::::::::::::::::: 6.8.3 Prognostic Scoring Systems in MDS:::::::::::::::::: 6.8.4 Other Prognostic Markers in MDS::::::::::::::::::::: 6.9 Best Supportive Care (BSC) of Patients with MDS ::::::: 6.9.1 Transfusion of Red Blood Cells and/or Platelets:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.9.2 Erythropoietin (EPO) :::::::::::::::::::::::::::::::::::::::: 6.9.3 G CSF and Combination Treatment of EPO with G CSF :::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.9.4 Thrombopoietin (TPO) and TPO Mimetics :::::::: 6.9.4.1 PEG rHuMGDF ::::::::::::::::::::::::::::::::: 6.9.4.2 Recombinant Human TPO (rHuTPO)::::::: 6.9.4.3 Romiplostim (AMG531, Nplate)::::::: 6.9.4.4 Oral TPO Mimetics Eltrombopag and AKR 501 (YM477):::::::::::::::::::::: 6.9.5 Other Drugs for Palliative Amelioration of Cytopenia :::::::::::::::::::::::::::::::::::::::::::::::::::::

154 155 156 156 158 160

160 161 161 161 162 162 162 162 166 167 167 172 172 172

173 174 175 178 178 178 178 179 180 181 181 181 181 182

x

6.10

6.11

6.12

6.13

6.14

6.15 6.16

6.17

6.18

6.19

Contents

6.9.6 Iron Chelation Therapy (ICT):::::::::::::::::::::::::::: 182 6.9.6.1 Deleterious Sequelae of Iron Overload in MDS Patients :::::::::::::::::: 182 6.9.6.2 What are the Goals of ICT?:::::::::::::::: 183 6.9.6.3 In Whom Should ICT Be Considered? ::::::::::::::::::::::::::::::::::::::: 183 6.9.6.4 When Should ICT Be Initiated and for How Long? :::::::::::::::::::::::::::: 183 6.9.6.5 Monitoring of Body Iron Stores in MDS:::::::::::::::::::::::::::::::::::::::::::::: 183 6.9.6.6 Currently Available Iron Chelators:::::: 184 Low Dose Palliative Chemotherapy in MDS ::::::::::::::: 185 6.10.1 Low Dose Melphalan :::::::::::::::::::::::::::::::::::: 185 6.10.2 Low Dose Cytosine arabinoside (Ara C) :::::::: 185 Treatment of MDS with Curative Intention ::::::::::::::::: 185 6.11.1 Myeloablative Chemotherapy and Allogeneic Stem Cell Transplantation (SCT) ::::::::::::::::::: 185 6.11.2 When to Transplant in the Course of Disease? ::::::::::::::::::::::::::::::::::::::::::::::::::: 186 6.11.2.1 Factors Associated with Allogeneic SCT Outcome::::::::::::::::::::::::::::::::: 187 6.11.3 Reduced Intensity Conditioning (RIC) ::::::::::: 187 6.11.3.1 Patient Selection for RIC :::::::::::::::: 188 6.11.4 Induction of a T cell Response Against the Malignant Clone :::::::::::::::::::::::::::::::::::::: 188 6.11.5 AML like Chemotherapy in MDS:::::::::::::::::: 189 6.11.6 High Dose Chemotherapy (HDCT) with Autologous Stem Cell Rescue:::::::::::::::::::::::: 189 Epigenetic Therapies: DNA Methyltransferase Inhibitors ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 190 6.12.1 Hypermethylation in MDS::::::::::::::::::::::::::::: 190 6.12.2 Hypomethylating Agents :::::::::::::::::::::::::::::: 191 6.12.2.1 5 Azacitidine (Vidaza):::::::::::::::::: 191 6.12.2.2 5 Aza 200 Deoxycytidine (Decitabine) (Dacogen) :::::::::::::::::::::::::::::::::::: 192 6.12.3 Histone Deacetylase Inhibitors (HDAC I) and Combination Therapy with Other Epigenetic Drugs or Differentiation Inducer ATRA (Vesanoid)::::::::::::::::::::::::::::::::::::::::::::::::::: 193 Immunosuppressive Treatment in MDS :::::::::::::::::::::: 193 6.13.1 Treatment with Anti thymocyte Globulin (ATG) ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 193 6.13.2 Immunosuppressive Treatment with Cyclosporin A (CyA) :::::::::::::::::::::::::::::::::::: 194 6.13.3 Treatment of MDS Associated Autoimmune Manifestations::::::::::::::::::::::::::::::::::::::::::::::: 195 Targeting Bone Marrow Microenvironment in MDS:::: 195 6.14.1 Thalidomide:::::::::::::::::::::::::::::::::::::::::::::::::: 195 6.14.2 Lenalidomide (Revlimid)::::::::::::::::::::::::::::: 195 6.14.3 Direct Targeting of TNF a: Infliximab and Ethanercept ::::::::::::::::::::::::::::::::::::::::::::::::::: 196 6.14.4 Antiangiogenetic Therapies ::::::::::::::::::::::::::: 197 Induction of Differentiation Retinoic Acids:::::::::::::: 197 Molecular Therapies Using Kinase Inhibitors ::::::::::::: 197 6.16.1 Farensyltransferase Inhibitors (FTIs): Tipifarnib (Zarnestra) and Lonafarnib (Sarasar) ::::::::: 197 6.16.2 FLT3 Antagonist Tandutinib (MLN518/ CT53518):::::::::::::::::::::::::::::::::::::::::::::::::::::: 198 Targeting NF kB:::::::::::::::::::::::::::::::::::::::::::::::::::::::: 198 6.17.1 Bortezomib (Velcade):::::::::::::::::::::::::::::::::: 198 6.17.2 Arsenic Trioxide (Arsenox) ::::::::::::::::::::::::: 198 Modulation of Pro Apoptotic Cytokines with Pentoxiphylline, Dexamethasone and Ciprofloxacine ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 199 MDS Subtypes Associated with Certain Cytogenetic Features::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 200

6.19.1 5q Syndrome :::::::::::::::::::::::::::::::::::::::::::::: 6.19.2 MDS with Isolated del(20q)::::::::::::::::::::::::::: 6.19.3 Monosomy 7 Syndrome::::::::::::::::::::::::::::::::: 6.19.4 MDS with Isolated Trisomy 8:::::::::::::::::::::::: 6.19.5 17p Syndrome::::::::::::::::::::::::::::::::::::::::::::: 6.19.6 3q21q26 Syndrome:::::::::::::::::::::::::::::::::::::::: 6.20 MDS Variants :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.20.1 Therapy Related MDS::::::::::::::::::::::::::::::::::: 6.20.2 Hypocellular or Hypoplastic MDS ::::::::::::::::: 6.20.3 Hyperfibrotic MDS:::::::::::::::::::::::::::::::::::::::: 6.20.4 Familial MDS ::::::::::::::::::::::::::::::::::::::::::::::: 6.21 Simplified Treatment Algorithm for MDS ::::::::::::::::::

7

200 201 201 201 202 202 202 202 205 205 205 206

Chronic Myelomonocytic Leukemia (CMML) ::::::::::::::::::::::::::::::::::::::: 223 Lisa Pleyer, Daniel Neureiter, Victoria Faber, and Richard Greil

7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8

8

Introduction to CMML Problems in Classification ::::: Epidemiology of CMML:::::::::::::::::::::::::::::::::::::::::::::: Molecular Biology of CMML :::::::::::::::::::::::::::::::::::::: Cytogenetics of CMML ::::::::::::::::::::::::::::::::::::::::::::::: Clinical and Laboratory Features of CMML ::::::::::::::::: Diagnosis of CMML:::::::::::::::::::::::::::::::::::::::::::::::::::: Prognostic Factors of CMML::::::::::::::::::::::::::::::::::::::: Treatment of CMML ::::::::::::::::::::::::::::::::::::::::::::::::::: 7.8.1 Best Supportive Care and Control of Myeloproliferation for CMML :::::::::::::::::::::::::: 7.8.2 Intensive Chemotherapy for CMML :::::::::::::::::: 7.8.3 Curative Treatment Options for CMML ::::::::::::: 7.8.3.1 Allogeneic Stem Cell Transplantation :::::::::::::::::::::::::::::::::: 7.8.3.2 Reduced Intensity Conditioning :::::::::: 7.8.4 Hypomethylating Agents in CMML::::::::::::::::::: 7.8.4.1 Azacitidine (Vidaza)::::::::::::::::::::::::: 7.8.4.2 Decitiabine (Dacogen):::::::::::::::::::::: 7.8.5 Other Treatment Options :::::::::::::::::::::::::::::::::::

223 224 224 225 225 226 227 227 227 228 228 228 229 229 229 229 230

Rare Clonal Myeloid Diseases ::::::::::::::::::: 235 Thomas Melchardt, Lukas Weiss, Lisa Pleyer, Daniel Neureiter, Victoria Faber, and Richard Greil

8.1 Chronic Clonal Disorders of Mast Cells ::::::::::::::::::::::: 8.1.1 Epidemiology ::::::::::::::::::::::::::::::::::::::::::::::::::: 8.1.2 Course of Disease and Prognosis ::::::::::::::::::::::: 8.1.3 Pathophysiology and Molecular Biology:::::::::::: 8.1.4 Cytogenetics ::::::::::::::::::::::::::::::::::::::::::::::::::::: 8.1.5 Clinical Presentation ::::::::::::::::::::::::::::::::::::::::: 8.1.6 Diagnosis and Classification of Mastocytosis ::::: 8.1.6.1 Classification of Mastocytosis::::::::::::: 8.1.6.2 Diagnostic Work up of a Patient with Suspected Mastocytosis :::::::::::::::::::::: 8.1.7 Differential Diagnosis ::::::::::::::::::::::::::::::::::::::: 8.1.8 Indications for Treatment and Therapeutic Options::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 8.1.8.1 Treatment of Symptoms Related to Mast Cell Degranulation::::::::::::::::::::: 8.1.8.2 Treatment of Cutaneous Mastocytosis :::::::::::::::::::::::::::::::::::::: 8.1.8.3 Treatment Options in Indolent Systemic Mastocytosis:::::::::::::::::::::::: 8.1.8.4 Treatment of Aggressive Systemic Mastocytosis ::::::::::::::::::::::::::::::::::::::

236 236 236 236 237 237 238 238 239 239 240 240 241 241 241

Contents

8.2 Chronic Clonal Eosinophilic Disorders and the Idiopathic Hypereosinophilic Syndrome ::::::::::: 8.2.1 Idiopathic Hypereosinophilic Syndrome (IHES) ::::::::::::::::::::::::::::::::::::::::::::: 8.2.1.1 Epidemiology::::::::::::::::::::::::::::::::::::: 8.2.1.2 Pathophysiology::::::::::::::::::::::::::::::::: 8.2.1.3 Cytogenetics :::::::::::::::::::::::::::::::::::::: 8.2.1.4 Clinical Presentation of IHES (Idiopathic Hypereosinophilic Syndrome) ::::::::::::::::::::::::::::::::::::::::: 8.2.1.5 Diagnosis of IHES ::::::::::::::::::::::::::::: 8.2.1.6 Treatment :::::::::::::::::::::::::::::::::::::::::: 8.2.2 Clonal Eosinophilic Diseases::::::::::::::::::::::::::::: 8.2.2.1 Myeloid and Lymphoid Neoplasms with Eosinophilia and Abnormalities of PDGF RA, PDGF RB or FGF R1 ::::::::::::::::::::::::::::::::::::::::: 8.2.2.2 Chronic Eosinophilic Leukemia (CEL), not Otherwise Specified:::::::::::::::::::::: 8.2.3 Causes of Reactive Eosinophilia :::::::::::::::::::::::: 8.2.3.1 Infections as Causes of Reactive Eosinophilia ::::::::::::::::::::::::::::::::::::::: 8.2.3.2 Drug Induced Reactive Eosinophilia ::::::::::::::::::::::::::::::::::::::: 8.2.3.3 Non Malignant Diseases Associated with Eosinophilia ::::::::::::::::::::::::::::::: 8.2.3.4 Malignant Clonal Diseases Associated with Eosinophilia ::::::::::::::::::::::::::::::: 8.2.4 Acute Eosinophilic Leukemia (AEL) ::::::::::::::::: 8.3 Disorders of Basophilic Granulocytes ::::::::::::::::::::::::::: 8.3.1 Reactive Polyclonal Basophilia:::::::::::::::::::::::::: 8.3.2 Clonal Basophilia Accompanying Other Myeloproliferative Neoplasms ::::::::::::::::::::::::::: 8.3.3 Acute Basophilic Leukemia::::::::::::::::::::::::::::::: 8.4 Chronic Neutrophilic Leukemia (CNL)::::::::::::::::::::::::: 8.4.1 Differential Diagnosis of Neutrophilia ::::::::::::::: 8.5 Chronic Clonal Histiocytic Diseases ::::::::::::::::::::::::::::: 8.5.1 Rosai Dorfman Syndrome (Sinus Histiocytosis with Massive Lymphadenopathy)::::::::::::::::::::::: 8.5.1.1 Epidemiology::::::::::::::::::::::::::::::::::::: 8.5.1.2 Clinical Features of Rosai Dorfman Syndrome :::::::::::::::::::::::::::::::::::::::::: 8.5.1.3 Diagnosis of Rosai Dorfman Syndrome :::::::::::::::::::::::::::::::::::::::::: 8.5.1.4 Histopathological Findings of Rosai Dorfman Syndrome:::::::::::::::::: 8.5.1.5 Treatment of Rosai Dorfman Syndrome :::::::::::::::::::::::::::::::::::::::::: 8.5.2 Langerhans Cell Histiocytosis (LCH) (Histiocytosis X, eosinophilic granuloma, Abt Letterer Siewe disease or Hand Sch€ uller Christian disease) :::::::::::::::::::::: 8.5.2.1 Epidemiology of LCH :::::::::::::::::::::::: 8.5.2.2 Prognosis and Course of Disease of LCH :::::::::::::::::::::::::::::::::::::::::::::: 8.5.2.3 Clinical Presentation :::::::::::::::::::::::::: 8.5.2.4 Diagnosis of LCH :::::::::::::::::::::::::::::: 8.5.2.5 Treatment :::::::::::::::::::::::::::::::::::::::::: 8.5.3 Malignant Histiocytosis::::::::::::::::::::::::::::::::::::: 8.5.3.1 Histiocytic Sarcoma ::::::::::::::::::::::::::: 8.5.3.2 Tumors of Langerhans Cells ::::::::::::::: 8.5.3.3 Follicular Dendritic Cell Sarcoma :::::::::::::::::::::::::::::::::::::::::::: 8.5.3.4 Interdigitating Dendritic Cell Sarcoma::::::::::::::::::::::::::::::::::::::::::::: 8.5.3.5 Treatment ::::::::::::::::::::::::::::::::::::::::::

xi

241 241 242 242 242

242 243 243 243

243 245 245 246 246 246 246 247 247 247 248 248 248 249 249 250 250 250

9

De novo Classic Paroxysmal Nocturnal Hemoglobinuria (PNH) (Marchiafava–Micheli Syndrome) :::::::::::::::::::::::::::::::::::::::::::::::::: 259 Lisa Pleyer and Richard Greil

9.1 Epidemiology of PNH:::::::::::::::::::::::::::::::::::::::::::::::::: 9.2 Pathophysiology and Molecular Biology of PNH :::::::::: 9.2.1 Pathomechanism of Hemolysis ::::::::::::::::::::::::: 9.2.2 Pathomechanism of Hemoglobinuria and Hemosiderinuria:::::::::::::::::::::::::::::::::::::::::::::::: 9.2.3 Pathomechanism of Thrombotic Tendency ::::::::: 9.2.4 Pathomechanism of Dystonias, Abdominal Pain and Systemic or Pulmonary Hypertension:::::::::: 9.3 Functional Defects of GPI Deficient Hematopoietic Cells:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 9.4 Clinical Features and Disease Complications of PNH ::: 9.5 Diagnosis, Laboratory Findings and Diagnostic Tests in PNH:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 9.5.1 Laboratory Findings :::::::::::::::::::::::::::::::::::::::::: 9.5.2 Diagnostic Tests:::::::::::::::::::::::::::::::::::::::::::::::: 9.6 Differential Diagnosis of PNH ::::::::::::::::::::::::::::::::::::: 9.6.1 PNH Cells in Normal Individuals and After Therapy with Campath :::::::::::::::::::::::::::::::::::::: 9.7 Cytogenetics in PNH ::::::::::::::::::::::::::::::::::::::::::::::::::: 9.8 Risk Factors in PNH :::::::::::::::::::::::::::::::::::::::::::::::::::: 9.9 Treatment of PNH Current State of the Art:::::::::::::::: 9.9.1 Treatment of Anemia and Other Cytopenias in PNH ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 9.9.2 Treatment of Thrombotic Events in PNH ::::::::::: 9.9.3 Targeted Treatment Complement Inhibition :::: 9.9.3.1 Inhibition of Terminal Complement C5 and MAC Formation ::::::::::::::::::::: 9.9.3.2 Exogenous Replacement of GPI Linked Proteins::::::::::::::::::::::: 9.9.4 Immunosuppression :::::::::::::::::::::::::::::::::::::::::: 9.9.5 Allogeneic Stem Cell Transplantation for PNH:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 9.9.6 Perioperative Management of PNH Patients :::::::: 9.9.7 Avoidance of Drugs Known to Induce Hemolytic Anemia :::::::::::::::::::::::::::::::::::::::::::: 9.9.8 Management of Pregnancy in Women with PNH :::::::::::::::::::::::::::::::::::::::::::::::::::::::::

259 260 262 263 264 264 265 266 267 267 267 267 268 269 269 269 270 270 272 272 272 273 273 274 274 274

250 251

10

251

251 251 251 251 252 253 253 254 254

Clonal Bone Marrow Failure Overlap Syndromes:::::::::::::::::::::::::::::::::::::::::::::::: 281 Lisa Pleyer, Daniel Neureiter, and Richard Greil

Introduction ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: MDS/PNH Overlap Syndromes :::::::::::::::::::::::::::::::::: Aplastic Anemia (AA) and AA Overlap Syndromes :::: 10.3.1 Aplastic Anemia:::::::::::::::::::::::::::::::::::::::::::: 10.3.2 AA/PNH Overlap Syndromes :::::::::::::::::::::::: 10.3.3 AA/MDS Overlap Syndromes:::::::::::::::::::::::: 10.4 T cell Large Granular Lymphocyte Leukemia (T LGL) and T LGL Overlap Syndromes :::::::::::::::::::: 10.4.1 T LGL :::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 10.4.2 T LGL/MDS Overlap Syndromes::::::::::::::::::: 10.4.3 T LGL/PNH Overlap Syndromes ::::::::::::::::::: 10.4.4 T LGL/AA and T LGL/Pure Red Cell Aplasia (PRCA) Overlap Syndromes ::::::::::::::::::::::::::

10.1 10.2 10.3

281 282 283 283 283 284 285 285 286 286 286

254 254 254

List of Contributors ::::::::::::::::::::::::::::::::::::::::::::: 289 About the Editors:::::::::::::::::::::::::::::::::::::::::::::::: 291

1

Introduction to Classic Chronic Myeloproliferative Disorders (CMPDs) – Molecular and Cellular Biology Lisa Pleyer and Richard Greil

Contents 1.1 Pathogenetic Role of the JAK2V617F Mutation Definition of JAK2V617F+ CMPDs with Common Pathogenesis and Natural Disease Evolution from ET to PV to post-ET/PV-MF vs. JAK2V617F2 CMPDs:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2 1.1.1 The Clonal Stem Cell Nature of Classic CMPDs :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2 1.1.2 JAK2V617F is an Acquired Somatic Mutation :::::::: 4 1.1.3 Timing of the JAK2 Mutation Relationship Between its Emergence and Clonal Hematopoiesis: JAK2V617F is an Early, but not the Earliest Event in the Transformation Process :::::::::::::::::::::::::::::: 4 1.1.4 JAK2 Mutations in Murine Systems Disease Phenotype and Biologic Consequences ::::::::::::::::: 4 1.1.5 Gene Dosage and the Role of JAK2 Mutations in the Generation of Different Types of CMPD::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 5 1.1.6 JAK2 Mutations, Signaling Aberrations and Consequences for Cell Biology::::::::::::::::::::::::::::: 7 1.1.7 Altered Downstream JAK2 Signaling and STAT Phosphorlyation States for the Discrimination Between Classic CMPD Entities:::::: 8 1.2 Other Important (Epi)genetic Factors Functionally Equivalent to the JAK2V617F Mutation ::::::::::::::::::::::::: 8 1.3 Therapeutic Targeting of the JAK2 STAT Signaling Axis ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 9

Philadelphiachromosome-negative chronic myeloproliferative disorders (CMPDs) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), subsumed as the classic CMPDs, as well as the following disorders: chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia (CEL) and the hypereosinophilic syndrome (HES), clonal basophilic disorders and unclassifiable CMPDs. The diagnosis and management of CMPDs has been difficult in the past due to several reasons. (1) Significant phenotypic mimicry exists among classic CMPDs on the one hand, as well as between classic CMPDs and non-clonal benign and malignant hematopoietic disorders on the other hand. (2) The initial lack of clonal molecular diagnostic markers in the pre-JAK2 era, as well as the previous lack of clear-cut diagnostic criteria and an adequate classification system, has often led to misclassification of chronic myeloproliferative disorders. CMPDs share several features, namely (a) involvement of a multipotent hematopoietic progenitor cell with dominance of the transformed clone over normal hematopoiesis, (b) autonomous proliferation of at least one cell line in the absence of a definable stimulus, (c) growth factor independent colony formation in vitro on the one hand, and growth factor hypersensitivity on the other, (d) varying rates of thrombocythemia with subsequent thrombohemorrhagic diathesis and (e) transformation to acute myeloid leukemia (AML) or development of secondary bone marrow fibrosis. The latter phenomena are of great concern due to the lack of effective therapies for these conditions. Cytogenetic abnormalities of chromosomes 1, 8, 9, 13 and 20, as well as genetic mutations in the JAK2 or MPL locus (equivalent to the gene encoding for the thrombopoietin (TPO) receptor) (see below), epigenetic abnormalities as well as increased PRV-1 mRNA and impaired megakaryocyte and platelet MPL expression are also shared features of the classic CMPDs (see also Tables 1.1 1.3).

2

L. Pleyer and R. Greil

Table 1.1: Diagnostic histological bone marrow features in CMPDs (merged from multiple reports found in the literature) Diagnosis

RTh (%)

ET (%)

CIMF-0 (%)

Increased cellularity Megakaryopoiesis Maturation defects Nuclear lobulation Naked nuclei Small forms Giant forms Bulbous nuclei Dense clusters Erythropoiesis Quantity " Left shift Myeloid stroma Reticulin fibres " Granulopoiesis Left shift

29

10

97

50 80 20 50

20 50 G10 80 100

CIMF-1 (%)

PV (%) 100

50 80

G10

50 80 20 50

20 50 50 80

50 20 50 10 10 10

G10

10 20

# G10

80 100 80 100

80 100

10 20

80 100

Pleomorphic size distribution 80 100

50 80

80 50 80 20 20 20

50 80

RTh Reactive thrombocytosis; ET essential thrombocytosis; PV polycythemia vera; CIMF chronic idiopathic myelofibrosis (CIMF 0 1 can be subsumed as early stage PMF in the new classification)

Table 1.2: Karyotypic abnormalities in CMPDs (merged from multiple reports found in the literature) CMPD

20q2 (%)

13q2 (%)

Abn 1 (%)

+8 (%)

+ 9p (%)

Abn 12 (%)

27/7q2 (%)

25/5q2 (%)

9p24 (JAK2) (%)

1q2 (%)

3p2 (%)

3q2 (%)

PV ET MDS MMMa

17 10 8 22

6 5 Rare 28

6 9 3 17

13 5 15 21

10 4 Rare 50

6 1 5 3 9

6 3 20 11

6 5 30 6

74 97 32 57 5 35 50

25

24

22

a

MMM Myeloid metaplasia with myelofibrosis: former nomenclature, which now corresponds to PMF and post ET/PV MF

Except for early stage PMF, all CMPDs are characterized by overproduction of mature blood cells with a predominance of one or more myeloid cell lineages. Due to defective post-translational processing at the stem cell level the TPO receptor (MPL) is poorly glycosylated and poorly expressed on platelets [1]. This results in (a) enhanced sensitivity to TPO with increased proliferation and (b) in resistance to apoptosis, which also conveys a proliferative advantage. In addition, elevated serum TPO levels are observed in the majority of patients with ET/PV/PMF/post-ET-MF/post-PV-MF, which further enhance clonal myeloproliferation and/or induce stromal cell production of fibrogenic, osteogenic and angiogenic cytokines. Phenotypic mimicry and disease overlap between ET, PV and early phase myelofibrosis can cause problems in diagnosis and differential diagnosis. This will be discussed in detail in the following chapters. Tables 1.1 1.3 give an overview of diagnostic histological bone marrow features, karyotypic abnormalities as well as genetic and

phenotypic features in the classic CMPDs and their most relevant differential diagnoses, respectively.

1.1 Pathogenetic Role of the JAK2V617F Mutation – Definition of JAK2V617F+ CMPDs with Common Pathogenesis and Natural Disease Evolution from ET to PV to post-ET/PV-MF vs. JAK2V617F2 CMPDs 1.1.1 The Clonal Stem Cell Nature of Classic CMPDs There is long-standing evidence for the clonal nature of classic CMPDs [2, 3], and this clonogenic capacity is present despite the absence of hematopoietic growth factors such as erythropoietin (EPO) [4]. The discovery of an acquired somatic gain-of-function mutation of tyrosine

Chap. 1

Molecular and Cellular Biology of CMPD

3

Table 1.3: Genetic and phenotypic features in CMPDs (merged from multiple reports found in the literature) Entity

RTh ET

PV

SE

CIMF-0 CIMF 1 3

CML

MDS 5q-/RARS-T

PLT > 500 109/L PLT > 1,000 109/L EMC formation Increased sensitivity to TPO Serum TPO MPL # Erythrocytes EEC formation Hematocrit PRV 1 gene overexpression Serum erythropoietin History of hemorrhagic complications at diagnosis History of thrombotic complications at diagnosis Erythromelalgia Clonality

"" " No No " No $ No $ No $ $

98% 45% """ Yes " Yes $ "/"" $ 21 67% $ 20%

46% 35% "" Yes " Yes N/" """ N/" 91 100% # 13%

87% 33% " Yes " Yes N/# " N/# þ/

" " No

9%

32% 5% No n.d. n.d. n.d. $ No $ No $ $

No No $ No " No " /þ $/"" $ 13%

$/"

16%

19%

$/"

1%

$

$

No No

Yes No Monoclonal No

No

JAK2V617F (PCR) heterozygous JAK2V617F (PCR) homozygous LAP score Ph 1 chromosome Resistance to TGFb due to

0% 0% $ No

71% 21 35% "" No TbetaRII#

0% 0% $ No

45% 6 21% N/" " No TbetaRII#

Grade 3 4 " BM microvessel density

n.d.

Yes Oligo/ monoclonal 49 ( 93)% 4% N/" No Smad4# TbetaRII# 12%

No Monoclonal

No Monoclonal

33%

n.d.

13%

43% 14% " n.d. n.d. n.d. # " # 50 67% $

No Monoclonal

70%

## ## No $/""

20% in atypical CML 2% 0% 50 60% ## $ Yes No TbetaRII# n.d.

n.d.

RTh Reactive thrombocytosis; SE secondary erythrocytosis; PLT platelet count; EMC endogenous megakaryocytic colony; N within the normal range; EEC endogenous erythroid colony; MPL thrombopoietin receptor; PRV 1 polycythemia rubra vera 1; LAP leukocyte alkaline phosphatase, TGFb transforming growth factor b; CML chronic myeloid leukemia; TPO thrombopoietin; BM bone marrow; n.d. not determined

kinase JAK2V617F located on chromosome 9 further substantiated the now generally accepted view, that a common hematogeneic progenitor cell is at the origin of all classic CMPDs. JAK2V617F is found in all CMPD subtypes, but is less prevalent in AML with antecedent PVor myelofibrosis (36%), megakaryocytic leukemia AML-M7 (18%), and other entities such as Ph-negative CML (19%), CMML (13%) and MDS (5 15%) [5]. Most patients with MDS bearing the JAK2 mutation belong to the subtypes MDSRARS-T and MDS-5q-syndrome, in whom the mutation can be found in up to 60% of patients [6 8] (see respective sections in MDS chapter). The positive predictive value of a JAK2V617F PCR test for the diagnosis is extremely high (almost 100%) [9]. JAK2V617F is found in 23 72%, 65 99% and 39 57% in patients with ET, PV and myelofibrosis, respectively [10, 11]. The high degree of variability depends on the method used to detect the JAK2V617F mutation, with RT-PCR being more sensitive than allele-specific PCR and DNA-resequencing. The JAK2V617F mutation has also been demonstrated in B- and

T-lymphoid compartments [12], supporting the stem cell, or at least early progenitor cell, as the level of acquisition of the mutation [13, 14]. This mutation has also been directly detected in hematopoietic stem cell isolates of CMPD patients [12]. However, not all hematopoietic cells in a patient carry this mutation. In fact, JAK2V617F expression in different blood cell lineages seems to be dependent on the type of CMPD. For example, JAK2 mutations seem to occur in erythroid progenitors of most PV patients, whereas they occur only rarely in erythroid progenitors of ET patients [15]. However, the clonal stem cell character is not limited to JAK2V617F þ CMPDs. In the few cases of PVas well as the larger fraction of ET and PMF patients negative for the JAK2V617F mutation, X-chromosome inactivation studies (XCIP) reveal the presence of a dominant clone in the marrow capable of autonomous growth [11, 16, 17]. In conclusion, there is now overwhelming evidence for the clonal nature of CMPDs which is further substantiated by the various mouse models mentioned below.

4


1.1.2 JAK2V617F is an Acquired Somatic Mutation

(iv)

The JAK2 mutation is an acquired somatic mutation not found in the germline configuration and is acquired at the level of the hematopoietic stem cell or very early progenitor cell. This also holds true for the rare familial cases of CMPDs. The study of a large cohort of CMPD-families revealed genetic heterogeneity regarding the presence of JAK2V617F mutation, thus not lending support to the existence of germline JAK2V617F, indicating that this mutation is acquired, even in familial cases [18]. (v)

1.1.3 Timing of the JAK2 Mutation – Relationship Between its Emergence and Clonal Hematopoiesis: JAK2V617F is an Early, but not the Earliest Event in the Transformation Process Several lines of evidence suggest that development of clonal hematopoiesis may precede the acquisition of JAK2V617F, suggesting that mutation of JAK2 is not the earliest event of transformation, and thus not the diseaseinitiating incident, in classic CMPDs. (i)

(ii)

(iii)

In a recent analysis performed in a Chinese hospital, 36/3.935 randomly chosen patients were found positive for JAK2V617F, in the absence of other criteria sufficing for the diagnosis of a CMPD [19]. The authors concluded that the occurrence of JAK2V617F may be a prelude to a myeloproliferative disorder, but that its mere presences per se cannot be used to diagnose CMPDs. When taking a closer look at the patients bearing JAK2V617F, 21/35 evaluable patients had WBC H 8.000/ml, 7/35 had PLT counts H 350.000/ml, and 5/15 patients had a history of cerebral ischemia, thrombosis or coronary heart disease. It will be extremely interesting to pursue the follow up data, and to see whether, and how many of these patients will develop CMPDs. In line with the above, JAK2 mutations have been shown in patients with de novo AML [20] and in the majority of patients with otherwise unexplained Budd Chiari syndrome [21], thus raising the question of a pre-existing but unmasked myeloproliferative disorder [22]. Not all cells of clonal origin carry the JAK2V617F mutation. Using XCIP (X-chromosome inactivating patterns) clonality analysis, the percentage of JAK2V617F þ granulocytes and platelets was often markedly lower than the percentage of clonal granulocytes and platelets [11, 23].

(vi)

5 10% of patients with CMPDs carry deletions of chromosome 20q. When deletions of chromosome 20q were used as an autosomal, X-chromosome independent clonality marker, a similar discrepancy was found between the percentage of cells carrying JAK2V617F and del(20q) [23, 24], thus pointing to an earlier acquisition of the 20q deletion [23]. This suggests, that at least in some patients with CMPDs, the gain-of-function mutation in JAK2 occurs on the background of already existing clonal hematopoiesis. This pre-JAK2phase is caused by an as yet unknown (epi)genetic event. Recently, TET2 (ten-eleven-translocation) mutations have been proposed to be a pre-JAK2 event. In 3/4 patients with a JAK2V617F þ CMPD who subsequently developed AML, the evolving AML is JAK2V617F , suggesting that the leukemia cell [24]. Thus, arose in a JAK2V617F JAK2V617F AML could have developed from (a) a normal stem cell not part of the original clone; (b) a cell that had some other initiating mutation prior to JAK2, implicating an ancestral abnormality preceding the acquisition of the JAK2V617F mutation, or (c) a JAK2V617F þ cell with subsequent reversion to V617F , although the last model seems very unlikely.

1.1.4 JAK2 Mutations in Murine Systems – Disease Phenotype and Biologic Consequences The biologic consequences of JAK2V617F mutations have been clearly demonstrated by murine experiments using either retrovirally transfected hematopoietic stem cells in bone marrow transplantation settings or transgenic animals [10]. Retroviral mouse models, using mouse JAK2 cDNA in which the mutation had been introduced, have demonstrated a PV-phenotype which often progresses to post-PV-MF, but thrombocytosis is absent in these mice [25 28]. Lack of thrombocytosis is thought to be due to the inherent JAK2V617F overexpression of retroviral models (see below). These models usually display polycythemia with erythrocytosis, leukocytosis and transformation to myelofibrosis. However, the degree of leukocytosis and development into myelofibrosis varies, and is probably dependent on the genetic background and gene dosage among other factors [26 28]. Very recently a JAK2V617F mutant transgenic mouse model using human JAK2V617F has been generated [29]. These mice developed a phenotype resembling ET, with moderate neutrophilia and marked thrombocytosis that has never been observed with retroviral models. Through induction of the mutated

Chap. 1


5

Fig. 1.1 Gene dosage effect of JAK2 on the phenotypic evolution of CMPDs. Heterozygous somatic JAK2V617F mutation with slightly increased kinase activity seems sufficient for activation of the TPO receptor MPL with increased megakaryocytic proliferation and cyto kine hypersensitivity of platelets in ET and early PV mimicking ET (fruste PV). Homozygous JAK2V617F with pronounced kinase activity occurs due to a second mitotic recombinatorial event in a stem cell already carrying the mutation, resulting in loss of heterozy gosity (LOH) of 9p. Homozygosity of JAK2V617F is probably neces

sary for activation of the EPO and G CSF receptors, leading to trilineage megakaryocytic, erythroid and granulocytic myeloproli feration. Myeloid metaplasia and secondary myelofibrosis with the clinical pictures of overt classical PV, PMF and post PV myelofibrosis are the consequence. The clone carrying the homozygous mutation eventually outcompetes the cells that are only heterozygous, resulting in sustained high levels of JAK2 mutant protein. This is though to promote disease progression and contribute to leukemic transforma tion. *denotes an activated state of the respective receptor

transgene expression, and thus control over the ratio of expression levels of mutated transgene to the endogenous mouse JAK2, a variable phenotype ranging from (a) thrombocytosis only, to (b) bilineage disease involving thrombopoiesis and granulopoiesis (ET-like), to (c) the full trilineage PV-like phenotype was observed [29]. The phenotypes (a) and (c) were induced by JAK2V617F/JAK2wt (wildtype) ratios ofG1 or ¼1, respectively. High-levels of JAK2 with JAK2V617F/JAK2-wt H1 appear to be inhibitory to megakaryopoiesis. This is demonstrated by normal or decreased platelet counts in retroviral murine CMPDmodels as well as the lower on average platelet count observed in PV patients as compared to ET patients [29]. One possible explanation for this phenomenon is that JAK2V617F downregulates MPL expression [1, 30]. The above-mentioned murine model systems implicate and underline a gene dosage effect of the mutated JAK2 gene, and this seems to play an essential role in CMPD disease phenotype and biology (see Fig. 1.1).

This gene dosage effect on the CMPD phenotype of the whole spectrum of classic CMPDs is reinforced and conformed by similar findings in humans (see Fig. 1.1, [29] and see below). Presence of homozygous JAK2V617F is associated with significantly higher WBC count and Hb-levels with less requirement for transfusions, implicating a masked erythroid phenotype, as well as a higher rate of evolution of ET towards PVand PV to pPV-MF, respectively. Analyses of bone marrow hematopoietic stem cells shows homozygosity for the JAK2V617F mutation in up to 90% of patients with PV [31 33]. This is in stringent contrast to ET, where the number of homozygous patients is 1 2% and hematopoietic progenitor cells show similarly low amounts of homozygosity [15]. This homozygosity results from mitotic recombination at chromosome 9 [34]. Homozygosity leads to a doubling of gene dosage and increased downstream signaling (see Fig. 1.1). The degree of homozygosity increases with time in PV but not in ET [15]. Apart from being correlated with disease progression, homozygosity for JAK2V617F is associated with a poorer prognosis and highly aggressive forms, implicating prognostic relevance. However, there does not seem to be a correlation with a higher thrombotic risk or leukemic transformation [35], with additional molecular events being seemingly necessary for the latter. Since mitotic recombination rates also vary in normal controls, genetic and environmental factors may exert pressure on the rate of development of mitotic recombination and the

1.1.5 Gene Dosage and the Role of JAK2 Mutations in the Generation of Different Types of CMPD The recent data from the above-mentioned mouse models clearly point to the role of JAK2V617F gene dosage for the development of the relevant phenotype of CMPD evolving as a result of the JAK2V617F mutation.

6


development of features more typical for ET or PV, respectively [22, 36]. Persuasive evidence implicates a distinct disease entity, that must be differentiated from CMPDs not carrying the JAK2 mutation, as well as novel insights into a common pathogenesis, whereby the interrelationships between ET, fruste PV, PV and secondary myelofibrosis are seen as a continuous progression of the same disease at different stages, rather than as separate entities. Disease progression can readily be explained by sequential occurrence of heterozygous and homozygous JAK2V617F mutations. Further confirmation of the presumptive common JAK2V617F-associated pathogenic mechanism is furnished by several other lines of evidence: (i)

(ii)

The acquisition of thrombopoietin receptor (np1) defects has frequently been observed during disease progression of ET towards PV and MF. Transition from hetero- to homozygosity for JAK2V617F parallels an enhanced JAK2V617F gene dosage effect on both granulocyte activation as well as CD34 þ counts. A high JAK2V617F burden therefore seems to have a mobilizing effect on CD34 þ hematopoietic stem cells [37]. This is in accordance with the increased number of CD34 þ hematopoietic precursor cells found in all CMPDs, which increases along with the proliferative capacity of the individual CMPDs, from ET to early phase PV, to PV with large spleen and finally secondary MF (e.g., [38] and Fig. 4.1 in Chap. 4).

Fig. 1.2 PCR-detection of JAK2 mutational status. Cross: de notes no template control; Red circles: denote patients with JAK2 wt; (a) and (b) patients with a small proportion of mutated alleles, i.e., a small clone within a predominant wildtype background; (c)

In conclusion, this unique mutation not only provides a proliferative advantage, but also mediates abnormal trafficking of the hematopoietic clone, as well as disease progression, providing the rational for determination of JAK2-status by quantitative PCR for diagnostic and/or prognostic purposes, as well as for monitoring disease progression in patients known to be heterozygous (see Fig. 1.2). As mentioned in 1.1.4., not only the presence of JAK2V617F is important however, but especially its ratio to wildtype JAK2 determines the CMPD phenotype, as has elegantly been demonstrated in a transgene mouse model using the human JAK2 gene [29, 39]. In mice engineered to express JAK2V617F at levels lower than, equal to, or higher than the endogenous wt-JAK2, phenotypes resembling ET, PV with erythrocytosis, neutrophilia and thrombocytosis, and PV without thrombocytosis developed, respectively [29]. These results further confirm the already assumed effect of JAK2 gene dosage on disease phenotype and progression. Lower levels of JAK2 are associated with thrombocytosis and ET, whereas higher levels are necessary for significant splenomegaly and a PV phenotype. Even higher levels lead to normal platelet numbers and progression to myelofibrosis. Very recently it was shown that strong activation of JAK2V617F stimulates homologous recombination, centrosome and ploidy abnormalities, induces a mutator phenotype and resistance against genotoxic agents [40]. This JAK2V617F induced genetic instability may well be responsible for the phenotypic heterogeneity of CMPD features as well as disease evolution to secondary leukemia [40].

and (d) patients with approximately equal numbers of wildtype and mutated alleles; (e) patient with predominance of cells bearing the JAK2V617F mutation, implicating the presence of a homozygous subclone

Chap. 1


7

The JAK2 mutation occurs within the JH2 pseudokinase domain which is usually considered a negative autoregulator of the JAK2 kinase function [41]. This molecular aberration activates a number of autonomous signal transduction events along the EPO-R, TPO-R and G-CSF-R pathways. Due to the constitutive JAK2 activation, these pathways are no longer dependent on, and thus no longer

under the control of, growth factors. In addition, the mutated JAK2 seems to escape the negative regulation by suppressors of cytokine signaling (SOCS) proteins. Janus kinases (JAKs) are emerging as integral parts of cytokine receptor complexes, and wildtype (wt) as well as mutated JAK2 molecules require the growth factor receptors as scaffolds for the docking of downstream effectors [42, 43]. This scaffold function of cytokine receptors is essential for the transforming and signaling activities of mutated JAK2V617F [43], and is explained as follows: JAK2V617F

Fig. 1.3 JAK2- and MPL-signaling: Differential effect of various mutations. (1) Normal signaling of wildtype (wt) JAK2. (2) Ligand independent increased signaling of JAK2V617F and JAK2 exon 12 mutant proteins. (3) Cytokine hypersensitivity of JAK2V617F and JAK2 exon 12 mutant proteins. (4) Presence of MPL mutations further increase JAK2 signaling. (1) Normally cytokine ligands, such as EPO, which is used as an example here, bind their receptive receptors, which then results in the phosphorylation of JAK2. JAK2 is a monomeric cytosolic protein which is inactive when not bound to a cytokine receptor. Phosphorylation of JAK2 leads to the recruit ment of STAT proteins, which are inturn phosphorylated and activated. Binding leads to downstream signaling of several signaling cascades, including the PI3K AKT mTOR and Ras Raf MEK ERK pathways. SOCS or SHP 1 mediate negative regulation of JAK2 signaling. Mutant JAK2 however might be able to escape this negative feedback mechanism. (2) JAK2V617F

and JAK2 exon 12 mutant kinases can bind cytokine receptors and are phosphorylated in the absence of ligand and result in ligand independent activation of downstream pathways, which is several fold stronger than activation by wildtype JAK2. Two mutant JAK2 molecules must be close to each other, and they must have bound to their cognate cytokine receptors in order to allow for autophop sphorylation and ligand independent activation. (3) Even low levels of cytokine ligands can result in a dramatic increase in V617F or exon 12 mutant JAK2 signaling. This phenomenon is termed cytokine hypersensitivity. The phenomena described in (2) and (3) are thought to be mediated by favorable steric changes of JAK2 which enable easier access to phosphorylation sites. (4) Various MPL mutants are also able to increase JAK2 signaling, especially in the presence of ligand (in this case thrombopeietin (TPC)). Occasionally, a concomitant JAK2V617F mutation is present, which complements the MPL mutation and further enhances JAK2 signaling

1.1.6 JAK2-Mutations, Signaling Aberrations and Consequences for Cell Biology

8

is a monomeric protein and is inactive in the absence of cognate cytokine receptors. Two JAK2V617F molecules must be physically close to each other in order for the adjacent JAK2V617F kinase to phosphorylate and thus activate the other, even in the absence of cytokine binding. This physical proximity is given, when two JAKV617F molecules bind to cell surface bound growth factor receptors [43] (see Fig. 1.3). In addition to its kinase activity for cytokine receptor signaling, JAK2 is also an essential subunit that binds cytokine receptors such as EPO-R in the endoplasmatic reticulum, thus promoting EPO-R cell surface expression [44]. Additionally, wt JAK2 profoundly affects TPO-receptor (MPL) stability, availability and recycling function, thereby promoting higher cell surface TPO-R expression [42]. Platelets and megakaryocytes from patients with CMPDs exhibit lower TPO-R levels, with most of the receptors being immature and dysfunctional [1]. The activating point mutation JAK2V617F is thought to be responsible for, or at least to contribute to, this down-regulation of TPO-R cell surface levels in myeloproliferative diseases. This may be due to defective TPO-R processing/recycling, while cytokine-hypersensitivity is probably due to conformational changes brought about by ligand binding. These structural changes possibly allow JAK2V617F to assume a more favorable orientation, rendering the molecule more accessible to phosphorylation and thus activation [44]. Enhanced JAK signal transducer and activation (STAT) signaling is thought to result in the commonly observed (transcription factor mediated) overexpression of antiapoptotic Bcl-xL in erythroid cells of PV patients [45]. Mutated JAK2 also significantly influenced genes, such as NF-E2, which orchestrate erythroid differentiation [46, 47]. Antiapoptotic effects are also induced by activation of the PI3KAkt and the MAPK-ERK pathways [48] (see Fig. 1.3). This decrease in the rate of apoptosis may also be caused by an increased death receptor resistance, a system which is used for tuning the erythropoietic drive in normal erythropoiesis [49, 50]. However, mutant JAK2 also drives the cells through the cell cycle, thus promoting proliferation of the mutated clone, an effect caused by upregulated cyclin-D2 and reduced levels of p27 [51]. As a result of the reduced rates of apoptosis and increases in cell cycle transit, the hematopoietic compartment of the bone marrow is expanded (hypercellular) in CMPDs.

1.1.7 Altered Downstream JAK2 Signaling and STAT-Phosphorlyation States for the Discrimination Between Classic CMPD Entities In CMPDs, the phosphorylation status and expression of pSTAT3 and pSTAT5 is deeply altered, and ET, PV, as well


as MF are specifically associated with three distinct abnormal patterns of pSTAT3/pSTAT5 [52]. In this regard, the phosphorylation status of the downstream signaling molecules STAT3 and STAT5 in bone marrow cells may further help in discriminating classic CMPDs among each other as well as against secondary erythrocytosis or thrombocytosis [52], although this is not (yet) applicable to routine everyday practice. Moderately increased pSTAT3 and pSTAT5 expression was observed in secondary forms of thrombocytosis and erythrocytosis, where it may reflect the chronic stimulation along the TPO and EPO receptor, respectively [52]. In contrast, uniformly and strongly increased pSTAT3 and pSTAT5 expression typically occur in PV, while reduced expression of both STATs is typical of idiopathic myelofibrosis. The high pSTAT5 expression observed in PV has been explained by its ability to upregulate and cooperate with Bcl-xL in inducing erythroid differentiation, whereas the impaired STAT activation in myelofibrosis could have a role in facilitating marrow fibrosis induced by cytokine release, as STAT3 is a known anti-inflammatory response mediator [52]. In contrast to PV, ET is characterized by increased pSTAT3 and reduced pSTAT5 [52, 53]. This is in good correlation to the established role of STAT3 in the regulation of early stage megakaryopoiesis and thrombopoiesis, which is presumed to be mediated by expansion of megakaryocytic progenitor cells [54]. Interestingly, these patterns of STAT phosphorylation were not influenced by the presence of the JAK2V617F mutation [52]. This is in line with the inability of JAK2-regulated events, such as increased rates of PRV-1 mRNA content [55] or endogenous erythroid colony (EEC) growth [4], to differentiate between the diverse myeloproliferative conditions, be they of clonal or secondary nature [56]. This points to the pathogenetic involvement of other molecular events, supposedly leading to the activation of the same signaling pathways as JAK2V617F. In accordance, several such novel mutations have been recently discovered (see below).

1.2 Other Important (Epi)genetic Factors Functionally Equivalent to the JAK2V617F Mutation There are several other mutations which lead to constitutive activation of the JAK2 STAT5 pathway and manifest in CMPD phenotypes, and are therefore seen as functionally similar, if not equivalent to the JAK2V617F mutation. These include the following: (1)

Several JAK2 exon 12 mutations which have only been described in PV so far (see respective section

Chap. 1

(2)

(3)

(4)

(5)

(6)


in PV chapter (3.3.4.)) and are associated with a predominantly erythroid phenotype with lower WBC and PLT counts. Various mutations in the MPL gene which encodes the thrombopoietin receptor also lead to enhanced JAK2-dependent signaling and have been observed in patients with ET, post-ET-MF and myelofibrosis in blast crisis, but not in PV [57] (see respective sections in chapters on ET (2.3.7.), PV (3.3.) and PMF (4.3.)). MPL mutations may occur concurrently with JAK2V617F, suggesting functional complementation [57]. MPL mutations segregate primarily with the phenotypes of thrombocytosis, extramedullary disease and myelofibrosis. JAK2T875N is a novel activating mutation that results in a myeloproliferative disease with features of megakaryoblastic leukemia in a murine bone marrow transplantation model [58]. JAK2T875N, like JAK2V617F, is a constitutively active tyrosine kinase that activates downstream effectors including STAT5. In mice, this mutation leads to megakaryocytic hyperplasia as well as increased reticulin fibrosis of bone marrow and spleen [58]. Given the fact that JAK2 exon 12 mutations and JAK2T875N have similar in vitro and in vivo effects as JAK2V617F, the predominance of the JAK2V617F allele is surprising and as yet unclear [10]. Several translocations involving JAK2, leading to a constitutively active JAK2-fusion protein, such as t(9;15;12)(p24;q15;p13) ETV6-JAK2 [59], t(8;9) (p23;p24) PCM1-JAK2 [60 62] and t(9;22)(p24; q11.2) BCR-JAK2 [63], have been described in chronic myeloid malignancies, most notably in atypical chronic myeloid leukemia. The translocation t(8;9)(p23;p24) resulting in the fusion gene PCM1-JAK2 seems to be associated with a more aggressive clinical course and may be associated with myelodysplastic features [64]. Whereas JAK2V617F probably leads to decreased negative autoregulation of JAK2 [60, 62, 64], chimeric PCM1-JAK2 constitutively activates JAK2, explaining the stronger oncogenic potential. In fact, the translocation may even influence the erythroid lineage to the point that it leads to overt erythroid leukemia [65]. Furthermore, several single nucleotide polymorphisms (SNPs) in the JAK2 gene or the EPO-R have been associated with PV, and sometimes ET (see [66] and respective section in PV chapter (3.3.5.)). Finally, epigenetic alterations, such as hypermethylation and epigenetic inactivation of SOCS1 and 3 or SHP-1 (Src homology 2-containing protein tyrosine

9

phosphatase-1), all of which are negative regulators of the JAK STAT pathway, may complement mutation of the JAK2 kinase. Inactivation of SOCS proteins results in the loss of negative regulation of physiological regulation of JAK2 activity, which further enhances JAK STAT signaling [67] (see Fig. 1.3). SOCS1, SOCS3 or SHP-1 inactivation due to hypermethylation has been found in up to 15%, 41% and 7% of CMPD patients, respectively [67, 68]. Importantly, this hypermethylation-mediated silencing of JAK2-inhibitors was independent of, and could co-occur with, positive JAK2 mutational status [68] and seems to occur more frequently in patients with PMF, post-ET/PV-MF or postCMPD-AML [67, 68]. Therefore, epigenetic silencing of negative JAK2 regulators seems to act as an alternative or complementary mechanism to JAK2 mutations. However, others have found upregulations of SOCS1 [69], SOCS2 [70] and SOCS3 [71]. This was interpreted as a compensatory upregulation of a natural negative feedback loop, in a futile attempt to counteract the elevated activity of mutated JAK2. It has even been hypothesized, that JAK2 can overcome the negative regulation by SOCS and possibly even exploit the compensatory overexpression to potentiate its own myeloproliferative capacity [71]. In line with this argumentation, it has been suggested that therapeutic inhibition of SOCS3 might selectively attenuate JAK2V617F, but not wildtype JAK2 [10]. In light of these seemingly conflicting propositions, further results concerning epigenetic modification of negative JAK2 regulators are eagerly awaited.

1.3 Therapeutic Targeting of the JAK2–STAT Signaling Axis (Table 1.4) The dominant effect of JAK2 mutations on pathologic signaling and biologic consequences, as well as the appearing role of JAK2 mutations on the evolution and prognosis of the diseases, are strong arguments in favor of therapeutically targeting mutated JAK2 kinases in CMPDs. Pre-clinical experiments with a number of JAK2 selective or non-selective drugs have shown various degrees of efficacy in primary cells from patients with CMPDs as well as in mouse xenograft models (for review see [73]). Phase I/II trials have been initiated with several JAK2 inhibitors (see Table 1.4 and Tyrosine Kinase Inhibitor section in PMF chapter (4.13.6.1.)).

10


Table 1.4: JAK2 under fire in the development of targeted therapy Inhibitor

JAK2 selective

Company

Phase of development

INCB018424

Yes

Incite Corporation

* * * * *

XL019

Yes

Exelixis

* *

TG 101348 CEP 701

Yes No

TareGen Cephalon

* * * * * * * * * * *

AT 9283

MK 0457 (VX 680)

No

No

Astex Therapeutics

*

Merck

*

*

* * *

I/II, recruiting II, recruiting II, recruiting II, recruiting II, recruiting I, active/n.r. I, active/n.r. I, recruiting II, recruiting II, recruiting II, recruiting I/II, recruiting II, completed III, recruiting I, recruiting II, completed II, terminated II, completed I, recruiting I/II, recruiting

I, suspendeda II, suspendeda I, suspendeda II, terminateda

I, terminateda a * I, terminated Pre clinical *

AG 490

No

WP 1066

?

Cyt387 G€o6976

Yes No

Sigma Calbiochem Callisto Pharmaceuticals Cytopia Calbiochem

TG 101209 Erlotinib

Yes No

TargeGen Inc. Roche

Indication

Primary target

ClinicalTrials identifier

PMF/ET/PV M. myeloma * Prostate CA * Psoriasis * Rh. arthritis * PMF/ET/PV * PV PMF * PMF * ET/PV * AML * AML ( þ CTX) * AML * ALL ( þ CTX) * Neuroblastoma * Psoriasis * M. myeloma * Prostate CA * Lymphoma * PMF/AML/ALL * CML/MDS

JAK2

NCT00509899 NCT00639002 NCT00638378 NCT00617994 NCT00550043 NCT00522574 NCT00595829 NCT00631462 NCT00494585 NCT00586651 NCT00494585 NCT00469859 NCT00030186 NCT00557193 NCT00084422 NCT00236119 NCT00242827 NCT00081601 NCT00443976 NCT00522990

* *

* * * *

* *

ALL/MDS/CML CML/ALLT315I þ CML/Ph þ ALL NSCLC (þ dasatinib) Advanced CA Advanced CA

JAK2 FLT3 JAK2

Aurora A Aurora B JAK2 Bcr/Abl Flt3 Aurora JAK2 Bcr/Abl

NCT00111683 NCT00405054 NCT00500006 NCT00290550 NCT00104351 NCT00099346

JAK2 JAK3 JAK2/STAT3

Pre clinical Pre clinical Pre clinical Pre clinical Pre clinical

JAK2

FDA approved for 2nd line NSCLC

JAK2 JAK2 Flt3 JAK2 EGFR TK

n.r. Not recruiting; CTX chemotherapy; CA cancer Terminated or suspended due to QT prolongations

a

Many questions are open concerning the side effect profile. Side effects on the JAK3 gene should be minimized because severe inhibition of this gene is associated with a severe combined immune deficiency [73, 74]. In this sense, JAK3-selective inhibitors are currently being employed in clinical trials for the prevention of acute rejection in organ transplant recipients (e.g., ClinicalTrials.gov Identifier: NCT00483756). In contrast, selectivity for the V671F mutant over the wildtype allele may not be necessary since in vitro data with colonies of CMPD patients have shown stronger inhibitory effects

on both JAK2V617F as well as MPLW515L mutated colonies, as compared to wildtype colonies [75]. Treatment with these drugs has led to a survival benefit in mice [75], as well as promising preliminary data in humans enrolled in early phase clinical trials (see Tyrosine kinase inhibitor section in PMF chapter (4.13.6.)). In addition, JAK2 inhibitors seem to be primarily effective in reducing proliferation of clonal colonies. If this proves true, JAK2 inhibitors could also be used for effective molecular targeted therapy in patients with CMPDs, regardless of whether the causative mutations occur at the cytokine

Chap. 1


receptor level or involve JAK2 itself, and quite likely also in patients without currently detectable mutations.

References [1] Moliterno AR, Hankins WD, Spivak JL (1998) Impaired expression of the thrombopoietin receptor by platelets from atients with polycythemia vera. N Engl J Med 338: 572 580 [2] Adamson JW, Fialkow PJ, Murphy S, Prchal JF, Steinmann L (1976) Polycythemia vera: stem cell and probable clonal origin of the disease. N Engl J Med 295: 913 916 [3] Fialkow PJ, Faguet GB, Jacobson RJ, Vaidya K, Murphy S (1981) Evidence that essential thrombocythemia is a clonal disorder with origin in a multipotent stem cell. Blood 58: 916 919 [4] Prchal JF, Axelrad AA (1974) Letter: bone marrow responses in polycythemia vera. N Engl J Med 290: 1382 [5] Jelinek J, Oki Y, Gharibyan V et al. (2005) JAK2 mutation 1849GHT is rare in acute leukaemias but can be found in CMML, Philadelphia chromosome negative CML, and mega karyocytic leukaemia. Blood 106: 3370 3373 [6] Ingram W, Lea NC, Cervera J et al. (2006) The JAK2 V617F mutation identifies a subgroup of MDS patients with isolated deletion 5q and a proliferative bone marrow. Leukaemia 20: 1319 1321 [7] Gattermann N, Billiet J, Kronenwett R et al. (2007) High frequency of the JAK2 V617F mutation in patients with thrombocytosis (platelet countH600109/L) and ringed side roblasts more than 15% considered as MDS/MPD, unclassifi able. Blood 109: 1334 1335 [8] Zipperer E, Wulfert M, Germing U, Haas R, Gattermann N (2008) MPL 515 and JAK2 mutation analysis in MDS pre senting with a platelet count of more than 50010(9)/l. Ann Hematol 87: 413 415 [9] Michiels JJ, Berneman Z, Van Bockstaele D, van der PM, De Raeve H, Schroyens W (2006) Clinical and laboratory features, pathobiology of platelet mediated thrombosis and bleeding complications, and the molecular etiology of essential throm bocythemia and polycythemia vera: therapeutic implications. Semin Thromb Hemost 32: 174 207 [10] Levine RL, Pardanani A, Tefferi A, Gilliland DG (2007) Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders. Nat Rev Cancer 7: 673 683 [11] Levine RL, Belisle C, Wadleigh M et al. (2006) X inactivation based clonality analysis and quantitative JAK2V617F assess ment reveal a strong association between clonality and JAK2V617F in PV but not ET/MMM, and identifies a subset of JAK2V617F negative ET and MMM patients with clonal hematopoiesis. Blood 107: 4139 4141 [12] Lasho TL, Mesa R, Gilliland DG, Tefferi A (2005) Mutation studies in CD3þ , CD19þ and CD34þ cell fractions in myeloproliferative disorders with homozygous JAK2(V617F) in granulocytes. Br J Haematol 130: 797 799 [13] Delhommeau F, Dupont S, Tonetti C et al. (2007) Evidence that the JAK2 G1849T (V617F) mutation occurs in a lymphomye loid progenitor in polycythemia vera and idiopathic myelofi brosis. Blood 109: 71 77 [14] Ishii T, Bruno E, Hoffman R, Xu M (2006) Involvement of various hematopoietic cell lineages by the JAK2V617F muta tion in polycythemia vera. Blood 108: 3128 3134 [15] Scott LM, Scott MA, Campbell PJ, Green AR (2006) Progenitors homozygous for the V617F mutation occur in

11

[16]

[17]

[18]

[19]

[20]

[21]

[22] [23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

most patients with polycythemia vera, but not essential throm bocythemia. Blood 108: 2435 2437 Antonioli E, Guglielmelli P, Pancrazzi A et al. (2005) Clinical implications of the JAK2 V617F mutation in essential throm bocythemia. Leukaemia 19: 1847 1849 Kiladjian JJ, Elkassar N, Cassinat B et al. (2006) Essential thrombocythemias without V617F JAK2 mutation are clonal hematopoietic stem cell disorders. Leukaemia 20: 1181 1183 Bellanne Chantelot C, Chaumarel I, Labopin M et al. (2006) Genetic and clinical implications of the Val617Phe JAK2 mutation in 72 families with myeloproliferative disorders. Blood 108: 346 352 Xu X, Zhang Q, Luo J et al. (2007) JAK2(V617F): preva lence in a large Chinese hospital population. Blood 109: 339 342 Scott LM, Campbell PJ, Baxter EJ et al. (2005) The V617F JAK2 mutation is uncommon in cancers and in myeloid malignancies other than the classic myeloproliferative disor ders. Blood 106: 2920 2921 Patel RK, Lea NC, Heneghan MA et al. (2006) Prevalence of the activating JAK2 tyrosine kinase mutation V617F in the Budd Chiari syndrome. Gastroenterology 130: 2031 2038 Campbell PJ, Green AR (2006) The myeloproliferative dis orders. N Engl J Med 355: 2452 2466 Kralovics R, Teo SS, Li S et al. (2006) Acquisition of the V617F mutation of JAK2 is a late genetic event in a subset of patients with myeloproliferative disorders. Blood 108: 1377 1380 Campbell PJ, Baxter EJ, Beer PA et al. (2006) Mutation of JAK2 in the myeloproliferative disorders: timing, clonality studies, cytogenetic associations, and role in leukemic trans formation. Blood 108: 3548 3555 Bumm TG, Elsea C, Corbin AS et al. (2006) Characterization of murine JAK2V617F positive myeloproliferative disease. Cancer Res 66: 11156 11165 Lacout C, Pisani DF, Tulliez M, Gachelin FM, Vainchenker W, Villeval JL (2006) JAK2V617F expression in murine hemato poietic cells leads to MPD mimicking human PV with second ary myelofibrosis. Blood 108: 1652 1660 Wernig G, Mercher T, Okabe R, Levine RL, Lee BH, Gilliland DG (2006) Expression of JAK2V617F causes a polycythemia vera like disease with associated myelofibrosis in a murine bone marrow transplant model. Blood 107: 4274 4281 Zaleskas VM, Krause DS, Lazarides K et al. (2006) Molecular pathogenesis and therapy of polycythemia induced in mice by JAK2 V617F. PLoS. ONE. 1: e18 Tiedt R, Hao Shen H, Sobas MA et al. (2008) Ratio of mutant JAK2 V617F to wild type JAK2 determines the MPD pheno types in transgenic mice. Blood 111: 3931 3940 Moliterno AR, Williams DM, Rogers O, Spivak JL (2006) Molecular mimicry in the chronic myeloproliferative disor ders: reciprocity between quantitative JAK2 V617F and Mpl expression. Blood 108: 3913 3915 Baxter EJ, Scott LM, Campbell PJ et al. (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloprolif erative disorders. Lancet 365: 1054 1061 James C, Ugo V, Le Couedic JP et al. (2005) A unique clonal JAK2 mutation leading to constitutive signalling causes poly cythaemia vera. Nature 434: 1144 1148 Levine RL, Wadleigh M, Cools J et al. (2005) Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with my elofibrosis. Cancer Cell 7: 387 397

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[34] Kralovics R, Passamonti F, Buser AS et al. (2005) A gain of function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 352: 1779 1790 [35] Pemmaraju N, Moliterno AR, Williams DM, Rogers O, Spivak JL (2007) The quantitative JAK2 V617F neutrophil allele burden does not correlate with thrombotic risk in essential thrombocytosis. Leukaemia 21: 2210 2212 [36] Holt D, Dreimanis M, Pfeiffer M, Firgaira F, Morley A, Turner D (1999) Interindividual variation in mitotic recombination. Am J Hum Genet 65: 1423 1427 [37] Passamonti F, Rumi E, Pietra D et al. (2006) Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34þ cells into peripheral blood in myeloproliferative disorders. Blood 107: 3676 3682 [38] Thiele J, Kvasnicka HM, Diehl V (2005) Bone marrow CD34þ progenitor cells in Philadelphia chromosome nega tive chronic myeloproliferative disorders a clinicopathologi cal study on 575 patients. Leuk Lymphoma 46: 709 715 [39] Xing S, Ho WT, Zhao W et al. (2008) Transgenic expression of JAK2V617F causes myeloproliferative disorders in mice. Blood 110: 5109 5117 [40] Plo I, Nakatake M, Malivert L et al. (2008) JAK2 stimulates homologous recombination and genetic instability: potential implication in the heterogeneity of myeloproliferative disor ders. Blood 112(4): 1402 1412 [41] Saharinen P, Takaluoma K, Silvennoinen O (2000) Regulation of the JAK2 tyrosine kinase by its pseudokinase domain. Mol Cell Biol 20: 3387 3395 [42] Royer Y, Staerk J, Costuleanu M, Courtoy PJ, Constantinescu SN (2005) Janus kinases affect thrombopoietin receptor cell surface localization and stability. J Biol Chem 280: 27251 27261 [43] Lu X, Levine R, Tong W et al. (2005) Expression of a homodimeric type I cytokine receptor is required for JAK2V617F mediated transformation. Proc Natl Acad Sci USA 102: 18962 18967 [44] Huang LJ, Constantinescu SN, Lodish HF (2001) The N terminal domain of Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin re ceptor. Mol Cell 8: 1327 1338 [45] Silva M, Richard C, Benito A, Sanz C, Olalla I, Fernandez Luna JL (1998) Expression of Bcl x in erythroid precursors from patients with polycythemia vera. N Engl J Med 338: 564 571 [46] Labbaye C, Valtieri M, Barberi T et al. (1995) Differential expression and functional role of GATA 2, NF E2, and GATA 1 in normal adult hematopoiesis. J Clin Invest 95: 2346 2358 [47] Goerttler PS, Kreutz C, Donauer J et al. (2005) Gene expres sion profiling in polycythaemia vera: overexpression of tran scription factor NF E2. Br J Haematol 129: 138 150 [48] Zeuner A, Pedini F, Signore M et al. (2006) Increased death receptor resistance and FLIPshort expression in polycythemia vera erythroid precursor cells. Blood 107: 3495 3502 [49] Greil R, Anether G, Johrer K, Tinhofer I (2003) Tuning the rheostat of the myelopoietic system via Fas and TRAIL. Crit Rev Immunol 23: 301 322 [50] Greil R, Anether G, Johrer K, Tinhofer I (2003) Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools. J Leukoc Biol 74: 311 330 [51] Walz C, Crowley BJ, Hudon HE et al. (2006) Activated JAK2 with the V617F point mutation promotes G1/S phase transi tion. J Biol Chem 281: 18177 18183 [52] Teofili L, Martini M, Cenci T et al. (2007) Different STAT 3 and STAT 5 phosphorylation discriminates among Ph negative


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chronic myeloproliferative diseases and is independent of the V617F JAK 2 mutation. Blood 110: 354 359 Heller PG, Lev PR, Salim JP et al. (2006) JAK2V617F mutation in platelets from essential thrombocythemia patients: correlation with clinical features and analysis of STAT5 phos phorylation status. Eur J Haematol 77: 210 216 Kirito K, Osawa M, Morita H et al. (2002) A functional role of Stat3 in in vivo megakaryopoiesis. Blood 99: 3220 3227 Teofili L, Martini M, Luongo M et al. (2002) Overexpression of the polycythemia rubra vera 1 gene in essential thrombo cythemia. J Clin Oncol 20: 4249 4254 Vannucchi AM, Guglielmelli P, Antonioli E et al. (2006) Inconsistencies in the association between the JAK2(V617F) mutation and PRV 1 over expression among the chronic mye loproliferative diseases. Br J Haematol 132: 652 654 Pardanani AD, Levine RL, Lasho T et al. (2006) MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood 108: 3472 3476 Mercher T, Wernig G, Moore SA et al. (2006) JAK2T875N is a novel activating mutation that results in myeloproliferative disease with features of megakaryoblastic leukaemia in a murine bone marrow transplantation model. Blood 108: 2770 2779 Peeters P, Raynaud SD, Cools J et al. (1997) Fusion of TEL, the ETS variant gene 6 (ETV6), to the receptor associated kinase JAK2 as a result of t(9;12) in a lymphoid and t(9;15;12) in a myeloid leukaemia. Blood 90: 2535 2540 Bousquet M, Quelen C, De MV et al. (2005) The t(8;9)(p22; p24) translocation in atypical chronic myeloid leukaemia yields a new PCM1 JAK2 fusion gene. Oncogene 24: 7248 7252 Reiter A, Walz C, Watmore A et al. (2005) The t(8;9)(p22;p24) is a recurrent abnormality in chronic and acute leukaemia that fuses PCM1 to JAK2. Cancer Res 65: 2662 2667 Bousquet M, Brousset P (2006) Myeloproliferative disorders carrying the t(8;9) (PCM1 JAK2) translocation. Hum Pathol 37: 500 502 Griesinger F, Hennig H, Hillmer F et al. (2005) A BCR JAK2 fusion gene as the result of a t(9;22)(p24;q11.2) translocation in a patient with a clinically typical chronic myeloid leukae mia. Genes Chromosomes. Cancer 44: 329 333 Heiss S, Erdel M, Gunsilius E, Nachbaur D, Tzankov A (2005) Myelodysplastic/myeloproliferative disease with erythropoi etic hyperplasia (erythroid preleukaemia) and the unique translocation (8;9)(p23;p24): first description of a case. Hum Pathol 36: 1148 1151 Murati A, Gelsi Boyer V, Adelaide J et al. (2005) PCM1 JAK2 fusion in myeloproliferative disorders and acute erythroid leukaemia with t(8;9) translocation. Leukaemia 19: 1692 1696 Pardanani A, Fridley BL, Lasho TL, Gilliland DG, Tefferi A (2008) Host genetic variation contributes to phenotypic diversity in myeloproliferative disorders. Blood 111: 2785 2789 Jost E, do ON, Dahl E et al. (2007) Epigenetic alterations complement mutation of JAK2 tyrosine kinase in patients with BCR/ABL negative myeloproliferative disorders. Leukaemia 21: 505 510 Capello D, Deambrogi C, Rossi D et al. (2008) Epigenetic inactivation of suppressors of cytokine signalling in Philadel phia negative chronic myeloproliferative disorders. Br J Hae matol 141(4): 504 511 Bock O, Hussein K, Brakensiek K et al. (2007) The suppressor of cytokine signalling 1 (SOCS 1) gene is overexpressed in Philadelphia chromosome negative chronic myeloprolifera tive disorders. Leuk Res 31: 799 803

Chap. 1


[70] Usenko T, Eskinazi D, Correa PN, Amato D, Ben David Y, Axelrad AA (2007) Overexpression of SOCS 2 and SOCS 3 genes reverses erythroid overgrowth and IGF I hypersensitivi ty of primary polycythemia vera (PV) cells. Leuk Lymphoma 48: 134 146 [71] Hookham MB, Elliott J, Suessmuth Y et al. (2007) The myeloproliferative disorder associated JAK2 V617F mutant escapes negative regulation by suppressor of cytokine signal ing 3. Blood 109: 4924 4929 [72] Pardanani A (2008) JAK2 inhibitor therapy in myeloprolifera tive disorders: rationale, preclinical studies and ongoing clini cal trials. Leukaemia 22: 23 30

13

[73] Russell SM, Tayebi N, Nakajima H et al. (1995) Mutation of JAK3 in a patient with SCID: essential role of JAK3 in lymphoid development. Science 270: 797 800 [74] Macchi P, Villa A, Giliani S et al. (1995) Mutations of JAK3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature 377: 65 68 [75] Pardanani A, Hood J, Lasho T et al. (2007) TG101209, a small molecule JAK2 selective kinase inhibitor po tently inhibits myeloproliferative disorder associated JAK2V617F and MPLW515L/K mutations. Leukaemia 21: 1658 1668

2

Essential Thrombocythemia (ET) Lisa Pleyer, Victoria Faber, Daniel Neureiter and Richard Greil

Contents 2.1 Epidemiology of ET :::::::::::::::::::::::::::::::::::::::::::::::::: 2.2 Course of Disease and Prognosis of ET :::::::::::::::::::: 2.3 Cellular and Biological Abnormalities Observed in ET ::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.3.1 Monoclonality Versus Polyclonality in ET :::::::: 2.3.2 Endogenous Megakaryocytic Colony (EMC) Formation and Endogenous Erythroid Colony (EEC) Formation:::::::::::::::::::::::::::::::::: 2.3.3 Overexpression of the PRV 1 Gene ::::::::::::::::::: 2.3.4 Decreased cMPL Expression and Elevated Serum Thrombopoietin (TPO) Levels ::::::::::::::: 2.3.5 Quantitative and Qualitative Defects in Platelets and Leukocyte Biology in ET (and PV) :::::::::::::::::::::::::::::::::::::::::::::::: 2.3.6 JAK2V617F Mutations and Role of Allelic Burden in ET ::::::::::::::::::::::::::::::::::::::::::::::::::: 2.3.7 Thrombopoietin Receptor Gene (MPL) Mutations in ET ::::::::::::::::::::::::::::::::::::::::::::::: 2.4 Cytogenetics in ET :::::::::::::::::::::::::::::::::::::::::::::::::::: 2.5 Clinical Presentation and Disease Complications of ET ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.6 Diagnosis and Differential Diagnosis of ET ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.7 Pathophysiology of Thrombosis and Microcirculatory Disturbances in ET (and PV) :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.8 Pathophysiology of Hemorrhagic Complications in ET (and PV):::::::::::::::::::::::::::::::::: 2.9 Risk Factors for Thrombotic Events in ET/PV ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.10 Risk Factors for Myeloid Disease Progression to PV, Post-ET-MF and/or Leukemic Transformation ::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.11 Indication for Treatment and Choice of Drugs in Patients with ET ::::::::::::::::::::::::::::::::::::::::::::::::::: 2.11.1 Acetylic Salicylic Acid (ASA, aspirin) ::::::::::: 2.11.2 Platelet Reducing Agents Current State of the Art ::::::::::::::::::::::::::::::::::::::::::::: 2.11.2.1 Hydroxyurea :::::::::::::::::::::::::::::::::: 2.11.2.2 Anagrelide ::::::::::::::::::::::::::::::::::::: 2.11.2.3 Interferon a (IFN a)::::::::::::::::::::::: 2.11.2.4 Pipobroman :::::::::::::::::::::::::::::::::::: 2.11.2.5 Busulphan :::::::::::::::::::::::::::::::::::::: 2.11.2.6 Radiophosphorus 32P :::::::::::::::::::::: 2.11.3 Treatment of Bleeding Events and Indications for Platelet Apheresis :::::::::::: 2.11.4 Life Style Modifications and Control of Other Risk Factors ::::::::::::::::::::::::::::::::::::

2.11.5

16 16 16 16

17 17 17

17 18 19 20 20 21

25 26 27

28 29 31 32 32 34 35 35 36 36 37 37

Effect of Therapeutic Strategies on Re thrombosis :::::::::::::::::::::::::::::::::::::::::: 2.11.6 Should the Site of Occurrence of the Thrombotic Event Have an Influence on Preventive Treatment Strategy? ::::::::::::::::: 2.11.7 Should JAK2V617F Positivity Influence the Choice of Cytoreductive Therapy? ::::::::::: 2.11.8 Antithrombotic Prophylaxis for Elective Surgery in ET (and PV)::::::::::::::::::::::::::::::::: 2.12 ET in Pregnancy ::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.12.1 Course of Pregnancies in Women with ET:::::: 2.12.2 Prediction of Pregnancy Outcome :::::::::::::::::: 2.12.3 Management and Treatment of Pregnant Women with ET :::::::::::::::::::::::::::::::::::::::::::: 2.12.3.1 General Considerations ::::::::::::::::::: 2.12.3.2 Antiaggregatory Platelet Therapy During Pregnancy::::::::::::::::::::::::::: 2.12.3.3 Cytoreductive Therapy During Pregnancy :::::::::::::::::::::::::::::::::::::: 2.12.3.4 Relevance of Periodic Platelet Apheresis in Pregnancy::::::::::::::::::: 2.12.3.5 Recommendations for Treatment of Pregnant Women with ET :::::::::: 2.13 Childhood ET:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.14 Familial, Hereditary Thrombocytosis ::::::::::::::::::::::: 2.15 Rare ET Varients :::::::::::::::::::::::::::::::::::::::::::::::::::::: 2.15.1 Philadelphia Chromosome (Ph) Positive ET:::::::::::::::::::::::::::::::::::::::::::: 2.15.2 Bcr Abl Positive Ph Negative ET ::::::::::::::::::

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38 38 38 39 39 39 39 39 40 40 40 41 41 42 42 42 43

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Essential Thrombocythemia (ET) is a chronic myeloid disorder with megakaryocytic proliferation in the bone marrow resulting in a persistent increase in platelets in the peripheral blood, with ensuing thrombohemorrhagic symptoms. Furthermore, mild leukocytosis, lack of hepatosplenomegaly and excellent prognosis with only rare transformation to acute leukemia are typical characteristics of this disease.

2.1 Epidemiology of ET The annual incidence rate of ET in Western Europe and the United states is 1.5 per 100,000 inhabitants when adjusted to a standard population, with the incidence being approximately twofold higher in females [1]. The prevalence is 30 per 100,000, reflecting the excellent long-term prognosis of the disease when adequately managed. The median age at diagnosis is 55 70 years. Median survival as from diagnosis is approximately 19 22 years [2]. Thus, the mortality of patients with ET is not significantly higher than that of the general population, reflecting an indolent nature of the disease. However, while confirming the excellent prognosis of ET patients in the first decade of the disease, which was in the range of normal controls [3], others have documented a deterioration of prognosis and decline in survival thereafter [2].

2.2 Course of Disease and Prognosis of ET Life expectancy is mainly affected by disease-related complications. Although seemingly paradox, both arterial and/or venous thromboses as well as hemorrhagic complications occur in ET. Thromboemoblic events, rank second in the causes of mortality after leukemic or myeloid transformation [4, 5]. Arterial complications account for 60 70% of the events. Reported rates for thromboses range between 11% and 25% in retrospective analyses [3], and the average risk for thrombotic episodes per patient year is approximately 6.6% [6]. The rate for cardiovascular events ranges from 1.9% to 3% per patient year in prospective trials [7]. In a retrospective analysis of 494 patients (PV/ET 235/259) with previous arterial (67.6%) or venous thrombosis (31%) or both (1.4%), the first thrombotic event was cerebrovascular disease in 191 cases, followed by venous thromboembolism (160/ 494), acute coronary syndrome (106/494) and peripheral arterial thrombosis (44/494) [8]. Recurrence after the index thrombotic event during a median observation time of 5.3 years showed recurrent arterial or venous thrombosis in approximately 61% and 40% of patients, respectively, whereas major bleeding complications were documented in 5.4% of patients [8].

L. Pleyer et al.

Risk factors for overall survival include age 60 years, hemoglobin levels less than normal, and a leukocyte count 15109/l [9]. Overall survival differs significantly for these three risk groups and amounts to 278, 200 and 111 months, respectively, for the low-, intermediate- and high-risk group, respectively (see 2.9. and 2.10.).

2.3 Cellular and Biological Abnormalities Observed in ET 2.3.1 Monoclonality Versus Polyclonality in ET Essential thrombocythemia is a more heterogenous disease than the other classical myeloproliferative disorders, both in terms of its molecular pathobiology and its clinical presentation. Monoclonality may not necessarily be a universal indispensable trait in ET, as X-chromosome inactivation pattern (XCIP) analysis indicates polyclonal myelopoiesis in a portion of patients (e.g., [10]), which sets ET apart from other CMPDs. The analysis of XCIPs indicates a clonal origin in roughly two-thirds of cases [10 13]. Cases with polyclonal myelopoiesis also exist and present with an overlapping range of clinical features [10]. These findings may reflect pitfalls in the interpretation of clonality tests [14] as well as difficulties in clinical diagnosis of a disease spectrum which is somewhat heterogeneous, and also has overlapping features with secondary thrombocytosis. Furthermore, it must also be taken into account that these analyses were performed in the pre-JAK2-era. In addition to the obvious limitation of XCIP-analysis to female patients, there are concerns pertaining to age-dependent unbalanced X-chromosome skewing. Since ET patients with clonal disease have a considerably higher risk for vascular complications [10, 12, 15], assessment of clonality used to be of importance in the pre-JAK2-era. Importantly, monoclonal myelopoiesis was significantly correlated with the development of thrombosis, as 32% of patients with monoclonal hemopoiesis presented with thrombosis, compared to 6% of polyclonal subjects [16]. Unfortunately, and contrary to former beliefs, impaired expression of cMPL in bone marrow megakaryocytes, overexpression of PRV-1, as well as the ability to form endogenous megakaryocytic colonies (EMCs) and endogenous erythroid colonies (EECs), while being hallmarks of ET, are not clustered according to mono- or polyclonality of myelopoiesis in ET. Therefore, they cannot be used as a substitute for XCIP-analysis in order to determine monoclonality, although this is rarely necessary in clinical practice (discussed below and in [16]).

Chap. 2

Essential Thrombocythemia

2.3.2 Endogenous Megakaryocytic Colony (EMC) Formation and Endogenous Erythroid Colony (EEC) Formation The formation of EMCs and EECs is a hallmark of classic CMPDs, and the diagnostic value in the pre-JAK2 era has been demonstrated. Growth-factor-independent megakaryopoiesis has been associated with the JAK2V617F mutation [17]. The genes PRV1 and NF-E2, which are involved in EEC formation, are clearly regulated by JAK2 [18, 19]. EMC and EEC formation have been found in 78% and 33% of patients with ET, respectively, when performed with bone marrow progenitors [20]. In ET, assessment of EEC and EMC formation capacity may be helpful in vascular risk evaluation, and especially so for JAK2 mutation negative patients [21]. Furthermore, assessment of EEC in the bone marrow or peripheral blood may be helpful in discriminating early or masked PV, or in the prediction of polycythemic evolution in patients with ET ([22] and respective section in the PV chapter (3.3.2.)). Interpretation of conflicting results correlating clinical features with the capacity for EMC/EEC formation must however be viewed with caution, as currently various methods to measure these phenomena exist [23].

2.3.3 Overexpression of the PRV-1 Gene Approximately 50% of all patients with ET show elevated PRV-1 (polycythemia rubra vera-1) expression, and this seems to be correlated with, and restricted to, EEC formation [24]. Interestingly, an inverse correlation between PRV1 levels and methylation of this gene have been found [25]. Additionally, an inverse correlation with the presence of the JAK2 mutated allele burden seems to exist, at least in PV, while results in ET or myelofibrosis are rather inconsistent [18, 25 27]. Importantly, PRV-1 mRNA overexpression seems to discriminate two types of essential thrombocythemia. PRV-1 positivity is correlated with a pathophysiologically distinct ET subtype that tends towards phenotypic disease progression to PVand is associated with a significantly higher number of microcirculatory or thromboembolic events [24, 28]. In fact, as many as 40% of EECpositive and PRV-1-positive ET patients develop PV during long-term follow-up, whereas none of the PRV-1 negative patients showed such disease progression [24]. However, conflicting data concerning the use of PRV-1 as a surrogate marker of thrombotic risk, exists [16]. It has been suggested, that a reduction in PRV-1 expression may be used for monitoring treatment efficacy in patients with ET [29], although this hypothesis is strongly challenged by others who argue, that the observed changes result from altered gene expression or neutrophil release and do not reflect an effect on disease activity [30].

17

The JAK2 mutation was very highly correlated with the ability to form EECs as well as with PRV-1 overexpression in patients with ET, PV or primary or secondary myelofibrosis. Thus, these genetic and biologic features seem to define a distinct subgroup of CMPD patients [27].

2.3.4 Decreased cMPL-Expression and Elevated Serum Thrombopoietin (TPO) Levels Reduced platelet content of the TPO-receptor MPL is frequently observed in ET. Structural or numerical arrangements of PRV-1, TPO or cMPL genes however, have not been found in patients with ET [31]. Serum TPOlevels in ET are unexpectedly normal or elevated, which may be the result of increased bone marrow stromal production of TPO or decreased ligand clearance associated with reduced platelet cMPL expression (e.g., [32, 33]). While mutations within the TPO or PRV1 genes have not yet been reported [31], mutations within the MPL gene which encodes the TPO receptor are rare and observed in about 1% of cases (see e.g., [34, 35] and below). A heterogenous expression pattern of cytoplasmic MPL distribution in the bone marrow with presence of a significant percentage of weakly stained or cMPL negative megakaryocytes, has been correlated with a sixfold increased risk of thrombosis, compared to that of patients with a uniform cMPL pattern [36]. Decreased cMPL expression was however not correlated with an enhanced rate of thromboembolic complications in a retrospective analysis [28].

2.3.5 Quantitative and Qualitative Defects in Platelets and Leukocyte Biology in ET (and PV) Quantitative and qualitative defects in platelets and leukocyte biology in ET (and PV) will be further alluded to in the section on pathophysiology of thrombosis (2.7.) and bleeding symptoms (2.8.). In this section it should merely be foreclosed, that ET patients with vasomotor symptoms and microvasculature disturbances display shortened platelet survival, increased plasma-levels of platelet activation markers b-thromboglobulin- (b-TG) and platelet factor 4- (PF4), as well as endothelial cell damage marker thrombomodulin (TM) and a 3- to 30-fold increased urinary thromboxane B2 (TXB2) excretion, all of which indicate platelet-mediated thrombotic processes as the cause for the observed symptoms. The high shear rate of blood flow in arterioles contributes to the localization of intravascular platelet aggregation and activation, with consecutive release of platelet derived growth factor (PDGF), which accounts for the fibromuscular intimal proliferation of erythromelalgia (Fig. 2.1a b).

18

a

L. Pleyer et al.

c

b

Fig. 2.1a Scheme of normal cellular content of a blood vessel. This is a scheme only, and obviously the number of cellular components is dramatically reduced for demonstrative reasons. b Scheme of pathophysiology of erythromelalgia in ET. PLT Platelet; PMN polymorphonuclear granulocyte; PDGF platelet derived growth factor; TXB2 thromboxane B2; TM thrombomodu

lin; PF4 platelet factor 4; B TG basic thromboglobulin; vW von Willebrandt. c Erythromelalgia. The photograph shows the pres ence of erythromelalgia of the hands in a patient with essential thrombocythemia. This condition is associated with burning pain in the feet or hands accompanied by erythema, pallor, or cyanosis, in the presence of palpable pulses

2.3.6 JAK2V617F Mutations and Role of Allelic Burden in ET

negative for the mutation thus pointing to the origin from a common ancestral pre-JAK clone [40, 41]. This is not only of importance for understanding the orchestration of the neoplastic process per se, but apparently also has therapeutic impact for the application of JAK-specific kinase-inhibitors in the various phases of the disease (see respective sections in Introduction to Classic CMPDs (Chapter 1) and Primary Myelofibrosis (Chapter 4.13.6.1), as well as, e.g., [42]). In rare instances, i.e., 1% of patients with ET with JAKV617F negativity, mutations within the MPLW515 gene locus, which codes

The exploration of JAK2V617F mutations has altered the scenery significantly. JAK2 mutations affect the hematopoietic stem cells in classic CMPD patients [37]. In line with this, JAK2V617F mutations are not only detected in cells of the myeloid lineage, but also occur in nonmyeloid cells such as B and T cells (e.g., [38]) and natural killer cells (NKCs) [39]. Of note, blasts from JAK2V617F mutated patients in leukemic transformation are usually

Chap. 2


for the thrombopoietin receptor, may occur [41, 43]. Although MPLW515 mutations may coexist with JAK2V617F mutations in PMF, no such data are yet available concerning ET (reviewed e.g., in [34, 35]) (for more details see 2.3.7.). The JAK2V617F mutation occurs in 50 60% of adult patients with ET [44] but with a lower frequency in childhood ET (i.e., 20% and 38% [45, 46]). Mostly, the mutation is heterozygous and homozygosity (resulting from mitotic recombination) is observed in only 2 4% of patients with ET [47 51]. As already mentioned in the introduction to classic CMPDs section, homozygous cases are more likely to experience disease progression to post-ET-MF. The level of JAK2V617F allele burden is in the low range with only 25% of patients with ET showing more than 25% mutant alleles. This is in good correlation with the data obtained in animal models (see Section 1.1.4 in introduction to classic CMPDs chapter). The presence of the JAK2V617F mutation and the allelic burden significantly impact on the biology and clinical presentation of the disease. Patients bearing the mutation typically present with significantly higher white blood cell counts, hemoglobin concentrations and serum alkaline phosphatase levels as well as an elevated frequency of EEC, but lower median platelet counts, when compared to patients with the wild type gene [52]. Clinically, increased allelic JAK2V617F burden has been reported to correlate with increased age, palpable splenomegaly, arterial or venous thrombosis at diagnosis and symptoms from microvessel disease [52, 53]. All but age retained significance in multivariate analysis. In addition, a higher gene dosage of mutated JAK2 was observed, when comparing JAK2V617F mRNA/cDNA of platelets with granulocytes [17, 54, 55]. This higher allelic JAK2V617F burden in platelets versus granulocytes seems restricted to ET, as it was not observed in other myeloproliferative disorders. The impact of the JAK2V617F mutation is further underlined by the observation of increased levels of PRV1, which is directly regulated by JAK2, and erythropoietinindependent endogenous erythroid colonies (EEC) levels in homozygous cases [52]. The rare JAK2V617F homozygous cases of ETwere characterized by a nearly 4-fold and 1.5-fold higher incidence of cardiovascular events than wild type cases or heterozygous cases, respectively [56]. At present, the development and stability of the JAK2 mutation status over time is unclear. Only few cases with a time-dependent increase in mutational load have been reported [57, 58]. No increase in the JAK2 mutant clone burden was observed in longitudinal analyses after 47 months [59] and none of the JAK2V617F wild type ET patients became positive after 77 months [40]. Apparently, there is a marked longitudinal stability of the JAK2 mutational status and allelic burden.

19

Surprisingly, and contrary to what one would intuitively expect, JAK2V617F did not impact either survival or leukemic transformation rate in a retrospective survey of 605 ET patients [9].

2.3.7 Thrombopoietin Receptor Gene (MPL) Mutations in ET The thrombopoietin receptor MPL is one of several JAK2 cognate receptors and is essential for myelopoiesis in general and for megakaryopoiesis in particular. Mutations in TPO (e.g., MPLS505N) are associated with familial thrombocytosis [60 62]. Exon 10, codon 515 mutations (MPL515L/K, MPLK39N) in the transmembrane component of MPL are observed in up to 10% of JAK2V617F negative PMF and in a few patients (1%) with ET, post-ET-MF and myelofibrosis in blast crisis [63 65]. MPLS204P lesions have also been observed [66]. Interestingly, MPL mutations are not observed in patients with PV [64], which is in accordance with findings in murine models with overexpression of the mutant MPL gene (see below and [63]). Transfection of the mutated gene induces autonomous hematopoietic progenitor cell growth and activates signaling cascades along the JAK2-STAT, MAPK and PI3K pathways, as does the JAK2V617F mutation. In mouse systems, transplantation of MPLW515L mutated stem cells causes a lethal myeloproliferative disorder characterized by all clinical features of PMF, including thrombocytosis, marked splenomegaly due to extramedullary hematopoiesis, and myelofibrosis. However, these mice lack the erythrocytosis typical of JAK2 mutations [63]. Thus, in contrast to JAK2-exon-12 and -14 mutations which are usually associated with erythrocytosis, MPL-mutations segregate primarily with the phenotypes of thrombocytosis, extramedullary disease and myelofibrosis. Activation of JAK-STAT signaling via MPL mutations thus plays an important pathogenetic role in a subgroup of patients with CMPDs. MPL codon 515 mutations are not only detected in granulocytes and monocytes, but also in B- and T-lymphocytes, as well as in NKCs, albeit at lower levels [67, 68]. This implicates that these acquired mutations occur in a lympho-myeloid progenitor cell, but are predominant in cells belonging to the myeloid lineage. Although MPL mutations are mostly detected in JAK2V617F negative CMPD patients, they can occur concurrently with the JAK2V617F mutation [64], suggesting that these mutations may functionally complement each other. Homologous recombination causes homozygosity for the MPL mutation in 13% of patients [49], and this is often associated with additional, unfavorable cytogenetic alterations [69]. Patients with MPLW515L/K mutations

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differ from the respective wild type patients by older age, more severe anemia, and a higher need for transfusions [35]. The expression level of these mutated genes seems to remain constant during the course of the disease [70].

2.4 Cytogenetics in ET To date no specific cytogenetic marker has been identified in ET. Chromosomal abnormalities in ET are rare, with merely 5 15% of patients showing an abnormal karyotype at diagnosis, depending on the detection method used. Trisomies of chromosome 8 and 9 as well as deletions in 13q14 and 20q12 are the most commonly observed abnormalities in untreated patients [71] (see also Table 1.2 in Introduction to Classic CMPDs (Chapter 1 and Summary Box 1). A recent report documents the rare occurrence of del(5)(q13q33), monosomy 14 and 17 as well as trisomy 13 at the time of diagnosis [72]. Cytogenetics should always be performed in the initial evaluation of patients in whom ET is suspected [73]. If no further morphologic or clinical signs of MDS are present, these chromosomal changes should not lead to the diagnosis of MDS [74]. It has been suggested by several groups, that trisomy 8, deletion of 17p as well as der (1;7)(q10;p10), the incidence of which seem to be elevated after chemotherapeutic treatment, are associated with disease progression and leukemic transformation [75, 76]. Of note, the predictive value of sequential cytogenetic analysis of the bone marrow should be stressed, as de novo appearance of cytogenetic changes are often exhibited prior to, and highly associated with, disease transformation [76]. Obviously, repeated bone marrow examinations will only be performed when relevant changes indicative

Summary Box 1 Cytogenetic findings in ET *

* * * *

*

JAK2V617F mutations: Adults 40 50% Children 20 40% JAK2V617F homozygosity 2 4% MPL mutations 1 2% Aberrant cytogenetics 5 15% Del 17 and 1,7 (q10;p10) in patients at risk for transformation JAK2-mutated cases exclude reactive causes. Further evaluation of other MPD is however essential, particularly CML.

of disease progression in the differential blood count occur, such as a sudden drop or drastic increase in neutrophil-, red blood cell- or platelet count, or the appearance of blast cells.

2.5 Clinical Presentation and Disease Complications of ET In approximately half of the patients the diagnosis of ET is established in previously asymptomatic patients in whom the thrombocytosis was observed by chance, e.g., during preparation for a surgical intervention. The fraction of initially symptomatic patients may further depend on the institution in which the patients are seen. In symptomatic patients, the type of symptoms may vary over a wide range and over a broad spectrum of severity (for details see Table 2.1). In contrast to several other myeloproliferative disorders, constitutional or hypermetabolic symptoms such as fever, nocturnal sweating and excessive weight loss, are highly uncommon in ET. Physical findings are usually limited to mild splenomegaly. In symptomatic patients, vasomotor symptoms such as erythromelalgia, transient visual disturbances, cerebrovascular ischemia may be predominant. Erythromelalgia [77], although rare, is pathognomonious for ET (and PV) and is experienced as an intense burning or throbbing pain in the hands and/or feet, often accompanied by warmth and mottled erythema which may resemble livedo reticularis (see Fig. 2.1c). Pulses typically remain palpable, as usually only the smallest vessels are affected. The pain tends to be exacerbated by heat and exercise, and relieved by cold exposure. This phenomenon is caused by platelet thrombi, arterial endothelial swelling and fibromuscular proliferation [78] (see Fig. 2.1b). Initially, symptoms are intermittent due to spontaneous dispersion of the thrombi. If not adequately treated however, chronic changes can lead to permanent arterial occlusions sometimes resulting in progression to gangrene and necrosis of the digits. A myriad of mainly non-specific cerebrovascular ischemic symptoms may occur (see Table 2.1). Both erythromelalgia as well as cerebrovascular ischemia respond well to acetylic salicylic acid (ASA, aspirin) [79]. A thrombotic event or cerebral stroke may be the presenting symptom leading to a routine blood analysis and, hopefully to a referral to a hematologist, once the elevated platelet count has been perceived. In young females, recurrent miscarriages or fetal growth retardation [80] may lead to the first physician contact resulting in laboratory evaluation and awareness of elevated platelet counts. Multiple placental infarctions by platelet thrombi resulting in placental insufficiency are thought to be the cause.

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Table 2.1: Incidence of typical clinical features at diagnosis Incidence of presenting symptoms in patients with ET Asymptomatic (45%) Palpable mild splenomegaly (35%) Vasomotor symptoms (13%) * Pulsatile headache * Lightheadedness * Vertigo * Syncope * Seizures * Organic mental syndrome * Atypical chest pain * Livedo reticularis * Acral paresthesia or numbness * Ischemic attacks of digital arteries * Erythromelalgia * Disabling intense burning throbbing pain of hands and feet * Associated with erythema, warmth, congested extremities and peeling of skin * Possible progression to ischemic acrozyanosis, ulcer or gangrene * Typically palpable arterial pulsations * In rare cases these symptoms can also involve internal organs * Transient visual disturbances * Amaurosis fugax * Scintillating scotoma * Occular migraine * Diplopia * Hemianopsia * Blurred vision History of, or presentation with, thrombotic events (21%) * Superficial thrombophlebitis * Deep vein thrombosis * Splanchnic, hepatic or portal vein thrombosis * Pulmonary embolism * Typical and atypical TIA (with transient mono or hemiparesis, transient postural unsteadiness or unstable gait, dysarthria) * Ischemic stroke * Retinal artery occlusions * Femoral artery occlusion * Coronary artery ischemia and myocardial infarction * Abrupt, complete occlusions of digital arteries with progression of red, congested, warm fingers to cold, livid blue ones * Back and upper abdominal pain due to adrenal microvascular thrombosis * Priapism History of, or presentation with, hemorrhagic events (9%) * Gum bleeding * Epistaxis * Gastrointestinal bleeding (melaena, hematemesis, chronic occult blood loss) * Skin bleedings (bruises, subcutaneous hematomas, ecchymosis/ suggillation) * Secondary bleeding after trauma or surgery * Hemarthrosis (very rare) History of recurrent abortions or fetal growth retardation (due to placental infarctions) (11%)

21

Hepatic or portal vein thrombosis resulting in Budd Chiari syndrome are rare complications of ET. However when they do occur, they are often (31 60%) associated with JAK2 positivity [81 84]. In this setting, JAK2V617F represents a sensitive marker for latent, occult or forme fruste of underlying ET or PV [85 88]. This finding has recently been extended to non-splanchnic venous thrombo-embolisms, in whom JAK2V617F with a history of recurrent, unprovoked thrombosis in the absence of overt CMPD was found in 2% [88]. A very recent publication reports a prevalence of JAK2V617F or JAK2 exon 12 mutations in more than 45% of patients with splanchnic vein thrombosis [86]. In many incidences, marked thrombocytosis has existed, and sadly even been documented by physicians, often years prior to the occurrence of the first thrombotic event. Thus, increased physician awareness of the clinical relevance and potential complications of elevated platelet counts, especially when accompanied by neutrophilia with a slight to moderate left shift, is essential. Bleeding manifestations in ET (and any other CMPD or state in which extreme thrombocytosis H1,500,000/ml exists) most often occur in superficial locations either spontaneously or after minimal trauma. Primarily the skin and mucocutaneous membranes are involved. Hemorrhagic complications do not routinely occur (11%) [89], but when they do, ecchymosis, epistaxis, gingival bleeding and menorrhagia are predominant [90], whereas gastrointestinal hemorrhages are seldom, but may be severe [91]. Intraarticular, retroperitoneal or deep muscular hematomas have been reported on rare occasions [92 94]. Typically, vascular ischemic symptoms will precede bleeding symptoms for many years. Thrombotic tendency persists as long as platelet counts are H400,000/ml. At platelet counts H1,000,000/ml thrombosis and bleeding frequently occur in sequence. Severe bleeding events are often associated with starkly elevated platelet counts (usually H1,500,000/ml) which may result in an acquired type II-like von Willebrand syndrome with characteristic absence of high and intermediate von Willebrand factor (vWF) multimers (for details, see 2.8.) and/or functional platelet defects such as those mentioned above (2.3.5.).

2.6 Diagnosis and Differential Diagnosis of ET An algorithm for the diagnostic work-up of suspected ET is presented in Fig. 2.2. Currently, ET is not sufficiently cytogenetically or morphologically defined and is the most difficult to define entity among the CMPDs. Until very recently ET was diagnosed by excluding causes of

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Fig. 2.2 Algorithm for diagnostic work-up for patients with suspected ET. NSCLC Non small cell lung cancer; SCLC small cell lung cancer; CIBD chronic inflammatory bowel disease; GIT gas

trointestinal; EPO erythropoietin; ALP alkaline leukocyte phospha tase; *e.g.: CRP, ESR (erythrocyte sedimentation rate), fibrinogen, ferritin, procalcitonin; **according to WHO/ECMP criteria

reactive thrombocytosis (detailed in Table 2.2) and by excluding the presence of other CMPDs. In fact, less than 10% of cases with isolated thrombocytosis reflect a hematologic disorder compatible with the diagnosis of ET [34]. It is important to keep in mind, that the extent of thrombocytosis cannot be used as a criterion for discerning a primary from a reactive process, since PLT H1,000,000/ml are by no means unusual among patients with solid neoplasia, in particular lung cancer, or with inflammatory bowel disease [34]. The prevalence of thrombocytosis in patients with malignant disease in general is very high, and reaches 53% for patients with primary lung cancer, especially in late stage disease [95]. Furthermore, TPO levels are generally increased in secondary thrombocytosis, as a result of increased levels of acute phase reactants which induce the expression of TPO in liver cells [96]. IL-6 is thought to play a predominant role in the latter mechanism [97]. In contrast to ET, thrombosis prophylaxis is probably not

required in secondary thrombocytosis, except for cases with additional prothrombotic risk factors [98]. ET diagnostic criteria of the PVSG (Polycythemia Vera Study Group) are almost three decades old and somewhat arbitrary, ill-defined, incomplete and sometimes confusing. Bone marrow histomorphology with immunostaining, as one of the most powerful tools in distinguishing between thrombocytemic states and CMPD subtypes, was not included. Consequently prodromal stages of PV (latent-initial PV) without prodigious erythrocytosis, sustained reactive thrombocytosis (RTh) as well as prefibrotic stages of PMF were not excluded. Therefore only 22% of the patients diagnosed as ET by the PVSG-criteria can retrospectively be recognized as true ET [99]. According to the recent WHO (World Health Organization) and ECMP (European clinical, molecular, and pathological) criteria, bone marrow histology assessment should remain the gold standard criterion

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23

Table 2.2: Differential diagnosis of ET

for the diagnosis and staging of true ET, as well as the differentiation from secondary thrombocytosis [100]. Until very recently, the WHO/ECMP criteria for the diagnosis of ET were in use (see Table 2.3). The 2008 WHO proposal for the diagnosis of ET [73, 101] differs only slightly and requires the presence of four major criteria (Table 2.4):

Short-term reactive (spurious) thrombocytosis following * Physical exertion * Treatment of vitamin B12 deficiency * Allergic reactions * Tissue damage (surgical or otherwise) * Myocardial infarction * Acute pancreatitis * Acute bleeding or hemolysis * Rebound effect after treatment of ITP, myelosuppressive treatment or after ethanol induced thrombocytopenia * Reaction to vincristine, epinephrine, IL 1b or ATRA * Mixed cryoglobulinemia (temperature dependent apparent increase in leucocyte and PLT counts, attributable to cryo blobulin precipitate particles which are counted as WBCs or PLTs in automated cell counters) * Pseudothrombocytosis due to EDTA artifacts * RBC fragmentation due to hemolysis or burns (small cytoplasmic fragments are counted as platelets) Long-term persistent reactive thrombocytosis due to * Iron deficiency * Surgical or functional asplenia * Chronic infections, tuberculosis (marked granulocytic hyperplasia with left shift, neutrophilic vacuolization, toxic granulation, CRP", ESR", acute phase proteins"; megakaryopoiesis shows no gross anomalies and BM smears reveal small to medium sized cells with regularly lobulated nuclei) * Rheumatologic disorders, vasculitides * Systemic amyloidosis, inflammatory bowel disease, celiac disease, POEMS syndrome (target cells and Howell Jelly bodies, nuclear remnants that are normally removed by the spleen, in the PB smear) * Metastatic cancer, lymphoma * Chronic renal disease (renal failure, nephritic syndrome) Early primary myelofibrosis with accompanying thrombocytemia Latent (initial) PV and fruste PV CML (may also present with either isolated thrombocytosis or substantial bone marrow fibrosis) MDS with 5q syndrome associated with thrombocytosis

(i) The presence of thrombocytosis H400,000/ml (ii) Certain histomorphologic features of the various myeloid lineages (see Table 2.6 and Fig. 2.5) (iii) The absence of WHO criteria for other myeloproliferative disorders like chronic myeloid leukemia (CML), PV, PMF, or MDS (iv) Demonstration of JAK2V617F or another clonal marker, or in the absence thereof, lack of evidence of reactive thrombocytosis. Analysis of these major criteria is done by careful evaluation of peripheral blood smears (see Fig. 2.3 and Table 2.5), bone marrow cytology (see Fig. 2.4 and Table 2.5) and histologic evaluation of the marrow (see Fig. 2.5 and Table 2.6) are essential. Cytogenetics or FISH (fluorescence in situ hybridization) for Bcr/Abl translocation are not only helpful, but also mandatory for the differential diagnosis against CML with thrombocytosis, particularly in case dwarf megakaryocyte are observed. It has been proposed, that occult PV should be excluded in the iron-deficient patient by a trial with oral iron [100]. However, this should not be necessary in the overwhelming majority of patients as diagnosis can almost always be made with sufficient surety by other means (e.g., JAK2, typical features of bone marrow cytology and histology, as well as numerous other factors mentioned above). Furthermore, oral iron can lead to excessive erythroid proliferation in patients with PV even at low iron dosages, and should therefore be used with extreme restrictions and under strictly controlled conditions.

Table 2.3: WHO bone marrow features and European clinical, molecular and pathological (ECMP) criteria for the diagnosis of ET [100, 101] Clinical and molecular criteria

Pathological criteria (WHO)

C1

Persistent increase in PLT counts; ECP: H400,000/ml, WHO: H600,000/ml

P1

C2 C3 C4 C5

Presence of large or giant PLT in PB smear Normal values of hb, hct, ery, WBC differential Presence of JAK2V617F or MPL515 mutations Absence of Ph chromosome or any other cytogenetic fusion gene abnormality

P2

Increase of dispersed or loosely clustered, predominantly enlarged MK with mature cytoplasm and hyperlobulated nuclei No proliferation or immaturity of granulopoiesis; normal normoblastic erythropoiesis; no or borderline increase of reticulin (myelofibrosis grade 0)

According to WHO/ECMP criteria C1 þ P1 and P2 establish the diagnosis of true ET. A typical ET bone marrow picture (see Fig. 2.4a, b) excludes PV, PMF, CML, MDS, RARS Tand reactive thrombocytosis. PLT Platelets, hb hemoglobin; hct hematocrit; ery erythrocytes; WBC white blood cell count; Ph chromosome Philadelphia chromosome; MK megakaryocytes; PB peripheral blood

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Table 2.4: 2008 World Health Organization diagnostic criteria for ET (according to [101])

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Table 2.6: Typical histological findings in ET Typical histological findings in ET

2008 WHO major diagnostic criteria for ET (diagnosis requires meeting all 4 major criteria) (1) Platelet count H400,000 (450,000)/ml (2) MK proliferation with large and mature morphology; no or little granulocyte or erythroid proliferation (3) Not meeting WHO criteria for PVa, PMFb, CMLc, MDSd or other myeloid neoplasm (4) Demonstration of JAK2V617F or another clonal marker, or no evidence of reactive thrombocytosis

*

* *

*

a

Requires the failure of iron replacement therapy to increase hemoglobin level to the PV range in the presence of decreased serum ferritin. Exclusion of PV is based on hemoglobin and hematocrit levels; red cell mass measurement is not required (see also PV chapter) b Requires the absence of relevant reticulin fibrosis, collagen fibrosis, peripheral blood leukerythroblastosis, or marked hyper cellular marrow accompanied by megakaryocyte morphology that is typical for PMF (small to large MKS with an aberrant nuclear/ cytoplasmic ratio and hyperchromatic, bulbuous or irregularly folded nuclei and dense clustering) (see chapter on PMF) c Requires absence of Bcr Abl (see respective chapter on CML) d Requires absence of dyserythropoiesis and dysgranulopoiesis (see respective chapter on MDS) Table 2.5: Typical cytological features observed in ET Peripheral blood cytologic findings * Mild leukocytosis, generally G30,000/ml * Mild eosinophilia/basophilia * Normal RBC count * Thrombocytosis with giant platelets and bizarre forms and/or clumps of large, abnormal platelets * Circulating MKs and MK fragments * Increased mean platelet volume Bone marrow cytologic findings * Mild to moderate hypercellularity * Striking MK hyperplasia with clustering * Enlarged and hyperlobulated MKs * Erythroid and myeloid lines not remarkable

*

* *

Predominant growth of randomly dispersed or loosely clustered large to giant MK MKs with hyperlobulated, stag horn like nuclei No significant change in the distribution of neutrophil granulo or erythropoiesis * DD: in contrast to early stage PMF, where a pronounced proliferation and left shifting of neutrophil granulopoiesis is typically observed Regular ratio between nuclear size and lobulation as well as cytoplasmatic maturation * DD: in contrast to early stage PMF, which shows gross defects of MK maturation Reduced megakaryocytic MPL immunohistochemical staining in the bone marrow (BM) Increased BM angiogenesis Mild reticulin fibrosis can be observed in a minority of cases * encompasses less than 1/3 of the biopsy * patients without splenomegaly and leukerythroblastic reaction (DD: in contrast to PMF) * DD: in contrast to the reticulin fibers observed in ET, mature collagen fibres are found in the bone marrow of myelofibrosis patients

Table 2.7: Typical laboratory findings in ET Laboratory findings in ET *

*

* * * *

Visible buffy coat (the thickness of which can be used to estimate PLT count, with each mm being equivalent to 1 million PLT/ml) Pseudohyperkalemia: potassium release from aggregated platelets in patients with marked thrombocytosis, e.g., due to inadequate shaking during transport Endogenous megacaryocytic colony (EMC) growth EEC (endogenous erythroid colony) formation Reduced expression of MPL on platelets and MKs Overexpression of PRV 1 (polycythemia rubra vera) gene in peripheral blood granulocytes in 21 67%

RBC Red blood cell; MR megakaryocyte

Histology is essential to exclude the cellular phase/ prefibrotic phase of PMF or MDS. In patients negative for the JAK2 mutation and without concomitant causes of reactive thrombocytosis (see Table 2.2), the fulfillment of the first three criteria is considered sufficient for the diagnosis of ET. A consequent search for infectious, rheumatologic, autoimmune or neoplastic conditions as potential causes of persistent thrombocytosis, has to be carried out in every patient particularly when he/she is JAK2V617F negative. The routine work-up should include evaluation of potential infectious foci and/or exclusion of solid tumors. Mammography, chest-X-ray or CT-scan of the chest, panorama X-rays of the jaw-bones, ultrasono-

Fig. 2.3 Cytology of peripheral blood in ET. Impressive augmen tation of platelet counts in peripheral blood

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Fig. 2.4 Cytology of bone marrow aspirate in ET. Pronounced increase of (atypical) megakaryopoiesis, with large sheets of platelets

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types and could therefore represent a useful tool for distinguishing true ET from other CMPD subtypes as well as secondary thrombocytosis, if confirmed in multiinstitutional investigations [102]. In the extremely rare cases of erythromelalgia (see Fig. 2.1c), an aspirin test can be used for the diagnosis of ET. The long-lasting effect (3 days) of a single dose of aspirin (500 mg) causes irreversible COX-1 inhibition and promptly relieves erythromelalgic pain. This effect is highly specific for erythromelalgia in ET and PV, and is considered a diagnostic criterion by many hematologists, although this is not included in the WHO criteria. An example of an algorithm for diagnostic work-up that is currently used at our institute for patients with suspected ET is given in Fig. 2.2 (see p. 22).

2.7 Pathophysiology of Thrombosis and Microcirculatory Disturbances in ET (and PV)

Fig. 2.5 BM histology in ET. Predominant proliferation of mega karyopoiesis with large to giant megakaryocytes showing hyperlo bulated nuclei (HE staining 400)

graphy of the abdomen, gynecologic inspection, as well as gastroscopy and colonoscopy should be performed (see Fig. 2.2 Diagnostic work-up of suspected ET). It must be stressed that 30% of patients with non-small cell lung cancer (NSCLC) may present with significant thrombocytosis. Typical laboratory findings in patients with ET are summarized in Table 2.7. The presence of JAK2V617F mutations helps to differentiate ET against reactive cases of thrombocytosis [101], but clearly does not allow a firm differentiation against other JAK2V617F positive myeloproliferative disorders. As mentioned and further outlined in the Introduction to CMPDs chapter, the pSTAT3/pSTAT5 expression patterns are highly specific for the differing CMPD sub-

Microcirculatory symptoms such as headache, paraesthesia, neurologic and visual disturbances as well as erythromelalgia are typical features occurring in some, but not all ET patients. The proposed concept is that platelets in ET (and PV) are hypersensitive. Due to the existing high shear stress in the end-arterial circulation, platelets spontaneously activate, secrete their products and form aggregates mediated by vWF that transiently plug the microcirculation [103] (see Fig. 2.1b, p. 18). Increased hematocrit and or platelet levels lead to a narrowing in width of the mural plasmatic zone, which in turn displaces and exposes both erythrocytes and platelets to maximal vessel wall shearing forces, allowing greater platelet endothelial cell, as well as platelet platelet and platelet neutrophil interactions [104, 105]. This effect is more pronounced at high shear rates, as are observed in arterioles and capillaries, thus explaining the predominant location of microvasculatory symptoms. The transient and recurring nature of the symptoms in the initial phases of erythromelalgia is due to deaggregation of platelet thrombi and subsequent recirculation as exhausted, spent and defective platelets with the development of secondary storage pool disease [103]. Platelet-rich arteriolar thrombi paired with endothelial inflammation, intimal proliferation and increased platelet consumption during attacks, have been demonstrated in histological and laboratory work-up [106]. Platelet count per se does not seem to be the major or sole determinant of thrombotic risk. Rather, functional and structural platelet abnormalities seem to be of more relevance. Although platelet aggregation studies are often

26

abnormal, demonstrating either hypo- or hyperreactivity, and several platelet membrane protein abnormalities with either decreased (adrenergic receptor, GPIb, GPIIb/IIIa) or increased (GPIV) expression levels, no convincing correlations with thrombo-hemorrhagic complications have been found so far (reviewed in [4]). This is likely due to the known difficulties of ex vivo platelet manipulation as well as the coexistence of platelet hypo- and hyperreactivity within the same patient, and potential changes in platelet aggregation patterns occurring during the course of the disease within the same patient. Therefore, observations made at one time point are highly unlikely to correlate with previous, or predict future, thrombo-hemorrhagic events [4]. Currently, most single nucleotide polymorphisms (SNPs) in hemostatic genes encoding platelet receptors do not seem to play a role in the pathogenesis of thrombosis for patients with ET [107]. However, the presence of the PLA2 allele of GPIIIa seems to be associated with a higher risk for arterial thrombisis [107, 108]. This is thought to be due to a resulting aspirin resistant phenotype caused by a reduced sensitivity of platelets to the antithrombotic action of acetylic salicylic acid (ASA). This impaired ASA-sensitivity seems to be more prevalent in carriers of the PLA2 mutation [108]. Acquired storage pool deficiency, i.e., the decrease of platelet dense bodies in which the releasable pool of adenine nucleotides and 5HT are normally stored, results from platelet activation with resultant release of granule contents [109]. Increased plasma and urine levels of arachidone metabolites (thromboxane-B2), a-granule proteins (PDGF thromboglobulin, PF4) and membrane markers of platelet activation (P-selectin, thrombospoindin, GPIIb/IIIa), are seen as evidence for higher levels of platelet activation in patients with ET [4]. The pathogenesis of platelet activation has not been fully elucidated, but many factors are thought to contribute to this phenomenon. Among these are reduced lipooxygenase activity, interaction of abnormal rheologic phenomena or platelet leukocyte interactions, a priming effect mediated by elevated thrombopoietin levels, as well as JAK2V617F effects on surface localization of MPL (summarized in [5]). In fact, CMPD-specific defects in arachidonic acid metabolism leading to enhanced thromboxane A2 production by an as yet unknown mechanism, are hypothesized to be the reason why aspirin-mediated COX-1 inhibition alleviates microvasculature symptoms (e.g., [79]) and reduces the risk for thrombotic events [110, 111]. Leukocytosis, is also implicated in the pathogenesis of thrombotic complications (e.g., [112]), as is also adequately reflected by the well-established antithrombotic effect of myelosuppressive therapy (see 2.9. and 2.11.2.1.). As just described for thrombocytosis, not the absolute leuko-

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cyte count per se, but rather an enhanced state of leukocyte activation also plays a role in thrombosis generation (see also below in the section on risk stratification for thrombotic events in ET (and PV (2.9.))). Activated leukocytes promote a procoagulatory state by release of granule contents and formation of aggregates with activated platelets [113]. High plasma levels of neutrophil activation parameters (CD11b, ALP, elastase, myeloperoxidase) are common in ET (and PV), and correlate with markers of endothelial damage (thrombomodulin, vWF antigen) as well as hypercoagulation markers (thrombin antithrombin-complex, prothrombin fragments, D-dimer) [114, 115]. Circulating platelet leukocyte aggregates are also elevated in CMPD-patients, and have been associated with microvasculature disturbances or thrombotic events [113, 116]. Another beneficial effect of treatment with aspirin seems to be the reduction of leukocyte platelet aggregate formation on the one hand, and the reduction of neutrophil, as well as platelet-mediated leukotriene production on the other hand [113, 114, 117]. Additionally, these data offer an explanation for the observed benefit of pan-myelosuppression with hydroxyurea (HU) when compared to mono-lineage platelet reduction by anagrelide in the MRCPT-1 trial. Furthermore, hydroxyurea may suppress thrombotic events by downregulation of adhesion molecules [118] (see Sect. 2.11.2).

2.8 Pathophysiology of Hemorrhagic Complications in ET (and PV) Paradoxically, hemorrhages are the only clinical event that has been clearly associated with extreme thrombocytosis, and also occasionally occurs in secondary thrombocytosis [34]. At increasing platelet counts from below to above 1,000,000/ml, theore-thrombotic condition changes into an overt spontaneous bleeding tendency as a result of a functional vWF (von Willebrandt factor) deficiency that is caused by proteolysis of large vWF multimers [103]. A relationship between extreme thrombocytosis and loss of large vWF multimers has been established by several groups (e.g., [119]). vWF mediates initial adhesion of platelets to sites of vascular injury, as well as platelet aggregation. Thus loss or dysfunction results in a bleeding disorder. The exact mechanism of acquired vWF syndrome in CMPDs and other states associated with extreme thrombocytosis remains obscure. Decreased survival of large vWF multimers has been proposed to result from increased binding to platelets, or enhanced proteolytic cleavage by ADAMTS13, which has been proposed to result from conformational changes imparted by the high shear in microcirculation or interactions of platelet surface proteins with vWF

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(summarized in [4]). This is consistent with acquired type 2 von Willebrand syndrome, which is reversible by reduction of the platelet count to normal levels [103]. Aggravation of blood loss can be caused through inappropriate use of aspirin (in patients with PLTH 1,000,000/ml), anticoagulants, or unappropriately high doses of anagrelide, which may further enhance functional platelet defects. Some hemorrhagic complications, such as bleeding from esophageal or gastric varices may be the consequences of a prior thrombotic event (in this case from thrombosis of abdominal veins resulting in portal hypertension).

2.9 Risk Factors for Thrombotic Events in ET/PV Several risk factors may exist for thrombotic complications (Table 2.8). Patients with a prior thrombotic event had a 5.75 and 4.25 higher likelihood of developing a second arterial or venous thrombotic event, respectively, when compared to the cohort of patients without such a history of thrombosis [8]. As expected, age H60 years at the index event was associated with a significant increase in risk of recurrence, whereas the type of index event (arterial or venous) does not seem to play a role [8]. In ET, several groups have identified leukocytosis as an important independent risk factor for both inferior survival and thrombotic events (e.g., [2, 120, 121]). Leukocytosis and may be especially important in young patients, as the rate of re-thrombosis was significantly higher in those patients G60 years with an elevated leukocyte count (44.4% versus 18.5%) [8]. Importantly,

27

leukocytosis seems to increase the thrombotic risk of otherwise low risk ET patients by 3.3-fold, thus reaching a degree identical to that of high-risk patients [120]. Of note, slightly increased leukocyte counts (H8,700/ml) significantly increase the risk for arterial thrombosis, whereas leukocyte countsH15,000/ml seem to be required to enhance the risk for venous thrombosis [112]. This may explain the superior effect of hydroxyurea over anagrelide in the PT-1 trial since the former usually decreases leukocyte counts while anagrelide does not [7]. This may be due to a mitigation of the formation of thrombogenic activated leukocytes platelet aggregates [113, 114]. However, conflicting data exist [112], which is why, for the time being, leukocyte count at diagnosis should not be the sole factor influencing treatment decision. Perhaps leukocyte activation, which may be correlated with platelet activation and JAK2V617F mutational status [57, 122, 123], may be more important than the number of leukocytes per se. The role of JAK2V617F mutations in the occurrence of thromboembolic complications is not yet fully established and conflicting data exist. Overall, the risk for the occurrence of arterial or venous thrombosis (see Fig. 2.6) seems increased significantly for patients with ET (2.39-fold) or PV (3.63-fold) carrying the JAK2V617F mutation, compared with patients with JAK2 wild type (wt) ET [124]. The JAK2 mutation also seems to predispose to a higher frequency of thrombotic events, and especially so in patients younger than 60 years of age. In a large cohort, the incidence of thrombosis was 53%, compared to 6% in the JAK2 wild type patients [125]. These findings have been confirmed by a British study on 806 patients [126] and in an Italian trial in which patients with JAK2V617F

Table 2.8: Risk factors in ET (adapted from [34, 56, 120, 124]) Risk factors for thrombosis Established risk factors * AgeH60 years in patients not taking aspirin due to underlying vascular pathology (whereas age does not seem to be a risk factor in patients treated with aspirin) * History of vascular events at diagnosis (which is a much stronger risk factor for recurrent thrombosis than age) Potential risk factors * Leukocytosis V617F * JAK2 mutation V617F * JAK2 homozygosity Risk factors for hemorrhage * PLT H1,500,000/ml * Acquired von Willebrand disease type 2 * Use of ASA H325 mg/d or treatment with NSAR ASA Acetylic salicylic acid; PLT platelets; NSAR non steroidal antirheumatic drugs

Fig. 2.6 The occurrence of arterial or venous thromboembolisms in ETand PVaccording to the JAK2 mutation status [124]. Note: complications were either observed at presentation or during follow up of the patients

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homozygosity displayed a significantly higher rate of cardiovascular events (HR 3.97) than wild type (HR 1.0) or heterozygous patients (HR 1.49) [56], as well as by a Spanish group, which demonstrated a more than three fold increase in arterial thrombosis for JAK2V617F positive patients [127]. Although no such correlation was found in a cohort of 605 patients by others [112] and in a Taiwanese observation [128], it is probably wise to consider JAK2 mutations as a potential risk factor (Table 2.8 and [34]). In addition, increased risks of thromboembolic complications seem to occur in JAK2V617F positive women during pregnancy [129] (see 2.12.). Furthermore, disease duration seems to be of importance, as the risk for disease progression to PV or myelofibrosis as well as leukemic transformation seems low in the first decade after diagnosis (1.4% and 9.1%, respectively), but continuously increases during the second (8.1% and 28.3%, respectively) and third decades of the disease (24.0% and 58.5%, respectively) [2]. Obviously the well-defined cardiovascular risk factors such as smoking, arterial hypertension, hypercholesterinemia, arteriosclerosis, diabetes mellitus and obesity are also of importance in patients with ET and have an additional influence on the expected risk of thrombosis. They are usually considered to be of an intermediate risk category. Hypercholesterinemia and hypertension for example, increased the risk of major vascular complications by a factor of up to 3.7 [127, 130, 131], and smoking nearly doubled the cardiovascular risk [132]. However, published data are not in complete concordance as surprisingly, contrary data exist for smoking as an independent risk factor for thrombosis in ET [112, 131]. The mere presence of these factors does not represent an indication for cytoreductive therapy, however, an appropriate management of these reversible factors is mandatory [133], and the probable effect of life style factors on an increased risk of cardiovascular events should be communicated to the patient. The role of inherited or acquired thrombophilia is unclear. Italian guidelines for ET management recommend screening for thrombophilia [134], whereas the UK suggest not to do so [5]. The presence of thrombophilia, defined as deficiency of antithrombin, protein C or S, presence of Factor-V-Leiden, prothrombin G20210A, hyperhomocysteinemia, lupus anticoagulant or antiphospholipid antibodies, were associated with an elevated rate of thrombotic recurrences (42.8% versus 25%) [8]. Several genetic alterations are known to affect hemostatic or platelet proteins. Of these, single nucleotide polymorphisms (SNPs) in factor-V, PT G20210A and ZPI R67Stop are polymorphisms associated with venous thrombosis, whereas HPA-1 located in the aIIbb3 integrin, HPA-2 located in von Willebrand factor receptor,

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GPIa C807T and PSGL-1 are polymorphisms associated with arterial thrombosis. Surprisingly, the analysis of functional hemostatic polymorphisms in the above-mentioned loci did not reveal an influence in the occurrence of arterial thromboses in patients with ET or PV [127]. Interestingly, there does not seem to be a link between the initial platelet count and the risk for subsequent thrombosis in either ET or PV, or at least there is no conclusive data to date. Other factors of presumed relevance include monoclonal myelopoiesis, which was significantly correlated with the development of thrombosis, as 32% of patients with monoclonal hemopoiesis presented with thrombosis, compared to 6% of polyclonal subjects [16]. Furthermore, some data suggest a predictive value of low EPO levels for thrombosis in ET [21], and male gender is considered as a risk factor by several authors [3, 53, 131]. Surgical interventions may also represent a significantly elevated risk for both thrombotic and hemorrhagic complications (for more details see Sect. 2.11.8). Pregnancy is considered to be a risk factor for thrombotic and other complications in women with ET by most authors (e.g., [129, 137, 138]), especially, as pregnancy itself is a physiological hypercoagulable state (for more detailed information see section on Pregnancy in ET 2.12.). However, conflicting data exist, especially for low risk patients [136].

2.10 Risk Factors for Myeloid Disease Progression to PV, Post-ET-MF and/or Leukemic Transformation ET may transform into PV (2.7%), post-thrombocytemic MF (4%) or AML (1.4%), with M1, M2, M4 and M7 as reported FAB subtypes. This transformation is associated with a dramatic worsening of prognosis and life expectancy (see relevant chapters). The rate of transformation into myelofibrosis has been estimated to increase from 3% at 5 years to 8% at 10 years and 15% at 15 years [139]. The probability for this transformation increases with the duration of the disease and therefore is particularly important for younger patients. In an analysis of 126 young patients with ET, myelofibrosis developed in 3% within 10 years [140]. This probability was significantly increased in patients with grade 2 bone marrow reticulin fibrosis at diagnosis, although none of the patients fulfilled the criteria for prefibrotic PMF at presentation. Patients with abnormally high LDH levels at presentation tend to develop post-ET myelofibrosis more often [140]. In the largest single center analysis to date, 605 patients were analyzed for the rate of leukemic evolution and relevant risk factors [9]. Risk factors for leukemic trans-

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formation were hemoglobin levels less than normal and platelet counts 1,000,00/ml as well as age H60 years. According to the number of risk factors present, the subgroups with 0, 1 or 2 risk factors developed leukemia in 0.4%, 4.8% and 6.5% of cases, respectively. In a recently published retrospective analysis of 1,061 CMPD patients, 603 with ET and 458 with PV, three groups were identified and compared. The first group comprised patients with ET/PV who had survived at least 20 years without development of MDS/AML or secondary myelofibrosis, and the second and third group comprised patients who developed either MDS (group 2) or myelofibrosis (group 3) within a decade of first diagnosis. On multivariate analysis, only anemia, defined as less than normal levels of hemoglobin, was able to discriminate these groups in patients with ET, whereas leukocytosisH10,000/ml was associated with disease progression in patients with PV [141]. Surprisingly, the presence or absence of JAK2V627F mutation had no impact on the leukemic transformation rate in patients with ET [9]. This is in line with the finding that in 3 of 4 patients with a JAK2V617F þ CMPD who subsequently developed AML, the evolving AML is JAK2V617F , suggesting that the leukemia arose in a JAK2V617F cell [40] (for details see Introduction to CMPDs chapter). Others, however, have shown that strong activation of JAK2V617F induces genetic instability which may well be responsible for the phenotypic heterogeneity of CMPD features as well as disease evolution to secondary leukemia [142]. Treatment-related leukemic transformation is a matter of constant debate. Although the univariate analysis showed an increased rate for leukemic transformation in patients treated with cytotoxic therapy, this correlation vanished in multivariate analysis. Similar to previous reports from French trials, patients with blastic phase ET showed a predominance of chromosome 17 anomalies [143] and prior exposure to hydroxyurea. However, leukemic transformation also occurs in patients without any previous treatment [144]. The majority of reports argue against a relevant contribution of hydroxyurea to the transformation process. It is assumed, that as yet unknown intrinsic risk factors are responsible for treatment-independent leukemic transformation [134, 145]. However, 32P and the sequential use of several cytoreductive agents does seem to enhance the rate of leukemic transformation [143] (for a more detailed discussion see p. 33).

2.11 Indication for Treatment and Choice of Drugs in Patients with ET The indication for treatment and the choice of drugs is guided by, and must include, the ET-associated risk

29

factor profile for thrombosis. Factors such as age, prior thrombotic events and cardiovascular risk factors must be taken into consideration. In addition, the risk for hemorrhages and transformation into myelofibrosis or leukemia must be assessed and incorporated in the aggressiveness of the therapeutic approach. A risk-factor-adapted approach has been justified by prospective comparative and randomized trials. Furthermore, a more individualized patient approach is warranted, as the currently applied uniform pharmacological interventions likely result in a tendency for over-treatment of patients at low risk and treatment not aggressive enough in those at high-risk [111]. The principles of a treatment algorithm are depicted in Summary Box 2, Fig. 2.9 and Table 10a b. It has to be stated that the definition of risk factors, other than the ones mentioned above, the inclusion or exclusion of thrombophilia in the risk profile, and also the exact role of molecular risk factors, remains illdefined at present and must be considered an area of dynamic current research efforts. One should keep in mind, that the absolute platelet count more likely represents a risk factor for hemorrhage than for thrombosis and that the protective effect of cytoreductive therapy for thromboembolic events is not due to adequate platelet control alone [7]. The predominance of clinical risk factors over the platelet count is reflected by the range of initial platelet counts (208,000 2,320,000/ml) seen in patients considered to be at high-risk in the UK MRC PT-1 trial [7]. Furthermore, a prospective trial comparing low risk ET patients, defined as age below 60 years and no previous history of thrombosis or bleeding and with platelet counts G1.5 million/ml followed without cytoreductive treatment, with age-matched controls was unable to find differences in the rates of thromboses or hemorrhages [136]. In contrast, high-risk patients showed a significant benefit from cytoreductive therapy with HU, as the rate of thrombotic events was significantly reduced in a pivotal randomized Italian trial [146]. Similarly, the widespread use of acetylic salicylic acid (ASA, aspirin) in the lowest risk ET patients may have to be overthought [111]. On the other hand, the rate of thrombotic occurrences and recurrences in patients with ET and PV remains unacceptably high. Therefore, physicians awareness of the high-risk, as well as the correct assignment of individual patients to the appropriate risk group need to be heightened in order to avoid the overestimation of the neoplastic risk of HU in relatively young patients, and/or the potential risk of ASA-associated bleeding in patients with gastrointestinal symptoms. It must also be kept in mind, that some patients

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Summary Box 2 Essential thrombocythemia – treatment strategies (modified from [34, 134, 152, 265]) Essential thrombocythemia – Treatment 1. There are three major threats of ET patients: i.e., transformation in MF or AML, thrombosis or hemorrhage, all of which have to be taken into consideration in treatment and follow-up. 2. Antithrombotic prophylaxis: Low dose aspirin is the treatment of choice for all patients without an overt contraindication and independent of the platelet count in the range ofH400,000/ml 1,000,000/ml. Stop the drug in case PLT areH1,000,000/ml to avoid bleedings and re-substitute below this threshold. In case of gastric symptoms add a proton pump inhibitor. 3. Stop aspirin immediately in case of bleeding or at least 1 week prior to elective surgery with danger from bleeding complications. Stop aspirin 3 5 days prior to elective heparin prophylaxis except for emergency cases like myocardial infraction, angioplasty ischemic stroke, etc. Restart aspirin 1 day after the stop of heparin prophylaxis. 4. Advise the patient against self-administration of non-steroidal antiinflammatory drugs while on aspirin. 5. Consider platelet apheresis in cases of excessive thrombocytosis simultaneous bleeding in order to avoid significant hemorrhage. Begin cytoreductive therapy at the same time. 6. The cytoreductive treatment of choice is hydroxyurea due to the lower rate of MF, better control of thrombosis and lower rate of hemorrhages as compared to anagrelide. Younger patients and females in child-bearing age should be considered for interferon-a provided no contraindication exists. Pipobroman may be used in older patients. 7. The treatment algorithm may follow the subsequent line: Low risk (age G60a; no prior vascular event, no cerebrovascular risk factors): low dose aspirin. Intermediate risk (age G60a, no prior vascular event, with cardiovascular risk factors): low dose aspirin. High risk (either ageH60a and/or prior vascular events): low dose aspirin plus hydroxyurea. Leukocytosis may significantly increase the risk of otherwise low risk patients whereas the role of Jak2V617F allelic burden is still to be defined. The inclusion of the former risk factor in the treatment decisions is not yet fully defined and must be carefully individualized at present. 8. While there is no correlation between thrombocytes and thrombosis, such a correlation exists for hemorrhages if platelets are H1 million/ml. The target platelet count is 400,000/ml in patients with a history of thrombosis, 600,000/ml may be acceptable when age is the only risk factor [1]. 9. In pregnancy interferon-a is the treatment of choice if cytoreduction is indicated (see respective section in this chapter).

with ET/PV may demonstrate decreased platelet sensitivity to standard dose ASA [147]. Risk stratification with resulting therapeutic implications. Currently, the most widely used risk stratification uses age at diagnosis, prior history of a thromboembolic event and platelet counts H1,500,000/ml to stratify patients into a low, intermediate or high risk-group [89]. This risk classification

may be oversimplified and outdated however, as it does not take into account the progressive impact of age, neglects the role of classical cardiovascular risk factors (as demonstrated by Table 2.9) although a clear role has not been established for all of these in ET/PV patients. Furthermore, the widely accepted, but not guide-line implemented threshold of 1,000,000 platelets/ml commonly considered as an indication for cyto-

Table 2.9: Retrospective survey of risk factors for thrombotic events (adapted from [89]) Risk category

PLT G1.5106/ml

Age in years

Risk of major thrombosis in ET

History of thrombosis or bleeding

Coexistent cardiovascular risk factorsa

Low

Yes

No

Yes

1.7% 6.3% per patient year intermediate

No

Intermediate

G40 40 60 60 65

Yes

High

No

H60

15.1% per patient year

1 event of minor thrombosis Yes

a

Such as: arterial hypertension, arteriosclerosis, hyperlipidemia, smoking habit, etc.

Yes

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31

Table 2.10a: Risk stratification and possible guides for treatment of patients with ET (according to [11]) Score

Risk level

G1

Low

1 3

Moderate

3.1 5.5

High

H5.5

Very high

Suggested trt. for ET Phlebotomy Consider ASA (careful balancing of risk/benefit ratio) Phlebotomy Indication for ASA ASA strongly recommendeda Hydroxyurea ASA strongly recommendeda Hydroxyurea Consider more aggressive treatment

AR G1.5

1.5 3 3.1 6 6 10

a

Add proton pump inhibitor in case of history of gastrointestinal symptoms/bleeding ASA Acetylic salicylic acid; low dose, 50 100 mg daily AR Approximate absolute risk (%patients/year)

Table 2.10b: Scoring system for Table 2.10a Risk factor

Score

Age G40 Age 40 55 Age 56 65 Age H65 Hypertension Dyslipidemia PLT H1,000,000/ml WBC H12,000/ml Smoking Diabetes Past history of thrombosis

0 1 2.5 3.5 0.5 0.5 1 1 1.5 1.5 3.5

reduction by most hematologists, is ignored. Landolfi and Di Gennaro have proposed a novel simple prognostic score, which identifies four risk groups, that differ in approximate vascular risk levels ranging from G1.5 to 6 10% per year (see Table 2.10a, b) [111]. The authors provide a treatment recommendation algorithm based on the assignment of patients to one of these risk levels (see Table 2.10a, b).

2.11.1 Acetylic Salicylic Acid (ASA, aspirin) While the antithrombotic efficacy of ASA has not been explicitly tested in a prospectively randomized setting for ET patients, the protective effect of ASA demonstrated in PV subjects in the ECLAP trial [110], can likely be extended to ET subjects, and especially so to those bearing the JAK2V617F mutation [111].

Furthermore, there is currently no evidence suggesting a lower efficacy of ASA in JAK2V617F negative patients with ET [111]. While the risks and benefits of lowdose aspirin should be carefully balanced in the individual ET patients assigned to the lowest risk category (according to Table 2.10a, b), the so-called contraindications for aspirin therapy must be carefully weighed in patients at high or very high-risk [111]. In the latter groups, the absolute benefits of ASA are expected to be very high, and relatively safe use of ASA can be assumed if a proton pump inhibitor is coadministrated [148, 149]. When considering the literature one must be aware of the fact, that Americans tend to use higher dosages of ASA (325 mg/d), than Europeans (50 100 mg/d), which is likely to play a role in the incidence of hemorrhagic complications. In fact, in an early US-trial performed in 1986, PV patients received 900 mg ASA per day [150]. Not surprisingly, the study was terminated early because of an excess of gastrointestinal bleeding events [150]. Low-dose aspirin (100 mg/d) is highly effective in the treatment and secondary prevention of thrombotic and ischemic events. Treatment with aspirin results in the correction of all the above-mentioned laboratory parameters of platelet-mediated thrombotic processes (see Sect. 2.3.5). In particular, inhibition of platelet cyclooxygenase-1 by ASA is followed by correction of increased plasma levels of PF4, thrombomodulin and thromboglobulin, as well as correction of increased urinary levels of thromboxane metabolites [106]. This explains the complete relief and prevention of microvascular disturbances and major thrombosis in ET (and PV) patients with aspirin and not with coumarin [79, 106, 151]. However, acetylic salicylic acid may aggravate or elicit hemorrhagic events at platelet (PLT) counts above 1,000,000/ml. Therefore, low dose aspirin should be discontinued in patients whose PLT counts exceed this number. At this time point cytoreductive therapy should be commenced or dose escalated. Reduction of PLT counts to less than 1,000,000/ml results in reappearance of intermediate-large vWF-multimers and the disappearance of bleeding symptoms (if they were present), and low dose aspirin should be recommenced at this stage. Correction of the PLT count to normal (i.e., G400,000/ ml) is associated with complete correction of the vWF multimeric pattern. Few reports exist on the role of JAK2V617F mutations on the efficacy of ASA. However, in the series of Passamonti et al. [129], ASA was unable to reduce the increased complication rate in pregnant patients carrying the mutation. Further studies are necessary in this regard.

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2.11.2 Platelet Reducing Agents – Current State of the Art

The principles of cytoreductive therapy and the indications when which drug should be commenced, are depicted in Fig. 2.9.

2.11.2.1 Hydroxyurea Hydroxyurea (HU) is a non-alkylating antineoplastic agent widely used in the treatment of myeloproliferative diseases. HU inhibits ribonucleotide reductase and consequently DNA synthesis, producing a megaloblastic blood picture. As the effect of hydroxyurea is downstream from effects of vitamin B12 and folic acid, no response to these agents will be observed. Currently HU is regarded as the first choice cytoreductive therapy according to evidence-based guidelines [134], suggestions from experts in the field (e.g., [146, 152]) and from results of the MRCPT-1 trial [7]. This seminal randomized clinical trial demonstrated the superiority of hydroxyurea over anagrelide in terms of prevention of thrombosis, while simultaneously bleeding events were lower in the HU-arm (see Fig. 2.7). This advantage for HU occurred despite the adequate and comparable lowering of platelet counts in the group of patients treated with anagrelide [152]. The better protection against thrombosis may be explained by the cytoreductive effect of the drug on erythrocytes and particularly leukocytes which both may be important in the pathogenesis of thrombosis in the disease [114]. The bleeding rate in patients treated with hydroxyurea plus aspirin was small and significantly lower than in the anagrelide plus aspirin group. This may be explained by

7 5.72

6

Hazard ratios

In erythromelalgia, ASA at dosages of 100 500 mg rapidly reduces the clinical symptoms, and discontinuation is followed by prompt recurrence of microvasculature circulation disturbances. Interestingly, other analgesics (e.g., sodium salicylate, glaphinine, acetaminophen) or platelet-inhibiting substances (dipyridamole, sulfapyrazinone) typically show no alleviation of erythromelalgic symptoms [79, 151]. This is explained by a direct interference of acetylic salicylic acid with the mechanism involved in the pathophysiology or etiology of erythomelalgia (see Fig. 2.1b). Furthermore, the generation of thrombin is not essential for the formation of platelet-rich thrombi [79, 106], which explains the inefficacy of warfarin derivatives or heparin in the prevention and treatment of arterial microvascular thrombosis.

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5 4 3

3.54 2.92 2.61

2.16 2 1 0 MF

arterial thrombosis

TIA

hemorrhages

GI hemorrhages

Fig. 2.7 Hazard ratio (HR) for disease complications in patients treated with anagrelide versus hydroxurea in the MRC PT-1 trial [7]. In addition, venous thromboembolisms occurred less frequently in the hydroxyurea group (HR 0.27). All differences were statistically significant. MF Disease progression to myelofi brosis; TIA transient ischemic attack; GI gastrointestinal

the additive effect of both drugs on platelet function [152]. More recently however, non-inferiority of anagrelide compared to HU in 258 newly diagnosed patients with high-risk ET has been demonstrated in the GCPconform randomized ANAHYDRET study [153], currently presented only in abstract form. However the follow-up period at the time of publication was only 12 months. Side effects and potential caveats of HU. Side effects are usually minimal in degree and include neutropenia, macrocytic anemia, oral (see Fig. 2.8) and leg ulcers [154, 155], skin lesions, nausea, diarrhea, [156, 157] rarely drug-fever [158, 159] or elevated liver function tests. The onset of action can be expected within 3 5 days. Similarly, the effect is short lived once the medication is stopped. Complete blood counts and liver function tests should be monitored frequently during the first 3 months of treatment initiation. Significant increases in MCV are not only expected, but also indicative of appropriate

Fig. 2.8 Side effects of hydroxyurea: oral ulcers. After nearly a decade of hydroxyurea, this patient developed painful ulcerous cheilitis

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33

Fig. 2.9 Algorithm for cytoreductive treatment indications for patients with ET (adapted from [111, 134])

drug action and patient compliance. Particularly in elderly patients however, caution is warranted, as they tend towards hydroxyurea-induced cytopenias in all three hematopoietic cell lineages, which can occur even at very low doses of 500 mg/d, and are sometimes severe and of prolonged duration. The use of HU is also of concern in patients previously treated with other cytotoxic agents, because of a probable increase in the rate of leukemic transformation due to sequential use of various cytoreductive agents. Hydroxyurea is currently contraindicated in pregnancy, women with child-bearing potential without adequate contraception, as well as women who are breast-feeding. Although the use of HU is convenient and its mode of action is quick, it may not always be adequately tolerated or even be inefficient. A recent international working party defined the criteria for challenging the further application of hydroxyurea in ET patients by the presence of at least one out of the following criteria [160] (Table 2.11): (i) platelet count H600,000/ml after 3 months of treatment with at least 2 g/day or 2.5 g/day in pts with H80 kg body weight, (ii) platelet count H400,000/ml and WBC G2,500/ml or Hb G10 g/dl at

Table 2.11: Definition of resistance/intolerance to hydroxyurea (HU) in patients with ET (according to [160]) Definition of resistance to HU PLT H600,000/ml after 3 months of at least 2 g/d (2.5 g/d in patients with H80 kg) PLT H400,000/ml and WBC G2,500/ml at any dose of HU PLT H400,000/ml and Hb G10.0 g/dl at any dose of HU Definition of intolerance to HU Presence of leg ulcers at any dose of HU Presence of unacceptable mucocutaneous manifestations at any dose of HU HU related fevers

any dose of hydroxyurea, (iii) presence of leg ulcers or other unacceptable mucocutaneous manifestations at any dose of hydroxyurea, and (iv) hydroxyurea-related fever. Is hydroxyurea leukemogenic? Although not directly genotoxic, HU may impair the repair of damaged DNA, raising a legitimate concern regarding leukemogenicity. There has been discussion, whether hydroxyurea in combination with alkylating agents may

34

increase the incidence of transformation to AML [145]. When used alone however, the transformational capacity of HU approaches nil, as demonstrated by the identical leukemic transformation rate in the hydroxyurea and anagrelide arm of the Harrison et al. trial [7]. Sterkers et al. observed an incidence of MDS or AML after treatment using HU alone or with other agents of 3.5% and 14%, respectively, and a high proportion of these patients demonstrate 17p deletions [143]. This led the authors to conclude that long-term treatment of ET patients with HU may increase the risk for MDS/ AML with p53 mutations, and that HU probably increases the leukemic risk of other cytoreductive treatments given in ET [143]. They follow that widespread and prolonged use should be reconsidered in asymptomatic ET patients. However, it is imperative to point out several shortcomings of this study: (i) the study was not randomized and was systematically biased as patients were grouped on the basis of treatment requirement. (ii) Allocation to different treatment groups may have been biased according to age and/or disease refractoriness, both of which may influence leukemic risk. (iii) Patient numbers were too small to allow statistically valid comparisons. (iv) Evolution to AML with 17p abnormality has also been observed in untreated ET [144]. In general, published reports on the association of HU and evolution to acute leukemia have been inconsistent, with treatment groups often not being biologically comparable, and the strength of the association has been relatively small. In addition it is extremely difficult to discern a potential true leukemogenic effect of HU from the natural course of the disease. This seems confirmed by the lower transformation rate into myelofibrosis observed in patients treated with HU, which only amounted to a third of that observed in the anagrelide group [7]. In addition, HU has been recommended for long-term use in children with thalassemia [161] and so far no elevated transformation rates to leukemia have been reported in this patient population.

2.11.2.2 Anagrelide Anagrelide is the only drug currently licensed for the treatment of thrombocytotic conditions in myeloproliferative disorders in the US [152]. In Europe, however, anagrelide is licensed by the EMEA only after resistance or intolerance to first-line therapy has been documented. Resistance/intolerance to HU as recently been defined as a consensus process [160] (see Table 2.11). Up to 10% of patients do not obtain the desired reduction of platelet number with the recommended dose of HU, thus exhibiting clinical resistance, whereas others

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develop unacceptable side effects, demonstrating clinical intolerance. The recently presented results of the above-mentioned ANAHYDRET trial demonstrate noninferiority of anagrelide compared to HU [153] and may lead to an application for first-line approval of the drug by the EMEA. Anagrelide inhibits platelet aggregation via platelet anticyclic AMP phosphodiesterase activity at higher doses and has a platelet-lowering effect at lower doses (2 mg/d) through inhibition of megakaryocyte maturation. The platelet inhibitory function is seen at doses higher than those usually needed for controlling thrombocytosis and should therefore not be a concern in treated patients. However, this effect may become relevant when higher doses of the drug are combined with aspirin [7], although no hemorrhages were found in a small study when the ASA dose was reduced to 50 mg [162]. Among 722 prospectively documented ET patients treated with anagrelide, no evidence of increased bleeding rates or disease progression was found during a 5-year follow-up period [6]. Some authors prescribe lower dose (50 mg) ASA for those patients receiving anagrelide, as compared to the 100 mg dosage usually prescribed for patients treated with HU. Red blood cells are also reduced to some degree, resulting in mild to moderate anemia after long-term use, with 24% of patients experiencing a more than 3 g/ dl decrease in hemoglobin level [163]. In a retrospective analysis of a multicenter international trial reviewing 3,660 patients, including 2,251 with ET and a maximum follow-up of 7 years, 67% of ET patients achieved adequate platelet control, which was defined as reduction of PLT to G600,000/ml or H50% from baseline [164]. In this trial 2.1% of ET patients developed AML, all of whom had previously been exposed to other cytoreductive agents. None of the ET or PV patients exposed solely to anagrelide developed AML during the treatment duration analyzed [164]. Importantly, anagrelide was found to be associated with an increased rate of secondary myelofibrosis, compared to hydroxyurea [7], after a median followup time of 39 months. The recent ANAHYDRET trial has so far not demonstrated an enhanced rate of disease progression, but the follow-up is much shorter (12 months) [153]. Short-term side effects include palpitations (27%), tachycardia and other arrhythmias (G10%), congestive heart failure (2%), headache, fluid retention, diarrhea, and nausea. Long-term therapy is associated with decreased reporting of initial side effects and the development of mild to moderate anemia [163]. Rare cases of severe hypersensitivity pneumonitis have been reported [165]. Overall, the therapeutic range is broad and the side effect profile

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is favorable. However, due to the above-mentioned data, anagrelide should not generally be viewed as the first therapeutic option, unless the patient has: (a) received prior chemotherapy with e.g., alkylating agents due to another malignancy. (b) anemia or leukopenia due to comorbidities, i.e., renal anemia or rare cases of co-occurent MDS, which must however be differentiated from RARS-T. (c) the propensity for recurrent infections or ulcers.

2.11.2.3 Interferon-a (IFN-a) Interferon-a suppresses the proliferation of hematopoietic as well as bone marrow fibroblast progenitor cells, antagonizes the action of cytokines involved in the development of myelofibrosis (e.g., platelet derived growth factor (PDGF), transforming growth factor b (TGF-b)). Starting doses between 6 and 70 million units/week were used, with most studies using 21 million units/week, i.e., 3 million units s.c./day initially, with tapering during maintenance [134]. Overall response rates of up to 85% were observed, with 54% of patients achieving complete platelet normalization [134]. According to a synopsis of six studies, only 15% of patients seem to be primarily IFN-a resistant [134]. Results from a phase II clinical trial show 75% response rates with 61% achieving complete hematological responses [166]. Higher initial platelet counts may require higher initial dosages and longer application of the drug [167]. Although the efficacy of the drug has been confirmed by many, clonal hematopoiesis often persists [168], and qualitative platelet abnormalities are not, or only partially corrected [169]. This is in line with clinical data as the effects steadily decrease after cessation of treatment in the vast majority, with only a small proportion of patients remaining in longterm remission [166]. Better tolerability has been observed with pegylated IFN-a, which only needs to be administered once weekly, while maintaining acceptable toxicity, tolerability and activity profiles [170], 84% remissions were obsered with no drop outs or substantial toxicity after 1 year of treatment in patients with ET (or PV) [171]. However, only limited effects on JAK2 mutational status have been observed after pegylated IFN-a-2b therapy, and while the drug results in a hematologic response in 79% of patients, decreases splenomegaly (from 22% to 6%), reduces disease-related symptoms (from 42% to 2%) [172] and lowers the percentage of circulating JAK2V617F positive cells, the malignant myeloproliferative clone remains present [171]. Furthermore, reversal of PRV-1 overexpression occurs only in approximately one-third of patients treated with pegylated IFN-a-2b,

35

suggesting a suppression of the malignant clone only in some [173]. However, neither HU nor anagrelide are able to achieve suppression or eradication of the malignant clone. Pegylated IFN-a-2a may be more effective in this regard, at least in patients with PV [174]. In the vast majority of patients the effects steadily decrease after cessation of treatment with only a small proportion of patients remaining in long-term remission. Oral IFN-a formulations have also been tested, were very safe, but of no appreciable clinical benefit for ET (or PV) patients [175]. It is important to stress, that IFN-a is the only therapy for CMPDs that has been shown to modulate abnormal biologic processes in a subset of patients. These documented biological effects include reversal of chromosome abnormalities, restoration of polyclonal hematopoiesis, suppression of EEC-growth, normalization of PRV-1 expression as well as rare complete suppression of JAK2V617F. Such biologic effects have thus far not been demonstrated for hydroxyurea or anagrelide (reviewed in [173]). However, side effects (fever, flu-like symptoms, weakness, myalgia, severe depression, local reactions at the injection sites, weight loss, hair loss, gastrointestinal and cardiovascular problems), as well as inconvenient dosing schedules lead to discontinuation of the drug in approximately one-third of the patients (e.g., [167]). IFN-a is not known to be leukemogenic or teratogenic and does not cross the placenta. It has successfully been used during pregnancy and is therefore considered the treatment of choice in patients requiring cytoreductive therapy during pregnancy and in young females in childbearing age [176 178].

2.11.2.4 Pipobroman (Vercyte) Pipobroman is a piperazine derivative with a chemical structure similar to alkylating agents, and is generally well tolerated with mild dose-related side effects (mainly gastrointestinal symptoms). Clinical activity in ET has been well documented by European centers [179 181], whereas the drug does not seem to be in widespread use on other continents. Complete hematological response rates of 92% within a median of 12 weeks at an initial dosage of 1 mg/kg/day, with no acute or chronic toxicity have been reported in an efficacy trial of pipobroman in patients with ET [179]. These good results have been confirmed by others who achieved 86 95% complete hematological responses (defined as PLTG400,000/ml in these studies), with excellent tolerability and without elevated rates of secondary malignancies or leukemic transformation after a median follow-up of 10 years

36

[180 182]. Remission continues over 10 years in 85% of patients, with a cumulative risk of thrombotic events of 18% at 15 years and a risk for acute leukemia of 6% at 15 years [181]. Dose may be tapered for maintenance to 0.2 1 mg/kg/day and according to platelet counts. The antiproliferative activity of pipobroman on bone marrow megakaryocytes seems particularly relevant in lowering the disease transformation rates to post-ET myelofibrosis, the risk of which (G4% at 10 years) is the lowest registered with available treatments [183]. Importantly, leukemic transformation rates were low (5.5% after a median of 153 months) in a group of 164 ET [184] patients treated with pipobroman as first-line therapy. However, physicians should be aware that rare cases of severe aplastic anemia related to pipobroman have been reported in the literature, some of which show spontaneous recovery within 6 months of discontinuation of the drug [185 188]. An immune-mediated suppression of hematopoiesis seems to be the underlying mechanism of pipobroman-induced pancytopenia and immunosuppressive treatment may lead to partial recovery in patients without spontaneous remissions [185, 187]. In conclusion, pipobroman is a well tolerated and simple to use drug that constitutes a valid alternative to hydroxyurea or anagrelide.

2.11.2.5 Busulphan Busulphan is a well-tolerated alkylating agent able to induce long-lasting remissions in CMPDs and appears to have a more specific action on megakaryocyric proliferation [189]. However, treatment with alkylating agents has been shown to carry a definite leukemogenic risk in myeloproliferative disorders (MPDs), including ET, which is why the drug is currently barely in use in this indication. However, when busulphan was used alone, the incidence of MDS/AML was only 3%, as compared to 17% when busulphan was combined with, or sequentially followed by, other cytoreductive agents [143]. In 37 ET patients with follow-up periods of up to 25 years, busulphan effectively reduced platelet counts to below 400,000/ml, which coincided with resolution of vascular occlusive symptoms, whereas hemorrhagic symptoms often remained unchanged at a median cumulative dose of 589 mg [190]. The rate of leukemic transformation was not higher than that expected during the natural course of disease for this patient cohort, which emphasizes the relative safety of long-term busulphan treatment in ET [190]. However, the progression to secondary myelofibrosis was noted in 24% of ET patients [190]. Busulphan-related leukemia has mainly been observed in patients who received a high cumula-

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tive dose, or were treated with a combination of various cytoreductive agents [189]. Based on the assumption that low dosage and short duration of busulphan therapy is important to prevent its potential leukemogenic effect, as has been stressed by the EORTC, an Israelian group performed a trial using a novel short-term treatment schedule comprised of a short single course with low cumulative dose of busulphan [191, 192]. In their more recent publication they describe 37 elderly (H60 years) ET patients treated with 4 mg/d for the first week, 2 mg/d for the next 3 weeks and 2 mg every other day until platelet counts below 400,000/ml were reached, and then treatment was stopped [191]. After just one such course, all patients responded with a prompt normalization of platelet count that lasted for months to years without the need to reinstitute treatment. Disappearance of thrombocytosis-related symptoms were observed in all patients and a considerable reduction of thrombotic complications occurred [191]. The median time for next treatment was 56 months, but was necessary only in 2/3 of patients [191]. Importantly, this short-term schedule reduces the potential leukemogenic effect of busulphan by reducing the cumulative exposure (median 124 mg). In this trial, no leukemic transformation was observed, although three cases of transition to myelofibrosis were noted, one of which may have been due to sequential application of busulphan and hydroxyurea [191]. In a previous trial, similar results were demonstrated, and two women with recurrent abortions had successful pregnancies after achievement of normal platelet counts, and delivered healthy babies [192]. Considering these data, the current mindset that busulphan should only be used as a last line agent in very old patients who are simultaneously refractory or intolerant to hydroxyurea, anagrelide and interferon-a, or in whom all of these agents are contraindicated and/or ineffective, may have to be revised in the future. In fact, this shortterm protocol seems feasible and has been proposed for elderly patients on the one hand, as well as for young women of child-bearing age on the other hand, as it provides long chemotherapy free periods and the possibility of pregnancy with no teratogenic risk [191, 192]. Nevertheless, the indication for busulphan should be seen as restricted. Further experience as well as long-term observations are needed before widespread use can be recommended.

2.11.2.6 Radiophosphorus 32P Most data concerning the use of radiophosphorus in CMPDs are derived from PV patients, which is why this substance will be primarily discussed in that chapter (see 3.10.3.4.). Suffice it to say, that this substance is obsolete

Chap. 2


in ET for several reasons: (i) In 366 consecutive patients with ET or PV, oral administration of 32P resulted in a reduced 10 year overall survival rate (51% versus the expected 66%) [193]. (ii) The incidence of MDS/AML after treatment with 32P alone or in combination with other cytoreductive agents was 7% and 9%, respectively [143], and others have reported H10% leukemic transformation in 32P treated patients after 10 years [193]. (iii) The increased risk of MDS/AML or lymphoma reached a value of 30% after 20 years in a prospective analysis of 682 cases of ET and PV [194]. (iv) Importantly, these staggering rates of leukemogenicity do not seem to be dose-dependent [194]. (v) The risk of carcinoma was 15% at the 10th year [194]. (vi) Patients having received 32P followed by hydroxyurea maintenance (in order to reduce the cumulative does of 32P) had an alarming 19% risk of leukemia and a 29% risk of carcinoma at 10 years [194]. However, when 32P is the sole treatment modality, it does not seem to be more leukemogenic than hydroxyurea or busulphan [143, 193], and leukemic transition was only noted in 5/230 patients with ET or PV by others [195]. Nowadays however, there is only rarely the necessity or a reasonable indication for this drug in ET.

2.11.3 Treatment of Bleeding Events and Indications for Platelet Apheresis In case of extremely high platelet counts (H1,000,000/ ml [196]) in the symptomatic patient associated with overt thrombosis, cerebral ischemia, or other clinical symptoms of stasis, immediate drastic platelet reduction should be obtained by thrombapharesis [197, 198]. Furthermore, this procedure should be used in asymptomatic highest risk patients with extremely high platelet counts in order to prevent acute severe sequelae [197, 198]. In addition, PLT apheresis is recommended when bleeding associated with massive thrombocytosis is present [196]. Secondary von Willebrandt syndrome necessitates immediate platelet apheresis in addition to the administration of a plateletreductive agent. Importantly, desmopressin is contraindicated in vWF syndrome IIb due to a potential aggravation of the defect. Platelet apharesis has successfully been employed in controlling hemorrhagic complications during surgery associated with ET in a patient with subdural hematoma [199]. Platelet apharesis should be combined with a cytoreductive agent, and has effectively been employed as initial treatment in CMPD patients with symptomatic thrombocytosis [200]. Furthermore, the procedure has successfully been used for the management of high-risk pregnant patients with ET [201, 202].

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2.11.4 Life Style Modifications and Control of Other Risk Factors Life style modifications should be advised, including (a) cessation of smoking, as it is not only thrombogenic, but also alters the inhibition of in vivo platelet action of ASA, and (b) consequent weight reduction in overweight patients, as obesity has also been associated with thrombosis in ET. In patients with comorbidity of atherosclerosis or cardiovascular risk factors (hypertension, diabetes, dislipidemia) appropriate therapy with antihypertensive drugs, antidiabetics and/or statins should be used for risk reduction according to standard criteria.

2.11.5 Effect of Therapeutic Strategies on Re-thrombosis Re-thromboses generally occur in the same district as that of the first thrombotic event [8, 110], thus raising the question of whether secondary prevention strategies should be differentiated according to the site of occurrence of the first thrombotic event. It is currently not clear, whether the same recommendations that apply to the general population, also apply to patients with ET (or PV). In fact, ASA has an efficacy in preventing venous and arterial cerebrovascular events in ET/PV not found in other clinical conditions. This is thought to be due to the proposed pathophysiological role of thromboxane overproduction in the thrombophilic state of patients with ET/PV [203]. Thromboxane levels are throttled by ASA in patients with PV [204], but not by low molecular weight heparin, and unfractionated heparin even leads to a significant increase in urinary thromboxane metabolite levels [205]. In a multicenter cohort of 494 ET/PV patients, a significant reduction of the risk for re-thrombosis was only achieved by cytoreductive therapy (multivariable hazard ratio: 0.53), whereas the use of antiaggregatory agents such as ASA or phlebotomy as the only means of treatment merely resulted in a borderline reduction of recurrent thrombosis [8]. This may seem surprising at the first glance, especially when considering that antiplatelet agents have been shown to reduce thrombotic events in patients without CMPDs (e.g., [206]). However, one must remember, that thrombus-formation in ET/PV patients is influenced by factors not present in the normal population, e.g., by the interplay between higher levels of activated platelets and leukocytes as well as polymorphonuclear aggregates. Of note, acetylsalicylic acid seems to mitigate these effects [113]. In a prospective trial using hydroxyurea, a reduction in both platelet count and rate of thrombosis could be demonstrated [146]. In a subsequent trial comparing anagrelide plus aspirin versus hydroxy-

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urea plus aspirin, hydroxyurea consistently demonstrated a greater protective effect for strokes, transient ischemic attacks, acute myocardial infarctions as well as vascular death, than anagrelide, despite an equivalent long-term control of platelet counts [7]. Merely for the prevention of venous thrombotic events, did anagrelide seem to be the more efficacious agent [7]. Thus, the benefit of cytoreductive therapy may well lie in a general myelosuppressive effect with concomitant reduction of leukocytosis. Importantly, combined treatments seem more effective than single agent strategies. Combination of cytoreductive treatment with either an antiplatelet agent or oral anticoagulants led to an even higher protection against rethrombosis [8].

2.11.6 Should the Site of Occurrence of the Thrombotic Event Have an Influence on Preventive Treatment Strategy? The answer to this question may well be yes, but further data are necessary. In patients with the first thrombotic event occurring in the venous system significant prevention of re-thrombosis was independently achieved by both long-term use of oral anticoagulants (68% risk reduction) and by antiplatelet agents (58% risk reduction for antiplatelet agents) [8]. In patients with a history of acute coronary syndrome or any other peripheral arterial thrombotic event, cytoreductive therapy was particularly effective (70% and 53% risk reduction, respectively), whereas the benefit of cytoreduction was not statistically significant in patients with previous cerebrovascular disease [8]. In the latter cohort of patients, antiplatelet agents were found to be highly effective in preventing re-thrombosis (67% risk reduction). To summarize, in ET/PV patients with a venous thromboembolism, both long-term treatment with antivitamin K agents or low dose aspirin after a conventional short-term period of oral anticoagulation seem effective and safe. As cytoreductive treatment halves the incidence of re-thrombosis, particularly in patients with arterial events and/or acute coronary syndrome, and as the occurence of a thrombotic event places the patient into the high risk group, initiation of cytoreductive therapy is indicated. It is likely that more aggressive antithrombotic therapy, based on the combination of ASA with clopidogrel for patients with previous myocardial infarction, or with dipyridamole in patients with previous TIA or stroke, may have an additional protective effect, but this remains to be demonstrated in prospectively randomized trials. In favor of this hypothesis is the documented inhibition of leukocyte platelet adhesion as well as platelet-mediated leukocyte activation by clopdiogrel [207].

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2.11.7 Should JAK2V617F Positivity Influence the Choice of Cytoreductive Therapy? The relevance of JAK2V617F for current CMPD therapies is currently unclear. If the generally assumed hypothesis that JAK2V617F represents an additional risk factor proves true, JAK2V617F allele burden may become an indication for earlier initiation of cytoreductive therapy. JAK2V617F positive ET patients have been demonstrated to be more sensitive to hydroxyurea than to anagrelide, in that they obtained a better degree of cytoreduction and had a lower rate of arterial thrombosis than patients treated with anagrelide [126]. As mentioned above, JAK2V617F positivity or exon 12 mutations are found in up to 75% of patients with splanchnic vein thrombosis [85 88]. Whether or not the detection of such a mutation indicates the need for cytoreductive treatment, even in the absence of an overt myeloproliferative disease, remains speculative. However, transjugular intrahepatic portosestemic shunts (TIPS) are highly effective in patients with acute or subacute Budd Chiari syndrome, including those with overt ET, uncontrolled by medical therapy [208, 209].

2.11.8 Antithrombotic Prophylaxis for Elective Surgery in ET (and PV) The risk of ET complications may be particularly important when patients are undergoing surgical interventions. In a retrospective survey by GIMEMA comprising patients with ET/PV, symptomatic deep venous thrombosis (1.1%/7.7%) and arterial thrombosis (5.3%/1.5%) rates remain high, despite effective control of hematocrit by phlebotomy and cytoreduction and administration of standard antithrombotic prophylaxis. The increased bleeding risk of 10.5% was observed, with an unexpectedly high incidence of major bleeding and a clear trend for an increased incidence in patients receiving antiplatelet therapy or heparin [135]. It has been suggested, that antithrombotic prophylaxis prior to surgery be restricted to patients with PV, whereas antiplatelet drugs may be the optimal choice in patients with ETand several arterial risk factors [135]. Patients with low risk ET may not require any additional antithrombotic prophylaxis, as surgery was not associated with thrombosis in these patients [136]. In a large group of ET (150) and PV (105) patients, the majority of which was treated with cytoreductive therapy and/or phlebotomy prior to surgery and showed near normal platelet and leukocyte counts and hemoglobin values and who also received antithrombotic prophylaxis, 5.1% of major and 2.5% of minor surgeries were accompanied by an episode of deep venous thrombosis [135]. This is

Chap. 2


equivalent to an at least five-fold increase over the normal control groups. While surgery per se is no risk factor for arterial thrombosis, the risk of ET patients nearly doubled, particularly when cardiovascular disease was also present. However, these risk factors dramatically decrease about 1 month after surgery, arguing against the necessity for long-term prophylaxis in patients undergoing surgery.

2.12 ET in Pregnancy All women with ETof child-bearing age should adequately be counselled concerning the potential dangers and complications of the disease during pregnancy and potential consequences for the child.

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disease complications before pregnancy, or by the use of specific therapy during pregnancy [129, 138, 210, 213, 215, 216]. In patients with multiple pregnancies the outcome of the subsequent pregnancy does not seem to be predicted by the first [210]. The rate of successful pregnancies in women who had previous miscarriages is however significantly lower (48% failure rate, compared to 35% failure rate in women without prior spontaneous abortions) [134]. Currently, merely JAK2V617F positivity has been established as an independent predictor of pregnancy complications and fetal loss in women with ET in multivariate analysis [129]. JAK2V617F positive women had a twofold elevated risk of developing pregnancy complications than JAK2V617F negative women [129]. These important results, which may have therapeutic implications, remain to be corroborated by other groups.

2.12.1 Course of Pregnancies in Women with ET Maternal complications occur in 9%, while fetal complications occur in approximately 40% of pregnancies, and the rate of live births is 50 64% [129]. Pregnant women with ET have a substantially increased risk of spontaneous abortion (up to 59%) compared with the expected risk in the general population (15%) [138, 210]. The risk is especially high during the first trimester (80% of all abortions) and is equivalent to a threefold increase for unsuccessful outcome of pregnancies [80, 138, 210]. Later obstetric complications are infrequent and include intrauterine fetal death (5%), premature delivery (8%), pre-eclampsia (2 4%), fetal growth retardation (4%), and placental abruption (3.6%) (e.g., [80, 210, 211]). The latter seems to be associated with villous placental infarctions, which is thought to be related to thrombocytosis [80]. Post-partum thrombotic episodes occur in about 5% of pregnancies and include venous thrombosis, pulmonary embolism, sagital sinus thrombosis, transient ischemic attacks and Budd Chiari syndrome [134]. Spontaneous decreases in PLT counts occur frequently during pregnancy. Drops from a median of 1,100,000/ ml to 600,000/ml, or 850,000/ml to 500,000/ml have been well documented [138, 210, 212], and can also be confirmed by our experience. The degree of platelet reduction has been associated with pregnancy outcome [212] and is thought, that this phenomenon may be related to placental and/or fetal production of interferon-like substances [213, 214].

2.12.2 Prediction of Pregnancy Outcome Pregnancy outcome is not predictable by preconception platelet counts, preconception leukocyte counts, history of

2.12.3 Management and Treatment of Pregnant Women with ET 2.12.3.1 General Considerations If pregnancies are being planned and cytoreductive therapy is necessary in females of child-bearing age with child-bearing potential and -intention, interferona should be given as first-line cytoreductive treatment [134]. Patients already receiving anagrelide and/or hydroxyurea should be switched to interferon-a in time. In all women with menstrual delay, anagrelide or hydroxyurea should be withheld until results of a pregnancy test are available [134]. Our proposal for a treatment algorithm for pregnant women with ET is depicted in Fig. 2.10 (p. 40).

2.12.3.2 Antiaggregatory Platelet Therapy During Pregnancy Whereas therapy with acetylic salicylic acid (ASA) prior to conception does not seem to influence pregnancy outcome in ET, i.e., live birth versus abortion or stillbirth, treatment with ASA during pregnancy seems to have a beneficial effect on obstetric complications and positively influence pregnancy outcome [138, 210, 213, 216 218], although conflicting data exist [210, 215]. Among 461 pooled cases from the literature, 74% of women with ET treated with ASA during pregnancy had successful pregnancies, compared to only 55% (80/145) of women not receiving ASA [134]. While these data have resulted from retrospective or prospective analysis, and were not generated by randomized clinical trials, the use of antiaggregatory agents such as ASA should thus be seriously considered [80].

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Fig. 2.10 Possible treatment algorithm for pregnant women with ET (modified from [218])

Antiplatelet therapy is recommended for pregnant women with a previous history of a microvascular event or at least one previous spontaneous abortion. Thrombosis prophylaxis with low molecular weight heparin is recommended for the third trimester in patients with a previous history of thrombosis. Patients with thrombotic events during pregnancy should receive heparin at therapeutic dosages and oral anticoagulation for at least 6 weeks during puerperium. In high-risk patients, the additional use of low molecular heparin or IFN-a should be considered (see below and e.g., [217]).

2.12.3.3 Cytoreductive Therapy During Pregnancy The Italian consensus recommends the use of cytoreductive therapy in women with a history of major bleeding or thrombosis, presence of cardiovascular risk factors, when the platelet count is H1,000,000/ml, or when there is a history of familial thrombophilia [134]. Fetal outcome seems improved by treatment with interferon-a, which has been successfully and safely used in many women with ET [176 178, 219 222]. Based on these data, interferon-a is currently considered to be the best therapeutic option in pregnant women with ET necessitating cytoreductive therapy. Interferon-a has also been successfully combined with ASA [178]. Maternal use of interferon-a has however been associated with intrauterine growth retardation, drug-induced neonatal lupus and transient thrombocytopenia in extremely rare instances [223]. Despite the plethora of reports on uncomplicated pregnancies during treatment with interferon-a, no clinical trials have been conducted

in this setting and therefore no definitive data exist regarding the safety of interferon-a in pregnancy. In very small cohorts of females treated with hydroxyurea at conception or during pregnancy, no malformation and only one stillbirth in a woman with simultaneous eclampsia was reported (summarized in [134]). Nonetheless, hydroxyurea is a DNA inhibitor and should be considered contraindicated during pregnancy. One case of successful gestation during continued treatment with anagrelide has been reported [224]. However, this drug is capable of crossing the placenta and its teratogenic potential is unknown. Its use should therefore be avoided in females with the potential or intention for pregnancy. In two women with repeated abortions, successful pregnancies with delivery of healthy children after achievement of remission with a short course of busulphan have been documented [192]. This substance should however not be used outside of clinical trials and of course not within the time period of conception.

2.12.3.4 Relevance of Periodic Platelet Apheresis in Pregnancy Only rare reports on the use of platelet apharesis in pregnant women with ET exist [201, 202]. Currently prophylactic therapy or prophylactic platelet apharesis during pregnancy or delivery does not seem warranted in asymptomatic women [210]. Periodic platelet pheresis should however be considered in pregnant women with platelet countsH1,000,000/ml, with careful monitoring of both fetal and maternal circulations [202].

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2.12.3.5 Recommendations for Treatment of Pregnant Women with ET The management of patients during pregnancy is still controversial and a challenge which requires experienced hematologists and their tight collaboration with obstetricians. The decisions have to be based on the previous history of the patient in terms of thromboembolic complications, the actual risk constellation and results of previous pregnancies. Risks for the patient and the fetus have to be discussed critically and frankly with the patient and should be adequately documented. Pregnancies should be considered as high risk pregnancies and accompanied by very close follow-up of blood counts and physical examinations, as well as by frequent obstetric control visits with sonographic examinations of the placental blood flow and control of fetal growth. Large, multicentric, prospective clinical trials would be necessary in order to establish the best conduct and the ideal therapeutic approach in pregnant women with ET, but so far evidence-based data resulting form phase III clinical trials is lacking. Currently, IFN-a is generally considered as the drug of choice in high-risk patients (previous thrombohemorrhagic events, PLTH1,000,000/ml) where platelet reduction seems indicated, as well as in women with a history of recurrent abortions. Low dose aspirin with or without heparin can be additionally considered to prevent placental thrombosis, but obviously requires close monitoring [225]. Current recommendations for treatment of pregnant women with ET are summarized in Fig. 2.10.

2.13 Childhood ET Primary sporadic thrombocytosis is extremely rare in childhood, with an incidence of 1 4 per 10 million children per year [226] and is mostly diagnosed during the second decade of life [98]. To date only 100 cases of childhood ET have been published in the scientific literature [227]. The molecular and biological features of pediatric ET patients differ from adult ET and familial ET. In particular, polyclonal [45], rather than monoclonal, hematopoiesis is common, the capacity to form EEC is present less often, erythropoiesis and granulopoiesis seem normal in histopathological examinations, EPO and TPO levels are usually normal, the JAK2V617F mutation seems to be a rare event, and the incidence of cytogenetic abnormalities or mutations in MPL or TPO genes is significantly lower, if they occur at all [45, 226, 228, 229]. In 12 children diagnosed with ET, the JAK2V617F mutation could not be detected in either peripheral blood leukocytes or in separated platelets or granulocytes and

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Table 2.12: Presence of myeloproliferative and genetic markers in sporadic ET, familial ET and childhood ET [141, 169 171] Marker JAK2V617F JAK2 Exon 12 TPO mutations MPL mutations EEC formation PRV-1 mRNA Monoclonal hematopoiesis Low serum EPO levels Elevated TPO levels EPOR mutation

Adult ET þþ /þ þ þ þþ þ(þ) þþ(þ) /þ þþ n.a.

Childhood ET þ(þ)

þ þ(þ)

n.a.

Familial ET þ n.a. þþþ þþþ þ /þ /þ n.a. þþ n.a.

n.a. Not assessed

merely rare colonies among EECs were observed to bear the JAK2V617F mutation [227]. The presence of molecular, myeloproliferative and genetic markers of childhood ET in comparison to familial ETand adult ETare depicted in Table 2.12. In addition to differing genetic and biological features, children with ET also present with a different clinical picture than their adult counterpart, in that they seem to have a milder course of disease. In particular, the incidence of thromboembolic or bleeding complications seems especially low in children with ET [227], but when they do occur, then almost exclusively in children who present with platelet counts well over 1,000,000/ml. Others report thromboembolic complications at a rate similar to that of adults, affecting one-third at diagnosis and one-fifth at follow-up [98]. In light of the above depicted, the WHO diagnostic criteria for ET cannot be used for the diagnostic screening of childhood ET, which requires a specific set of diagnostic criteria [230]. These should exclude familial forms due to inherited molecular defects and consider that pathogenetic alterations commonly found in adult ET patients are detectable only in a minority of the children [230]. Thus, in children with ET, other pathogenetic mechanisms must be involved, and most likely molecular defects functionally similar to JAK2V617F remain to be detected [226]. For treatment of childhood ET, anagrelide or interferon-a may be preferred over hydroxyurea as the discussion about its potential leukemogenicity is ongoing [231] (see hydroxyurea section above (2.11.2.1.)). However, an increased vulnerability to the rare side effect of anagrelide-mediated anemia has been suggested [232].

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2.14 Familial, Hereditary Thrombocytosis Hereditary thrombocytosis is a genetically heterogeneous condition that may be due to autosomal dominant activating mutations in the TPO gene, leading to more efficient translation and subsequently higher plasma TPO-levels, or to an Ser505Asn activating mutation in the transmembrane domain of the TPO receptor MPL [62, 229, 233, 234]. These mutations result in increased expression of TPO, sustained intracellular signaling or disturbed regulation of circulating TPO. MPLG1238T is another mutation associated with altered protein expression of MPL and familial thrombocytosis, thus far only observed in African American descendants and termed MPL Baltimore [65]. This polymorphism is transmitted in an autosomal dominant pattern with incomplete penetrance, is associated with a moderate to extreme elevation of platelet counts, depending on the heterozygous or homozygous status, respectively [65]. However, in most cases the disease causing mutation remains unknown (e.g., [235]). Often the mode of inheritance seems to be autosomal dominant [236], or autosomal dominant with variable penetrance [237]. Merely one publication describes a recessive, possibly X-linked trait in an Arab family [238]. The true prevalence of hereditary thrombocytosis is possibly underestimated as it is often asymptomatic and generally not systematically sought for. Thus far, there is no evidence, that familial thrombocythemia has a more aggressive course of disease than spurious ET [236, 239, 240]. In fact, it has been proposed that familial thrombocythemia represents a different disease from ET with a more benign course [241], although self-limiting leukemoid reactions may occur [242]. Prominent thrombocytosis, bone marrow megakaryocytic hyperplasia and splenomegaly seem to be prevailing features [236, 243], whereas cytogenetic abnormalities are rare [240, 243, 244]. In the absence of specific treatment recommendations for patients with hereditary thrombocytosis, it seems wise to stick to the treatment algorithm of spurious ET patients (see respective sections above Summary Box 2, Table 2.10a b and Fig. 2.9) with special consideration of familial history of thromboembolic events and the individual risk profile of the patients.

2.15 Rare ET Variants 2.15.1 Philadelphia Chromosome (Ph)-Positive ET Philadelphia chromosome (Ph)-positive ET has been repeatedly described in the literature (e.g., [245, 246]

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and see below). This disease entity must be differentiated from CML with thrombocytosis, as well as from true ET. Patients typically present with pronounced thrombocytosis and no evidence of CML in the peripheral blood. Normal values of hemoglobin, a completely normal differential white blood cell count, megakaryocytic hyperplasia of uniformly small-sized megakaryocytes with non-lobulated or sparsely lobulated nuclei, with no increase or abnormalities of granulopoiesis or erythropoiesis, a normocellular marrow, as well as normal to elevated LAP-levels (leukocyte alkaline phosphates) and a nonenlarged spleen are common features of Ph-positive ET and are seen as diagnostic clues to the diagnosis of this borderline entity [245, 247]. These megakaryocytic morphological features are in clear contrast to the clustered, mature and enlarged, hyperploid megakaryocytes found in Ph-negative true ET, and also differ from most cases of reactive thrombocytosis. In the latter, the increased number of bone marrow megakaryocytes usually have hypersegmented nuclei, are of normal size and do not display clustering phenomena. In CML with thrombocytosis micromegakaryocytes with sparsely lobulated nuclei are characteristic (see CML chapter). Furthermore, CML is characterized by a low LAP (leukocyte alkaline phosphates) score, and an obligate transition into accelerated phase and ultimately lymphoid or myeloid blast crisis, when left untreated. Ph-positive ET seems to have a rather poor prognosis with a frequent incidence of thrombotic or hemorrhagic events, which are rare in Ph-positive CML [245]. Additionally, Ph-positive ET has a high tendency for disease progression to Ph-positive classic CML, as well as a high-risk for progression to myleofibrosis and/or blastic transformation [245, 248 250]. In contrast, the tendency for blastic transformation in true ET is extremely low. Considering all of the above, the presumption currently prevails that both Ph-positive ET and Ph-positive thrombocythemia associated with CML are early manifestations of the chronic stable phase of CML [245 247, 249, 251, 252]. This is further substantiated by the fact that quantitative indices of bone marrow morphology in Phpositive ET, in particular the small size of megakaryocytes, more closely resemble CML than ET [253]. The differences in initial clinical presentation are thought to be due to other genetic changes [246]. As some cases of CML can present in an identical fashion as ET, the Polycythemia Vera Study Group recommends mandatory routine testing for the presence of the Philadelphia chromosome [251]. Only in the absence of this translocation, can the diagnosis of true ET be made. The correct differentiation between these some-

Chap. 2


times overlapping entities is magnified in importance by the availability of targeted therapy for CML patients, without which a significant reduction in overall survival can be expected. Patients with Ph-positive ET have been successfully treated with imatinib.

2.15.2 Bcr–Abl Positive Ph-Negative ET True ET is characterized by the absence of the Philadelphia chromosome. On the molecular level however, several groups have demonstrated a high frequency of a positive Bcr Abl transcript status in peripheral blood in up to 63% of Philadelphia chromosome-negative ET patients [254 257], although these results could not be recapitulated by others [258, 259]. Whether ET expressing Bcr Abl transcripts might be considered a variant form of ET [253 255, 257, 260] or of CML [258, 260 264] has raised controversies for more than two decades. Bcr Abl positive Ph-negative ET, has been proposed to represent a separate disease category, which does not show disease progression to CML, acceleration or blast crisis. During a short follow-up period of 22 43 months, no discriminatory clinical or laboratory characteristics could be found between Bcr Abl positive or negative Ph-negative ET patients, except for higher patient age in the former subgroup [254, 255]. Furthermore, the lack of difference between Bcr Ablpositive ET and true ET with respect to bone marrow cellularity and megakaryocytes, as well as the absence of clinical features of CML, long-term uneventful follow-up, the occasional disappearance of Bcr Abl transcripts in sequential analysis, and the exclusion of masked t(9;22) by interphase-FISH indicate, that Bcr Abl positive Phnegative ET is most likely a variant of ET and not a forme fruste of CML [253, 257]. As the mere finding of Bcr Abl transcripts in the peripheral blood or bone marrow of these patients does not seem to exclude the diagnosis of ET, nor influence the course of disease, and therefore may lack clinical significance. It has been suggested, that therapeutic decisions should not be based on PCR results, when the Ph-chromosome cannot be demonstrated by cytogenetic or FISH analysis [257].

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3

Polycythemia Vera (PV) Lisa Pleyer, Daniel Neureiter, and Richard Greil

Contents Epidemiology of PV :::::::::::::::::::::::::::::::::::::::::::::::::: Should ET and PV be Considered as the Same Disease?::::::::::::::::::::::::::::::::::::::::::::::::: 3.3 Pathophysiology and Molecular Biology of PV::::::::: 3.3.1 Overview of the Role of JAK2V617F Mutations in PV::::::::::::::::::::::::::::::::::::::::::::::: 3.3.2 Overexpression of the PRV 1 Gene in PV ::::::::: 3.3.3 Other Molecular Features Implicated in the Pathogenesis of PV:::::::::::::::: 3.3.4 Exon 12 Mutations in JAK2V617F Negative PV::::::::::::::::::::::::::::::::::::::::::::::::::::: 3.3.5 Single Nucleotide Polymorphisms (SNPs) in JAK2 and EPO R Contribution of Host Genetic Variation to CMPD Phenotype ::::::::::::: 3.4 Cytogenetics in PV :::::::::::::::::::::::::::::::::::::::::::::::::::: 3.5 Clinical Features and Symptoms Occurring in PV::: 3.6 Disease Complications::::::::::::::::::::::::::::::::::::::::::::::: 3.7 Diagnosis of Polycythemia Vera (PV):::::::::::::::::::::::: 3.8 Differential Diagnosis of Polycythemia Vera :::::::::::::::::::::::::::::::::::::::::::::::: 3.8.1 Absolute Polycythemia/Erythrocytosis :::::::::::::: 3.8.2 Relative and Spurious/Apparent Polyglobulia:::: 3.8.3 Idiopathic Erythrocytosis (IE)::::::::::::::::::::::::::: 3.9 Risk Stratification of Patients with PV ::::::::::::::::::::: 3.10 Treatment of PV:::::::::::::::::::::::::::::::::::::::::::::::::::::::: 3.10.1 Phlebotomy:::::::::::::::::::::::::::::::::::::::::::::::::::: 3.10.2 Antiaggregatory Therapy :::::::::::::::::::::::::::::::: 3.10.3 Indications for Treatment and Choice of Cytoreductive Drugs in Patients with PV :::: 3.10.3.1 Hydroxyurea::::::::::::::::::::::::::::::::::: 3.10.3.2 Interferon a :::::::::::::::::::::::::::::::::::: 3.10.3.3 Pipobroman :::::::::::::::::::::::::::::::::::: 3.10.3.4 Other Cytoreductive Agents only Rarely Used Nowadays ::::::::::::::::::: 3.10.4 Allogeneic Bone Marrow Transplantation in PV ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 3.10.5 Future Treatment Possibilities JAK2 Inhibitors ::::::::::::::::::::::::::::::::::::::::::::::::::::::: 3.1 3.2

52 52 52 52 53 53 54

54 54 56 57 58 63 63 65 66 67 68 68 68 69 70 70 70 70 71 71

3.11 3.12

Polycythemia Vera in Pregnancy :::::::::::::::::::::::::::::: 71 Childhood Polycythemias/Erythrocythosis:::::::::::::::: 72 3.12.1 Primary Familial and Congenital Polycythemia ::::::::::::::::::::::::::::::::::::::::::::::::: 72 3.12.2 Sporadic Pediatric Non Familial PV ::::::::::::::: 72 3.12.3 Familial Polycythemia Vera :::::::::::::::::::::::::::: 73 3.12.4 Congenital Secondary Erythrocytosis :::::::::::::: 73 3.12.4.1 High Affinity Hemoglobin Variants :::::::::::::::::::::::::::::::::::::::::: 73 3.12.4.2 Congenital 2,3 Bisphosphoglycerate (BPG) Deficiency ::::::::::::::::::::::::::: 74 3.12.4.3 Polycythemias due to Abnormal Hypoxia Sensing::::::::::::::::::::::::::::::::::::::::::: 74

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3.1 Epidemiology of PV The annual incidence of PV is 2 per 100,000 inhabitants [1], with the median age at diagnosis being 59 70 years. The age-standardized prevalence of PV in the state of Connecticut is 22 per 100,000 [2]. Only 5% of the patients areG40 years at diagnosis. Several studies report a higher incidence in males. The prevalence is 30/ 100,000. The incidence of thrombosis is 18 per 1,000 person-years [3]. If left untreated, the survival time of patients with PV is 2 years and patients predominantly die from cardiovascular and/or cerebrovascular events. In patients treated with low-dose aspirin, cardiovascular mortality accounts for 45% of deaths, whereas hematologic transformation was the cause of death in 13% of cases [4]. If managed adequately, the life-span is increased significantly, but still depends on the efficacy and the type of treatment used, in particular whether or not potentially leukemogenic alkylating agents were prescribed. Even when optimally managed, overall life expectancy remains reduced when compared with the general population, especially in patients younger than 50 years. Although median survival of patients G50 years is 23 years, their life expectancy is markedly lower than that of the general population due to disease progression to leukemic transformation (generally not before 10 years post-diagnosis) or post-PV myelofibrosis [5]. When leukemic transformation occurs, outcome is poor, with a median survival of 2.9 months, independent of treatment strategy chosen (best supportive care or intensive chemotherapeutic treatment) [6]. PV patients older than 50 years of age have a 1.6-fold higher mortality rate than the general population, whereas those younger than 50 years have a 3.3-fold elevated mortality rate [3]. The overall mortality rate of 3.7 deaths per 100 persons per year results from a moderate risk of cardiovascular death and a high risk of death from noncardiovascular causes, mainly disease transformation [4]. A history of thrombosis has been proclaimed to be the main predictor of death [3].

3.2 Should ET and PV be Considered as the Same Disease? ET and PV share many similarities regarding disease course and survival as well as origin from a multipotent hematopoietic progenitor cell, relatively normal cellular maturation, accumulation of cells of the myeloid lineage, genotypic, molecular and phenotypic mimicry, and a tendency to evolve into each other or develop myelofibrosis (e.g., [7]). As such, the question arises as to whether these diseases are separate entities, different manifesta-

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tions of the same disease or a combination of both (see also Introduction to CMPDs chapter). Recently, it has been acknowledged, that the JAK2V617F mutation divides ET patients into two subtypes, with the JAK2V617F positive group presenting with a clinical phenotype very similar to PV. JAK2V617F positive ET patients generally tend to have higher neutrophil counts as well as higher hematocrit and hemoglobin levels than their JAK2 negative counterparts (e.g., [8]). The striking differences in clinical features between JAK2V617F positive and negative patients have been recently confirmed by others [9]. Furthermore, JAK2V617F positivity as well as allele burden predicts chemosensitivity to hydroxyurea in both ET and PV [8, 10]. In fact, JAK2V617F positive patients with ET have rates of thrombotic complications that almost reach those of patients with PV (see Chap. 2.9). This means, that the presence of JAK2V617F seems more important than the distinction between the disease entities ET and PV. Therefore, many authors consider JAK2V617F positive ET and PV to be variations of the same disease that form a biological continuum, in which the degree of erythrocytosis is modified by additional factors such as genetic background and other, as yet unidentified, biological parameters (e.g., [8]). This is in line with data generated by JAK2V617F transgenic mouse models (see respective section (1.1.4.) in introduction to CMPDs chapter) [11].

3.3 Pathophysiology and Molecular Biology of PV The pathophysiology of CMPDs in general, and of ET in particular, has already been discussed extensively in Chapters Introduction to classic CMPDs and Essential thrombocythemia, and the interested reader is referred to these sections for further details. Here, only the points of particular interest or relevance for PV will be briefly recapitulated.

3.3.1 Overview of the Role of JAK2V617F Mutations in PV As already mentioned and elaborated on extensively in the Chapter Introduction to classic CMPDs, activating JAK2V617F mutations are observed in the overwhelming majority of PV patients (H95%). Ample evidence supports a gene dosage effect, in that PV patients with homozygous JAK2V617F mutations have a more severe clinical phenotype than patients heterozygous for JAK2V617F (see Fig. 1.1 in Introduction to classic CMPDs chapter). JAK2V617F homozygote PV patients

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display a significantly higher hemoglobin level at the time of diagnosis, alongside with an increased incidence of pruritus (69% versus 38%) and a higher rate of fibrotic transformation (23% versus 2%) [12]. Mutant allele burden, i.e., the ratio of JAK2V617F to total JAK2 (JAK2V617F þ wild type JAK2), directly correlates with leukocyte count, spleen size, thrombosis risk and need for treatment in a recent analysis of PV patients [13, 14]. In PV, a time-dependent increase in JAK2V617F allele burden has been recognized, whereas this does not seem to occur in ET [15]. Testing for JAK2 mutations can be successfully performed on peripheral blood, bone marrow aspirate as well as bone marrow biopsy specimen and yields concordant results across specimen types [16]. However, 9pLOH does not completely segregate with the PV phenotype, indicating more than one (epi)genetic event is necessary for disease evolution/manifestation. Numerical gain and amplification of JAK2 has also been observed in PV, primarily in patients bearing the JAK2V617F mutation, and appears important in the pathogenesis of PV, whereas JAK2 rearrangements seem to primarily occur in MDS or AML [17]. Other JAK2 mutations associated with PV include JAK2C618R and JAK2C616Y, which are typically missed by allele-specific methods searching for the classic JAK2V617F mutation. Several other JAK2 mutations have been detected in patients with MDS, AML or ALL [18]. JAK2 exon 12 mutations are discussed separately below (Sect. 3.3.4).

3.3.2 Overexpression of the PRV-1 Gene in PV Overexpression of granulocyte PRV-1 gene, a member of the urokinase-type plasminogen activator superfamily and an allele of the CD177 gene, is observed in nearly all PV patients and coincides with endogenous erythroid colony (EEC) formation and growth factor independent proliferation. Very recently mutated JAK2V617F has been postulated to induce PRV-1 overexpression, with the latter leading to increased cell proliferation [19, 20]. PRV-1 positivity was thought to play a critical role in the pathogenesis of CMPDs in general, and also seemed to define a pathophysiologically distinct subgroup of ET at higher risk for the development of thromboembolic or microcirculatory events, as well as for disease progression to PV [21]. However, recent evidence reveals that PRV1 may not be able to discriminate between primary and secondary erythrocytosis and thrombocytosis, as originally thought [22]. Expression of PRV-1 may be increased in both patients with myeloproliferative disorders (also depicted schematically in Fig. 1.3 in Introduction to classic CMPDs chapter) and in some patients with elevated neutrophil counts secondary to acute infections and se-

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vere burns, as well as in healthy subjects given G-CSF [23]. In line with this, PRV-1 expression also closely correlates with leukocyte alkaline phosphatase scores [23]. Therefore CD177 mRNA expression may simply be a marker of increased or activated myelopoiesis, rather than a cause of CMPDs [22]. However, when looking at each disease entity separately, JAK2V617F positivity in patients with PV, but not ET or PMF, was significantly associated with PRV-1 overexpression [24]. An alleledose-dependent effect of JAK2V617F on granulocyte PRV-1 expression seems confirmed [25]. In PV patients, a concordance of increased PRV-1 expression and presence of JAK2V617F was found in 85%, of increased PRV-1/ JAK2V617F/EEC in 63%, and of PRV-1/JAK2V617F/EEC/ low Epo levels in 45%, indicating the superiority of JAK2V617F mutation screening, compared with the PRV-1 assay, for distinguishing PV from secondary erythrocytosis [25, 26]. Treatment with interferon or hydroxyurea significantly reduces and often normalizes increased PRV-1 expression levels [27]. Thus, JAK2V617F status and PRV-1 mRNA expression level appear to be suitable markers for monitoring treatment efficacy in PV patients, although this is still a matter of debate [28].

3.3.3 Other Molecular Features Implicated in the Pathogenesis of PV Recent preliminary results demonstrate that peripheral blood cells from patients with PV have distinct microRNA signatures, which seem to correlate with JAK2V617F allele burden [29]. It remains to be elucidated whether these PV-specific microRNA signatures have diagnostic and or prognostic significance. It is thus tempting to speculate, that dysregulation of microRNAs whose physiological function is to regulate hematopoiesis, may contribute to the pathophysiology of PV. GATA-1, a lineage specific transcription factor (TF), plays an essential role in normal hematopoiesis, and, along with erythropoietin, induces antiapoptotic BclxL [30, 31]. Furthermore, direct physical interaction of GATA-1 with FOG-1 is essential for normal erythroid and megakaryocytic maturation. Thus, upregulation of these factors in ET/PV does not come as a surprise (also depicted schematically in Fig. 1.3 in Introduction to classic CMPDs chapter) [32]. The anemia-inducing side effect of ACE-inhibitors first implicated a role for the renin-angiotensin system in regulating erythropoiesis, although the controlling mechanisms have as yet not been fully elucidated. ACE knockout mice develop anemia, which is fully reversible upon infusion of angiotensin-II, elegantly demonstrating that angiotensin-II directly mediates erythro-

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poiesis. Whether or not a deregulation in this system plays a pathophysiologic role in PV remains to be demonstrated.

3.3.4 Exon 12 Mutations in JAK2V617F Negative PV – Association with a Predominantly Erythroid Phenotype with Lower WBC and PLT Counts Several gain of function JAK2 exon 12 mutations are associated with a predominantly erythroid phenotype with lower WBC and PLT counts. These mutations have been identified in 27 80% of JAK2V617F negative sporadic cases of CMPDs presenting with erythrocytosis, i.e., the rare cases of JAK2V617F negative PV [33 36]. Exon 12 mutations may result in amino acid (AA) substitutions (e.g. K539L), deletions of AA-residues 537 through 543, or duplications from AA-residue 547 onwards to the JH2 pseudokinase domain [35]. The most frequent JAK2 exon 12 mutations include H538QK539L, K539L, F537K539delinsL, E543-D544del, N542-E543del, R541E543delinsK and I540-E543delinsMK (e.g., [37, 38]). In common with the JAK2V617F mutation which involves exon 14, exon 12 mutations confer EPO-independent autonomous growth as well as Epo-hypersensitivity of bone marrow colonies both in vitro and in vivo, and give rise to a myeloproliferative phenotype in a murine model of retroviral bone marrow transplantation [33]. Patients with JAK2 exon 12 mutations present with erythrocytosis, low serum Epo levels, hypercellular bone marrows as the result of erythroid hyperplasia, as well as mild megakaryocytic atypia [34], cytogenetic abnormalities, splenomegaly, and occasionally transformation to myelofibrosis [33], all of which are features of true PV. However, JAK2 exon 12 mutations seem to define a distinctive myeloproliferative syndrome, in that most patients have isolated erythrocytosis with more subtle involvement of other lineages. In contrast, most patients with JAK2V617F positive PV also demonstrate various degrees of leukocytosis and thrombocytosis [33, 35, 36]. So far the occurrence of JAK2 exon 12 and JAK2V617F mutations within the same clone seems to be mutually exclusive. However two independent clones; one with exon 12 mutation and a second one with JAK2V617F, have been found in individual patients [39]. This finding of clonal heterogeneity is compatible with the hypothesis that additional clonal events involving loci other than JAK2 are involved in the pathogenesis of PV. It is currently not clear how exon 12 mutations result in unregulated JAK2 activity, or why exon 12 mutations are more invariably associated with increased erythropoiesis (as they have not been found in ET patients so far) than

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JAK2V617F. However, compared with V617F mutations, exon 12 mutations result in stronger ligand-independent signaling through JAK2, higher levels of JAK2 are generated, resulting in elevated phosphorylation levels of downstream molecules such as ERK [33]. The absence of exon 12 mutations in patients with ET is in accordance with the widely accepted view that low levels of JAK2signaling favor thrombocytosis, whereas more-active JAK2 signaling is necessary for erythrocytosis. This may also explain why the homozygosity often detected for JAK2V617F mutation has not been detected for exon 12 mutations so far. Of note, JAK2 exon 12 mutations have also been detected in rare cases of familial PV [35], suggesting that a genetic predisposition to the acquisition of any type of JAK2 mutation is inherited, not just for JAK2V617F.

3.3.5 Single Nucleotide Polymorphisms (SNPs) in JAK2 and EPO-R – Contribution of Host Genetic Variation to CMPD Phenotype In search for genetic factors, other than JAK2V617F mutations, that result in enhanced JAK-STAT signaling, one can assume, that either germline variations in the form of SNPs or acquired mutations in cytokine receptors relevant for JAK2-mediated signal transduction, might be of importance. In fact, it has just recently been demonstrated that host genetic variation contributes to phenotypic diversity in myeloproliferative disorders. Three SNPs in the JAK2 gene (rs10758669, rs3808850, rs109747) and as well as a SNP in the EPO-R gene (rs318699) were significantly associated with PV, but not with ET or PMF, whereas three additional JAK2 SNPs (rs704636, rs10815148, rs12342421) were identified to be significantly, but reciprocally, associated with PVand ET, but not PMF [40]. All PV-alleles demonstrated a significant association with leukocytosis and a trend towards higher hemoglobin-levels, and all ET-alleles as well as some of the PV-alleles (rs10758669, rs3808850, rs109747) were significantly associated with JAK2V617F [40]. These SNPs remained associated with PV or ET, even after adjusting for JAK2V617F status. In a comparative analysis using the HapMap population as control, highly significant differences in genotype frequency were found in CMPD patients at six SNP loci within the JAK2 gene, but not within EPO-R, MPL or GCSF-R genes [40].

3.4 Cytogenetics in PV Approximately 15 44% of patients with PV show clonal abnormalities. Trisomy 8, trisomy 9 and del(20q) are the

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most frequently observed genomic alterations constituting 80% of the mutations, followed by rearrangements of 13q, as well as abnormalities of chromosome 1q, 5 and 7 [41 49] (see also Table 1.2, p. 2). The only parameter that was significantly associated with abnormal cytogenetics at diagnosis in a retrospective series of 137 PV patients, was age H60 years [44]. In this series, neither JAK2V617F allele burden nor a history of thrombosis or hemorrhages were significantly associated with adverse cytogenetics [44]. The biologic significance of clonal chromosome abnormalities in PV is still a matter of debate. The clear,

a

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non-random pattern of chromosome involvement with preference for chromosomes 1, 5, 8, 9, and 20, indicates that specific genes within these loci have an influence on initiation and propagation of the disease. However, initial presence of an abnormal karyotype seems only weekly (if at all) associated with the development of leukemia [44, 49]. In contrast, clonal evolution with acquisition of novel chromosomal anomalies during the course of the disease, seems unequivocally associated with imminent disease progression. In this sense, abnormal karyotypes are generally accepted to be strongly associated with disease progression in PV and occur in

b

Oral contraceptives should be avoided

Fig. 3.1a Scheme of normal blood cell content in a blood vessel. This is a scheme only, and obviously the number of cellular components is dramatically reduced for demonstrative reasons. b Pathophysiology of propensity to thromboembolic complications in PV

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up to 85% of these patients. The occurrence of complex anomalies involving chromosomes 5q and 7q is seemingly associated with terminal phase PV [6, 46 50]. Complete or partial trisomy of chromosome 1q, sometimes associated with translocations to chromosome 9 and/or trisomy 9p, can appear at any stage of the disease and seems to occur at a higher frequency in patients with transformation to post-PV-myelofibrosis and/or leukemia [45]. Interphase FISH (fluorescence in situ hybridization) analysis or DNA analysis by comparative genomic hybridization of blood granulocytes can be used in the monitoring of PV as an adjunct to conventional marrow cytogenetics [42, 43]. However, blood granulocytes are not always a reliable surrogate for the detection of cytogenetic changes in bone marrow myeloid cells. In routine clinical practice, cytogenetics may be helpful in the initial evaluation of those cases in which differential diagnosis against CML may be difficult. Cytogenetic analysis should routinely be performed when disease progression or transformation into secondary myelofibrosis or AML becomes clinically apparent. Otherwise, cytogenetics are not routinely assessed in PV.

b

a

c

d

Fig. 3.2 a d Variants of facial plethora in PV

3.5 Clinical Features and Symptoms Occurring in PV Complaints are usually the result of the extremely increased cell turnover in the bone marrow and/or the increased blood viscosity due to the elevation in hematocrit and the consequently disturbed blood flow (see Fig. 3.1a, b). Erythrocytosis itself can cause microcirculatory disturbances as it leads to elevation in hematocrit and is a major determinant of blood viscosity. This results in reduced blood flow, e.g., in the cerebrum, due to higher viscosity, but also likely due to compensatory adjustments resulting from an increased arterial oxygen content as a direct consequence of polyglobulia [51, 52]. Thus, patients with PV are often characterized by facial plethora (ruddy cyanosis, 67%) (see Fig. 3.2a d) and injection of conjunctival vessels and/or engorgement of the veins of the optic fundus. Further clinical investigation reveals palpable splenomegaly (70%) and hepatomegaly (40%). Among the most frequent symptoms are headache and weakness (48%), dizziness (43%), excessive sweating (33%) and acute gouty arthritis (5 20%) [53]. Temperature-dependent pruritus of varying degrees, typ-

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ically aggravated by bathing or showering, is a characteristic feature of the disease and often the patients chief complaint (43%), although the pathogenic mechanism is as yet not fully elucidated. Symptoms may be so pronounced that patients resort to regional sponging of small areas of the body as the exposure to water is so irritating. The triggering factor seems to be a sudden decrease in the temperature of the skin, e.g., after a hot bath or shower [54]. Increased blood and urine levels of histamine have been reported in patients with myeloproliferative diseases in general, and PV in particular, in various stages of the diseases. In PV patients elevated histamine levels are associated with a 12-fold, 7-fold and 4-fold increase in incidence of urticaria, pruritus and upper gastrointestinal distress, respectively, as compared to those PV patients with normal histamine levels [55]. A combination of adenosine diphosphate emerging from erythrocytes, and catecholamines released from adrenergic vasoconstrictor nerves when the skin is cooling down, is thought to stimulate aggregation of platelets in the cooling skin, with release of pruritogenic prostaglandins and serotonin [54]. In line with this hypothesis, acetylic salicylic acid (ASA, aspirin) has been shown to alleviate symptoms [54]. Whereas phlebotomy alone fails to reduce blood or urinary histamine levels, cytoreductive therapy does so effectively, thus reflecting the decrease in histamine producing white blood cells [55]. Furthermore, treatment with cyprohepatidine, but not several other histamine antagonists, alleviates pruritus and recurrent urticarial attacks in most patients [55]. Gastrointestinal symptoms are also common in PV, with a high incidence of epigastric distress, peptic ulcer disease and gastroduodenal erosions. These symptoms are mainly caused by alterations in the blood flow of the gastric mucosa due to altered blood viscosity, and/or increased histamine release from tissue basophils. Erythromelalgia is a rare complication/presenting symptom commonly associated with elevated platelet counts [56], but when present, pathognomonic for both PV and ET (for details and pathophysiologic mechanism see chapter on ET (2.7.)). Hurting acral paresthesias, which respond dramatically and rapidly to low-dose aspirin, are predominant, compatible with a pathophysiologic role for prostaglandins [57, 58]. This treatment response eo ipso may serve as a diagnostic clue for the presence of an underlying CMPD.

3.6 Disease Complications In PV associated with thrombocytosis, increased hematocrit and whole blood viscosity aggravate the plateletmediated microvascular syndrome of thrombocytemia, leading to transient visual disturbances (e.g., amaurosis

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fugax, scintillating scotoma, ophthalmic migraine), transient ischemic attacks and/or major arterial and venous thrombotic complications (for details on the pathophysiology see Chapter Introduction to CMPDs and Pathophysiology of thrombosis and microcirculatory disturbances in ET (and PV) section in the Essential thrombocythemia chapter (2.7.), as well as Fig. 3.1a and b). These effects are largely responsible for the deterioration in survival as compared to age-matched controls. In fact, the 15-year cumulative risk for thrombosis is 27% [3]. Major thrombotic events occur in 9% of patients during a median follow-up of 2.8 years [59]. Budd Chiari syndrome, portal, splenic or mesenterial vein thrombosis are examples of major thrombotic events often associated with, and may be the first manifestation of, PV, especially in young women [60]. In fact, underlying occult PV should be searched for and suspected in all patients with these diagnoses, particularly in women under the age of 45 [60 62]. Spinal cord compression from extramedullary hematopoiesis within the spinal epidural space is a rare complication with high mortality rates in patients PV [63, 64]. Irradiation with laminectomy in addition to cytoreductive treatment must be performed immediately (see Sect. 4.13.9 in PMF chapter). Similar to ET, hemorrhages can also occur in PV when the platelet counts exceed 1,000,000/ml, but the incidence is less frequent (2%) [59] (for details see section Pathophysiology of hemorrhagic complications in ET (and PV) (2.8.) in chapter on Essential thrombocythemia). PV may transform into acute myeloid leukemia with a frequency of 5.3% after a median of 14 years. Transformation into myleofibrosis occurs in up to 5.1% of cases after a median of 13 years [3] and this may only be an intermediate step on the way to full-blown acute leukemia (for more details see Risk factors for myeloid disease progression section of chapter on Essential Table 3.1: Risk for disease complications in classic CMPDs (adapted from [3]) Incidence (per 1,000 patient years) Thrombosis Leukemia Myelofibrosis Solid cancer 15 year cumulative risk Thrombosis Leukemia Myelofibrosis Solid cancer

ET (%)

PV (%)

11.6 1.2 1.6 4

17.9 5.3 5.1 5.8

17 2 4 8

27 7 6 9

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thrombocythemia (2.10.) and Table 3.1). Indeed 14% of these secondary myelofibrosis cases eventually develop AML. Refractory anemia which persists even after cessation of myelosuppressive drugs, an excessive increase in MCV in the absence of increased hydroxyurea dosages, as well as increased myeloid blasts in the differential blood count and/or increases in LDH-levels should raise suspicion of such a transformation. Age H70 years and sequential use of several cytoreductive drugs predicted the risk of acute leukemia, whereas a long disease duration and hematocrit levels above 50% seemed to predict a higher risk for myelofibrosis [59].

3.7 Diagnosis of Polycythemia Vera (PV) Typical laboratory findings in patients with PV are summarized in Table 3.2 and Fig. 3.3, whereas cytological findings in the peripheral blood and histological findings can be found in Table 3.3 and Fig. 3.4a d. Several CMPD/PV classifications have been formulated and updated within the last decade by the PVSG (Polycythemia Vera Study Group), the WHO (World Health Organization) and other groups [65 73] (e.g., see Tables 3.4 3.6), all of which remain imperfect, reflecting the phenotypic, genetic and molecular mim-

Fig. 3.3 Effect of red blood cell content on erythrocyte sedimentation rate (ESR). Left: dramatically reduced ESR, polcythe mia vera (Hkt 63%); middle: normal ESR; right: dramatically enhanced ESR; anemic patient

Table 3.2: Laboratory findings in PV Routine laboratory findings in patients with PV V617F * JAK2 mutation (H97%) * Maximally suppressed EPO levels are very characteristic Patients with normal or enhanced erythropoietin levels must be carefully evaluated for secondary causes of polyglobulia * Arterial O saturation H92% 2 * elevated serum ALP * Serum vitamin B12H900 pg/ml or unbound serum B12 binding capacityH2200 pg/ml * Absent storage iron (94%) Additional laboratory findings, not routinely assessed * EEC formation (100%) is the hallmark of PV and the most sensitive of all assays for the diagnosis of PV * Overexpression of PRV 1 gene in peripheral blood granulocytes (97 100%) * Overexpression of Bcl xL on erythroid cells * Reduced expression of MPL on platelets and megakaryocytes * Fourfold increase of serum concentrations of IGF binding protein 1, which directly stimulates erythroid progenitors via IGF 1R * Up to threefold increase in tyrosine phosphatase kinase activity EEC Endogenous erythroid colony formation; EPO erythropoie tin; ALP alkaline leukocyte phosphatase; MPL thrombopoietin receptor; IGF insulin like growth factor

Table 3.3: Cytologic and histologic findings in PV Cytological findings in the peripheral of patients with PV * Hct", Hb", red cell mass" * Normal RBC morphology (unless spent phase or additional iron deficiency) * Normoblasts * ThrombocytosisH400,000/ml (60%) * Mild to moderate leukocytosisH12,000/ml * ANCH10,000/ml in the absence of fever or infection (40%) * Mild basophilia Histological findings in the bone marrow of patients with PV * Moderate to marked hypercellularity (94%) * Trilinear proliferation pattern (panmyelosis) * Increased, loosely scattered, hyperlobulated megakaryocytes Pleomorphic size distribution Without maturation defects DD: in contrast to early stage PMF * Dilated sinusoids with intravascular hematopoiesis * Decreased or absent iron stores * Increased reticulin (in a minority of patients) DD: in contrast to PMF, where mature collagen is found Hct Hematocrit; Hb hemoglobin; RBC red blood cell; ANC abso lute neutrophil count; PMF primary myelofibrosis

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a

b

c

d

Fig. 3.4 Bone marrow histology in PV. Trilineage proliferation and hypercellularity associated with slight dominance of erythro poiesis and megakaryopoiesis, as well as dilated sinusoids (HE stain

ing: a 100, b 400). Pleomorphic aspects of megakaryocytes with small,medium, and largesized forms in a dispersed orclustered pattern (c HE staining, 400); (d NASD reaction, 400)

icry among CMPDs and the apparent difficulty in achieving consensus as to which criteria are effectively indispensable for correctly differentiating PV from apparent erythrocytosis. In this regard, the latest WHO diagnostic guidelines (see Table 3.6), which are heavily based on morphologic criteria, have been severely criticized [74]. The following and Figs. 3.5 and 3.6 will try to provide a guidance through the confusing maze of existing diagnostic criteria for PV (Tables 3.4 3.6). Polcythemia vera is the most common CMPD and is a trilinear disease with hyperproliferation of varying degrees of all myeloid lineages. Elevation of the hematocrit as well as hemoglobin and red blood cell count of often microcytic hypochromic character, is the crucial finding to be expected. Absolute erythrocytosis is the hallmark of the disease, without which the diagnosis cannot be established. However, erythrocytosis alone does not often occur in PV (0 17%) and is more often accompanied by leukocytosis (16 30%), thrombocytosis (16 30%) or both (38 57%) [53, 75]. Whereas microcytic erythrocytosis may be an important clue to the presence of an increased red blood cell mass, hemoglobin or hematocrit values alone, cannot be used as surrogate markers for the presence of erythrocytosis, as plasma volume expansion and/or

splenomegaly can mask true increases in red cell mass (reviewed in [74]). A normal hematocrit or hemoglobin level, does not necessarily signify a normal red cell mass, and the hematocrit of blood taken from a peripheral vein will not accurately reflect total body hematocrit, due to differing volume distribution of red blood cells in the microvasculature, as compared to that in large vessels [76]. Indeed, red cell mass and plasma volume determinations identified erythrocytosis in 46.5% of patients initially considered to have ET by the WHO hemoglobin criteria. In patients bearing the JAK2 mutation, this proportion rose to 64%. Other markers of PV, such as low serum erythropoietin levels or EEC formation, could not distinguish between patients with or without erythrocytosis [73]. A high hematocrit may simply be due to plasma volume contraction, and a hematocrit of H60% is necessary to distinguish plasma volume contraction from absolute erythrocytosis, when used as the sole parameter [77]. In PV, the plasma volume usually does not shrink with the development of erythrocytosis, and may even expand, particularly in women, thus masking an absolute increase in red cell mass [74]. Consequently, PV can also present as isolated leukocytosis, isolated thrombocytosis, or resemble the hyperproliferative phase of PMF, and as such, diagnosis based solely on clinical grounds can be misleading. It must also be

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Table 3.4: Diagnosis of PVaccording to updated PVSG, WHO and ECP criteria (according to, e.g., [71, 72]) Major criteria A1 Early PV RCM normal and Hct in the upper limit of normal (Hct 45 51% in males, Hct 43 48% in females) (ECP) Classic PV Hct H51%/H48% in males/females or: RCM " H25% above mean normal value or: Hb H18.5 g/dl in males, H16.5 g/dl in females A2 Absence of secondary erythrocytosis (WHO, ECP) Normal arterial oxygen saturation H92% (PVSG) A3 Splenomegaly on palpation (PVSG, WHO) CT or ultrasound H12 cm (ECP) A4 Clonal evidence other than Ph þ or BCR/ABL (WHO, ECP) A5 Spontaneous EEC formation (WHO, ECP) Minor criteria B1 Platelets H400,000/ml B2 Granulocytes H10,000/ml or leukocytes H12,000/ml B3 Bone marrow biopsy with PV picture (WHO, ECP): increased cellularity with trilineage myeloproliferation and clustering of small to giant (pleomorphic) MK Bone marrow biopsy disregarded (PVSG) B4 Low serum erythropoietin (WHO, ECP) Elevated ALP score (PVSG) Diagnosis is certain in the following scenarios * A1 þ A2 þ any other from A (WHO) * B3 plus any other criterium (ECP) * Manifest PV: increased RCM (ECP) * Latent early stage PV: RCM normal (ECP) * A1 þ A2 þ A3 (PVSG) * A1 þ A2 þ B1 þ B2 (PVSG) RCM Red cell mass; Hct hematocrit; EEC endogenous erythroid colonies; ALP alkaline leukocyte phosphatase; PVSG Polycythemia Vera Study Group; WHO World Health Organization; ECP European Clinical and Pathological

kept in mind, that the definition of normal values means that 2.5% of the normal population will have values that exceed, and 2.5% will have levels below the 95% confidence limits of reference values. This makes the diagnosis in borderline cases more difficult. Furthermore, normal regulation of hematocrit levels following exercise and other factors can cause variation in laboratory findings, and these normal range variations have to be considered, making repeat testing imperative. A young marathon athlete, for example, will naturally have a much higher hematocrit level than a 70-year old bed-ridden citizen.

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Considerable ambivalence concerning the necessity of red cell mass measurements and plasma volume determinations exists in the scientific community. The former can be measured by with 51Cr-labeled erythrocytes or 125Ilabeled serum albumin, two equivalent methods [78]. Several experts are convinced that these measurements should remain mandatory for the correct diagnosis of PV [74], at least in the absence of palpable splenomegaly, thrombocytosis or leukocytosis. However, this is although not currently recommended in the revised WHO diagnostic criteria for PV [70]. Others however argue, that red cell mass and blood volume measurements initially devised as study eligibility criteria by the PVSG became accidentally endorsed as diagnostic criteria without any systematic evaluation for diagnostic accuracy [79, 80]. In line with this, red cell mass exceeded the 98 99% limits of the reference range in 76%, 57%, 22% and 20% of patients with PV, ET, spurious/apparent polycythemia, and secondary polycythemia respectively, and decreased plasma volume was rarely seen in any of these disease entities [81]. In fact, 24% of PV patients had normal red cell mass in this retrospective analysis. Red cell mass measurement had 76% sensitivity in the diagnosis of PV and 79% specificity in distinguishing PV from other causes of polycythemia, and had no additional diagnostic value [81]. Furthermore, obesity remains a significant confounding factor in result interpretation, despite various methods used to compensate for body composition [82]. As red cell mass has been significantly correlated with both hemoglobin and hematocrit levels, which are therefore often used as substitutes, many hematologists seldom or never use red cell mass measurements in their diagnostic workup of suspected PV, also due to the availability of more biologically relevant tests (e.g., [83]). Therefore, these measurements seem inadequate to specifically differentiate between above mentioned conditions, and one might conclude that these cumbersome, time-consuming and costly tests are fraught with multiple level imprecisions, suboptimal in diagnostic accuracy and thus no longer warranted for the diagnosis of PV [80, 81]. In our opinion, JAK2 positive patients with ET should be treated as PV as soon as the critical hematocrit levels are exceeded (in the absence of other definable causes), thus making a potential additional information gained by an elevated red cell mass measurement clinically and therapeutically irrelevant. A positive JAK2 mutation assay only proves the presence of a CMPD, not necessarily PV. In addition, as mentioned above, up to 5% of true PV cases either test negative for known JAK2 mutations (V617F, exon 12), or may be associated with an allele burden too low to be measured. According to the WHO criteria, such cases are identified by three biologically relevant minor crite-

Chap. 3

Polycythemia Vera

61

Table 3.5: Diagnosis of PV according to updated WHO and ECP criteria [71] Clinical (C) criteria for suspected PV

Pathological (P) criteria diagnostic for PV

C1 Classical PV: HbH18.5 g/dl/H16.5 g/dl male/female; Hct H51%/H48% male/female

P1 BM biopsy with PV picture: increased cellularity with trilineage myeloproliferation and clustering of small to giant (pleomorphic) MK P2 BM biology: spontaneous EEC formation

C2 Early/latent PV: Hct 45% 51% male; Hct 43% 48% female C3 Low plasma Epo level C4 Persistent increase in PLT counts, Grade I: 400 1,500109/L; Grade II: H1,500109/L C5 Splenomegaly on palpation (PVSG, WHO) Splenomegaly in CT or ultrasound H12 cm (ECP) C6 Granulocytes H10,000/ml in absence of fever or infection or: leukocytes H12,000/ml and/or: increased ALP score or: increased PRV 1 C7 PLT mediated microvascular ischemic/thrombotic complications C8 Symptoms of hypervolemia C9 Pruritus, fatigue, upper abdominal complaints C10 Absence of any cause of secondary erythrocytosis

P3 molecular biology: presence of hetero or homozygous JAK2V617F mutation WHO diagnostic criteria for PV:

True PV P1 þ P2 þ P3; Classical PV P1 or P2 þ P3 þ C1; Early PV mimicking ET an ET Eri picture (cellularity G60%) or ET/PV BM picture þ C3 þ C4 (cellularity 60 80%) ECP diagnostic criteria for PV: Stage 1: masked/early PV: P1 þ C2 þ any other C criterion; Stage 2: erythrocytemic stage of PV: P1 þ P2 þ P3 þ C1 þ C3 and none of the others; Stage 3 and 4: classical PV: C1 þ P1 þ P2 or: C1 þ P2 þ any other criterion

RCM Red cell mass; Hct hematocrit; EEC endogenous erythroid colonies; EPO erythropoietin; ALP alkaline leukocyte phosphatase; BM bone marrow; WHO World Health Organization; ECP European Clinical and Pathological Table 3.6: Diagnosis of PV according to WHO revised criteria 2008 (according to [70]) Major criteria (1)

HbH18.5 g/dl (men) HbH16.5 g/dl (women)

or

Hb or HctH99th percentile of reference range for age, sex or altitude of residence

or

HbH17 g/dl (men) HbH15 g/dl (women) if associated with a sustained increase of 2 g/dl from baseline that cannot be attributed to correction of iron deficiency

or

Elevated red cell massH25% above mean normal predicted value Presence of JAK2V617F or similar mutation

(2) Minor criteria (1) (2) (3)

Bone marrow trilineage myeloproliferation Subnormal erythropoietin level EEC growth

Diagnosis of PV requires Meeting both major criteria and one minor criterion * Meeting the first major criterion and two minor criteria *

Hb Hemoglobin; Hct hematocrit; EEC endogenous erythroid colony; WHO World Health Organization

ria: CMPD consistent bone marrow histology, serum erythropoietin levels below the normal reference range and presence of endogenous erythroid colonies (EEC) (see Table 3.6). The bone marrow morphologic criteria proposed by the WHO have been criticized, based on the observation that 13% of PV patients do not have hypercellular marrow at diagnosis. Another point of criticism is that substantial interobserver variability for the histologic features exists, in addition to the apparent inability of these criteria to correctly distinguish the prefibrotic cellular phase of PMF from ET or PV (reviewed in [74]). While a maximally suppressed erythropoietin level is typical for PV, it is not always present. Furthermore, patients with ET may also present with equally low levels of erythropoietin [84 86]. Similarly, patients with secondary or hypoxic erythrocytosis only have elevated erythropoietin levels when hypoxia is severe, and often have normal serum erythropoietin levels. Therefore, a normal serum erythropoietin level does not exclude the diagnosis of PV, and a high erythropoietin level is not a conditio sine qua non for secondary erythrocytosis [87]. While EEC formation in the presence of erythrocytosis confirms an autonomous nature of the disease, EEC are

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Sustained Hct > 45% (women), Hct > 48% (men) with or without PLT > 400,000/μl

Laboratory workup History of smoking habit? Daily fluid intake? Capillary blood gas analysis Determination of serum EPO and ALP level JAK2 mutation screening BCR-ABL screening (Met-Hb) (MPL515 mutation screening) (pSTAT3 and pSTAT5 expression levels)

V617F-positive AND EPO suppressed

V617F-positive BUT EPO normal/elevated

V617F-negative BUT EPO suppressed

V617F-negative AND EPO normal/elevated

PV highly likely

PV likely

PV possible

PV unlikely

Bone marrow biopsy* encouraged, but not necessary

Bone marrow biopsy* recommended for confirmation

Bone marrow biopsy* + JAK2 exon 12 mutation screening

Bone marrow biopsy* + JAK2 exon 12 mutation screening

Search for causes of secondary polycythemia Consider congenital polycythemia with VHL mutation Chest X-ray Sonography of the abdomen Doppler duplex of renal arteries CT of cerebrum Lung function analysis Echocardiography

(emphysema? cardiomegaly?) (renal cysts? tumor?) (stenosis?) (arterio-venous-malformations?) (COPD? OSAS?) (intracardial shunts?)

Fig. 3.5 Algorithm for diagnostic workup for patients with suspected PV according to WHO diagnostic criteria (modified from [70]). including cytogenetic analysis; Hct Hematocrit; EPO erythropoietin; ALP alkaline leukocyte phosphatase; COPD chronic obstructive pulmonary disease; OSAS obstructive sleep apnea Sustained Hct > 45% (women), Hct > 48% (men) with or without PLT > 400,000/μl

Red cell mass & plasma volume measurement

Elevated red cell mass & normal plasma volume

O2 saturation >93%

PV

No absolute erythrocytosis

65a (and no CI)

Experimental trt.

Failure

BSC

Failure

FDA approved

Fig. 6.25 Simplified treatment algorithm for MDS. * Favorable ESA risk profile: serum erythropoietin levels G500 IU/l. ESA Erythropoietin stimulating agents; CI contraindications; BSC best supportive care

dyskeratesis congenita, Shwachman-Diamond syndrome, neurofibromatosis type 1, Bloom syndrome, Dubowitz syndrome, hereditary neutropenias, or the rare familial platelet disorder with propensity to AML (FDP/AML) [489]. Partial or complete monosomy 7 seems to be present in most known MDS/AML families [490]. So far, several culprit genes have been detected, whereby germline mutations in RUNX1 result in a familial platelet disorder with propensity to myeloid malignancy, and inherited mutations of CEBPA predispose to AML [487]. ZNF140 and MNDA are downregulated in some MDSfamilies [491]. However, most genetic causes still remain obscure.

6.21 Simplified Treatment Algorithm for MDS Finally, a general overview of treatment sequences in MDS is given in Fig. 6.25.

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Myelodysplastic Syndromes

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[454] Maciejewski JP, Risitano A, Sloand EM, Nunez O, Young NS (2002) Distinct clinical outcomes for cytogenetic abnormalities evolving from aplastic anemia. Blood 99: 3129 3135 [455] Sloand EM, Kim S, Fuhrer M et al. (2002) Fas mediated apoptosis is important in regulating cell replication and death in trisomy 8 hematopoietic cells but not in cells with other cytogenetic abnormalities. Blood 100: 4427 4432 [456] Jary L, Mossafa H, Fourcade C, Genet P, Pulik M, Flandrin G (1997) The 17p syndrome: a distinct myelodysplastic syn drome entity? Leuk Lymphoma 25: 163 168 [457] Marisavljevic D, Rolovic Z, Panitic M et al. (2004) Chromosome 17 abnormalities in patients with primary myelodysplastic syndrome: incidence and biologic signifi cance. Srp Arh Celok Lek 132: 10 13 [458] Yamamoto K, Nagata K, Tsurukubo Y, Morishita K, Hamaguchi H (2000) A novel translocation t(3;22)(q21; q11) involving 3q21 in myelodysplastic syndrome derived overt leukemia with thrombocytosis. Leuk Res 24: 453 457 [459] Hirabayashi K, Kawakami H, Kodaira H (2003) 3q21q26 syndrome with minor BCR/ABL type Ph chromosome. Rinsho Ketsueki 44: 1166 1171 [460] Jotterand BM, Parlier V, Muhlematter D, Grob JP, Beris P (1992) Three new cases of chromosome 3 rearrangement in bands q21 and q26 with abnormal thrombopoiesis bring further evidence to the existence of a 3q21q26 syndrome. Cancer Genet Cytogenet 59: 138 160 [461] Suzukawa K, Parganas E, Gajjar A et al. (1994) Identification of a breakpoint cluster region 30 of the ribophorin I gene at 3q21 associated with the transcriptional activation of the EVI1 gene in acute myelogenous leukemias with inv(3) (q21q26). Blood 84: 2681 2688 [462] Testoni N, Borsaru G, Martinelli G et al. (1999) 3q21 and 3q26 cytogenetic abnormalities in acute myeloblastic leuke mia: biological and clinical features. Haematologica 84: 690 694 [463] Lahortiga I, Vazquez I, Agirre X et al. (2004) Molecular heterogeneity in AML/MDS patients with 3q21q26 rearrange ments. Genes Chromosomes. Cancer 40: 179 189 [464] Wang HG, Reed JC (1998) Bc1 2, Raf 1 and mitochondrial regulation of apoptosis. Biofactors 8: 13 16 [465] Pedersen Bjergaard J, Andersen MT, Andersen MK (2007) Genetic pathways in the pathogenesis of therapy related myelodysplasia and acute myeloid leukemia. Hematology. Am Soc Hematol Educ Program 2007: 392 397 [466] Praga C, Bergh J, Bliss J et al. (2005) Risk of acute myeloid leukemia and myelodysplastic syndrome in trials of adjuvant epirubicin for early breast cancer: correlation with doses of epirubicin and cyclophosphamide. J Clin Oncol 23: 4179 4191 [467] Curtis RE, Boice JD Jr, Stovall M et al. (1992) Risk of leukemia after chemotherapy and radiation treatment for breast cancer. N Engl J Med 326: 1745 1751 [468] Le Deley MC, Suzan F, Cutuli B et al. (2007) Anthracyclines, mitoxantrone, radiotherapy, and granulocyte colony stim ulating factor: risk factors for leukemia and myelodys plastic syndrome after breast cancer. J Clin Oncol 25: 292 300 [469] Smith RE (2003) Risk for the development of treatment related acute myelocytic leukemia and myelodysplastic syn drome among patients with breast cancer: review of the literature and the National Surgical Adjuvant Breast and Bowel Project experience. Clin Breast Cancer 4: 273 279 [470] Leone G, Pagano L, Ben Yehuda D, Voso MT (2007) Therapy related leukemia and myelodysplasia: susceptibility and incidence. Haematologica 92: 1389 1398

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[471] Pedersen Bjergaard J, Pedersen M, Roulston D, Philip P (1995) Different genetic pathways in leukemogenesis for patients presenting with therapy related myelodysplasia and therapy related acute myeloid leukemia. Blood 86: 3542 3552 [472] Levine EG, Bloomfield CD (1992) Leukemias and myelo dysplastic syndromes secondary to drug, radiation, and en vironmental exposure. Semin Oncol 19: 47 84 [473] Whitlock JA, Greer JP, Lukens JN (1991) Epipodophyllotoxin related leukemia. Identification of a new subset of secondary leukemia. Cancer 68: 600 604 [474] Bloomfield CD, Archer KJ, Mrozek K et al. (2002) 11q23 balanced chromosome aberrations in treatment related mye lodysplastic syndromes and acute leukemia: report from an international workshop. Genes Chromosomes. Cancer 33: 362 378 [475] Maschek H, Kaloutsi V, Rodriguez Kaiser M et al. (1993) Hypoplastic myelodysplastic syndrome: incidence, morphol ogy, cytogenetics, and prognosis. Ann Hematol 66: 117 122 [476] Nand S, Godwin JE (1988) Hypoplastic myelodysplastic syndrome. Cancer 62: 958 964 [477] Tuzuner N, Cox C, Rowe JM, Bennett JM (1994) Bone marrow cellularity in myeloid stem cell disorders: impact of age correction. Leuk Res 18: 559 564 [478] Tuzuner N, Cox C, Rowe JM, Watrous D, Bennett JM (1995) Hypocellular myelodysplastic syndromes (MDS): new pro posals. Br J Haematol 91: 612 617 [479] Barrett J, Saunthararajah Y, Molldrem J (2000) Myelodysplastic syndrome and aplastic anemia: distinct entities or diseases linked by a common pathophysiology? Semin Hematol 37: 15 29 [480] Wong KF, So CC (2002) Hypoplastic myelodysplastic syn drome a clinical, morphologic, or genetic diagnosis? Cancer Genet Cytogenet 138: 85 88 [481] Huang TC, Ko BS, Tang JL et al. (2008) Comparison of hypoplastic myelodysplastic syndrome (MDS) with normo / hypercellular MDS by International Prognostic Scoring System, cytogenetic and genetic studies. Leukemia 22: 544 550 [482] Konoplev S, Medeiros LJ, Lennon PA, Prajapati S, Kanungo A, Lin P (2007) Therapy may unmask hypoplastic myelo dysplastic syndrome that mimics aplastic anemia. Cancer 110: 1520 1526 [483] Matsui WH, Brodsky RA, Smith BD, Borowitz MJ, Jones RJ (2006) Quantitative analysis of bone marrow CD34 cells in aplastic anemia and hypoplastic myelodysplastic syndromes. Leukemia 20: 458 462 [484] Kasahara S, Hara T, Itoh H et al. (2002) Hypoplastic myelodysplastic syndromes can be distinguished from acquired aplastic anaemia by bone marrow stem cell expression of the tumour necrosis factor receptor. Br J Haematol 118: 181 188 [485] Lambertenghi Deliliers G, Orazi A, Luksch R, Annaloro C, Soligo D (1991) Myelodysplastic syndrome with increased marrow fibrosis: a distinct clinico pathological entity. Br J Haematol 78: 161 166 [486] Maschek H, Georgii A, Kaloutsi V et al. (1992) Myelofibrosis in primary myelodysplastic syndromes: a retrospective study of 352 patients. Eur J Haematol 48: 208 214 [487] Owen C, Barnett M, Fitzgibbon J (2008) Familial myelodys plasia and acute myeloid leukaemia a review. Br J Haematol 140: 123 132 [488] Kumar T, Mandla SG, Greer WL (2000) Familial myelodys plastic syndrome with early age of onset. Am J Hematol 64: 53 58

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[505] Cremin P, Flattery M, McCann SR, Daly PA (1996) Myelodysplasia and acute myeloid leukaemia following adjuvant chemotherapy for breast cancer using mitoxantrone and methotrexate with or without mitomycin. Ann Oncol 7: 745 746 [506] Travis LB, Andersson M, Gospodarowicz M et al. (2000) Treatment associated leukemia following testicular cancer. J Natl Cancer Inst 92: 1165 1171 [507] Felix CA (1998) Secondary leukemias induced by topoisom erase targeted drugs. Biochim Biophys Acta 1400: 233 255 [508] Thiede C, Koch S, Creutzig E et al. (2006) Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML). Blood 107: 4011 4020 [509] Stirewalt DL, Kopecky KJ, Meshinchi S et al. (2001) FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia. Blood 97: 3589 3595 [510] Christiansen DH, Andersen MK, Pedersen Bjergaard J (2003) Methylation of p15INK4B is common, is associated with deletion of genes on chromosome arm 7q and predicts a

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7

Chronic Myelomonocytic Leukemia (CMML) Lisa Pleyer, Daniel Neureiter, Victoria Faber, and Richard Greil

Contents 7.1 Introduction to CMML Problems in Classification:::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 7.2 Epidemiology of CMML :::::::::::::::::::::::::::::::::::::::::::: 7.3 Molecular Biology of CMML::::::::::::::::::::::::::::::::::::: 7.4 Cytogenetics of CMML:::::::::::::::::::::::::::::::::::::::::::::: 7.5 Clinical and Laboratory Features of CMML ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 7.6 Diagnosis of CMML::::::::::::::::::::::::::::::::::::::::::::::::::: 7.7 Prognostic Factors of CMML :::::::::::::::::::::::::::::::::::: 7.8 Treatment of CMML ::::::::::::::::::::::::::::::::::::::::::::::::: 7.8.1 Best Supportive Care and Control of Myeloproliferation for CMML ::::::::::::::::::::: 7.8.2 Intensive Chemotherapy for CMML ::::::::::::::::: 7.8.3 Curative Treatment Options for CMML :::::::::::: 7.8.3.1 Allogeneic Stem Cell Transplantation:::: 7.8.3.2 Reduced Intensity Conditioning :::::::::: 7.8.4 Hypomethylating Agents in CMML:::::::::::::::::: 7.8.4.1 Azacitidine (Vidaza)::::::::::::::::::::::::: 7.8.4.2 Decitiabine (Dacogen):::::::::::::::::::::: 7.8.5 Other Treatment Options ::::::::::::::::::::::::::::::::::

7.1 Introduction to CMML – Problems in Classification 223 224 224 225 225 226 227 227 227 228 228 228 229 229 229 229 230

The term chronic myelomonocytic leukemia (CMML) indicates that all cells of the myeloid lineage are involved (myelo-), but emphasizes the prominence of monocytoid features (-mono-). The hallmarks of CMML are peripheral monocytosis H1,000/ml, with G20% bone marrow blasts and the presence of bone marrow dysplasia. CMML shares clinical and biological features with both myelodysplastic syndromes (MDS) and chronic myeloproliferative diseases (CMPDs), and may take on predominantly myelodysplastic (MD-CMML) or myelprolifearative (MP-CMML) characteristics (e.g., Ref. [1]). There is a dynamic evolution through increasing monocyte counts in approximately one-third of patients (see Fig. 7.1). MDSRA patients may develop peripheral monocytosis during the course of their disease and ultimately progress to CMML [2]. Approximately one-fifth of patients with MDS present with a monocyte count of above 10% in the peripheral blood without fulfilling the FAB/WHO criteria for the diagnosis of CMML. A high incidence of disease progression to CMML has been reported for this subgroup [3] (see Fig. 7.1). The similarities and differences between CMML and MDS as well as CMPDs vary, depending on the different forms of phenotypic appearance CMML may take. The fundamental biological characteristic feature shared by CMML and classic CMPDs is the (hyper)sensitivity of hematopoietic progenitors to growth factors, although the pathways mediating this most likely differ, as does lineage specificity. The main difference between CMML and other classical CMPDs however, is the presence of ineffective hematopoiesis, which frequently manifests as anemia and/or thrombocytopenia. The spectrum of diseases defined as CMML has defied several attempts of classification (see below). This in turn, has hindered the development of effective treatment, as the diagnosis of CMML has often been

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RA/RARS with monocytosis >10% monocytes but < 1 ,000/μl in PB

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33%

Myelodysplastic CMML

33%

>1,000/μl mono in PB

Myeloproliferative CMML > 1,200/μl mono in PB

Fig. 7.1 Static and dynamic classification of CMML (adapted from Ref. [4]). Approximately 20% of patients with MDS present with a monocyte countH10%, but do not fulfill the WHO criteria for the diagnosis of CMML. 1/3 of these patients progresses to MD CMML and 1/3 of these may go on to progress to MP CMML. RA Refractory anemia; RARS refractory anemia with ringed sideroblasts; CMML chronic myelomonocytic leukemia; PB peripheral blood

an exclusion criterion for clinical trials in MDS/ CMPDs [4]. Initially, CMML was classified as one of two dysmyelopoietic syndromes recognized by the French American British group (FAB) in their classification of acute leukemias in 1976 [5]. Dysplasia is usually present in the bone marrow, which is why CMML was then classified as a myelodysplastic syndrome. Since then, much discussion has been conducted centered on whether MDCMML and MP-CMML represent distinct subgroups or merely different stages of the same disease [6, 7]. The suggested amount of white blood cells (WBC) serving as a cut-off between the two entities, varies from 12,000/ml (IPSS) [8] to 13,000/ml (FAB) (reviewed in Ref. [4]). However, a dynamic evolution of MD-CMML to MPCMML has been reported in approximately one-third of patients [6, 9] (see Fig. 7.1). This, among other facts, led to the separation of CMML from MDS and other myeloproloferative diseases. In the novel classification CMML was placed within a separate nosological group of mixed Myelodysplastic/Myeloproliferative Disorders by the World Health Organization (WHO) [10]. CMML has been split into CMML-1 with G10% bone marrow blasts, and CMML-2 with 11 20% bone marrow blasts, in recognition of the importance and prognostic significance of bone marrow blast percentage for the course of the disease. In addition, a new category of CMML with eosinophilia was created (see Table 7.2, p. 5).

7.2 Epidemiology of CMML The continuum of monocytosis in the context of a dysplastic marrow, together with the disease progression through RA/RARS with monocytosis, MD-CMML and MP-CMML (see below and Fig. 7.1), creates a problem for the interpretation of snap-shot assessed epidemiological data. It is with this in mind, that the following numbers should be interpreted. The median age of presentation is 70 73 years, and is thus similar to that of myelodysplastic syndromes [11, 12]. Median survival is approximately 2 years [11]. A tendency for older age at presentation was found

for the myeloproliferative subtype (MP-CMML), whereas a stronger male preponderance seemed to be present for the myelodysplastic subtype (MD-CMML) [7, 13]. CMML seems to be less prevalent in the Asian population than in western countries [14, 15]. Approximately 20 30% of CMML patients experience transformation into AML after 5 years [7], and when it does, blast crisis is invariably myeloid. Only very rare reports of transformation to acute lymphoblastic leukemia exist [16].

7.3 Molecular Biology of CMML As already mentioned above, hematopoietic progenitor cells are hypersensitive to growth factors, including IL-6 and GM-CSF in patients with CMML (and JMML (juvenile myelomonocytic leukemia)) [17 20], a feature which is shared with other chronic myeloproliferative diseases, and sets CMML apart from MDS. Furthermore, progenitor growth patterns set CMML apart from various subtypes of MDS, in that granulocytic/monocytic colony forming units (GM-CFU) are normal or high. Spontaneous granulocytic/monocytic colony growth is frequently observed in vitro [17, 18, 21]. Whilst CMML resembles other CMPDs at the cellular level, more differences than similarities may be found at the molecular level. Although dysregulation of signal transduction pathways is a common feature, mechanisms differ. In contrast to the classic CMPDs, activation of the JAK/STAT pathway and mutations in the JAK2 gene are rare ( 10%) in CMML [22, 23]. When JAK2V617F mutations do occur in CMML however, they are associated with the myeloproliferative CMML subtype, splenomegaly and significantly higher hemoglobin levels and neutrophil counts than in CMML patients not bearing the JAK2V617F the mutation [23]. Rather, activation of the RAS-pathway via mutations of NRAS and KRAS genes is common in CMML and JMML [24], but less frequent in MDS, and lacking in other CMPDs [25 28]. RAS activation is a key promoter of myeloproliferation, at least in vitro [29] and

Chap. 7

Chronic Myelomonocytic Leukemia (CMML)

in various murine models [30 32]. Occurrence of mutations in NRAS and KRAS oncogenes is significantly higher in MP-CMML compared to MD-CMML [9], and may be associated with disease progression to AML [33]. A recent report found RAS pathway mutations in 46% of MP-CMML, but no such mutations in MD-CMML [24]. The same authors and others found RUNX1 (runt-related transcription factor 1) alterations in both MP-CMML and MD-CMML in 37 38% of cases, sometimes co-occurring with RAS mutations [24, 34]. CMML patients bearing RUNX1 mutations had a trend of higher risk for progression to AML, especially when the mutation occurred in the C-terminal region, with the median time to AML progression being 6.8 months versus 28.3 months for CMML patients with or without C-terminal RUNX1 mutations [34]. Deregulated apoptosis also plays a role in CMML, a hypothesis which is supported by the fact that mice deficient in the proapoptotic BH3-only protein Bid develop CMML which bears the closest resemblance clinically and morphologically to human adult CMML of all animal models described so far [35]. In JMML, mutations in RAS and PTPN11, an activator of the RAS pathway, occur in 11% and 34%, respectively [36, 37]. Mutations in the RAS regulatory protein NF1 have also been reported, mainly in JMML [38, 39]. In adult CMML however, mutations of PTPN11 are infrequent (10%) [24, 40]. Mutations of the TET2 (ten-eleven translocations) gene, which is widely expressed in hematopoietic cells, but with currently unknown function, are thought to be a pre-JAK2 event and to play an important role in the pathogenesis of classic CMPDs (e.g., Ref. [41]). However, they are only found in less than 10% of CMPD patients [42]. TET2 mutations frequently occur in CMML (37 50%) and sAML developed from MDS/ CMPD (32%), but less often in typical MDS (10 23%) [42 45]. The frequency of this mutation in this putative myeloid regulatory gene in CMML suggests an important role in the pathogenesis and prognosis of this disease. The presence of a TET2 mutation seems to be associated with a favorable prognostic outcome (4.1-fold reduced risk of death, independent factor in multivariate analysis) in MDS patients [44], presumably to the higher Hb levels observed in these patients. Interestingly however, presence of TET2 may be an adverse event in CMML patients as an association with a lower overall survival rate has been reported in a series of 88 CMML patients [45]. Angiogenesis, with a possible autocrine role for VEGF (vascular endothelial growth factor), has been recognized to play an important role in the biology of

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CMML. Clinical trials with agents targeting angiogenesis are underway (e.g., ClinicalTrials.gov identifier NCT00022048, NCT00509249, NCT00022048).

7.4 Cytogenetics of CMML No genetic predisposition has been identified for CMML so far, and no relevant etiological differences can be found between MD-CMML and MP-CMML. Cytogenetic aberrations are found in 20 42% of CMML patients (reviewed in [4]). While chromosome 7 abnormalities and trisomy 8 are the most frequently found karyotypic changes in CMML, complex karyotypes are less frequent in CMML than in MDS subtypes [46]. Monosomy 7, indicative of a very aggressive disease course, or del(7q), has been associated with MDS-type CMML. Rare occurrence of der(9)t (1;9)(q11;q34) as a sole abnormality in CMML has been reported [50]. A rare CMML subtype associated with eosinophilia and translocations of 5q33 (involving the PDGF-beta gene (platelet derived growth factor beta)) with various partners including the ETV6/TEL gene, resulting in constitutively activated PDGF-beta, has been reported. Balanced translocations include t(9;12), t(5;7) and t(5;12)(q33;p13). Deregulated proliferative signalling as well as dysregulated tyrosine kinases and eosinophila caused by these translocations, may be effectively inhibited by imatinib mesylate [47 49]. CMML appears underrepresented in therapy-related MDS [51 53].

7.5 Clinical and Laboratory Features of CMML Descriptions of the clinico-biological characteristics of patients with CMML have been published more than 2 decades ago [54]. Basically, symptoms associated with cytopenias, namely fatigue resulting from anemia, propensity for infections due to neutropenia, bleeding episodes related to thrombocytopenia, are shared with various MDS subtypes (see MDS chapter for details). In contrast to MDS however, patients with CMML, especially the myeloproliferative subtype, more often present with significant splenomegaly (approximately 20% [55]) and/or B-symptoms, i.e., nocturnal sweat, weight loss and fever, reflecting a catabolic state [4]. There does not seem to be a difference in the occurrence of spleen enlargement between CMML-1 and CMML-2 [55]. Some patients may also present with, or develop skin infiltrations. Pleuropericardial effusions are rare,

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Table 7.1: WHO criteria for the diagnosis of CMML (adapted from [112])

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a

WHO criteria for the diagnosis of CMML Major criteria * Persistant peripheral blood monocytosis H1,000/ml * Absence of Philadelphia chromosome or Bcr/Abl fusion gene * G20% myeloblasts þ monoblasts þ promonocytes in peripheral blood or bone marrow * Dysplastic changes in one or more myeloid lineages Minor criteria * Acquired clonal cytogenetic abnormality in bone marrow cells * Persistent monocytosis H3 months, after exclusion of all other causes of monocytosis For the diagnosis of CMML all 4 major criteria must be present. In the absence of myelodysplasia, or when only minimal myelodys plasia is present, the diagnosis of CMML can be made when the first 3 criteria and at least one of the minor criteria are fulfilled

but potentially life threatening complications of CMML. They may develop during uncontrolled leukocytosis, even in the absence of other sites of extramedullary hematopoiesis [56]. These effusions may result in pericardial tamponade and are poorly responsive to conventional chemotherapy, intracardial instillation of mitoxantrone or other forms of treatment [56, 57]. LDH (lactate dehydregeinse) levels may be higher in MP-CMML than in MD-CMML [6]. Hemoglobin levels also tend to be higher in MP-CMML than in MD-CMML.

b

Fig. 7.2a CMML cytology of peripheral blood. Starkly elevated numbers of monocytic cells. b CMML cytology of peripheral blood. Monocytic cells with promonocytes

7.6 Diagnosis of CMML The defining laboratory criterion for CMML is a persistent, otherwise unexplained monocytosis H1,000/ml. Table 7.1 sums up the WHO diagnostic criteria for CMML. For the diagnosis of CMML all 4 major criteria must be present. In the absence of myelodysplasia, or when only minimal myelodysplasia is present, the diagnosis of CMML can be made when the first 3 criteria and at least one of the minor criteria are fulfilled. Importantly the exclusion of an underlying Bcr-Abl driven oncogenesis is an essential component of the diagnostic work-up of patients with suspected CMML [58]. Bone marrow morphological features typically include dysplastic, hypercellular marrow with variable excess of blasts and an increased monocytic/promonocytic component (see Figs. 7.2a, b and 7.3). CMML-1 is defined by G10% bone marrow blasts and CMML-2 by 11 20% bone marrow blasts (see Table 7.2). Variable fibrosis may

20 μm

Fig. 7.3 CMML bone marrow histology. CMML showing granulopoetic hyperplasia with dominance of the monocytic cell lineage visualized by immunohistochemistry (immunohistochem istry with CD68, 400)

Chap. 7


Table 7.2: WHO classification criteria for CMML (adapted from [112]) WHO criteria for the classification of CMML CMML-1 * Bone marrow blasts G10% * Peripheral blood blasts G5% CMML-2 * Bone marrow blasts 10 19% * Peripheral blood blasts 5 19% * OR: Presence of Auer rods when blasts in peripheral blood and bone marrow G20% CMML-1 or CMML-2 with eosinophilia * Above criteria for CMML 1 or CMML 2 AND * Peripheral blood absolute eosinophil count H1,500/ml

also be present. Distinction from atypical CML may prove problematic (see Chapter 5.9) [9].

7.7 Prognostic Factors of CMML Prognostic factors of adult CMML have been reviewed as early as 1988 [59]. Prognosis in the myeloproliferative variant of CMML is generally worse compared to dysplastic CMML (reviewed in Refs. [4] and [9]). Most large single center retrospective studies report shorter overall survival (11 17 months) and slightly higher AML-transformation rates (17 52%) for MP-CMML than for MDCMML (16 31 months and 15 40%, respectively) (reviewed in [4]). Although median survival for CMML-2 (12 months) seems lower than for CMML-1 (20 months), no statistical difference in overall survival could be found in a cohort of 41 CMML patients [55]. Many single factors have been identified as negative prognostic indicators in univariate analysis, including mature monocyte counts in peripheral blood (H5,000/ml) or marrow, bone marrow monocytic nodules, age H60 years, neutrophil count (G2,000/ml), lymphocyte count (G1,000/ml), severe anemia (G6 g/dl), low platelet count and presence of circulating immature myeloid cells [55, 60, 61]. However, only lymphocyte and neutrophil count remained significant upon multivariate analysis [55]. Others have found partly contradictory results, with lymphocyte counts H2,500/ml and less severe anemia (G12 g/ dl) being predictive of poor survival [61]. Presence of bone marrow monocytic nodules has also been associated with resistance to intensive chemotherapy [60]. Interestingly, LDH-levels, gender and presence of abnormal cytogenetics do not seem relevant for prognosis [55, 61, 62]. In the past, CMML patients were often risk assessed with prognostic scoring systems developed for MDS, i.e., the international prognostic scoring system (IPSS)

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Table 7.3a:

D€ usseldorf scoring system for CMML [65, 66]

A score of 1 is allocated to each of the following parameters * Bone marrow blasts 5% * LDH H200 U/l * Hb 9 g/dl * PLT 100,000/ml

Table 7.3b: Risk stratification of CMMl patients according to the D€ usseldorf score [65] Risk group Low risk Intermediate risk High risk p value

Score points 0 1 2 3 4

Cumulative 2-year survival 91% 52% 9% 0.00005

Risk of AML 0% 19% 54% G0.05

[63]. Several prognostic scores have been developed for CMML, including the modified Bournemouth score [64], the D€usseldorf score [65, 66], the Spanish score [67] and the MD Anderson prognostic score [61, 63]. The latter however, was of limited value in community-based settings [66, 68]. In general, the D€usseldorf scoring system (see Table 7.3a, b) seems most useful to predict prognosis, but currently there is no agreement of prognostic factors for CMML due to conflicting results and limited patient numbers of most studies. However, none of the known scoring systems seems to be able to define risk groups within the MP-CMML subtype [7].

7.8 Treatment of CMML Therapy of CMML still remains challenging and unsatisfactory. So for no strategy has proven effective in prolonging survival. Effective treatment and targeted therapies have been hampered by the paucity of clinical trials looking specifically at CMML. Indications for treatment include presence of B-symptoms, symptomatic organ involvement (e.g., massive splenomegaly resulting in gastrointestinal symptoms, or presence of splenic infarctions, renal dysfunction, pulmonary involvement and/or presence of effusions), increasing blast counts, hyperleukocytosis and leukostasis, and/or worsening cytopenias.

7.8.1 Best Supportive Care and Control of Myeloproliferation for CMML Until recently, best supportive care (BSC) for the complications of bone marrow failure, with growth

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factors and/or red blood cell transfusions and/or platelet transfusions, was the mainstay of treatment in most patients with the myelodysplastic CMML subtype (MD-CMML). Control of myeloproliferation is necessary in patients with the myeloproliferative subtype (MP-CMML) with excessive leukocyte counts and/or rapidly rising leukocyte numbers, constitutional symptoms and/or symptomatic hepatosplenomegaly. Hydroxyurea (hydroxycarbamide, HU) is the treatment of choice for cytoreduction in CMML. In a randomized trial comparing HU with etoposide in 105 patients with advanced CMML, HU gave higher response rates and better survival than etoposide [69]. HU effectively reduced leukocyte counts in 84% of patients and even resulted in reduction in red blood cell transfusion requirements and/or increases in baseline hemoglobin in approximately one-third of patients [69]. However, oral etoposide demonstrated a response rate of 70% in a small series and might therefore be an alternative to HU [70], but this substance is not commonly used in CMML. Low dose cytarabine has also been used for cytoreduction in CMML [71], but even in combination with HU, responses were only partial and survival was generally poor. Thus, despite effective control of myeloproliferation, the consequences of ineffective hematopoiesis usually remain the most significant clinical problem. The topoisomerase-I inhibitor topotecan (hycamptin), alone or in combination with chemotherapy is also effective, but response durations are short [72, 73]. The use of toptotecan was encouraged by its activity in acute leukemias, as well as the knowledge that the target of the drug, topoisomerase-I, is present on all cells, regardless of the cell cycle phase. This is a factor to be considered in the slowly cycling MDS cells [74]. In a trial including 30 patients with CMML, topotecan was applied at a dosage of 10 mg/m2 i.v. for 5 days for up to 2 induction courses, with reduced doses for the followings cycles in responding patients [72]. Complete remission (CR) was seen in 33% of CMML patients, with significant side effects being severe mucositis (23%), diarrhea (17%), fever of unknown origin (85%) and documented infections (47%), undoubtedly related to the high dose of the drug [72]. Most patients responded after the first cycle, up to 10 cycles were given without cumulative toxicity, and median duration of CR was 7.5 months, with median survival being 10.5 months [72]. Dose reduction to 1.5 mg/m2 per day for 5 days in a maintenance treatment setting abolished most of the non-hematologic side effects [74]. Evaluation of the effectiveness of oral topotecan and its combinations seems particularly interesting [77 80].

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7.8.2 Intensive Chemotherapy for CMML Intensive chemotherapy alone is of little benefit, within the small numbers of cases reported. Combination of low dose topotecan (1.5 mg/m2 per day for 5 days) with cytosine arabinoside (Ara-C) (1 g/m2 per day for 5 days) led to a CR rate of 44% in CMML patients, even in those with unfavorable karyotype, with median duration of CR being 33 weeks and median survival being 44 weeks [75]. Importantly, these results have less induction mortality (G10%) than intensive chemotherapy regimens such as idarubicin/high dose AraC or FLAG (fludarbine, Ara-C þG-CSF) but with comparable responses [75, 76]. However the median survival sill remains low, with only 41 44 weeks for 27 patients with advanced CMML treated with topotecan monotherapy or combinations of topotecan and cytarabine [75]. Furthermore, these forms of treatment were not only associated with tedious application regimens necessitating hospitalization for several days, but also with numerous side effects including grade 3 and 4 mucositis and/or diarrhea (approximately 20%) as well as neutropenic infections (approximately 50%) [75, 81].

7.8.3 Curative Treatment Options for CMML 7.8.3.1 Allogeneic Stem Cell Transplantation Allogeneic stem cell transplantation, the only curative option, is only available to a minute number of patients, and outcome still remains suboptimal, with a disease free survival of 18 20% at 5 years [82, 83], even in those patients eligible for this toxic procedure. When compared with other myeloproliferative diseases such as ET, PV or PMF, patients with CMML fare a lost worse after allogeneic stem cell transplantation using conventional transplant regimens [84]. Three reports utilizing myeloablative regimen for CMML patients report disease free survival rates of 18 41% with relapse incidences ranging from 23 to 63% [82, 85, 86]. Transplantation early in the course of the disease and having few or no comorbidities seems to predict for better outcome. However, relapse remains the main cause of death. Therefore, an allogeneic transplantation should only be considered in younger patients with high-risk disease and without significant comorbidities, when a matching bone marrow donor is present. Similar to cases of advanced high-risk MDS, the role of reduction of the malignant/ dysplastic clone prior to transplantation has not been clarified so far.

Chap. 7


7.8.3.2 Reduced Intensity Conditioning Non-myeloablative reduced intensity conditioning (RIC) may be an option for CMML patients who are not suitable candidates for conventional myeloablative conditioning due to age or comorbid conditions. RIC regimen rely primarily on a graft-vs.-tumor effect to confer remissions. RIC has not been studied sufficiently in CMML patients yet, as most analyses only include a very low number CMML patients. A recent trial, with 7 included CMML patients among 141 patients with other myelodysplastic entities, reported the results of RIC with low-dose total body irradiation (TBI) fludarabine [87]. The authors demonstrated a 3-year RFS (relapse file survived) of 27% with a relapse incidence of 41% and a 3 year non-relapse mortality rate of 32% for all patients. The 3-year RFS and 3-year OS was 43% for patients with CMML [87]. Relapse was the leading cause for treatment failure. When retrospectively comparing RIC with myeloablative stem cell transplantation, comparable survival outcomes were observed, with decreased relapse rates in the myeloablative group, but at the expense of higher NRM (non-relapse mortality) [88, 89]. The lower NRM but higher relapse rate among RIC patients reinforces, that some degree of cytoreduction is necessary to control disease prior to establishing a graft-vs.-tumor effect. Therefore, primarily immunosuppressive conditioning regimens offering only minimal cytoreduction, such as low-dose TBI and/or fludarabine, may have contributed to the higher relapse rates observed in the RIC studies [87]. If a donor cannot be identified, AML-like chemotherapy with autologous stem cell or marrow transplant should be considered [11].

7.8.4 Hypomethylating Agents in CMML Only small numbers of CMML patients, all of the myelodysplastic subtype (MD-CMML), were included in the large clinical trials conducted with the hypomethylating agents azacitidine (e.g., Ref. [90]) and decitiabine [91]. The results of these trials led to the FDA approval of both substances for all types of MDS and CMML. Decitabine has not been approved by the EMEA, and in Europe, azacitidine is only approved for CMML with 10 19% bone marrow blasts and without myeloproliferation.

7.8.4.1 Azacitidine (Vidaza ) The FDA approved azacitidine for all MDS subtypes, AML with less than 30% blasts and for all types of CMML in 2004 (version 05-18-04 http://www.fda.gov/ cder/foi/label/2004/050794lbl.pdf). Four years later,

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azacitidine was approved with more restrictions by the EMEA in Europe, namely for high-risk MDS (defined by IPSS sore intermediate 2), AML with 20 30 blasts and multi-lineage dysplasia and CMML with 10 29% bone marrow blasts and without myeloproliferation (Vidaza EU Summary of Product Characteristics, available at http://www.emea.europa.eu/humandocs/PDFs/ EPAR/vidaza/H-978-PIen.pdf). In a retrospective analysis of 39 CMML patients treated with azacitidine, the largest reported cohort to date, overall response rates of 41% were seen, including 15% complete responses (CR) [92]. Average survival in responders versus nonresponders was 23.5 months versus 7.5 months [92]. Recommended treatment with azacitidine is a minimum of 4 6 28-day cycles (75 mg/m2 d1-7 applied subcutaneously). Separation of Kaplan Meier curves occurs permanently after completion of 3 cycles of azacitidine, approximately 75% of responses are seen by cycle 4 and 90% of responses by cycle 6 [93]. Treatment should be continued for as long as the benefit persists. More convenient dosing regimen have been tested in phase II randomized settings [94, 95]. Administration of azacitidine for six cycles at 75 mg/m2 s.c. per day on a 5 2 2 (5 days on, 2 days of, 2 days on), 5 2 5 (5 days on, 2 days of, 5 days on) or 5 day basis, repeated every 4 weeks revealed, that all 3 alternative dosing regimens yielded responses and toxicities consistent with the currently approved regimen (7 days) [95]. Another phase II nonrandomized study reported the outcome of 22 patients treated with 5-day azacitidine given intravenously [96]. This trial revealed similar partial response (PR) and complete response rates to what has been reported for the 7-day regimen, but with shorter overall survival, which was attributed to a higher percentage of patients with neutropenia. One must stress however, that most patients included in these studies were lower IPSS risk MDS patients, as compared to the mainly higher-risk MDS and CMML patients included in the trials that led to the approval of the 7 day regimen. The common, but usually harmless injection site reactions may be accompanied by pruritus, erythema and indurations, and may occasionally be painful. These local reactions usually persist for 2 3 days and can be alleviated in 6/10 patients by immediate topical application of evening primrose oil [97].

7.8.4.2 Decitiabine (Dacogen) Decitabine has also been approved by the FDA for the treatment of patients with all subtypes of MDS, including CMML on a schedule of 15 mg/m2 administered via i.v. infusion every 8 h for 3 days (135 mg/m2 per course), to be repeated every 6 weeks [98]. Other trials have

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revealed similar response rates when decitabine is applied intravenously at a dosage of 20 mg/m2 once a day for 5 consecutive days (100 mg/m2 per course), repeated every 4 weeks [99, 100]. Decitabine is an active substance in CMML [101]. Overall response rates of up to 73% have been reported in a small series of 11 patients [101]. Complete and overall response rates in the few patients (18 and 19) with CMML were 50 58% and 68 76%, respectively, and the 2-year survival was 48 percent [98, 100, 102]. When decitabine was given for more than a median of 9 cycles, complete response rates of 58% were achieved in patients with CMML, with overall response rates of 69% [100]. A review of 3 trials in which a total of 31 CMML patients where treated with decitabine however, revealed lower overall response rates of 45% (25% CR þ PR, and an additional 19% of patients had hematologic improvement) [103]. Outcome of patients post decitiabine failure is poor, with an overall survival of 4.3 months [104]. Adverse events included nausea and vomiting (42%), pneumonia (21%) and diarrhea (11%) [103]. A recent meta-analysis reveals the inferiority of decitiabine versus azacitidine, and that the overall survival benefit observed for azacitidine could not be established for decitabine [105, 106]. The authors assume this to be at least partly due to the shorter duration of treatment of decitabine (administered for a median of 3 4 cycles) as compared to azacitidine (administered for a median of 9 cycles) [105]. Importantly, the demethylating ability of both azacitidine and decitabine can be completely blocked by just one 500 mg tablet of hydroxyurea (hydroxycarbamide), which is a ribonucleotide reductase inhibitor and induces cell cycle arrest [107]. Therefore, concurrent treatment with HU is contraindicated when treating patients with azacitidine or decitabine. However, this antagonistic effect can be avoided with sequential treatment [107]. As the half-life of HU is only 6 h, it is sufficient to pause treatment on the day before the next treatment cycle with a hypomethylating agent is initiated. For further details on epigenetic approaches in general, and demethylating agents and potential combination partners in particular, see respective section in the MDS chapter (Chap. 6.12).

7.8.5 Other Treatment Options Imatinib mesylate (Glivec) should be considered in patients with CMML and presence of fusion genes involving TGF-b and/or PDGFR-b, as significant and durable responses have been shown in this subset of CMML patients [47 49, 108].

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Experimental agents with possible efficacy in CMML include farensyltransferase inhibitors and antiangiogeneic agents. Targeted therapy with farensyltransferase inhibitors lonafarnib or tipifarnib (Zarnestra), which inhibit RAS activation, seems promising in preliminary trials [109 113]. No specific studies of iron chelation therapy in CMML patients exist. The reader is referred to the appropriate section in the MDS chapter (Chap. 6.9.6).

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8

Rare Clonal Myeloid Diseases Thomas Melchardt, Lukas Weiss, Lisa Pleyer, Daniel Neureiter, Victoria Faber, and Richard Greil

Contents 8.1 Chronic Clonal Disorders of Mast Cells :::::::::::::::::::: 236 8.1.1 Epidemiology :::::::::::::::::::::::::::::::::::::::::::::::::: 236 8.1.2 Course of Disease and Prognosis :::::::::::::::::::::: 236 8.1.3 Pathophysiology and Molecular Biology::::::::::: 236 8.1.4 Cytogenetics :::::::::::::::::::::::::::::::::::::::::::::::::::: 237 8.1.5 Clinical Presentation :::::::::::::::::::::::::::::::::::::::: 237 8.1.6 Diagnosis and Classification of Mastocytosis:::::::::::::::::::::::::::::::::::::::::::::::: 238 8.1.6.1 Classification of Mastocytosis::::::::::::: 238 8.1.6.2 Diagnostic Work up of a Patient with Suspected Mastocytosis :::::::::::::: 239 8.1.7 Differential Diagnosis :::::::::::::::::::::::::::::::::::::: 239 8.1.8 Indications for Treatment and Therapeutic Options :::::::::::::::::::::::::::::::::: 240 8.1.8.1 Treatment of Symptoms Related to Mast Cell Degranulation::::::::::::::::: 240 8.1.8.2 Treatment of Cutaneous Mastocytosis:::: 241 8.1.8.3 Treatment Options in Indolent Systemic Mastocytosis:::::::::::::::::::::::: 241 8.1.8.4 Treatment of Aggressive Systemic Mastocytosis :::::::::::::::::::::::::::::::::::::: 241 8.2 Chronic Clonal Eosinophilic Disorders and the Idiopathic Hypereosinophilic Syndrome ::::::: 241 8.2.1 Idiopathic Hypereosinophilic Syndrome (IHES) :::::::::::::::::::::::::::::::::::::::::::: 241 8.2.1.1 Epidemiology::::::::::::::::::::::::::::::::::::: 242 8.2.1.2 Pathophysiology::::::::::::::::::::::::::::::::: 242 8.2.1.3 Cytogenetics :::::::::::::::::::::::::::::::::::::: 242 8.2.1.4 Clinical Presentation of IHES (Idiopathic Hypereosinophilic Syndrome) ::::::::::::::::::::::::::::::::::::::::: 242 8.2.1.5 Diagnosis of IHES ::::::::::::::::::::::::::::: 243 8.2.1.6 Treatment :::::::::::::::::::::::::::::::::::::::::: 243 8.2.2 Clonal Eosinophilic Diseases:::::::::::::::::::::::::::: 243 8.2.2.1 Myeloid and Lymphoid Neoplasms with Eosinophilia and Abnormalities of PDGF RA, PDGF RB or FGF R1::::: 243 8.2.2.2 Chronic Eosinophilic Leukemia (CEL), not Otherwise Specified:::::::::::::::::::::: 245 8.2.3 Causes of Reactive Eosinophilia ::::::::::::::::::::::: 245 8.2.3.1 Infections as Causes of Reactive Eosinophilia ::::::::::::::::::::::::::::::::::::::: 246 8.2.3.2 Drug Induced Reactive Eosinophilia:::::: 246 8.2.3.3 Non Malignant Diseases Associated with Eosinophilia ::::::::::::::::::::::::::::::: 246

8.2.3.4 Malignant Clonal Diseases Associated with Eosinophilia ::::::::::::::::::::::::::::::: 8.2.4 Acute Eosinophilic Leukemia (AEL) :::::::::::::::: 8.3 Disorders of Basophilic Granulocytes :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 8.3.1 Reactive Polyclonal Basophilia::::::::::::::::::::::::: 8.3.2 Clonal Basophilia Accompanying Other Myeloproliferative Neoplasms :::::::::::::::::::::::::: 8.3.3 Acute Basophilic Leukemia:::::::::::::::::::::::::::::: 8.4 Chronic Neutrophilic Leukemia (CNL) ::::::::::::::::::::: 8.4.1 Differential Diagnosis of Neutrophilia :::::::::::::: 8.5 Chronic Clonal Histiocytic Diseases::::::::::::::::::::::::::: 8.5.1 Rosai Dorfman Syndrome (Sinus Histiocytosis with Massive Lymphadenopathy):::::::::::::::::::::: 8.5.1.1 Epidemiology::::::::::::::::::::::::::::::::::::: 8.5.1.2 Clinical Features of Rosai Dorfman Syndrome :::::::::::::::::::::::::::::::::::::::::: 8.5.1.3 Diagnosis of Rosai Dorfman Syndrome :::::::::::::::::::::::::::::::::::::::::: 8.5.1.4 Histopathological Findings of Rosai Dorfman Syndrome:::::::::::::: 8.5.1.5 Treatment of Rosai Dorfman Syndrome :::::::::::::::::::::::::::::::::::::::::: 8.5.2 Langerhans Cell Histiocytosis (LCH) (Histiocytosis X, eosinophilic granuloma, Abt Letterer Siewe disease or Hand Sch€ uller Christian disease) ::::::::::::::::::::: 8.5.2.1 Epidemiology of LCH :::::::::::::::::::::::: 8.5.2.2 Prognosis and Course of Disease of LCH :::::::::::::::::::::::::::::::::::::::::::::: 8.5.2.3 Clinical Presentation :::::::::::::::::::::::::: 8.5.2.4 Diagnosis of LCH :::::::::::::::::::::::::::::: 8.5.2.5 Treatment :::::::::::::::::::::::::::::::::::::::::: 8.5.3 Malignant Histiocytosis:::::::::::::::::::::::::::::::::::: 8.5.3.1 Histiocytic Sarcoma ::::::::::::::::::::::::::: 8.5.3.2 Tumors of Langerhans Cells ::::::::::::::: 8.5.3.3 Follicular Dendritic Cell Sarcoma::::::: 8.5.3.4 Interdigitating Dendritic Cell Sarcoma::::::::::::::::::::::::::::::::::::::::::::: 8.5.3.5 Treatment ::::::::::::::::::::::::::::::::::::::::::

246 247 247 247 248 248 248 249 249 250 250 250 250 251 251

251 251 251 251 252 253 253 254 254 254 254 254

236

The classical myeloproliferative neoplasms such as essential thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF) and chronic myeloid leukemia (CML) are relatively rare disorders. Due to the long life span of most patients with these diseases however, the prevalence is quite high, so that patients with these diseases are commonly seen in hematological outpatient departments. In contrast to these disorders, many other extremely rare myeloid malignancies are known. Useful epidemiological data are rare, and most publications are retrospective case reports of very few patients. Therefore, appropriate phase-3 trials are uncommon. Consequently, treatment recommendations are almost all based on recommendations from experts in the field. New molecular techniques such as polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH) are becoming important in detecting possible molecular targets for existing or novel therapeutics, for example in idiopathic hypereosinophila (see Sect. 8.2). Therefore, a better molecular understanding of this rare entities is required to more fully understand the mechanisms behind the etiology of these diseases, and should also help to develop new treatment strategies to further improve current therapies.

8.1 Chronic Clonal Disorders of Mast Cells Mastocytosis is defined by the clonal proliferation and accumulation of neoplastic mast cells, which infiltrate one or more organs. Many subtypes of mastocytosis exist and are categorized by distribution, manifestation and course of disease.

8.1.1 Epidemiology The incidence of mastocytosis is not exactly known due to its rarity [1]. Cutaneous mastocytosis represents the most frequent form of mastocytosis. The disease occurs more often in early childhood, and may resolve spontaneously by the time of puberty. In adults however, cutaneous mastocytic lesions are usually often associated with systemic involvement of some sort and rarely involute [2].

8.1.2 Course of Disease and Prognosis The prognosis of patients with indolent systemic mastocytosis (SM) or cutaneous mastocytosis (CM) is

T. Melchardt et al.

Table 8.1: Risk stratification of independent of stage of disease [17]

systemic

mastocytosis

Factors associated with a higher risk of disease progression * * * * * * * *

Lower platelet count Elevated LDH High alkaline phosphatase Low hemoglobin levels Qualitative changes in red blood cell and/or white blood cells Hepatosplenomegaly Older age at onset of systemic symptoms Absence of cutaneous lesions (especially UP)

good, with a normal life expectancy due its indolent course [3 5]. Few patients may progress to more aggressive categories and in patients with systemic mastocytosis with an associated non-mast cell lineage clonal hematological disorder (AHNMD), such as idiopathic myelofibrosis or myeloid leukemia [6], prognosis is determined by the non-mast cell lineage disorder. Aggressive systemic mastocytosis shows a variable course of disease with a possible rapid decline and survival in mast cell leukemia (MCL) also remains very poor [5]. Clinical features reported to be associated with an increased risk of death due to disease progression include older age, elevated LDH or cytopenia. These factors seem to be independent of disease stage [3] (see Table 8.1).

8.1.3 Pathophysiology and Molecular Biology The pathogenesis is largely unknown, with no established risk factors for the development of clonal mastocytosis, and only rare familial occurrence. Mast cell disorders are defined by a clonal proliferation of mast cells and tissue infiltration of various organs [6]. Clinical symptoms often arise due to release of stored mediators including histamine, heparin, leukotrienes, prostaglandins, proteases and cytokines [8] such as SCF, chemokines, IL-5, IL-6, IL-13, and IL-16 [15], and possibly also IL-4 and IL-5 [16] (see Fig. 8.1). In anaphylactic reactions, the mediator-release is triggered when, adjacent receptors, occupied by receptor-bound IgE, are cross-linked by antigens [8] (see Fig. 8.1). In patients with mastocytosis many mediators act independently of IgE and also initiate a rapid release of these mediators, causing typical clinical symptoms. Stem cell factor (SCF) is known to play an important role in expansion of mast cells and it is still the only known mast cell growth factor [9]. Its receptor (KIT,

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Rare Clonal Myeloid Diseases

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CD2

Mast Cell Cytoplasm

Cold Cold/Heat Stress

CD25

Food Alcohol

?

NSAIDs Opiates

TLR IgE FcR I SCF

Mediator production Growth/Survival Migration/Adhesion

KIT

Allergic Reaction Anaphylaxis Vomiting/Diarrhea Flushing Hypotension

Nucleus

Production, release or degranulation of stored

Histamin Serotonin Tryptase Chemokines Cytokines Leukotrienes Prostaglandines

Fig. 8.1 Triggers and symptoms due to mediator release in mastocytosis. FceRI high affinity IgE Fc receptor; FcyRIIb IgG Fc receptor; TLR Toll like receptor; CD cluster of differentiation

antigen; SCF stem cell factor; NSAIDs non steroidal antiinflamma tory drugs

CD117) is constitutively expressed on the mast cell surface. In contrast to most other hematopoietic stem cells, which lose KIT early in their development, mast cells retain its expression throughout their life-span. Dimerization of the KIT receptor, mediated, e.g., by SCF, induces activation of many important molecules for mast cell growth and survival such as SRC-family members, phospholipase C or phosphatidylinositol 3kinase [10] (see Fig. 8.1). Mutation of CD117, especially D816V, is seen in more than 90% of all cases of systemic mastocytosis and leads to constitutive pro-survival signalling [11]. Apart from the D816V mutation, many other KIT mutations are described in mastocytosis [6]. Unfortunately, imatinib shows only activity in patients without the D816V mutation [12, 13]. Neoplastic mast cells in systemic mastocytosis also express CD2 and CD25, which are not usually seen on healthy mast cells [14]. Therefore, these expression markers are used for diagnostic purposes when mast cell disorders are suspected.

8.1.5 Clinical Presentation

8.1.4 Cytogenetics Additionally, various rare (G5% of all cases) gene defects are described in mastocytosis possibly contributing to pathogenesis of disease [6]. Aberrations involving the PDGFRA gene [6] or the translocation (4;5)(q21.1; q31.3) involving PDGFRB have been described in mastocytosis [15].

Symptoms in mastocytosis are caused by the triggered release of mediators from mast cell granules or due to mast cell organ-infiltration. Flushing, hypotension, pruritus, diarrhea, nausea and many other unspecific complaints are caused by release of mediators stored in mast cells (see Table 8.2 and Fig. 8.1) [6, 7]. These symptoms can be observed in localized as well as systemic disease and may even lead to anaphylactic reactions resulting in anaphylactic shock in some patients. Accumulation and infiltration of mast cells in the skin are the most frequent findings in patients with mastocytosis. Cutaneous infiltration (see Fig. 8.2a, b) can result in multifocal skin disease termed urticaria pigmentosa (UP) which is characterized by heterogeneous lesions such as brown macules or papules [17]. Table 8.2: Mediator released symptoms disorders [16, 25] * * * * * * * *

Flushing Hypotension Tachycardia Headache Pruritus Diarrhea Nausea Abdominal cramping

in

mast

cell

238

T. Melchardt et al.

a

50 μm

b

50 μm

c

Diffuse infiltration of the skin which results in less obvious clinical signs is the pathognomonic feature of diffuse cutaneous mastocytosis, whereas single mastocytoma lesions can also occur. All forms of lesions may show a positive Darier sign (urticaria and erythema induced by scratching) [17]. The most common hematological abnormality in systemic mastocytosis is cytopenia, which may be caused by bone marrow (see Fig. 8.2c, d) or splenic involvement. Complications may also derive from mast cell infiltration of the gastrointestinal tract, resulting in unspecific gastro-intestinal complaints such as malabsorption, usually associated with the intake of certain foods. Mast cell infiltration of liver and spleen may lead to hepatosplenomegaly and may ultimately result in liver cirrhosis, portal hypertension, gastro-esophageal varicces and ascites. Skeletal infiltration may cause lytic or osteosclerotic bone lesions [18]. Osteopenia and osteoporosis are also rare complications of mastocytosis [19] and are thought to be mediated by mast cell mediators and cytokines promoting osteoclast activity [20].

8.1.6 Diagnosis and Classification of Mastocytosis 8.1.6.1 Classification of Mastocytosis

50 μm

d

Mastocytosis, shown in a representative biopsy, is classified into cutaneous or systemic mastocytosis (SM) and solid mast cell tumors upon extent of organ involvement according to the WHO classification 2008 [17] (see Table 8.3). Cutaneous mastocytosis is categorized in urticaria pigmentosa also called macopapular cutaneous mastocytosis, diffuse cutaneous mastocytosis and solitary mastocytoma of the skin [17] (see Table 8.4). On the other hand, the diagnosis of systemic mastocytosis requires involvement of the bone marrow or another extracutaneous organ (major criterion), as well 3

20 μm

Fig. 8.2a Cutaneous mastocytosis: Cutaneous mastocytosis with diffuse and scattered aggregates of mast cells in the papillary dermis (HE staining, 200). b Cutaneous mastocytosis: Cutaneous mas tocytosis with mast cell tryptase staining (200) emphasizing the perivascular and periadnexal localization of the mast cells. c Systemic mastocytosis: Bone marrow histology. Systemic mas tocytosis revealing well circumscribed lesions of mast cells with a dominant paratrabecular and perivascular localization, as well as a heterogeneous composition of lymphocytes, eosinophiles, fibro blasts and mast cells (NASD staining, 200). d Systemic masto cytosis: Bone marrow histology. Visualization of spindle shaped mast cells by immunohistochemistry (CD 117 staining, 400)

Chap. 8


Table 8.3: Criteria for the diagnosis of cutaneous and systemic mastocytosis [17] Cutaneous mastocytosis (CM) Skin lesions demonstrating clinical findings of mastocytosis and typical histological infiltrates of mast cells in a skin biopsy. In addition, a diagnostic prerequisite of CM is the absence of features sufficient to establish diagnosis of SM * Systemic mastocytosis (SM) The diagnosis of SM can be made when the major criterion and one minor or at least three minor criteria are present Major criteria Multifocal, dense infiltration of mast cells (15 mast cells in aggregates) detected on sections of bone marrow and/or other extracutaneous organ(s) Minor criteria 1. In biopsy sections of BM or other extracutaneous organs 25% of the mast cells in the infiltrate are spindle shaped or have atypical morphology or, of all mast cells in BM aspirate smears, 25% are immature or atypical 2. Detection of an activating point mutation at codon 816 of KIT in BM, blood or another extracutaneous organ 3. Mast cells in BM, blood or other extracutaneous organs express CD2 and/or CD25 in addition to normal mast cell markers 4. Serum total tryptase persistently exceeds 20 ng/ml (unless there is an associated clonal myeloid disorder, in which case this parameter is not valid) *

BM Bone marrow Table 8.4: to [17])

Subclassification of cutaneous mastocytosis (according

1. Urticaria pigmentosa (UP)/maculopapular cutaneous mastocytosis (MPCM) 2. Diffuse cutaneous mastocytosis 3. Solitary mastocytoma of the skin

as the presence of at least one minor criterion (see Table 8.3). Minor criteria have been defined as (i) more than 25% of all infiltrating mast cells being spindle-shaped, atypical or immature, (ii) presence of an activating point mutation at codon 816 of KIT, (iii) additional expression of CD2 or CD25 on mast cells, or (iv) serum tryptase levels over 20 ng/ml [17]. Alternatively, in the absence of the major criterion, at least three minor criteria are required for the diagnosis of systemic mastocytosis (see Table 8.3). After the establishment of the diagnosis of systemic mastocytosis, the mastocytosis variant should be determined using established B and C findings. B findings such as hepatomegaly or a high serum tryptase level, but without C findings establish the diagnosis of indolent or smoldering systemic mastocytosis [17]. Bone marrow dysfunction, impaired liver function or malabsorption, defined as C findings define aggressive systemic mastocytosis. Mast cell leukemia, diagnosed by

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bone marrow infiltration of more than 20%, and mast cell sarcoma are also highly aggressive forms of systemic mastocytosis [17] (for details see Table 8.5). Some patients may fulfil diagnostic criteria of systemic mastocytosis and another clonal hematological disorder at the same time and should be categorized as systemic mastocytosis with associated non-mast cell lineage clonal hematological disorder (AHNMD) [17] (for details see Table 8.5). In most cases, myeloid neoplasms are reported as additional disorder. Eosinophilia in the peripheral blood or bone marrow however, is a known feature sometimes seen in cases of systemic mast cell diseases [21, 22]. FIP1L1/PDGFRA fusion genes have been reported in the peripheral blood cells of these patients and is associated with a response to imatinib [6, 23, 24].

8.1.6.2 Diagnostic Work-up of a Patient with Suspected Mastocytosis Diagnostic work-up and initial staging of a patient with possible mastocytosis should include a careful skin examination with biopsy of suspicious lesions, measurement of serum tryptase, a bone marrow aspirate and biopsy with mutational analysis of CD117. Additional staining of CD2 or CD25 on neoplastic mast cells by immunohistochemistry or by flow cytometry can be done to fulfill minor criteria for the diagnosis of systemic mastocytosis [6, 25]. A radiological skeletal survey, a chest X-ray and sonography of the liver and the spleen should be considered as routine diagnostic work-up, to determine possible organ involvement and/or damage due to mast cell infiltration. Depending on presenting symptoms, additional examinations may be necessary, such as gastrointestinal examination in the presence of malabsorption symptoms, to exclude mast cell infiltration or peptic ulcer disease due to release of mediators.

8.1.7 Differential Diagnosis Minor or more substantial increases in mast cell numbers are detected in tissues affected by various disorders such as IgE-associated conditions (asthma or urticaria), autoimmune diseases (rheumatoid arthritis, scleroderma, etc.), infectious diseases or neoplastic disorders [6]. Mast cells are also reported to be increased severalfold in tumor draining lymph nodes [26] or in lymphoproliferative disease [27]. Significant but smaller increases in mast cells are also reported in synovial tissues affected by rheumatoid arthritis [28] and in the

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Table 8.5:

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Criteria for variants of systemic mastocytosis (according to [17])

1. Indolent systemic mastocytosis (ISM) * Meets criteria for SM * No C findings (see below) * No evidence of an associated non mast cell lineage clonal hematological malignancy/disorder (AHNMD). In this variant, the mast cell burden is low and skin lesions are almost invariably present 1.1 Bone marrow mastocytosis As above for ISM, but bone marrow involvement, and no skin lesions 1.2 Smouldering systemic mastocytosis As above for ISM, but with 2 or more B findings and no C findings 2. Systemic mastocytosis with associated non-mast cell lineage clonal hematological malignancy/disorder (AHNMD) Meets criteria for SM and for any other hemtological neoplasm in the WHO classification 3. Aggressive systemic mastocytosis (ASM) Meets criteria for SM. One or more C findings. No evidence of mast cell leukemia. Usually without skin lesions 3.1 Lymphadenopathic mastocytosis with eosinophilia Progressive lymphadenopathy with peripheral blood eosinophilia, often with extensive bone marrow involvement and hepa tosplenomegaly, but usually without skin lesions. Cases with rearrangement of PDGFRA are excluded 4. Mast cell leukemia (MCL) Meets criteria for SM. BM biopsy shows a diffuse infiltration, usually compact, by atypical, immature mast cells. BM aspirate smear show 20% or more mast cells. In typical MCL, mast cells account for more than 10% or more of peripheral blood white cells. Rare variant: a leukemic mast cell leukemia with 10% of white blood cells being mast cells. Usually without skin lesions 5. Mast cell sarcoma (MCS) Unifocal mast cell tumor. No evidence of SM. Destructive growth pattern. High grade cytology 6. Extracutaneous mastocytoma Unifocal mast cell tumor. No evidence of SM. No skin lesions. Non destructive growth pattern. Low grade cytology B findings 1. BM biopsy showing 30% infiltration by mast cells (focal, dense, aggregates) and/or serum total tryptase level 200 ng/ml 2. Signs of dysplasia or myeloproliferation, in non mast cell lineage(s), but insufficient criteria for definitive diagnosis of a hematopoietic neoplasm (AHNMD), with normal or only slightly abnormal blood counts 3. Hepatomegaly without impairment of liver function, and/or palpable splenomegaly without hypersplenism, and/or lymphade nopathy on palpation or imaging C findings 1. BM dysfunction manifested by one or more cytopenia (ANCG1.0109/l, HbG10 g/dl or platelets G100109/l), but no obvious non mast cell hematopoietic malignancy 2. Palpable hepatomegaly with impairment of liver function, ascites and/or portal hypertension 3. Skeletal involvement with large osteolytic lesions and/or pathological fractures 4. Palpable splenomegaly with hypersplenism 5. Malabsorption with weight loss due to GI mast cell infiltrates

bone marrow of patients with chronic liver disease or renal insufficiency [29].

8.1.8 Indications for Treatment and Therapeutic Options 8.1.8.1 Treatment of Symptoms Related to Mast Cell Degranulation Due to lack of curative treatment options, lifestylemodifications for the prevention of mast cell degranulation and its resulting symptoms are of great importance. Different triggers and a huge interpatient variation in tolerances to these triggers are observed.

In general, exposure to heat, cold, stress, exercise, alcohol, hymenoptera stings (wasps, bees, hornets, etc.) and spicy food may cause degranulation in mast cells. Additionally, drugs such as opiates, non-steroidal antiinflammatory agents, general anesthetics and radiocontrast agents may also be problematic [30] (summarized in Table 8.6). H1 and H2 histamine receptor antagonists should be considered for treatment of cardiovascular or allergic symptoms, as well as for most skin specific symptoms (for a detailed review see [25]). Proton pump inhibitors or H2 histamine receptor antagonists, leukotriene antagonists and oral cromolyn sodium should be used for peptic ulcer disease, nausea and abdominal symptoms [25]. Malabsorption or

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Table 8.6: Possible triggers of mediator released symptoms in mastocytosis [16] * * * * * * * * *

Radiocontrast agents Opiates Non steroidal inflammatory agents General anesthetics Heat and cold Stress, exercise Alcohol Hymenoptera stings Spicy food

ascites may be treated with short term use of systemic steroids [31]. Acetylic salicylic acid is also recommended by some for symptoms such as flushing and tachycardia, although it may cause vascular collapse itself [15, 34]. It is mandatory, that all patients carry two doses of epinephrine in a self-injectable form with them, which should be available at all times for treatment of possible anaphylaxis due to massive release of histamines [25].

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area daily [33], can result in clinical benefit. Steroids may be added initially. Cladribine (2-Cda) has shown activity in patients with aggressive mastocytosis and may be also beneficial [34]. Splenectomy in case of hypersplenism and associated anemia and thrombocytopenia may be appropriate. Due to the common expression of the SCF-receptor KIT on mast cells, the use of KIT-targeting tyrosine kinase inhibitors (TKI), such as imatinib, has also been suggested. Unfortunately, the common D816V Kit mutation is associated with resistance against imatinib, which only shows activity and efficacy in patients without D816V [13]. Presence of FIP1L1/PDGRA fusion gene on the other hand, has been associated with response to imatinib [13]. New TK inhibitors are also currently under clinical investigation in patients, including patients with the D816V Kit mutation (ClinicalTrials.gov Identifiers: 00255346, 00233454, 00814073). In case of osteoporosis or osteolysis bisphosphonates are used for treatment [35]. In cases of severe osteoporosis IFN-a2b may be considered. Radiation therapy may be appropriate for patients with large lytic lesions, pathological fractures or resistant bone pain [16, 25].

8.1.8.2 Treatment of Cutaneous Mastocytosis Extensive cutaneous lesions can be treated with topical PUVA therapy (psoralen and ultraviolet A radiation therapy) or topical corticosteroids if needed. In severe cases systemic glucocorticoids may be considered [6].

8.1.8.3 Treatment Options in Indolent Systemic Mastocytosis Generally, indolent systemic mastocytosis needs no further specific treatment. Systemic treatment including IFN-a and steroids (for details see Sect. 8.1.8.4) may be needed in selected cases with rapid progression, for example in patients with progressive or symptomatic splenomegaly or other progressive B findings [6].

8.1.8.4 Treatment of Aggressive Systemic Mastocytosis Intensive treatment is indicated in cases of aggressive systemic mastocytosis. IFN-a can be considered in these patients. Two larger series are published reporting response and dosage in mastocytosis. IFN-a2b starting at a dose of 3 million IU s.c. three times a week and increasing to 3 5 million units per day [32], or increasing doses up to 5 million U/m2 body surface

8.2 Chronic Clonal Eosinophilic Disorders and the Idiopathic Hypereosinophilic Syndrome Blood eosinophilia as prerequisite for an eosinophilic disorder is defined as more than 600 eosinophilic granulocytes/ml [36]. Eosinophila is categorized into three groups as follows: * * *

Idiopathic eosinophilia (see Sect. 8.2.1) Clonal eosinophilia (see Sect. 8.2.2) Reactive eosinophilia (see Sect. 8.2.3)

There may be a range of cellular abnormalities regarding cell size, nuclear hypersegmentation or sparse granulation in all types of eosinophilia and thus these parameters are not very helpful diagnostic criteria [17].

8.2.1 Idiopathic Hypereosinophilic Syndrome (IHES) The diagnosis of idiopathic hypereosinophilic syndrome should be considered as a diagnosis of exclusion after a secondary eosinophilia can be considered as unlikely and no clonal marker is found.

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8.2.1.1 Epidemiology

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a

There is no evidence for inheritance and no predisposing factors are known. Idiopathic hypereosinophilic syndrome (IHES) was first described by Hardy and Anderson in 1968 [37]. The idiopathic hypereosinophilic syndrome (IHES) is reported to be more common in males than in females (9:1) and mostly affects patients between the age of 20 and 50 years [38]. These data may change in future due to the new classification which excludes many cases of hypereosinophilia, previously attributed to this group of diseases, but nowadays diagnosed as clonal eosinophilia.

8.2.1.2 Pathophysiology The role of eosinophils in inflammatory processes has not been clear for many years. They are able to produce a variety of different cytokines and chemokines. Eosinophils have the ability to release toxic granule proteins, oxygen free radicals and metalloproteases promoting fibrosis [39]. Transforming growth factors are also produced in significant amounts by eosinophils and are thought to play important roles in structural changes for example resulting in pulmonary airway remodelling in the lung [39]. The release of toxic proteins from degranulating eosinophils is responsible for the acute necrotic stage in eosinophilic mediated heart damage [40]. Release of tissue factor enhances procoagulant activity and is thus thought to be important for the pathogenesis of the thrombotic stage of cardiac disease. Replacement of such a thrombus by scar tissue would be a typical finding of the fibrotic stage [40].

8.2.1.3 Cytogenetics Evidence of clonality or reactive genesis as results from modern molecular diagnostics such as polymerase chain reaction or fluorescence in situ hybridization exclude the diagnosis of IHES. Therefore, increasing utilization of these new techniques revealed many cases formerly classified as idiopathic hypereosinophilia to be clonal diseases, necessitating reclassification.

8.2.1.4 Clinical Presentation of IHES (Idiopathic Hypereosinophilic Syndrome) The defining abnormality is sustained eosinophilia ( 1,500/ml) in the peripheral blood and bone marrow (see Fig. 8.3a, b). Patients may also present with unspe-

b

10 μm

Fig. 8.3 Hypereosinophilic syndrome cytology of peripheral blood. a Stark elevation of eosinophil count in peripheral blood smear. b Hypereosinophilia bone marrow histology. Normocellular hematopoesis with diffuse hyperplasia of eosino philic granulocytes in the circumstances of a hypereosinophilic syndrome (NASD reaction, 630)

cific systemic symptoms such as fever, night sweats or weight loss. The major affected organ systems of IHES are the cardiovascular (58%), cutaneous (56%), neurological (54%), pulmonary system (49%), the spleen (43%) as well as the liver (30%) [38]. The most common cardiac complications are restrictive cardiomyopathy and heart failure. Eosinophilia-mediated heart damage usually has three different stages. The initial stage is termed acute necrotic stage, which can only be diagnosed by myocardial biopsy. It is also thought to occur early in course of disease after a mean of 5 weeks [38]. The following thrombotic stage is characterized by the formation of thrombi in the ventricles after a mean of 10 months. The most advanced stage is the fibrotic stage and presents as restrictive cardiomyopathy [38].

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Cutaneous affections mainly comprise angioedematous and urticarial lesions or erythematous, pruritic papules and nodules. Neurological complications may be caused by thromboembolic occlusions resulting in embolic strokes or transient ischemic episodes, especially in patients with cardiac involvement. Peripheral neuropathy or central nervous system dysfunction resulting in encephalopathy or seizures may be other neurological complications [38]. In a report of 12 patients with hypereosinophilia and peripheral neuropathy, all afflicted patients had mononeuropathy multiplex or polyneuropathy with sensory symptoms as initial manifestation [41]. Chronic, non-productive cough can be a sign of cardiac or pulmonary involvement with infiltrates or bronchoconstriction [36, 38, 42].

8.2.1.5 Diagnosis of IHES According to WHO classification of 2008 the diagnosis of idiopathic hypereosinophilic syndrome (IHES) can be established if all following criteria are met [17]. *

*

*

*

Persistent blood eosinophilia 1,500 per ml for 6 months Exclusion of reactive (parasitic or allergic disease) eosinophilia Exclusion of any clonal hematological disorder or an aberrant T-cell population Signs or symptoms of end-organ dysfunction as a result of eosinophilia

In the absence of signs or symptoms of end-organ dysfunction, the diagnosis of idiopathic eosinophilia can be established [17].

8.2.1.6 Treatment Due to the rarity of this disease there is no consensus on the initial management of idiopathic hypereosinophilic syndrome. Nevertheless, the primary goal of any treatment should be to prevent further organ damage. In patients without clear evidence of end organ related damage, i.e., patients with idiopathic eosinophilia, a watchful waiting- strategy may be considered with close control of cardiac function [36, 42, 43]. However, if there is no spontaneous decrease in eosinophilia after several weeks of careful observation, we prefer the use of corticosteroids, after meticulous exclusion of any reactive causes of eosinophilia, in order to prevent early cardiac damage. When initial signs of endorgan damage, or clinical signs potentially related to eosinophilia, occur, and the criteria for IHES are fulfilled, immediate treatment should be initiated.

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Corticosteroids are the mainstay of therapy in patients with true idiopathic hypereosinophilic syndrome (i.e., chronic eosinophilia H1,500/ml lasting longer than 6 months with signs of endorgan dysfunction and after exclusion of clonal or secondary causes of eosinophilia, as opposed to idiopathic eosinophilia without endorgan dysfunction). High response rates of 70% (commonly used starting dose of 1 mg/kg/day of prednisolone) are typically observed [36, 44]. Dosage should be tapered according to eosinophil counts. However, despite good initial responses, relapse rates are high, especially when corticosteroids have been tapered too fast. However, most patients respond well to retreatment or dosereescalation. Hydroxyurea [44] and interferon-alpha [45, 46] have known clinical activity in pre-treated patients, and are useful second-line agents. Despite PDGF-R negativity, imatinib (400 mg per day) might be a possible third line therapy in those patients refractory to already mentioned treatment [36, 47]. Chemotherapy seems to be of minor importance, although many drugs such as chlorambucil, cladribine, vincristine, cytarabine or etoposide have been used, but without clinically relevant success [36]. Single cases have been reported for novel approaches using the monoclonal antibody alemtuzumab [48].

8.2.2 Clonal Eosinophilic Diseases Clonal eosinophilic diseases are characterized by the accumulation of eosinophils with a clonal marker in the peripheral blood and/or bone marrow. In recent years, there has been great improvement in the understanding of molecular pathogenesis and treatment of clonal eosinophilic diseases. Recently cytogenetic abnormalities of genes encoding platelet-derived growth factor receptor (PDGF-R) A and B or fibroblast growth factor receptor (FGF-R) 1 were identified [17]. Depending on the presence or absence of these genetic aberrations, the new 2008 WHO classification further subclassifies clonal eosinophilic diseases into (a) clonal eosinophilia (with the presence of PDGF-R and/or RGF-R aberrations) (see Sect. 8.2.2.1) and (b) chronic eosinophilic leukemia (by definition clonal disease, but without PDGF-R and/or FGFR aberrations) (see Sect. 8.2.2.2) [17].

8.2.2.1 Myeloid and Lymphoid Neoplasms with Eosinophilia and Abnormalities of PDGF-RA, PDGF-RB or FGF-R1 Many patients originally classified as chronic eosinophilic leukemia (prior to the new 2008 WHO classification),

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revealed genetic abnormalities of the PDGF-RA and B or FGF-R genes [17]. According to the current classification, these cases need to be reclassified as neoplasms with eosinophilia and abnormalities of PDGF-RA, PDGF-RB or FGF-R1 [17].

Epidemiology Clear data about incidence of these rare entities are not available. PDGF-RA-fusion genes are detected in about 60% of patients formerly classified as chronic eosinophilic leukemia [43]. The FIP1L1 PDGF-RA syndrome is reported to predominantly affect men with a peak incidence between 25 and 55 years [49].

Molecular Biology and Cytogenetics Like c-kit and FLT3, PDGF-R A and B are members of the class III receptor tyrosine kinases. There are four fusion products known involving the PDGF-RA-gene. The most common and best described is a microdeletion on chromosome 4q12, resulting in the FIP1L1 PDGF-R A fusion. This rearrangement results in a constitutively active tyrosine kinase that drives clonal proliferation of eosinophils involving several signalling pathways including phosphoinositol 3-kinase, ERK 1/2 and STAT5 [50, 51]. This fusion gene was detected in 9 of 16 patients (56%) treated for idiopathic hypereosinophilic syndrome, and importantly, seems associated with successful treatment with imatinib [52]. Rare point mutations in this gene such as the T674I variant of FIP1L1/PDGFRA associated with imatinib resistance similar to the resistance-inducing T315I mutation in Bcr Abl are also occasionally reported [53]. Other fusion genes involving PDGF-RA have been reported and include Bcr PDGF-RA resulting from t(14;22)(q12,q11), CDK5RAP2 PDGF-RA created by ins(9;4)(q33;q12q25) and KIF5B PDGF-RA [54 56]. Additionally, translocations of chromosome 5q involving PDGF-RB or translocations of chromosome 8p involving FGF-R1 are also detected in a minority of patients formerly diagnosed as chronic eosinophilic leukemia or idiopathic eosinophilic syndrome [50, 57, 58].

Clinical Presentation The clinical findings are similar to the idiopathic eosinophilic syndrome (see Sect. 8.2.1). Fatigue, splenomegaly and a high probability of eosinophilic endomyocarditis

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were the dominant clinical signs in 5 out of 8 patients in a series of patients carrying the FIP1L1 PDGF-RA fusion gene [59]. Rarely, hematologic neoplasms bearing abnormalities of PDGF-RA, PDGF-RB or FGF-R1 may present as acute myeloid leukemia, precursor T-lymphoblastic leukemia (especially PDGF-RA or FGF-R1) or chronic myelomonocytic leukemia with accompanying eosinophilia (especially PDGF-RB) [17].

Treatment Due to the new molecular findings discussed in detail above, the therapy also changed dramatically. Initially, after establishing the diagnosis and staging, it has to be evaluated whether end organ damage related to eosinophilia is present, which would represent a treatment indication. In patients without signs of end organ involvement, a strategy of watchful waiting may be considered [42, 43]. Others however tend to treatment without delay [50]. Up to now, there are no predictive markers to identify patients at a high risk for progression. Imatinib is considered as standard first line therapy in patients requiring therapy with a PDGF-RA or B aberration [43, 52, 60, 50]. Recommended starting dose is 100 mg per os daily. Some patients may require dose escalation up to 400 mg per day [43]. Treatment evaluation is thought to be sufficient by serial enumeration of eosinophil counts in order to adequately control disease response [43]. Molecular response can be evaluated by PCR or FISH analysis of the fusion gene product in specialized laboratories. Time to response is usually very short and molecular remissions are seen within few weeks [42, 43]. Some authors suggest a short course of systemic steroids prior to imatinib, especially in patients with cardiac involvement. This recommendation is mainly based upon the report of a patient with acute left ventricular failure within the first week of imatinib treatment (mediated by eosinophilic infiltration and degranulation). This patient responded well to high dose steroids [60, 61]. In patients refractory to imatinib, e.g., those bearing the mutation T6741I in the FIP1L1 PDGF-RA gene, 2nd generation tyrosine kinase inhibitors, especially nilotinib, may be considered [62]. For patients with FGF-R1 rearrangement, prognosis remains very poor and new tyrosine kinases such as PKC412, experimental drugs or allogeneic transplantation should be contemplated [43, 50]. Imatinib may not be useful in patients with FGF-R1 rearrangement [42, 50, 63].

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8.2.2.2 Chronic Eosinophilic Leukemia (CEL), not Otherwise Specified As mentioned before many patients originally classified to have chronic eosinophilic leukemia (CEL) had to be reclassified due to the presence of cytogenetic abnormalities of PDGF-R A and/or B (platelet-derived growth factor receptor) or FGF-R (fibroblast growth factor receptor) genes, according to the novel 2008 WHO classification. They are now listed as separate entities (neoplasms with eosinophilia and abnormalities of PDGF-RA, PDGF-RB or FGF-R1) [17]. Nowadays, diagnostic criteria of chronic eosinophilic leukemia, not otherwise specified include a sustained elevated eosinophil count higher than 1.5109/l, no genetical aberration typical for other myeloproliferative disorders especially involving PDGF-R or FGF-R genes and absence of signs of acute leukemia. Additionally, a clonal cytogenetic or molecular genetic abnormality or blast cells more than 2% in the peripheral blood or more than 5% in the bone marrow is required for diagnosis according to the WHO classification of 2008 after exclusion of any reactive cause of eosinophilia [17] (see Table 8.7). Karyotypic abnormalities such as þ 8 or i(17q) can be observed in cases of former hypereosinophilic syndrome and would now be classified as chronic eosinophilic leukemia, not otherwise specified according to the WHO classification of 2008 [17]. Due to the changes in the classification and diagnosis of CEL, epidemiologic data are sparse. The clinical findings are similar to the idiopathic eosinophilic syndrome (see Sect. 8.2.1) and no specific treatment recommendations currently exist. However, it seems reasonable to treat chronic eosinophilic leukemia in the same way as the idiopathic eosinophilic syndrome [61].

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8.2.3 Causes of Reactive Eosinophilia Many different pathological conditions may lead to reactive eosinophilia. Initial workup of patients presenting with eosinophilia should include a detailed case history including recent visits to foreign countries as well as the ownership of pets and physical examination. Furthermore, a profound work-up should include a bone marrow examination, sonography of lymph nodes and abdomen, an electrocardiogram as well as an echocardiography and a complete serum chemistry including autoantibody-screening and total IgE levels [43]. Elevated serum IgE levels are often found in patients with allergic diseases such as asthma, atopic dermatitis or allergic rhinitis [64], and may be used to distinguish allergic reasons of hypereosinophilia. IgE levels are also elevated in patients with parasitic infections. Repeated stool examinations for ova and parasites should be performed in all patients. Possible laboratory investigations for suspected parasitosis include serology for schistosomiasis, filariasis, strongyloidiasis and toxocariasis as anamnestically and clinically indicated [42]. These results should be used to reveal common nonhematological causes of eosinophilia, such as parasitic infections, drugs (see Table 8.8) or diseases with autoimmune or allergic etiology [36, 43]. An overview of nonmalignant causes of reactive eosinophilia is shown in Table 8.9 (adapted from [36]). Table 8.8: * * * * *

Table 8.7: Diagnostic criteria for chronic eosinophilic leukemia, not otherwise specified (CEL NOS) [17]

* * *

* *

* *

* *

*

Eosinophilia (eosinophil count H1.5109/l) No Ph chromosome or Bcr Abl1 fusion gene or other myeloproliferative neoplasm (PV, ET, PMF) or MDS/MPN (CMML or aCML) No t(5;12)(q31 35;p13) or other rearrangement of PDGFRB No FIP1L1 PDGFRA fusion gene or other rearrangement of PDGFRA No rearrangement of FGFR1 Blast cell count in peripheral blood and bone marrow less than 20% and no inv(16)(p13.1q22) or t(16;16)(p13.1;q22) or other feature diagnostic of AML Clonal cytogenetic or molecular genetic abnormality or blast cells more than 2% in the peripheral blood or more than 5% in the bone marrow

Drugs causing blood or tissue eosinophilia [42]

Dantrolene Penicillins, ampicillin, cephalosporins Ranitidine Tetracyclines Allopurinol Phenytoin Nonsteroidal anti inflammatory agents including Aspirin Beta blockers

Table 8.9: Non hematological (adapted from [36]) * * * * *

* *

reasons

of

eosinophilia

Infections (bacterial, viral or parasitic) Drugs Toxins (toxic oil syndrome, etc.) Allergy Autoimmune inflammatory conditions (Churg Strauss, eosinophilic fasciitis, etc.) Malignant tumors Endocrinopathies (Morbus Addison, etc.)

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8.2.3.1 Infections as Causes of Reactive Eosinophilia Worldwide, the most common cause of eosinophilia is reactive eosinophilia caused by helminthic infections of round-worms (nematodes), tape-worms (cestodes) or flukes (trematodes) [42]. Therefore, repeated microscopic stool examinations should be performed in all patients with eosinophila. Importantly, blood eosinophilia is only present after systemic exposure to parasites. Parasites, especially tape-worms and ascaris, in the intestinal lumen or in (Echinococcus) cysts in the lung or liver do not cause eosinophilia unless they are systemically introduced through tissue invasion or disruption of a cyst [42]. Bacterial infections with Bartonella henselae (resulting in cat scratch disease) or Brucellosis can also result in an increase of peripheral blood eosinophils [65]. Fungal infections, such as allergic bronchopulmonary aspergillosis or coccidioidomycosis have also been associated with an increase of eosinophilic granulocytes.

8.2.3.2 Drug-Induced Reactive Eosinophilia Drug-induced eosinophilia may be caused by many commonly used drugs, such as e.g., non-steroidal anti-inflammatory agents, antibiotics or allopurinol (see Table 8.8) [42]. Organs such as the kidney or the lung may also be involved in severe cases. Manifestations of drug-induced eosinophilia such as the DRESS syndrome (drug rash with eosinophilia and systemic symptoms) may be potentially fatal [42]. Rare fatal outcome is predominantly attributed to liver failure [66]. Clinical symptoms include fever, erythema, lymphadenopathy as well as putative involvement of the lung, liver or the heart. Drugs triggering this syndrome are reported to be allopurinol, cephalosporine, phenytoin, carbamazepine, phenobarbital, vancomycin, dapsone, sulfasalazine and sulfonamides. Onset of symptoms is reported to be 2 6 weeks after starting the causative drug. Systemic steroids should be considered as standard treatment after cessation of the trigger [42, 67, 68].

8.2.3.3 Non-Malignant Diseases Associated with Eosinophilia The Churg Strauss syndrome, also called allergic granulomatosis and angiitis is marked by (i) a prodromal phase with atopical features including asthma, (ii) an eosinophilic phase with prominent peripheral eosinophilia as well as infiltration of multiple organs and (iii) a vasculitic phase [69]. Eosinophilic fasciitis also often presents with elevated eosinophils, which accompanies typical symptoms such

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as symmetrical induration of the skin stiffness of extremities, fever and malaise. A biopsy is usually needed for diagnosis [70]. Toxic oil syndrome, first described in 1981, was caused by an unlabeled food oil, denatured with 2% aniline rapeseed oil, that was marketed as pure olive oil. Toxic oil syndrome was reported in more than 20,000 cases. Manifestations included fever, pulmonary symptoms and leukocytosis with eosinophilia. More than 1,500 deaths were described in this episode, almost all due to pulmonary involvement resulting in noncardiogenic pulmonary edema or pulmonary hypertension [71, 72].

8.2.3.4 Malignant Clonal Diseases Associated with Eosinophilia Lymphoid Hypereosinophilic Syndrome (LHES) [Eosinophiila with Aberrant T-cells] In alternative classifications the term lymphoid HES (LHES) is used for eosinophilia with aberrant T-cells [73] in opposite to myeloid HES, which formerly included chronic eosinophilic leukemia. These clonal T-cells have aberrant immunophenotypes, characterized, e.g., by cell surface expression of CD3þ CD4 CD8 or CD3 CD4þ [74]. These abnormal T-cells are thought to increase IgE synthesis and cause polyclonal hypergammaglobulinemia. Clinically, this variant subset is mainly characterized by cutaneous symptoms that dominate the clinical presentation. Infiltration is reported to be mainly by perivascular infiltrations of lymphocytes and eosinophils, with various degrees of epidermal involvement [74]. Nevertheless, a malignant potential of these clonal T-cells is likely, as has been shown in one series, in which 3 of 14 patients developed manifestations of cutaneous T-cell lymphoma and one was diagnosed with a Sezary syndrome [74]. Eosinophilia Associated with Chronic Myeloproliferative Diseases Eosinophilia may also be present in malignant hematologic disorders, including MDS or the classic CMPDs ET, PV, mastocytosis and/or PMF, as well as CML [42]. It has also been shown that eosinophils are part of the neoplastic clone in ETV6/ABL1 positive leukemias [75] and CML [76]. Eosinophilia Associated with Solid Tumors Eosinophilia may also occur in association with solid tumors [36]. Therefore, unexplained eosinophilia should

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also prompt a thorough search for the presence of solid tumors.

8.2.4 Acute Eosinophilic Leukemia (AEL) AEL is a very rare disease. This entity can develop in patients suffering from any hypereosinophilic syndrome or de novo and is defined by an increased blast percentage in bone marrow or peripheral blood. An exact percentage of bone marrow blasts required for diagnosis does not exist and it is not listed as an own entity in the WHO classification of 2008 [17]. Patients with AEL may develop signs of bronchospasm and endomyocardial fibrosis, similar to patients with any other hypereosinophilic syndrome. Hepatosplenomegaly is also described in these patients. Response to chemotherapy is reported to be similar to other forms of acute leukemia [17]. This rare condition has to be distinguished from secondary eosinophilia in patients with other forms of leukemia, e.g., acute myelomonocytic leukemia with inversion 16 or other abnormalities of chromosome 16 [78, 79].

8.3 Disorders of Basophilic Granulocytes Basophils are the smallest group of granulocytes and usually show absolute counts between 20 and 80/mL [80 82]. Together with mast cells, basophils are the main effectors of allergic and anaphylactic reactions. The crosslinking of high affinity IgE receptors (FceRI) on their cell surface leads to the release of histamine and other anaphylactic mediators from basophilic granules [83]. Moreover, basophils are thought to play an important role in the defence against parasitic infections [84] (see Fig. 8.4).

8.3.1 Reactive Polyclonal Basophilia A polyclonal reactive increase in basophil numbers may be seen in various inflammatory or immunologic processes, e.g., hypersensitivity accompanied by increased IgE-levels or in autoimmune disorders [85, 86].

Basophil

TLR

Cytoplasm

Allergen FcyRII FcRI

Glycoproteins

IgE

(helminthic/viral)

IgE production IL-3

CD123

Production, release or degranulation of stored

IL-3

Th2 response

IL-3 IL-4

IL-3

IL-13 ILIL-13

Release and degranulation

IL-4 IL-13

IL-4

IL-3 IL-13

IL-3

IL-4

Allergic Reaction Anaphylaxis

H

Interleukin 3 (IL-3) Interleukin 4 (IL-4) Interleukin 13 (IL-13)

LTC4

Vomiting Vomiting/Diarrhea Flushing Hypotension

Histamine (H) Leukotriene (LTC4)

Nucleus

IL-3

H H

LTC4 H

LTC4 H

Fig. 8.4 Triggers and symptoms due to mediator release in diseases associated with basophilia. Allergens or gylcoproteins derived from helminths or viruses can bind to prebound IgE on the surface of basophils. IgE crosslinking leads to release of histamine from preformed basophilic granules and induces production of leukotrienes and interleukins. In a feedback loop IL 3 can amplify this signal whereas ligation of FcyRII may block it. Bacterial and

viral components are recognized by Toll Like Receptors which in turn also leads to degranulation. Interleukin 4 and Interleukin 13 are responsible for modulating T cell response towards a Th2 response, resulting in heightened IgE production. Histamine and leukotrienes are the main effectors of systemic symptoms following basophil degranulation. FceRI high affinity IgE Fc receptor; FcyRIIb, IgG Fc receptor; TLR Toll like receptor; Th2 CD4 þ T helper cell Type 2

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8.3.2 Clonal Basophilia Accompanying Other Myeloproliferative Neoplasms

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Table 8.10: Diagnostic criteria for chronic neutrophilic leukemia (according to [17]) *

Chronic myeloproliferative neoplasms, such as polycythaemia vera (PV) [87] (see Chapter 3), may be accompanied by basophilia and slight increases in their absolute numbers are sometimes helpful to identify incipient disease. Recent evidence suggests that basophilia is at least in part composed of malignant clonal cells showing the characteristic JAK2 mutation [88]. Patients with chronic myeloid leukemia (CML) may present with substantial basophilia up to 20 90% [89]. Reports of Philadelphia-chromosome positive basophils in CML support their neoplastic origin [90] and patients with heightened basophil growth capacity are shown to have worse prognosis [91]. Therefore, basophil count has been incorporated into the Hasford prognostic score for survival in patients with CML treated with interferon-alpha [92] (see Chapter 5). Various types of acute myeloid leukemia (AML) may also show considerable basophilia, especially AML with t(6;9).

*

* *

* * *

*

8.3.3 Acute Basophilic Leukemia Although various types of acute myeloid leukemia (AML) may also show considerable basophilia, especially AML with t(6;9), acute basophilic leukemia although accounting for only G1% of all AML is an independent entity recognized by the WHO classification 2008 [17, 93]. As for other types of AML, induction therapy consists of a combination of cytarabine and an anthracycline, but due to its rarity, no specific treatment recommendations are existent for acute basophilic leukemia. Irrespective of its genesis, excessive basophilia can complicate management of the above-mentioned disorders by symptoms mainly caused by histamine or by other mediators released of dying basophils. In analogy to their pathophysiologic role in allergic reactions symptoms may include flushing, pruritus or hypotension [94, 95].

8.4 Chronic Neutrophilic Leukemia (CNL) Chronic neutrophilic leukemia (CNL) is a very rare myeloproliferative neoplasm. To date about 150 patients with this disorder have been reported [17]. Diagnosis is defined by sustained blood leukocytosis H25,000/ml with mostly mature forms of neutrophil granulocytes, hepatosplenomegaly, no evidence of any

Peripheral blood leukocytosis, WBC 25109/l Segmented neutrophils and band forms are H80% of white blood cells Immature granulocytes (promyelocytes, myelocytes, metamyelocytes) G10% of white blood cells Myeloblasts G1% of white cells Hypercellular bone marrow biopsy Neutrophilic granulocytes increased in percentage and number Myeloblasts G5% of nucleated marrow cells Neutrophilic maturation pattern normal Megakaryocytes normal or left shifted Hepatosplenomegaly No identifiable cause for physiologic neutrophilia or, if present, demonstration of clonality of myeloid cells by cytogenetic or molecular studies No infectious or inflammatory process No underlying tumor No Philadelphia chromosome or Bcr Abl1 fusion gene No rearrangement of PDF-RA, PDFG-RB or FGF-R1 No evidence of polycythemia vera, primary myelofibrosis or essential thrombocythaemia No evidence of a myelodysplastic syndrome or a myelodysplastic/myeloproliferative neoplasm No granulocytic dysplasia No myelodysplastic changes in other myeloid lineages Monocytes G1109/l

reactive cause of leukocytosis. Furthermore absence of any other myelodysplastic or myeloproliferative disorder and absence of molecular evidence for chronic myeloid leukemia (i.e., absence of Bcr Abl transcripts or Ph chromosome) is mandatory. Other molecular markers, such as JAK2 and rearrangement of PDGFRA, PDGF-RB or FGF-R1 may also not be present [17] (see Table 8.10). Bone marrow histology reveals granulocytic hyperplasia with pronounced hypercellularity and dominance of mature segmented granulocytes without blasts (Fig. 8.5). The best documented group of patients is a series of 12 cases published by the Mayo Clinic in 2005 [96]. All patients were negative for the Bcr Abl fusion gene and displayed no monocytosis or eosinophilia. The leukocyte alkaline phosphatase (LAP) score was elevated in the broad majority of the cases [96], in contrast to chronic myeloid leukemia (see also Table 8.12). However, ALP may be elevated in other chronic myeloid neoplasms (see Table 8.12) and therefore these must be excluded. Initial therapy with hydroxyurea showed a clinical response rate of 75% with a reduction of leukocyte count or spleen size [96]. Second line therapy consisted of low-

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Table 8.12:

Diseases with abnormal ALP levels [149 151]

Elevated ALP * Inflammatory disorders * Infections * Pregnancy * PV * CIMF (25%) Decreased ALP * CML * PNH * Hypophosphatemia * CIMF (25%) * Androgen abuse 20 μm

Fig. 8.5 Chronic neutrophilic leukemia bone marrow histology. CNL being characterized by pronounced hypercellularity of a packed hematopoiesis with dominance of segmented granulo cytes and without blasts (NASD reaction, 400)

Table 8.11: [42] * * * * * * * * * * * * * *

Non malignant reactive causes of neutrophilia

Infections [143] Smoking [98] Rheumatoid inflammation [144] Asplenia [145] Stress and exercise [146] Glucocorticoids [147] Lithium [148] G CSF Hereditary neutrophilia Asplenia Chronic idiopathic neutrophilia Sweets syndrome CD11/18 deficiency Pseudoneutrophilia due to maldistribution/demargination

dose cytarabine, 6-thioguanine, 2-chlorodeoxyadenosine, interferon-a or acute myeloid leukemia induction-type chemotherapy. Nevertheless, prognosis remained poor with a median survival of 2 years [96]. Similarly bad survival rates, with a median survival of 30 months were shown in another patient cohort. This cohort showed a high incidence of cerebral hemorrhages and/or clonal evolution during the course of disease [97]. More than 80% of the patients had a normal karyotype at diagnosis and in 25% of the cases clonal evolution with occurrence of novel cytogenetic aberrations during cytoreductive therapy was detected. Allogeneic transplantation has been performed in selected patients with disease free survival of more than 6 years in 2 of 5 patients [96].

8.4.1 Differential Diagnosis of Neutrophilia Neutrophilia is a common finding of many pathological processes in the body, aside from chronic neutrophilic leukemia and acute myeloid leukemia. It can arise due to various infectious diseases, chronic inflammation, exercise, drugs, asplenia or many other unspecific reasons in dependance of ALP levels (see Table 8.11). It is therefore necessary to exclude all secondary causes of neutrophilia in patients presenting with excess amounts of neutrophils. Mild to moderate neutrophilia is a common result of smoking. Studies showed a leukocyte count 27% higher in current smokers and this effect can remain for several years after cessation [98]. It was also shown that neutrophils were increased by the number of cigarettes smoked per day [99].

8.5 Chronic Clonal Histiocytic Diseases Monocytes, Langerhans cells and dermal and interstitial dendritic cells are the main groups summarized with the term histiocytes [100]. These cells arise from a common CD34-positive progenitor cell in the bone marrow and develop either along the CD14-negative or CD14-positive pathway, depending on the specific cytokine milieu in the bone marrow (see Fig. 8.6). CD14-positive cells have the ability to develop into macrophages or into interstitial dendritic cells, whereas CD14-negative precursor cells develop into Langerhans cells, which are specialized dendritic cells. Interdigitating dendritic cells also arise from the Langerhans cells. All these cells have the ability to process antigens, migrate to lymphoid organs to initiate immune responses, and express co-stimulatory molecules important for activation of lymphocytes [17, 100 102] (see Fig. 8.6).

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Myeloid stem cell

Mesenchymal

CD34+

stem cell

Monocyte

CD1a+

CD14+ CD1a-

CD14-

Macrophage CD68+, CD163+

Interstitial DC FXIIIa+, CD68+ DCSIGN+

Langerhans cells S100+, Langerin+

Follicular DC CD21+, CD23+, CD35+, Desmoplakin+

Fig. 8.6 Development of histiocytes (DC, dendritic cell)

In contrast to above-mentioned cells which derive from a myeloid stem cell, follicular dendritic cells derive from mesenchymal stem cells and reside in B-cell follicles, where they present encountered antigens to Bcells [103]. Histiocytic neoplasms are derived from histiocytes or dendritic cells and are among the rarest tumors affecting lymphoid tissues, comprising less than 1% of tumors of the lymphoid tissue [100, 104]. This heterogeneous group also includes Langerhans cell histiocytosis and malignant histiocytic disorders. Due to its clinical appearance Rosai Dorfman disease was formerly also classified as clonal disease of histiocytes and will also be discussed in this chapter, despite its polyclonal nature.

8.5.1 Rosai–Dorfman Syndrome (Sinus Histiocytosis with Massive Lymphadenopathy) The Rosai Dorfman Syndrome is a polyclonal benign disorder with occasionally malignant clinical presentation. This disease entity was first described by Rosai and Dorfman in 1969, who analyzed 4 histopathological cases, which were formerly diagnosed as malignant reticuloendotheliosis [105]. Causative virus infections, in particular Parvovirus B19 and EBV, are suspected, although until now no clear cause could be identified [106, 107]. So far, no cytogenetic abnormalities have been reported.

8.5.1.1 Epidemiology This rare disease occurs mostly within the first 3 decades of life, and its course is often self-limited with spontaneous remission within 9 18 months [108]. Nevertheless, cases with fatal outcome have been documented [109].

8.5.1.2 Clinical Features of Rosai–Dorfman Syndrome Typical findings include massively enlarged, painless cervical lymph nodes, which may present as isolated or generalized lymphadenopathy [106]. Lymphadenopathy may progress rapidly and is often accompanied by weight loss, fever and night sweats. Extranodal involvement is common and is found in about 43% of cases [106]. Possible sites of involvement include skin and soft tissue (16%), nasal cavity and paranasal sinuses (16%), eye, orbit, and ocular adnexa (11%), bone (11%), salivary glands (7%), central nervous system (7%), oral cavity (4%), kidney and genitourinary tract (3%), respiratory tract (3%), liver (1%) and tonsils (1%) [106].

8.5.1.3 Diagnosis of Rosai–Dorfman Syndrome Laboratory abnormalities are represented by the presence of signs of chronic inflammation, including anemia, neutrophilia, elevated erythrocyte sedimentation rate and polyclonal hypergammaglobulinemia [106].

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Bone marrow examination is usually without pathological findings. Histopathological examination of a tissue sample of an involved site, typically an enlarged lymph node, is essential for diagnosis [106].

8.5.1.4 Histopathological Findings of Rosai–Dorfman Syndrome Involved lymph nodes usually exhibit a high extent of fibrosis. The presence of phagocytic histiocytes with a variable number of intact lymphocytes within the cytoplasm, a phenomenon called lymphophagocytosis or emperipolesis, is the pathognomonic feature of the Rosai Dorfman Syndrome [106]. Positivity of S-100 protein on histocytes in immunohistochemistry is another characteristic feature. Moreover, histiocytes express macrophage markers (e.g., CD68, CD14, HAM 56, CD15, and EBM11) and antigens associated with phagocytosis (CD64), but lack markers of dendritic cell differentiation (CD21, CD23 or CD35) [106]. The most important histopathological differential diagnosis are Langerhans cell histiocytosis, histiocytic sarcoma, Hodgkin disease and due to positivity of S-100 protein metastatic melanoma.

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literature [114, 115]. Thalidomide has been used for cutaneous involvement [116].

8.5.2 Langerhans Cell Histiocytosis (LCH) (Histiocytosis-X, eosinophilic granuloma, Abt–Letterer–Siewe disease € ller–Christian disease) or Hand–Schu Langerhans cells reside in the mucosa and epidermis and derive from dendritic cells. After antigen contact, for example with invading pathogens, they migrate to the lymph nodes and present the uptaken pathogens to T-cells [101].

8.5.2.1 Epidemiology of LCH The historical terms histiocytosis-X, eosinophilic granuloma, Abt Letterer Siewe disease or Hand Sch€uller Christian disease are no longer commonly used. The median age at diagnosis is between 2 and 3 years and 90% of all cases are diagnosed before age of 30 years [117 119]. The incidence of this rare disease is about 5 to 8 cases per million children [17, 119] and with a preponderance for males (ratio of 3:1) [118, 119]. A genetical impact is not known, although one case of familial clustering has been reported [120].

8.5.1.5 Treatment of Rosai–Dorfman Syndrome Due to the rarity of this disease and the high rate of spontaneous regressions, no randomized trials have been conducted and thus treatment recommendations are based on case reports and expert opinions. Many patients do not require treatment. However, as already mentioned, several fatal cases have been documented in the literature [109]. Initially, watchful waiting for patients without local complications due to local lymph node masses is an accepted approach [110]. Surgical debulking if organ functions are compromised is an accepted approach [110]. Corticosteroids have also been used with success in case reports [110 112]. Patients with severe progressive cases have been treated with several chemotherapeutic regimens incorporating different substances such as cyclophosphamide and methotrexate, but the results are often poor. Only 2 of 12 patients responded to chemotherapy in a current report [110]. Radiotherapy also seems efficacious in some cases [110]. Successful treatment of a patient with Rosai Dorfman Syndrome diagnosed during a varicella zoster infection with acyclovir has been reported [113]. Single cases of effective treatment with IFN-a, which seems to be associated with long term survival, may be found in the

8.5.2.2 Prognosis and Course of Disease of LCH Survival has improved considerably over time, with 5year survival rates of 74% after first diagnosis for all patients in a British tumor registry. This improved survival is assumed to be caused by earlier diagnosis of the disease and better treatment of LCH over the last decades [121]. There were no deaths beyond 5 years among this cohort. Nevertheless, reports of late relapses after more than 10 years of relapse free survival, exist [122, 123]. Patients with involvement of high risk organs such as the lung, spleen or liver, are considered to have a poor prognosis with an overall survival of 25% after 5 years. Spontaneous resolution has also been reported in rare cases [124].

8.5.2.3 Clinical Presentation LCH may affect one or more organ systems, with the most common single sites of involvement being the bone, skin or lymph nodes. In children, LCH of the bone most frequently presents as a lytic lesion of the skull [125], which may be accompanied by pain and a tender spot.

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Other frequently involved sites are the extremities, ribs, vertebrae, pelvis or the jaw [124, 126]. Involvement of the calvarea must be sought for in patients presenting with seizures, hearing loss, recurrent otitis media, or in patients with polyuria and polydypsia as symptoms of diabetes insipidus, which is seen in up to 25% of patients [127]. LCH lesions of the skin can present as seborrheic, eczematoid or pustular dermatitis predominantly affecting the scalp. Involvement of organ sites other than the skin and bones mostly indicates multi-organ disease. Enlargement of the liver or spleen may complicate the course of disease due to dysfunctions resulting in (i) hypoalbuminemia and ascites, (ii) hemorrhagic diathesis due to reduced liver synthesis of clotting factors, or (iii) splenogenic pooling of blood cells resulting in cytopenia. Bone marrow involvement is mostly observed in patients with disseminated disease and is often accompanied by systemic symptoms (e.g., weight loss, nocturnal sweating and/or fever) [124]. Central nervous system involvement may cause dysarthria, dysphagia, ataxia, tremor or hyperreflexia [124]. Basically LCH may involve any organ system, but common patterns are often age dependent: LCH affecting multiple organs is much more common among children (50 70%) than adults (30%) [100].

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a

10 μm

b

8.5.2.4 Diagnosis of LCH Guidelines of the Histiocyte Society have been established for initial evaluation of patients with LCH [128]. LCH is defined as clonal neoplastic proliferation of Langerhans type cells which express CD1a, langerin and protein S100 (and see Fig. 8.7a c) [17]. Furthermore, Langerhans cells typically show Birbeck granules by ultrastructural examination [17]. Foremost, biopsy of a suspicious lesion is needed to confirm diagnosis by immunohistochemical staining for CD1a, anti-langerin and S100 protein. The electron microscopy examination of Langerhans cells searching for Birbeck granules is not routinely done, due to its rare availability and high costs. A complete skeletal radiographic survey and chest X-ray is mandatory in order to detect possible lytic bone lesions. Bone marrow biopsy is not routinely performed in baseline examination. In patients with suspected central nervous system involvement due to neurological symptoms MRI or CT scans of the brain and the spine are required. According to findings obtained by the above discussed work-up, patients should be stratified as indicated in recent guidelines [100]. Patients with a single affected organ system are stratified into unifocal or multifocal involvement

20 μm

c

20 μm

Fig. 8.7a Langerhans cell histiocytosis. Diffuse infiltrate of Langerhans cells with typical grooved nuclei (HE staining, 1000). Additionally, an enhanced amount of eosinophils, neu trophils and lymphocytes as well as histiocytes (including multi cleated forms) can be seen. b, c Langerhans cell histiocytosis: soft tissue infiltrate. Immunhistochemistry identified the Langerhans cells with typical co expression of CD1a (b: membraneous, 400) and S 100 (c: cytoplasmatic and nuclear, 400)

and patients with multi-organ disease are subcategorized depending on presence or absence of organ dysfunction.

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Clinical stratification of LCH based upon extent of disease Single-organ system disease

Unifocal

Multifocal

Multi-organ disease

No organ dysfunction

Organ dysfunction

Low risk: involvement of skin, bone, lymph node, and/or pituitary gland High risk: involvement of lung, liver, spleen, and/or hematopoietic system Fig. 8.8 Clinical stratification of Langerhans histiocytosis (LCH) based upon extent of disease

Liver dysfunction is defined by presence of one or more of the following signs: hypo-proteinemia (total protein G55 g/l, albumin G25 g/l), edema, ascites or bilirubinemia more than 15 mg/l. Pulmonary dysfunction is defined by presence of tachypnea or dyspnea, cyanosis, cough, pneumothorax or pleural effusion. Hematopoietic system dysfunction includes anemia (G10 g/dl, not caused by other reasons), leukopenia, neutropenia or thrombocytopenia [124]. Furthermore, patients with organ dysfunctions can be further classified in high risk involvement affecting lung, liver, spleen and hematopoietic system (see also Fig. 8.7) [100].

8.5.2.5 Treatment Since treatment modalities vary with stage of disease, correct stratification is a prerequisite (see also Fig. 8.8) [100]. Treatment of Asymptomatic Localized Disease Asymptomatic localized disease typically cutaneous disease does not require therapy, after involvement of other sites has been careful excluded. Patients with symptomatic cutaneous involvement requiring local treatment can be treated with topical steroids, topical nitrogen mustard or psoralen coupled with ultraviolet A light [129 131]. In general, isolated bone lesions are sufficiently treated with curettage [100]. Radiotherapy is an option for painful or inaccessible bone lesions [124]. Treatment of Multi-Organ Disease Multi-organ disease with or without organ dysfunction requires systemic therapy. Cortisone or single agent chemotherapy with cyclophosphamide or azathioprine

have been used as first-line treatment for many years [100, 122]. The Histiocyte Society conducted two clinical trials which demonstrated that therapy intensification improves results especially of high risk patients. In the LCH-I trial chemotherapy with vinblastine or etoposide for 24 weeks with initial corticosteroids were shown to be equivalent in all respects, including response at 6 weeks (49 57%) and 3-year overall survival (76 83%) [132]. However, further improvement of existing treatment options is necessary, especially for patients with high risk organ involvement. Therefore, in the LCH-II trial intensified treatment was tested incorporating 6 weeks of daily prednisone and weekly vinblastine and etoposide followed by continuation therapy with 6-mercaptopurine, vinblastine, prednisone and etoposide. Increased and rapid responses were observed with reduction of mortality rates from 44% to 27% in high risk patients [133]. Lack of response to chemotherapy during the first 6 weeks of induction chemotherapy was found to be a new negative predictor for survival and these patients should be considered for salvage regimens based on cladribine. Allogeneic stem-cell transplantation may be considered for selected patients [124, 132, 133].

8.5.3 Malignant Histiocytosis Due to the rarity of these cases no systematic clinical trials using uniform diagnostic criteria are reported. The International Lymphoma Study Group analyzed 61 cases of these extremely rare neoplasms and proposed the following classification which is similar to the WHO classification of 2008 [17, 134]: * *

* *

Histiocytic sarcoma (29% of all cases) Tumors of Langerhans cells including Langerhans cell tumor (28%) and Langerhans cell sarcoma (15%) Follicular dendritic cell sarcoma (21% of all cases) Interdigitating dendritic cell sarcoma (7% of all cases).

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Anaplastic and large-cell lymphoma can sometimes mimic malignant histiocytosis. In fact, the clinical syndrome of malignant histiocytosis is most often seen in patients with CD30þ anaplastic large cell lymphoma, but also in patients with T- or NK-cell Lymphoma, as well as angioimmunoblastic B-cell lymphoma. Therefore, the tumor cells should always be tested for T- and B-cell markers to exclude lymphoma. The malignant histiocytic syndrome is characterized by pancytopenia, hemophagocytosis, fever, reduced NK cell activity, disseminated intravascular coagulopathy, and potentially multi-organ-failure. As similar symptoms may be induced by infections with certain viral (i.e., EBV, HHV6, CMV, and parvovirus), bacterial (i.e., Mycobacterium tuberculosis and Salmonella species) or opportunistic (i.e., Aspergillus and Leishmania) pathogens or even drugs (i.e., phenytoin) these causes must be excluded.

8.5.3.1 Histiocytic Sarcoma Histiocytic sarcoma sometimes presents as a solitary mass but systemic disease with symptoms such as fever or weight loss also occurs. Reported manifestations include the skin, lymph nodes, the gastrointestinal tract and the liver [134, 135]. Morphology of this entity shows a diffuse pattern of large oval cells. Hemophagocytosis is occasionally seen. Immunohistochemistry shows positivity for histiocytic markers such as CD68 and concomitant lack of dendritic cell markers such as CD21 or CD35 as well as lack of Langerhans cell markers such as CD1a. Most cases are diagnosed in adults with an median age between 46 and 55 years [134, 135].

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8.5.3.3 Follicular Dendritic Cell Sarcoma The definitive histopathological diagnosis is challenging and requires complex analysis. Briefly, tumor cells show bizarre morphology and show typical immunohistochemical staining of normal follicular dendritic cells (CD21, CD23, and CD35). Electron microscopic analysis may also be needed to exclude interdigitating cell processes seen in interdigitating dendritic cell sarcoma [17]. The median age was 65 years in a recent report of 13 patients. The most frequent initial clinical finding was a painless mass, without systemic symptoms [134]. Often the course of disease is indolent [134].

8.5.3.4 Interdigitating Dendritic Cell Sarcoma This entity widely shares the histological appearance of follicular dendritic cell sarcoma. Lack of typical follicular dendritic cell markers and complex interdigitating cellular junctions are characteristic for this rare neoplasm [17, 136] (see Fig. 8.9). The largest published series consists of only 4 cases, and only 36 patients with this rare disease have been reported up to now [134, 139, 140]. Prognosis of this extremely rare disorder seems poor [140].

8.5.3.5 Treatment Only case reports or small series describe the treatment of the clinical syndrome malignant histiocytosis which must be differentiated from histiocytic sarcoma. In

8.5.3.2 Tumors of Langerhans Cells The 2008 WHO classification differentiates between the clinically aggressive Langerhans Sarcoma and the more benign classical Langerhans cell histiocytosis. Langerhans sarcoma is characterized by typical malignant cytological features (high mitosis rate, nuclear polymorphism, atypical mitosis) [17, 134, 136] and is a highly aggressive disease with a mortality rate of 61% in 13 reported cases [137]. Multi-organ involvement of skin, lymph nodes, bone, lung, bone marrow is characteristic [137]. However, although this entity is not recognized by the new WHO classification, most authors define an additional subgroup termed Langerhans tumor which has the cytological features of Langerhans cell histiocytosis, but a more aggressive clinical course [134, 138].

10 μm

Fig. 8.9 Interdigitating dendritic cell sarcoma. Focal para cortical lymph node infiltration of spindled to ovoid cells with relatively bland nuclei expressing S100 (immunohistochemistry, 1000), by negativity for CD1a and CD21 (not shown) in a case being confirmed by an external center of hematological reference

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patients with the clinical features of malignant histiocytosis it is essential to detect the underlying cause (often aggressive anaplastic or T/NK-cell lymphomas) and initiate the appropriate treatment. In most cases patients were treated with multiagent cytostatic regimens such as CHOP, ABVD or DHAP [136]. Localized disease, such as follicular dendritic cell sarcoma, can be treated with surgical excision [141]. As a novel agent, Thalidomide is reported to induce partial remission in a case of histiocytic sarcoma with recurrent disease after allogeneic bone marrow transplantation [142].

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9

De novo Classic Paroxysmal Nocturnal Hemoglobinuria (PNH) (Marchiafava–Micheli Syndrome) Lisa Pleyer and Richard Greil

Contents 9.1 Epidemiology of PNH :::::::::::::::::::::::::::::::::::::::::::::::: 259 9.2 Pathophysiology and Molecular Biology of PNH::::::: 260 9.2.1 Pathomechanism of Hemolysis :::::::::::::::::::::::: 262 9.2.2 Pathomechanism of Hemoglobinuria and Hemosiderinuria::::::::::::::::::::::::::::::::::::::::: 263 9.2.3 Pathomechanism of Thrombotic Tendency :::::::: 264 9.2.4 Pathomechanism of Dystonias, Abdominal Pain and Systemic or Pulmonary Hypertension ::::::::::::::::::::::::::::::::::::::::::::::::::: 264 9.3 Functional Defects of GPI-Deficient Hematopoietic Cells ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 265 9.4 Clinical Features and Disease Complications of PNH ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 266 9.5 Diagnosis, Laboratory Findings and Diagnostic Tests in PNH ::::::::::::::::::::::::::::::::::::: 267 9.5.1 Laboratory Findings ::::::::::::::::::::::::::::::::::::::::: 267 9.5.2 Diagnostic Tests ::::::::::::::::::::::::::::::::::::::::::::::: 267 9.6 Differential Diagnosis of PNH:::::::::::::::::::::::::::::::::::: 267 9.6.1 PNH Cells in Normal Individuals and After Therapy with Campath :::::::::::::::::::::::::::: 268 9.7 Cytogenetics in PNH :::::::::::::::::::::::::::::::::::::::::::::::::: 269 9.8 Risk Factors in PNH :::::::::::::::::::::::::::::::::::::::::::::::::: 269 9.9 Treatment of PNH Current State of the Art ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 269 9.9.1 Treatment of Anemia and Other Cytopenias in PNH::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 270 9.9.2 Treatment of Thrombotic Events in PNH :::::::::: 270 9.9.3 Targeted Treatment Complement Inhibition:::::: 272 9.9.3.1 Inhibition of Terminal Complement C5 and MAC Formation :::::::::::::::::::::::::: 272 9.9.3.2 Exogenous Replacement of GPI Linked Proteins:::::::::::::::::::::::::::::::::::::::::::::: 272 9.9.4 Immunosuppression :::::::::::::::::::::::::::::::::::::::::: 273 9.9.5 Allogeneic Stem Cell Transplantation for PNH ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 273 9.9.6 Perioperative Management of PNH Patients :::::: 274 9.9.7 Avoidance of Drugs Known to Induce Hemolytic Anemia ::::::::::::::::::::::::::::::::::::::::::: 274 9.9.8 Management of Pregnancy in Women with PNH::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 274

9.1 Epidemiology of PNH PNH, although a benign clonal stem cell myelopathy, is included in the myeloproliferative disorders by some [1]. PNH is mainly a disease of adults, but can be present in adolescence or childhood on rare occasions [2]. Overall both genders are affected in approximately equivalent numbers. However, some Asian studies report a strong male preponderance [3]. The median age at diagnosis is 30 years in Caucasians and 45 years in Asian patients [4]. The median survival time in PNH, which is a nonmalignant stem cell clonal myelopathy, used to be between 10 and 15 years from diagnosis [5, 6]. In a British cohort, 72% of patients had died 25 years after diagnosis, with the median age at the time of death being 56 years [5]. Others have reported significantly longer overall survival times of 25 years from diagnosis [4]. The main causes of death are either thrombosis or hemorrhage attributable to thrombocytopenia. With modern supportive methods however, the prognosis has probably improved. Spontaneous complete clinical remissions occur in up to 15% of all patients or 35% of patients who survive longer than 10 years after diagnosis [5] (see also Summary Box 1). Interestingly, analysis of a large cohort of 385 PNH patients from the United States or Japan revealed that Caucasian patients were typically younger at diagnosis, with more typical PNH symptoms including thrombosis, hemoglobinuria and infections, coinciding with a higher mortality rate and shorter overall survival. In contrast, Asian patients were older and presented with more bone marrow aplasia [4]. The authors try to explain these differences between white and Asian patients by hypothesizing that different viruses, which may be implicated in the pathogenesis of PNH, may be present in a different prevalence in the two ethnic groups. In both cohorts however, a larger PNH clone was associated with classical PNH symptoms, while a smaller clone was associated with aplasia. When sequential measurements in the same patient demonstrated a decrease in the size of the PNH clone this was usually followed by bone marrow failure [4].

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Summary Box 1 PNH – Epidemiology * Median age at diagnosis 30 years * Median survival time 10 15 years * Spontaneous complete remission 15% * Different courses in Asians vs. Caucasians PNH – Pathophysiology PIG-A mutations cause lack of membrane attachment of CD55, CD58, CD59, CD16a and others * H200 different mutations have been shown * PIG-A mutation prevents deletion of stem cells by cytotoxic T-cells due to lack of target antigens, thus selecting for outgrowth of a monoclonal PNH clone * PIG-A mutant cells are susceptible to the membrane attack complex. This results in intravascular hemolysis * Hemolysis is aggravated by nocturnal acidosis, trauma, infection, drugs * B- and T-cell deficiencies are common *

Clinical – Symptoms Intravascular hemolysis, hemoglobinuria, renal siderosis, tubular atrophy * Rate of thrombosis correlates with clone size * Pulmonary hypertension, esophageal spasms, erectile dysfunctions * Development of cytopenia and aplastic anemia * Development of MDS or AML *

9.2 Pathophysiology and Molecular Biology of PNH Somatic mutations in the PIG-A gene located on Xp22.1 result in defective biosynthesis of the glycosyl-phosphatidylinositol (GPI) anchor, which attaches many proteins to the cell membrane [7, 8]. Thus, PIG-A mutations in hematopoietic stem cells (HSC) lead to a lack of GPIanchored complement regulatory membrane proteins on the surface of all blood cell lineages. Therefore PNH cells typically lack CD55 (decay accelerating factor (DAF)), CD59 (membrane inhibitor of reactive lysis (MIRL)), CD58, CD16a (FcgR-IIIb), CD87 (uPAR), CD14, CD52, and/or CD109, to name but the most well known [9] (see also Summary Box 1). As the PIG-A gene is X-linked, while all other genes necessary for GPI-biosynthesis are located on autosomes, only one mutation is required in a stem cell

to generate a PNH phenotype, which explains why PIG-A mutations, rather than mutations in other GPIbiosynthetic genes, are found in all PNH patients. Close to 200 different somatic PIG-A mutations have been documented, with the majority of the mutations being unique [7]. Three distinct PNH cell populations, which often coexist in the blood cells of the same patient, can be discerned. Cells with normal expression, partial expression and complete deficiency of GPI-linked surface proteins are termed PNH type-I, type-II and type-III cells, respectively [10]. It is thought, that the intermediate type-II phenotype is due to partial inactivation of the PIG-A gene by missense mutation. Approximately 40% of patients have a combination of types I, II and III cells, which in itself implies and reflects the oligoclonal nature of the disease [11 13]. Multiple PIG-A mutations are found in approximately 10 20% of PNH patients [14]. PNH clones can be detected in the absence of hemolysis, the degree of which is mainly determined by the size and type of the PNH clone. It has been unequivocally demonstrated that the susceptibility of PNH erythrocytes to complement-mediated hemolysis is not due to mere CD55 deficiency, but requires the combined lack of several membrane proteins [15]. Consequently, PNH type-II erythrocytes with residual expression of 20% of CD59 are protected from intravascular hemolysis and have a normal life span of 100 days. In contrast, PNH type-III erythrocytes with complete deficiency of all GPI-anchored proteins have a life span that varies between 17 and 60 days [10]. GPI-deficient cells with PIG-A mutations frequently occur in normal individuals at low levels where they do not have an inherent growth advantage over their normal counterpart [16]. These PIG-A mutant colony forming cells are polyclonal, meaning that they have undergone neither clonal selection nor clonal expansion. Furthermore they seem to be regulated in a normal manner and do not display malignant traits, as PNH cells do not infiltrate or metastasis beyond the normal hematopoietic compartment. Therefore, classic PNH is a benign clonal stem cell myelopathy as there is limited expansion of PIG-A mutant clones in the absence of selective pressure. Mosaicism of normal and PNH cells in the peripheral blood is stable, no invasion of non-hematopoietic organs is observed, and mutant clones do not function autonomously. As mentioned above, spontaneous remissions can occur when the initiating toxic events declines (e.g., [5]). A hypothesis that is favoured by many claims that bone marrow injury, mediated by mechanisms similar to those relevant in the pathogenesis of aplastic anemia (AA), leads to attack of the normal hematopoi-

Chap. 9

De novo Classic PNH

261

Fig. 9.1 Pathophysiology of PNH. HSC Hematopoietic stem cells; BM bone marrow; TNFa tumor necrosis factor a; IFNg

interferon g; TGF b tumor growth factor b; MIP 1a macrophage inhibitory protein 1a

etic stem cells by specific cytotoxic lymphocytes (CTLs). In order for the PNH-clone to be able to evade this attack, either the target protein or an accessory molecule required for the immune destruction by CTLs must be GPI-linked. Under these circumstances, preexisting PIG-deficient CD34þ PNH cells would have a growth- and survival advantage by escaping from the immune system, leading to clonal selection (see Fig. 9.1). This is further confirmed by the finding that PIG-normal CD34þ cells of PNH patients show elevated cell-surface Fas-expression, which coincides with higher propensity for apoptosis [17]. This is suggestive of a targeted autoimmune process directed against CD34þ cells. In contrast, PNH-CD34þ cells evade the autoimmune attack and consequently do not receive proapoptotic stimuli, do not upregulate Fas and do not become apoptotic. A second pro-proliferative but non-transforming (epi) genetic event may be necessary [1] for preferential

clonal expansion, seemingly increased clonogenic potential [18] and ultimate domination of hematopoiesis (see Fig. 9.1). Some authors are convinced that PNH is a natural form of gene therapy, in which nature has accepted collateral damage in the form of hemolysis and thrombophilia, in order to escape immune-mediated bone marrow failure [19]. It is hypothesized, that the relatively high rate of spontaneous remissions is due to reduced intensity or burning out of the process triggering aplasia, thus positively selecting for PNH clones, over time. Therefore, loss of selective pressure for proliferation of GPI-deficient stem cells, which have the intrinsic capacity to evade immune attack, eventually results in a swing towards, and ultimate domination of, hematopoiesis stemming from remaining normal hematopoietic stem cells. A defect in the bone marrow stroma does not seem to be present or to be relevant in PNH [9].

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9.2.1 Pathomechanism of Hemolysis Erythrocytes are normally protected from spontaneous complement-mediated cell lysis by GPI-anchored pro-

teins that inhibit the assembly of membrane attack complex. Whereas CD55 controls the early part of the complement cascade by regulating the activity of the C3 and C5 convertases, CD59 inhibits the terminal memOsmotic swelling and lysis

Alternative pathway of complement activation

Intravascular hemolysis

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•NO-deficiency mediated PLT - adhesion & aggregation

•Endothelial dysfunction •Vascular permability •Adhesion molecules (ICAM, VCAM, E-selectin)

Proline, polyamines

•Collagen production •Smooth muscle proliferation •Airway remodelling

Fibrinolytic activityඏ

NO depletion

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Smooth muscle dystonia

•Hypertension •PAP ඍ •Local vasoconstriction

•Dysphagia •Abdominal pain •Erectile dysfunction

Intravascular thrombosis Serum uPAR Thrombin

TF

ADAMTS13 ඏ Factor XIII ඍ

Fig. 9.2 Pathophysiology of red blood cell (RBC) lysis and thrombotic complications in PNH: Deficiency of GPI anchored proteins, such as CD55 and CD59 leads to enhanced susceptibility of erythrocytes towards complement mediated RBC lysis via formation of the membrane attack complex (MAC), as sponata neous complement activation can no longer sufficiently be blocked. MAC leads to pore formation in the RBC membrane with consecutive osmotic swelling, lysis and release of free hemoglobin and erythrocyte arginase into the blood stream. Free hemoglobin scavenges nitric oxide (NO), whereas araginase ex pedites ornithine synthesis on the one hand, and promotes reactive oxygen species (ROS) production with further depletion of NO on the other. Ornithine plays an important role in endothelial and

smooth muscle cell function, thereby promoting air way remodel ling, pulmonary hypertension and intravascular thrombosis. MAC formation on thrombocyte surfaces also contributes to thrombo geneic tendency, in that it promotes platelet activation with granule secretion, as well as membrane blebbing which further enhances procoagulant surfaces. These effects are further enhanced by NO deficiency. Neutrophils also contribute to intravascular thrombosis via secretion of TF and increases of serum uPAR. n.gr. Neutro philic granulocytes; Nos nitric oxide synthase; TF tissue factor; uPAR urekinase plasminogen activator receptor; PAP pulmonary arterial pressure; PLT platelet; TNFa transcriptier factor a; TG triglycerides; ICAM intercellular adhesion molecule; VCAM vas cular cell adhesion molecule

Chap. 9

De novo Classic PNH

brane attack complex (MAC) formation (see Fig. 9.2). Deficiency in one or both of these proteins on the surface of erythrocytes deprives them of their capacity to inhibit spontaneous complement activation and consequently increases susceptibility to complement-mediated cell lysis. The membrane attack complex forms pores in the red blood cell membrane, resulting in increased in permeability, colloid osmotic cell swelling and lysis, with release of hemoglobin into the intravascular space (for further details see Fig. 9.2). This explains why the survival of PNH erythrocytes is shortened to 10% that of normal red blood cells [10]. The nature of hemolysis in PNH is intravascular, with no involvement of the reticuloendothelial system, which is why hepatosplenomegaly is not observed in PNH, in contrast to most other hemolytic anemias. Clone size often correlates with the degree of hemolysis and therefore also with the incidence of hemoglubinuria [20]. The abiding chronicity of complement-mediated hemolysis is broken from time to time by episodes of massively enhanced blood destruction. These hemolytic crises can be triggered by infections or anything leading to activation of the immune system, as well as by non-specific traumata such as a blow on the head or a surgical operation [21]. It is during such hemolytic crises that the nocturnal character of the disease is most prominent, although the word nocturnal is strictly speaking a misnomer, as increased hemolysis is related to sleep and not to the night time. During sleep hemolysis becomes more intense, plasma hemoglobin rises and the renal threshold for hemoglobin is surpassed. The first urine passed in the morning is typically dark, whereas the next specimen passed become progressively lighter, and by midday the urine is usually clear [21]. This circadian rhythm is diagnostic of PNH. In fact, the presence of PNH used to be established or ruled out by comparing urine hemoglobin levels at 8 a.m. and 8 p.m. [21]. This phenomenon remains measurable and diagnostic even during severe paroxysms, when hemoglobinuria persists throughout the whole day. If the patient sleeps during the day and remains awake at night, this pattern of hemoglobinuria is reversed [21, 22]. The acid pH-shift due to depression of the respiratory centre during sleep, with ensuing increases in carbon dioxide and decreases in pH, is the main culprit contributing to the nocturnal character of the disease [22].

9.2.2 Pathomechanism of Hemoglobinuria and Hemosiderinuria Although most of the globin of the hemoglobin is returned to the metabolic protein pool, proteinuria is

263

Fig. 9.3 Iron-loaded epithelial cell from urine sediment of a patient with PNH: Hemosiderinuria and hemoglobinuria (isolate blue specs), with a large iron granula loaded epithelial cell from the urine sediment (center) of a patient with PNH

always present to some extent in patients with significant hemolysis [23]. Plasma hemoglobin is normally filtered through the glomerulus and actively reabsorbed in the proximal tubulus, where it is catabolized to hemosiderin with release of iron. Kidney epithelial cells remove the iron molecule from the porphyrin ring, and return it to the body in the form of ferritin. Once the kidneys hemoglobin reabsorption capacity is exceeded, clinically significant hemoglobinuria occurs, and the kidneys capacity to metabolize hemoglobin to ferritin becomes rapidly saturated. Thus, iron begins to accumulate, hemosiderin is disgorged into the tubular lumen, siderotic epithelial casts may be found in the urine sediment (see Fig. 9.3) and hemosiderin deposition in proximal tubuli with ensuing defective renal reabsorption of small molecules, occurs [23]. Hyperaminoaciduria, glycosuria, hyperphosphaturia as well as bicarbonate and water loss may be the consequences thereof. Typically, the kidney becomes siderotic, whereas spleen and liver remain devoid of stainable iron, demonstrating even less than the normal concentration, which discerns PNH from most other severe hemolytic diseases, where iron is deposited in most organs. Chronic hemosiderinuria and/or hemoglobinuria used to lead to severe iron deficiency in PNH patients. Obviously these features of characteristic iron distribution and iron deficiency become void once the patient has received sufficient transfusions, which is why they are seldom found nowadays, and the contemporary PNH patients often suffer from the reverse problem, namely transfusion siderosis. During a severe hemolytic crisis the amount of hemoglobin filtered through the kidney can reach sufficient amounts to turn the urine black. Severe hemoglobinuria

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can last up to a week and acute renal failure may occur [23].

9.2.3 Pathomechanism of Thrombotic Tendency Reduced endothelial bioavailability of nitric oxide (NO) is thought to contribute to enhanced probabilities of thrombosis (and/or pulmonary hypertension) due to endothelial dysfunction as well as intimal and smooth muscle proliferation (reviewed in [24]). Furthermore, NO is known to inhibit platelet adhesion and aggregation. NO induces disaggregation of aggregated platelets through interaction with components of the coagulation cascade, which is the rational for, and mechanism of action of, NO donor drugs that increase systemic levels of NO [25] (see Fig. 9.2). Free plasma hemoglobin contributes to platelet activation and thrombosis via scavenging of NO, after the capacity of hemoglobin-scavenging haptoglobin has been exceeded. Erythrocyte arginase is released during intravascular hemolysis and further reduces systemic availability of NO by interfering with NO-production [24], in that it expedites the production of arginine to ornithine. Thereby arginine-mediated NO synthesis is hindered. As arginine is primarily synthesized in the kidney, patients with renal dysfunction demonstrate an additional impairment of de novo arginine synthesis, further decreasing the ratio of arginine to ornithine [24]. Additionally, under conditions of low arginine concentration, NOsyntethase (NOS) is uncoupled, producing ROS in lieu of NO. ROS in turn react with NO to produce peroxynitrite, thereby further reducing NO bioavailability (see Fig. 9.2). CD59-deficient platelets are 10 times more susceptible to attack and ensuing activation by complement. C5b-9 stimulates expression of membrane binding sites for factor Va which is paralleled by a corresponding 10fold increase in membrane-catalyzed prothrombinase activity [26]. Membrane attack complex assembly on the surface of platelets also results in the secretion agranule/dense-granule contents and the shedding of procoagulant vesicles from the platelet surface (see Fig. 9.2). These shed vesicles contribute to thrombotic tendency by providing the principal catalytic surface for assembly of procoagulant enzyme complexes [26]. The same authors explain the normal survival of PNH platelets, compared to the drastically reduced survival of PNH erythrocytes (G10% of normal red blood cells (RBCs)), by this ability of PNH platelets to shed nascent C5b-9 complexes by vesiculation (see Fig. 9.2). Others have demonstrated defective fibrinolytic activity secondary to


deficiency of GPI-anchored uPAR on PNH-affected monocytes and neutrophils with concomitantly elevated soluble uPAR serum levels. These factors may synergistically contribute to the development of thrombosis in PNH by inhibiting cell-associated fibrinolytic activity [27]. Liebman and Feinstein proposed that increased tissue factor (TF) secretion by complement-injured CD55- and CD59-deficient PNH monocytes and macrophages results in significant thrombin generation, thereby promoting thrombogenicity [28] (see Fig. 9.2). Last, but not least, PNH clone size significantly correlates with, and is predictive of, thrombotic events, with the cut-off being at 60% PNH-granulocytes of total absolute neutrophil counts (ANC). Patients with clone sizes below the cut-off did not develop thrombotic complications in a retrospective analysis, whereas 55% of patients with PNH clone sizes above the cut-off manifested with thrombosis (2/3 of which were fatal), as well as typical PNH-symptoms (abdominal pain, hemolysis, gastrointestinal spasms, erectile dysfunction) [20].

9.2.4 Pathomechanism of Dystonias, Abdominal Pain and Systemic or Pulmonary Hypertension The profound NO-depletion observed in patients with PNH leads to dystonia and spasms of the smooth musculature, resulting in dysphagia, esophageal spasms, abdominal pain, and erectile dysfunction [26, 29]. NO-consumption is also responsible for endothelial dysfunction as well as intimal and smooth muscle proliferation, resulting in vasoconstriction which leads to systemic systolic and diastolic, as well as pulmonary, hypertension (see Fig. 9.2). As proof of principle, these symptoms can be reversed by nitric oxide donors such as sodium nitroprusside, at least in mice [30]. Furthermore, elevated araginase levels are not only accounted for by release from erythrocytes during hemolysis, but also by induction of araginase synthesis by cytokines derived from lymphocytes [31]. The latter are generally acknowledged to play an important role in the pathophysiology of the initial (subclinical) bone marrow failure syndrome/toxic event leading to the selection of PNH hematopoietic stem cells in the first place. As mentioned above, elevated araginase levels ultimately result in reduced NO-production and bioavailability, as well as increased levels of ornithine (see Fig. 9.2). Ornithine is a precursor for the production of proline and polyamines required for the synthesis of collagen as well as cell proliferation, both of which are necessary for vascular remodelling process-

Chap. 9

De novo Classic PNH

es [32] occurring in pulmonary hypertension (see Fig. 9.2). As already described, arginine is primarily synthesized in the kidneys. Therefore, renal insufficiency results in a relative increase in ornithine, which explains the correlation between rising creatinine levels, and incidence (and perhaps severity) of pulmonary hypertension [24]. Additionally, cytotoxic lymphocyte-derived cytokines have also been implicated to increase triglyceride levels, which may also be induced by arginasemediated ornithine production. Elevated triglycerides, as well as elevated arginase levels per se, have been (univariately) associated with elevated expression of adhesion molecules, such as ICAM, VCAM and/or Eselectin [24], as well as endothelial dysfunction [33] (see Fig. 9.2). Thus endothelial dysfunction, implicated in both pathogenesis of elevated pulmonal arterial pressure as well as thrombogenic tendency, results from combined NO-depletion, a shift in arginine metabolism to ornithine production and subsequently increased levels of downstream ornithine metabolism (e.g., triglycerides, proline, polyamines). This has been further substantiated by association of the above-mentioned findings with clinical severity of pulmonary hypertension and mortality [24]. Considering these results, measurement of the ornithine/ arginine ratio might provide clinicians with an index of disease severity and prospective risk of complications. In summary, repetitive thromboembolisms in the pulmonary microvasculature, combined with endothelial dysfunction and hyperproliferation, increased collagen synthesis and pulmonary vascular remodelling, as well as vasoconstriction due to hemolysis-mediated NO-depletion lead to pulmonary hypertension as a late complication in PNH.

9.3 Functional Defects of GPI-Deficient Hematopoietic Cells In general the proportion of PNH monocytes closely parallels the proportion of PNH granulocytes. These GPI-deficient monocytes also display functional defects, such as reduced stimulation and activation by LPS, which significantly affects their cytokine production and costimulatory activity. However, this may be compensated by enhanced secretion of soluble CD14 [34]. Additionally, GPI-deficient monocytes are unable to undergo full differentiation to dendritic cells (DCs) in vitro [35]. The resulting immature, GPI-defective dendritic cells also show a severe impairment in activation and cytokine production, leading to a drastically reduced capability in delivering accessory signals for T-cells [35]. GPI-defi-

265

cient granulocytes are also functionally defective. They not only display reduced adhesion and migration [36], but also show impaired production of reactive oxygen species (ROS) with reduced occurrence of oxidative burst (although bacterial ingestion is paradoxically increased) [37]. As the circulating life span of PNH granulocytes is normal (in contrast to the reduced life expectancy of PNH erythrocytes), the proportion of GPI-deficient granulocytes more closely correlates with the proportion of PIG-A mutant HSC than the proportion of PNH erythrocytes. This is why the size of the PNH-clone is usually measured by the size of the PNH granulocyte clone, i.e., the percentage of PNH granulocytes of the whole granulocytic population. GPI-deficiency also leads to functional abnormalities in T-cells, in that they are sub-optimally stimulated by normal B-cells, showing lower proliferative rates and lower cytokine production [38]. Others have confirmed alterations in PNH T-cell activation, and have demonstrated defects in T-cell memory phenotype [39], lectin-dependent T-cell proliferation [40] and severe defects in TCR-dependent signalling [41]. In fact, even the nonGPI-deficient T-cells of PNH patients show functional changes, such as persistence of CD154 and consequently altered CD40-dependent signalling [41]. It has been suggested, that these so-called GPIþ PNH T-cells may be involved in biological mechanisms underlying immune-mediated disease pathogenesis. Interestingly, PNH T-cells comprise mainly na€ıve cells (CD45RAþ CD45RO ), whereas the remaining normal GPIþ T-cell population in the same patients is predominantly of the memory type (CD45RA CD45ROþ ) [39]. The same can be said for the respective B-cell populations in PNH patients. Residual normal B-cells in patients with large PNH clones are mostly of the memory phenotype (CD27þ ), and are thought to have been generated before the onset of PNH, whereas the GPI-deficient B-cell compartment is predominantly comprised by na€ıve Bcells (CD27 IgMþ IgG ) [42]. Disease duration can be correlated with the proportion of memory-type Tand B-cells, as this subset accumulates with time due to the conversion of normal na€ıve B-cells to the memory phenotype over the years. GPI-deficient T- and B-cells can be found long (up to 24 years) after the disappearance of other PNH cell lines due to their longevity. Therefore, the proportion of CD52-deficient T-cells [43] as well as CD48-deficient B-cells [42] have been proposed to correlate with the duration of the disease and may be useful as a distinct marker for the follow-up of clinical remission. Taking all of the above into account, ample explanations are readily available for the profound defects in the physiological crosstalk between innate and adaptive im-

266

munity and the susceptibility to infections observed in some PNH patients [34 43]. Furthermore, as was discussed in detail in 9.2.3., GPIdeficiency also plays an important role in the functional role of platelets and the pathogenesis of thrombosis. Interestingly, several high incidence blood group antigens such as Cromer Cartwright-, Holly Gregory-, John Milton Hagen- and Dombrock-antigens, reside on GPI-linked proteins such as decay accelerating factor (DAF, CD55) [44, 45]. Consequently, these antigens are not detectable on PNH type-III erythrocytes, although they are found in normal quantity on PNH type-I cells of the same patient.

9.4 Clinical Features and Disease Complications of PNH PNH is an acquired stem cell disorder characterized by intravascular non-malignant clonal expansion, Coombs negative intravascular hemolysis, hemoglobinuria, bone marrow hypoplasia and sometimes peripheral cytopenias, an increased risk for venous thrombosis, recurrent infections, severe lethargy, erectile dysfunction (35% of male patients), dysphagia and esophageal spasms (23%), and occasional leukemic transformation [46] (see also Summary Box 1). The most important disease complications have been compiled in Table 9.1. Recurrent abdominal pain results from thromboembolic events within the abdominal veins and occurs in 35% of patients during paroxysms [20]. Dyspnea is also a frequent symptom and is attributed to pulmonary arterial hypertension and/or anemia. There is a tight correlation between the size/dominance of the PNH clone and the occurrence and severity of hemolysis, paroxysms as well as thrombotic complications. Patients with less than 20% of GPI-deficient cells usually display evidence of hemolysis and hemosiderinuria but rarely have hemoglobinuria (e.g., [4]). Most patients with more than 60% of type-III cells have daily episodes of hemoglobinuria. Viral or bacterial infections activate complement, thereby triggering hemolytic crises [47 49]. Furthermore, iron substitution can also lead to waves of hemolysis. Thrombotic complications seem to occur in patients with more than 50% of GPI-deficient granulocytes [4]. Interestingly, thrombotic events seem to recur in the same organ or site, which is thought to reflect residual endothelial proliferation from the initial episode [50]. Such recurrent thrombotic episodes can ultimately lead to chronic organ insufficiencies such as cirrhosis hepatitis, hypersplenism, splenic rupture [51], mucosal ulcers [52] and severe bouts of abdominal pain.


Table 9.1: Incidence of disease complications in PNH Previous hematologic diseases [4] Previous MDS (5%) * Prior aplastic anemia (29 38%) Cytopenias at the time of diagnosis * Anemia (88 94%) [4] * Thrombocytopenia (80%) * Severe transfusion dependent thrombocytopenia G50,000/ml (48%) * Bleeding complications * Neutropenia G1.500/ml (45 72%) and neutropenic infections (9 18%) [4] Life-threatening or fatal recurring thrombotic complications (4 32%) in various locations [4] * Mesenteric veins * Hepatic vein thrombosis (asymptomatic to fatal Budd Chiari syndrome) Recurrent hepatic vein thrombosis ultimately leads to cirrhosis hepatis * Portal vein * Splenal vein May result in splenomagaly * Inferior vena cava * Splanchnic vessels Bouts of severe abdominal pain Mucosa ulceration * Cerebral veins * Dermal veins Erythema Purpura like lesions * Sinus cavernosus Priapism * Epididymis * Accounts for H1/3 of all deaths Renal dysfunction * Proteinuria * Hemoglobinuria * Hemosiderinuria * Hemosiderosis of the kidney (in the absence of iron deposition in liver or spleen) * Renal failure (10%) [4] Pulmonary arterial hypertension (50%) Paroxysms: Acute exacerbations of hemolysis * Transfusion dependent anemia * LDH levels up to 25 times that of normal * Severe hemoglobinuria lasting up to a week * Abdominal pain (35%) [20] * Episodes of dysphagia and esophageal spasms due to strong peristaltic waves Disease progression/transformation * Pancytopenia/hematopoietic failure (15 30%) * Progression to MDS (3.5 5%) * Progression to AML (0.6 5%) * Progression to aplasia (accounts for up to 10% of deaths) [5, 6, 53] * 29% have antecedent AA *

Approximately one-third of patients develop bone marrow failure during the course of the disease. Unfortunately, this seems to be a common terminal event in PNH, irrespective of symptoms or age at diagnosis [4].

Chap. 9

De novo Classic PNH

9.5 Diagnosis, Laboratory Findings and Diagnostic Tests in PNH PNH should be suspected in any patient with isolated defects of a single lineage (e.g., thrombocytopenia) or pancytopenia, especially when accompanied by reticulocytosis and clinical or laboratory signs of intravascular hemolysis. Furthermore, patients with repetitive thrombotic episodes should be screened for PNH. Unfortunately, there is currently no worldwide consensus on diagnostic criteria for PNH. The following however, should provide an unambiguous guideline for diagnosis.

9.5.1 Laboratory Findings Typical laboratory findings are summarized in Table 9.2. As mentioned above, although the deficiency of GPIanchored cell surface proteins is most obvious on red blood cells as it leads to anemia, hemosiderinuria and Table 9.2: Laboratory Findings in PNH Evidence of acquired hemolysis in the absence of a positive Coombs test * Elevated LDH, total and indirect bilirubin * High free plasma hemoglobin concentration (due to intravascular hemolysis) * Hemoglobinuria, hemosiderinuria, proteinuria * Depleted haptoglobin * Elevated reticulocytes Lack of GPI-linked surface proteins (CD55, CD59, CD52, CD24, CD48, CD66, uPAR, FcIIIRa) on hematopoietic cells * Erythrocytes * Granulocytes * Monocytes (deficiency in CD14, CD55, CD59, uPAR) * Platelets (CD55 and CD59) * Lymphocytes [54, 55] * Natural killer cells (NKC) * B cells * T cells Cytopenias * Granulocytopenia * Thrombocytopenia * Anemia * Lack of ALP in GPI deficient granulocytes [57] * Diminished erythrocyte acetylcholinesterase [58, 59] * Elevated levels of d dimer in patients with (recurrent) thrombotic events Lack of certain blood group antigens [44, 45] * Cromer blood group antigens * Cartwright antigens * Dombrock antigen * Holly Gregory antigen * JMH antigen [60] LDH Lactate dehydrogenase; ALP alkaline leukocyte phosphatase; GPI glycosyl phosphatidylinositol

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name-giving paroxysmal nocturnal hemoglobinuria, such deficiencies are present in many, if not all other hematopoietic cells, such as monocytes [35], granulocytes, B[42], T- [39], and natural killer cells (NKC) [54, 55], dendritic cells [35] and thrombocytes [56].

9.5.2 Diagnostic Tests It is important to keep in mind that detection of PNH clones may be difficult in patients with small fractions of PNH cells, or during or just after a hemolytic crisis, as most complement-sensitive PNH cells would have been destroyed. In the sucrose hemolysis test [61], often used as a convenient screening test for PNH, the patients red blood cells (RBCs) are incubated with fresh serum diluted in isotonic sucrose, which leads to complement activation. If GPI-deficient RBCs are present, complement-mediated hemolysis will occur. The HAM-test on the other hand, uses a pH-reduction of the added fresh serum to activate complement. According to the above, however, a single normal hemolysis test should not be considered strong evidence that a patient does not have the disease. In these circumstances, analysis of the urine for hemosiderinuria is a practical screening method. Although not specific for PNH, hemosiderinuria does not usually occur in other forms of hemolytic anemia. Complement lysis sensitivity tests are considered positive when more than 5% of RBCs are abnormally sensitive to complement-mediated hemolysis. Diagnosis can definitely be established by flow cytometric detection (see Fig. 9.4a, b) of deficiencies of GPI-linked proteins on the surface of RBCs, granulocytes or monocytes, using fluorescently labelled monoclonal antibodies to detect surface expression of CD55 and CD59. Currently the assessment of both granulocytes and erythrocytes is the standard practice used for routine diagnostic purposes, whereas other cell lineages are not routinely assessed for GPI-linked proteins. A positive flow cytometry test is usually defined as 3% GPI-deficient RBCs or polymorphonuclear cells.

9.6 Differential Diagnosis of PNH A negative Coombs test, absence of kryoglobulins or cold agglutinins, lack of splenomegaly as well as lack of foci of extramedullary hemaopoiesis are prominent features in PNH, setting this disease apart from several other forms of hemolytic anemia. The spleen may however enlarge during severe hemolytic crisis, after thrombosis of the lineal or vein or large hepatic veins, or after the patient has received multiple transfusions. Uni- or multi-

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a

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Fig. 9.4a Typical FACS analysis of erythrocytes in a patient with PNH; (left) gated total erythrocytes; (middle) 43.5% of patients erythrocytes are negative for CD55; (right) 43.1% of patients erythrocytes are negative for CD59; b Typical FACS

analysis of granulocytes in a patient with PNH; (left) gated total granulocytes; (middle) 43.5% of patients granulocytes are negative for CD55; (right) 43.1% of patients granulocytes are negative for CD59

lineage cytopenia(s), as well as hemosiderinuria, also help differentiate PNH from other diseases with increased hemolysis. Although hereditary eryhtroblastic multinuclearity with a positive acidified serum lysis test (HEMPAS) [62] is characterized by a positive HAMs test, as the name implies, it should not be easily confused with PNH. Firstly, HEMPAS cells behave as a uniform population in quantitative lysis tests, unlike PNH, where types I, II and III cells are present. Secondly, HEMPAS cells do not lyse in their own serum, as lysis of these cells occurs due to the presence of antibodies to unusual antigens on the surface of HEMPAS cells, which are lacking in the patients own serum [63 65]. Furthermore, the sucrose hemolysis test is negative and HEMPAS is not associated with cytopenias [63 65]. Inherited deficiency of CD55, the so-called Inab-phenotype [66], is another possible differential diagnosis one should consider, especially when flow cytometry-based screening methods are implemented. The Inab-phenotype is the null-phenotype of the Cromer blood group system which consists of 10 known antigens, all of which reside on CD55. Inab-erythrocytes are completely deficient in

CD55 and consequently Cromer-antigen expression. However, in contrast to the acquired defect in PNH type-III erythrocytes where all GPI-linked proteins are lacking on the cell surface, this seems to be the only protein lacking. Problems may arise, when differentiating between type-II PNH cells, with partial deficiency of GPIlinked proteins. However, Inab cells do not show the extreme sensitivity to complement-mediated hemolysis in in vitro assays [15, 66 68], and clinical differentiation should not prove difficult as the Inab-phenotype is not associated with significant hemolysis or other symptoms of PNH [66]. This is due to the presence of additional inhibitors of C3 convertases in human plasma. Furthermore, cases of acquired and transient deficiency of the Inab-phenotype associated with splenic infarctions have been described [69].

9.6.1 PNH Cells in Normal Individuals and After Therapy with Campath Small subclinical circulating fractions of PNH cells (G1%) can been detected in most normal individuals,

Chap. 9

De novo Classic PNH

leading to the speculation that PIG-A mutations in hematopoietic stem cells are common benign events [16, 70]. However, recent data reveal that most of these mutations detected in non-PNH patients are not derived from stem cells but arise in a more differentiated colony forming cell without self-renewal capacity [8, 71]. Furthermore, as described above, the presence of PIG-A mutation alone is insufficient for the development of overt PNH in the absence of an underlying aplastic process [72] (see also Fig. 9.1). Interestingly, antibody selection against a single GPI-linked protein, such as CD52, promotes the development of a PNH-like, GPI-deficient phenotype in lymphocytes [73 76]. The molecular involvement by mutation of the PIG-A gene is controversial and has been described in one report on B-CLL (chronic lymphocytic leukemia) patients treated with campath [72, 75]. Others propose a novel mechanism not involving PIG-A mutations [76]. Excessive TGFb (transforming growth factor b) production by B-CLL bone marrow stromal cells is thought to have an inhibitory effect on hematopoietic precursor cells [77]. Possibly, reduced capacity for mRNA production of PIG-anchored proteins synergizes with the excessive TGFb production of the B-CLL bone marrow stroma, which in turn synergizes with campath to increased selective pressure on pre-existing PNH clones [73]. Campath treatment results in complete depletion of CD52 positive cells from the peripheral blood, including T- and B-lymphocytes, natural killer cells and monocytes. CD52-negative lymphocytes emerge in 12% of patients after treatment [74]. When analyzed more closely, these CD52-negative cells failed to express other GPI-anchored proteins on their cell surfaces and were devoid of the capacity to synthesize GPI-precursors [76].

269

the PIG-A gene. However, existing data infer that MDS clones may arise within the PNH clone. It is thought, that during the course of the disease, singular PNH cells acquire additional cytogenetic abnormalities such as trisomy 8, resulting in a PNH/MDS clone which eventually and progressively replaces the PNH clone due to proliferative advantage [80, 81].

9.8 Risk Factors in PNH Factors associated with high-risk for thrombosis during the course of the disease include a history of a previous thrombotic event, infections, age over 54 years at diagnosis, European or United States origin [6], as well as size of the PNH clone [43]. In multivariate analysis poor survival is associated with the occurrence of thrombosis or severe infection, evolution to pancytopenia, MDS or acute leukemia, as well as age over 50 55 years and thrombocytopenia or severe leukopenia/neutropenia at diagnosis [4, 6]. Especially the development of thrombosis is felt to be a grave prognostic feature. In Japanese patients renal failure was a significant risk factor [4]. Diminuition in the fraction of CD59-negative granulocytes over time is significantly associated with the development of hematopoietic failure [4]. In general, a decreasing PNH-clone size is thought to reflect a decline in hematopoietic capacity by the PIG-A mutant clone. Marrow failure with aplasia would thus represent the end stage, in which the proliferative capacity of the PNH clones is exhausted, while normal hematopoiesis is continuously eliminated by the ongoing disease-initiating (auto)immune process. Risk factors observed for development of MDS or AML include abdominal pain crisis at presentation. Patients with antecedent AA however, tend to have a better overall survival [6].

9.7 Cytogenetics in PNH Although karyotypic abnormalities have been detected in up to 12 24% of patients with PNH (e.g., 13, 13q, 7, þ8, 18, þ21, 21, der(12)) and abnormal morphological bone marrow-features reminiscent of MDS seem to be a common feature in PNH (15.5 21.5%), these traits do not coincide with the presence of excess blasts or the development of PNH/ MDS (for details see Chap. 10.2) or leukemia [4, 78, 79]. There seems to be no specific cytogenetic abnormality for PNH, and neither mutational hot spots nor mutations specific for MDS/PNH could be found in

9.9 Treatment of PNH – Current State of the Art Disease monitoring is subsumed in Summary Box 2, whereas principles of treatment of PNH are summarized in Summary Box 3. When considering aggressive therapy one must bear in mind, that PNH can be considered as a natural form of gene therapy or natures way of treating bone marrow failure, in that GPI-deficiency enables the PNH cells escape (auto)immune attack. Correction of this genetic abnormality may reverse the benefit the patient gains

270


Summary Box 2: Disease monitoring in PNH * *

* *

*

At diagnosis flow cytometric analysis of both erythrocytes and granulocytes Flow cytometric follow-up for evaluation of fluctuations in clone size every 6 months for 2 years, thereafter annually Immediate flow cytometric workup in case of amelioriation or worsening Bone marrow histology, cytology and cytogenetics at diagnosis and at any time of significant changes in the course of disease Test for iron, ferritin, erythropoietin, hemosiderinuria, folate levels, vitamin B12, reticulocytes and haptoglobin on a regular basis

from having PNH hematopoiesis. Eradication of PNH hematopoiesis may result in complete lack of hematopoiesis, if the disease initiating event, which eliminates normal hematopoiesis, persists (for details, see Sect. 9.2 and Fig. 9.1) [53]. This would be in accordance with the above-mentioned diminuition in PNH-clone size, which typically precedes hematopoietic insufficiency. Furthermore, it should not be forgotten, that spontaneous longterm remissions can occur [5].

9.9.1 Treatment of Anemia and Other Cytopenias in PNH Anemia is alleviated by transfusion of red blood cells or thrombocytes. Substitution of folate is generally recommended. Iron substitution must be critically overthought, as this can trigger waves of hemolysis, due to the production and simultaneous release of large numbers of complement sensitive erythrocytes into the blood stream [87]. It should be noted, that attempts to elevate the production of RBCs in patients with PNH, i.e., with erythropoietin, androgen or corticosteroid therapy, have not been successful, and no controlled data exist to suggest clinical benefit, or even whether any potential benefit outweighs the known risks of such treatments. However, anecdotal reports exist, where androgen [88] or prednisone was shown to be beneficial, presumably due to diminishing of complement activation [89]. Growth factors such as erythropoietin and G-CSF may be used in patients with significant, clinically relevant accompanying cytopenias and recurrent infections. Importantly, it must be kept in mind, that in contrast to most other hemolytic anemias, the spleen is neither enlarged, nor contributes to the abnormal hemolytic process in PNH. Therefore, splenectomy will not lead to an improvement for the patient. Rather, early reports demonstrate a mortality of 25% for PNH patients who were splenectomized [21]. In fact, operative sur-

gery of any type should be avoided where possible due to the inherent risk of thrombotic complications (see Sect. 9.9.6).

9.9.2 Treatment of Thrombotic Events in PNH Acute thrombotic complications are treated similarly to venous thrombosis occurring in other settings. Many clinicians also use prednisone in the hope of reducing complement activation, which also plays a role in thrombosis initiation. Retrospective analyses of PNH patients suggest that warfarin prophylaxis is effective when granulocytic PNH clones comprise more than 50% of total granulocytes, the platelet count is H100,000/ml, and there are no contraindications for anticoagulation [83]. However, primary prophylactic oral anticoagulation with vitamin K antagonists, as is sometimes suggested for patients with large PNH-clones, must be viewed with caution for several reasons: (1) most patients with large PNH clones have concomitant thrombocytopenia, and (2) INR is often difficult to maintain within the therapeutic range of PNH patients. This is especially the case during paroxysms due to pending acute renal failure with altered pharmacodynamics of the oral anticoagulant, or nausea with vomiting and anorexia. (3) Due to these reasons, hemorrhage following oral anticoagulation is a feared complication in patients with PNH, and has been reported to be the cause of death in up to 50% of anticoagulated PNH patients [20]. Life long anticoagulation is recommended only after established venous thrombosis. Primary prophylaxis with oral anticoagulants should however be considered in patients at increased risk. Prophylaxis with LMW-heparin should be instituted in perioperative periods, during immobilization, or when an indwelling venous catheter is present. Some authors have even suggested prophylactic administration of LMW-heparin prophylaxis during

Chap. 9

De novo Classic PNH

271

Summary Box 3: Treatment of PNH (adapted from [82]) Treatment of Anemia * Clarify the contribution of hemolysis and impaired erythropoiesis to anemia * Clarify the presence of symptoms and their correlation with anemia for the indication of treatment * Corticosteroids (0.25 1.0 mg/kg prednisone/day) in case of hemolytic crisis or chronic hemolysis (matter of dispute, but sometimes highly efficient); carefully check for infections and protect against osteopenia * Androgens or Danazol (400 mg 2/day starting dose, 200 400 mg/day maintenance in chronic hemolysis) corticosteroids (often immediate effect observed) * Iron repletion can exacerbate PNH crises independent of the route of administration although the oral route may be preferred. If intravenous iron causes hemolysis, use transfusions for suppression of erythropoiesis or steroids to control hemolysis * Transfusions should be given when necessary * Folate supplementation (5 mg/day) is recommended * Eculizumab [Soliris ], now approved for patients with increased need for RBC transfusions thromboembolic complications; patients should be vaccinated against meningococcus 2 weeks prior to start of treatment and followed for signs of meningococcus infections (dosage: 600 mg every 7 days for the first 4 weeks, then 900 mg for the fifth dose 7 days later, then 900 mg every 14 days) Prophylaxis of thrombosis (main cause of death) Control for 50% threshold of granulocytes belonging to the PNH clone which increases the probability of thrombosis from 6% (below threshold) to 44% (beyond threshold) * Warafrin prophylaxis may be instituted in patients with a PNH clone H50% and no contraindication against warfarin, particularly in the US patients [83]. Thrombosis rate is lower in Europe and thus prophylaxis not deemed standard. The decision has to be individualized. * Adapt anticoagulant therapy to renal function and platelet counts. *

Treatment of thromboembolic events Heparin or low molecular weight heparin is standard of care * Thrombolysis or radiologic intervention in acute onset Budd Chiari syndrome [84, 85]. If the patient is thrombocytopenic, this is no absolute contraindication; discuss risks with the patient and substitute platelets simultaneously with thrombolysis [86] * Life long anticoagulation is indicated, once a thromboembolic event has occured either use high-intensity coumarins [INR 3.0 4.0] or sc LMWH (low molecular weight heparin) * If a thromboembolic event occurs during treatment with coumarins *

Stem cell transplantation Remains the only curative treatment, but consider that spontaneous complete remissions may occur and allogeneic transplantation is associated with significant morbidity and mortality * Indications may be * Development of bone marrow aplasia * Recurrent life-threatening thromboembolic disease * Refractory, tranfusion-dependent hemolytic anemia * Cure rate is in the range of 50 60%, and chronic graft-versus-host disease is in the range of 35% *

pregnancy and 4 6 week post-partum, as pregnancy in women with PNH has been associated with an elevated abortion rate [90 92]. There are no studies for protective effects of antiplatelet drugs such as aspirin or clopidogrel in PNH. Thrombolysis should be considered in patients with Budd-Chiari syndrome as well as large vein- or life-threat-

ening thromobis, if the incident occurred within the last 72 h. In patients with cerebral vein thrombosis however, this may be precarious, as a thrombotic stroke may potentially be converted into an even worse hemorrhagic one. Obviously, the administration of drugs associated with an increased risk of thrombosis, such as, e.g., oral contraceptives, is best avoided where possible. Tissue plas-

272


minogen activator has been suggested for abdominal thrombosis.

9.9.3 Targeted Treatment – Complement Inhibition 9.9.3.1 Inhibition of Terminal Complement C5 and MAC-Formation Eculizumab is the only FDA and EMEA approved therapeutic option for patients with PNH. Eculizumab is a humanized monoclonal antibody that specifically targets terminal complement C5 and prevents its cleavage, thereby preventing MAC formation as well as procoagulant activity of the split-product C5a. Through specific targeting of terminal complement, early complement activities critical for clearance of microorganisms and formation of immune complexes are preserved. Eculizumab is applied intravenously at a dosage of, e.g., 600 mg every 7 days for 1 month and 900 mg i.v. every 14 days, thereafter. Dramatic reduction in hemolysis as well as improvement in anemia, abdominal pain, dysphagia, erectile dysfunction as well as reduction of transfusion dependence by 51% have been documented in several phase-II clinical trials [93, 94] (see Table 9.3). Furthermore, this well-tolerated substance probably reduces the rate of thrombotic complications. However, systematic and prolonged blockade of complement function is expected to increase susceptibility to neisserial infections [95], which is why all patients are to be vaccinated against Neisseria meningitides at least 14 days prior to initiation of therapy with eculizumab. Additionally, therapy with eculizumab will increase the proportion of PNH erythrocytes, therefore raising the possibility of severe hemolysis if the therapy is interrupted.

9.9.3.2 Exogenous Replacement of GPI-Linked Proteins Soluble recombinant forms of the natural cell membrane regulators of complement activity are rapidly cleared

from the blood stream by the kidney due to their small size. Thus, replacement of complement regulatory proteins on PNH cells and platelets is another interesting therapeutic (see 9.6.) option. Hereditary deficiency of CD55 (Inab-phenotype) is not associated with significant hemolysis or other symptoms of PNH, due to the presence of additional inhibitors of C3 convertases in human plasma. This is why CD59 was chosen as the protein to be reconstituted. The ability of endogenous GPI-CD59 to insert into cell membranes is greatly reduced in the presence of serum, an effect likely due to the adsorption onto carrier proteins such as serum albumin. Therefore, recombinant human soluble CD59 (rhCD59-P, prodaptin) was synthetically modified by attachment of a soluble membrane-interactive peptide in imicking the GPI-anchor of endogenous CD59 [96, 97]. rhCD59-P attaches to the surface of PNH erythrocytes in vitro at levels sufficient to restore complement regulatory activity, thereby reducing complement-mediated lysis in murine models [96]. Potential caveats of this approach are the short periods in which this protection is sustained (3 days in vitro and 24 h in vivo). This would necessitate daily i.v. injections, which is not ideal for an outpatient-based therapeutic option. Others have used recombinant transmembrane forms of CD59 (CD59-TM) and demonstrated similar levels of protection against complement-mediated hemolysis [98]. However, this retroviral-gene-therapy-based approach faces the as yet insurmountable problems of gene therapy in general. Protein transfer of GPI-proteins via high density lipoproteins or RBC-derived microvessels to the GPI-deficient PNH cells may also be a feasible approach, but has only been done in vitro so far [99]. In this respect, the results of a recently completed study (ClinicalTrails identifier NCT00039923) are awaited with interest. In this study, the PNH cells are examined just after the patients have received a blood transfusion, in order to determine whether certain GPI-linked proteins in the transfused blood are transferred in to the patient’s blood cells. Earlier approaches included generation of CD59 and/or CD55 as (prodrug) Fc fusion proteins, which

Table 9.3: Hallmark trials in PNH Study name

Phase

Synopsis

Ref.

TRIUMPH

III

[93]

SHEPHERD

III

Double blind, placebo controlled; reduction in hemolysis and transfusion requirements, improved anemia, fatigue and QOL, most common side effects: headache and pyrexia Open label, no placebo arm; efficacy in a broader, more diverse population of PNH patients, with relaxed inclusion criteria 87% reduction in hemolysis, reduction of transfusion independence in 51%, improvement in QOL, without significantly enhanced occurrence of infections

[94]

Chap. 9

De novo Classic PNH

273

dramatically extended the half-life of these complement inhibitory factors, compared to their soluble counterparts, albeit deficits in function were also observed [100, 101].

9.9.4 Immunosuppression Immunosuppressive therapy with, e.g., ATG [102, 103] or cyclosporine-A [104, 105] may be effective, especially when signs of hematopoietic deficiency are present. The rational is to eliminate the (auto)immune process underlying the origin of the selective pressure, which allows PNH cells to thrive.

9.9.5 Allogeneic Stem Cell Transplantation for PNH Allogeneic stem cell transplantation is the only curative means of treatment to date [106 108]. Stem cell transplantation should only be considered in patients with aplastic complications or life-threatening thrombotic or hemolytic episodes, when no other treatment options are left and all conservative measures have been exhausted. The complications and morbidity of the transplantation procedure must always carefully be weighed with the lifethreatening nature of recurrent thromboses or refractory hemolysis. Kawahara and colleagues report promising results for a small group of 9 bone marrow transplanted PNH patients.

They document long-term survival of 5/6 PNH patients transplanted for aplastic complications. It was concluded, that marrow transplantation for aplastic complications of PNH seems to be successful, well tolerated and compatible with long-term survival, when an HLA-identical sibling or syngeneic donor is available [107]. These results were later confirmed by others, showing 7/7 patients alive and with complete hematologic recovery after transplantation with unmanipulated bone marrow from HLA-identical siblings [109], as well as 3/3 alive without signs of PNH [110]. Another larger report on 57 consecutive bone marrow transplants in patients with PNH between 1978 and 1995 reports a 2-year probability of survival of HLA-identical sibling transplants in 56%, whereas only 1/7 patients transplanted from a matched unrelated donor survived [108]. In this analysis a restoration of bone marrow function was observed in 50%. Two years later, Woodard demonstrated the feasibility of matched unrelated T-cell depleted donor transplantation for three PNH-related MDS and AA patients [110]. Similar observations were seen in Poland, where a matched unrelated donor transplant was performed successfully on 2/2 PNH patients, who remained PNH-free [116]. Overall the actuarial survival seems to be 50% 5 years post-bone marrow transplant, with the main cause of death being GvHD (graft-versus-host disease), and patients receiving TBI (total body irradiation) seem to be at the highest risk (unpublished data EBMT). Results from a review of the literature are summarized in Table 9.4.

Table 9.4: Bone marrow transplant results for patients with PNH Author

Year published

Number of patients

AA pre-BMT

Donor

Cond. regimen

Outcome

Ref.

Szer

1984

4

4

[111]

1985 1989

4 2

4

1992

9

6

All alive, no PNH Alive Alive with PNH 5/6 transplanted for underlying AA alive 2/3 with non AA alive, 1/3 alive with PNH

[106] [112]

Kawahara

yes yes yes no yes yes

All alive, no PNH

Antin Kolb

3 HLA identical sibling 1 twin HLA identical sibling 1 HLA identical sibling 1 twin 6 HLA identical sibling

yes yes yes yes yes yes

9 alive All alive, no PNH All alive, no PNH/AA Alive with PNH 4 alive, no PNH

[113] [109] [110] [114] [115]

yes

All alive, no PNH

[116]

2 syngeneic twin

Bemba Raiola Woodard Cho Lee

1999 2000 2001 2001 2003

16 7 3 1 5

Markiewicz

2005

2

6 0 3 2

1 HLA non identical HLA identical sibling HLA identical sibling MUD, T cell depleted Syngeneic donor 3 HLA identical sibling 2 unrelated, 1 antigen mismatched donor MUD

AA Aplastic anemia; BMT bone marrow transplantation; MUD matched unrelated donor

[107]

274

As discussed in detail above, the immune cell composition of the bone marrow microenvironment plays an essential role in the pathogenesis of the disease. Therefore, marrow ablative conditioning regimens are required in order to eliminate both the PNH clones as well as the bone marrow environment. Relapses occur in patients receiving transplants without prior conditioning [117, 118]. Relapse may be due to emergence of new PNH-clones, rather than anewed outgrowth of pre-existing ones [118], at least in some cases.


within the red blood cells. Additionaly, many compounds can oxidize hemoglobin to met-hemoglobin directly by means of a metabolic derivative or by generating O2 and H2O2 during their metabolism, causing drug-induced oxidant injury. Topical anesthetics such as benzocaine [121], lidocaine [122] or phenazopyridine [123], used either as spray, cream or bladder-analgesic, can cause severe met-hemoglobinuria and oxidative hemolysis. In addition, contaminants present in water used in hemodialysis [124], such as copper, zinc, chloramines, formaldehyde or nitrates, may result in hemolytic episodes.

9.9.6 Perioperative Management of PNH Patients Any surgical procedure for a patient with PNH is potentially a high-risk situation for the patient and should be closely coordinated by an experienced hematologist. Interdisciplinary cooperations with both the surgeon and the anesthesiologist are essential. Preoperatively, renal function and hematologic status must be assessed and optimized if possible. It must be seen to, that dehydration and hypoxia are minimized, and special attention must be paid to avoid anesthetic drugs that may activate complement (reviewed in [119]).

9.9.7 Avoidance of Drugs Known to Induce Hemolytic Anemia Many drugs can induce hemolytic anemia. Drug-induced hemolytic anemia can be mediated by four different mechanisms, three of which are immune-mediated. Drug-induced immune hemolytic anemia is a group of disorders characterized by antibody production against red blood cells and comprises warm autoimmune hemolytic anemia, cold autoimmune hemolytic anemia and paroxysmal cold hemoglobinuria. The three immune mechanisms involved are drug adsorption, drug-dependent antibody formation and autoimmune induction. Antibodies that are directed only against the drug bound to the surface of erythrocytes are characteristic of a drug adsorption reaction, whereas antibodies directed against a combination of the drug and red cell membrane components are characteristic of drug-dependent antibody formation [120]. Autoantibody production occurs when the drug stimulates production of antibodies that are directed primarily against intrinsic red cell membrane components. The fourth mechanism involves the nonimmunologic adsorption of proteins such as IgG and/or complement to the surface of erythrocytes [120]. Furthermore, many drugs may interact with metabolic pathways

9.9.8 Management of Pregnancy in Women with PNH Pregnancy in women with PNH represents a high-risk situation for both the mother and the child, with high rates of maternal morbidity and mortality, as well as fetal wastage and prematurity [125]. Although several case reports of successful PNH-pregnancies have been published [126 132], pregnancy should not be recommended in females with PNH [125, 133]. In the largest reported series of PNH pregnancies, the maternal mortality rate reached approximately 20%, almost half the infants were delivered preterm and the perinatal mortality rate was almost 10% [86]. Thirty percentage of pregnancies end in spontaneous abortion or still birth [127]. Fetal wastage occurs in 30% and prematurity rates are high [134]. Fetal death may occur during a maternal acute hemolytic crisis [135]. Major maternal complications include thromboembolism and infection [133], and are more frequent postpartum (30%) than ante-partum or intra-partum (16%) [133]. During pregnancy maternal complications related to PNH are seen in approximately three quarters of patients. Hepatic vein thrombosis resulting in Budd Chiarisyndrome is the most common thrombotic complication [134 136]. Minor maternal complications during pregnancy occur in 75% and consist mostly of increased need for red blood cell and platelet transfusions [133]. During puerperum acute hemolytic crises may be triggered by delivery [128, 135]. Post-partum abdominal crises [127] or cerebral thrombosis [130, 137] may occur. Neonatal complications may include isoimmune hemolytic anemia due to the multiple blood transfusions received before and during pregnancy [128]. If the woman insists on pregnancy, coordinated multidisciplinary treatment of mother and fetus by obste-

Chap. 9

De novo Classic PNH

tricians, neonatologists and hematologists throughout the pregnancy is mandatory. Frequent analysis of complete blood counts and early detection of infection are obligatory. Fetal growth must be closely monitored and signs or symptoms of preterm labour need to be carefully evaluated. In patients requiring therapeutic anticoagulation due to a previous thromboembolic episode or thrombophilia, oral anticoagulants should be switched to LMW-heparin during the first trimester, because of the known associations with embryopathy, still births, neonatal deaths, spontaneous abortions and premature delivery (e.g., [138]). A switch to unfractionated heparin prior to induction of labour or close to term should be considered due to the potential advantage of antagonisation by protamine, which may be of particular importance in women with low platelet counts with the risk of thrombocytopenic bleeding. Unfractionated heparin should be continued in the first few days after delivery before switching back to LMW-heparin for the rest of the post-partum period [125]. There are currently no guidelines for regarding prophylactic anticoagulation during pregnancy in women with PNH without requirement for anticoagulant therapy prior to pregnancy. Routine prophylactic anticoagulation both during pregnancy and the post-partum period is recommended by some [133]. Anticoagulation with LMW-heparin should be strongly considered, and should be continued for 6 weeks after delivery because of the starkly elevated risk of thromboembolism which reaches 30% during this time period [133]. Planned delivery at a hospital with expertise in managing obstetric high-risk patients with a hematologist on duty is mandatory. In order to minimize the hemorrhagic risk, maternal platelet counts should be held above 30,000/ml throughout the pregnancy and above 50,000/ml near term [125]. Hypertransfusion of anemia, keeping hemoglobin levels, e.g., above 10 g/dl is also essential to enable normal development of the fetus, as low hemoglobin levels in pregnancy are associated with low birth weight, preterm delivery and growth retardation [86, 139]. Folic acid should be substituted sufficiently and oral iron replacement may be considered. As described above, indication for intravenous iron substitution should be established with caution, as it may trigger hemolysis. Although thrombocytopenia may contraindicate spinal anesthesia, this option should be used whenever possible in order to minimize labour stress, pain and respiratory acidosis so as not to precipitate a hemolytic crisis [125, 133, 140, 141]. Furthermore, the possibility of superimposed preclampsia, eclampsia and/or HELLP syndrome must be considered. Although while differentiation between the origin of symptoms may be difficult, it is obviously important.

275

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De novo Classic PNH

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10

Clonal Bone Marrow Failure Overlap Syndromes Lisa Pleyer, Daniel Neureiter, and Richard Greil

Contents Introduction ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: MDS/PNH Overlap Syndromes:::::::::::::::::::::::::::::::: Aplastic Anemia (AA) and AA Overlap Syndromes:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 10.3.1 Aplastic Anemia::::::::::::::::::::::::::::::::::::::::::: 10.3.2 AA/PNH Overlap Syndromes:::::::::::::::::::::::: 10.3.3 AA/MDS Overlap Syndromes ::::::::::::::::::::::: 10.4 T-cell Large Granular Lymphocyte Leukemia (T-LGL) and T-LGL Overlap Syndromes:::::::::::::::: 10.4.1 T LGL:::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 10.4.2 T LGL/MDS Overlap Syndromes :::::::::::::::::: 10.4.3 T LGL/PNH Overlap Syndromes::::::::::::::::::: 10.4.4 T LGL/AA and T LGL/Pure Red Cell Aplasia (PRCA) Overlap Syndromes:::::::::::::

10.1 10.2 10.3

10.1 Introduction 281 282 283 283 283 284 285 285 286 286 286

Among acquired stem cell disorders aethiopathological links have been established between hypoplastic MDS, aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH) and T-cell large granular lymphocytic leukemia (T-LGL) (see Fig. 10.1). All these entities are bone marrow failure disorders1 in which oligoclonal T-cell-mediated immune responses are without doubt pathophysiologically relevant. These overlap syndromes seem to form some kind of disease-continuum, whereby each entity can occur on its own, or arise in the background of any of the other above-mentioned diseases. As an example, PNH may follow, or precede MDS, and MDS-clones as well as PNH-clones are often detectable in patients with aplastic anemia. It may well be that T-LGL represents one extreme end of this spectrum, characterized by maximal clonal/oligoclonal T-cell proliferation, as LGL-like immunodominant cytotoxic lymphocyte (CTL) clonotypes are found within the whole spectrum of this continuum of overlap syndromes [2]. It is generally accepted that T-cell-mediated immune attack is involved in the pathophysiology of bone marrow failure syndromes including LGL, AA, MDS and PNH, although it is currently unclear, whether this reflects an autoimmune attack directed against normal hematopoiesis, or an immune surveillance reaction instigated by dysplastic myeloid cells. These bone marrow failure syndromes are characterized by polyclonal CTL expansions, as well as immunodominant clonotypes, as determined by TCR (T-cell receptor) variable beta-chain CD3 region analysis. CTL expansions, leading to TCR Vb skewing, in bone marrow biopsy specimen are found in

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Nishimura and colleagues used the following scoring system to define bone marrow failure: each cytopenia was given one point; an additional point was added for severity if Hb wasG10 g/dl, WBC G3,000/ml or PLT G60,000/ml; two additional points were added for HbG6 g/dl, WBC G1,000/ml or PLT G20,000/ml; Total scores 4 points were classified as bone marrow failure [1].

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T-LGL PNH/LGL

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81% of patients with aplastic anemia and 97% of MDS patients, including both hypo- and hypercellular variants, respectively [3 5]. Although no correlation could be determined between clonality and disease severity, the decline of pathogenic CTL clones may be used as markers of disease activity as well as to monitor hematologic response to immunosuppressive therapy [5]. Suppression of hematopoiesis in hypoplastic MDS mediated by clonally expanded cytotoxic CD8þ T-cells, similar to the mechanism of progenitor inhibition in aplastic anemia, has been shown by several groups (e.g., [5, 6]) (see Chap. 6.3 and in particular 6.3.2 for details). Furthermore, MDS is often associated with autoimmune conditions, as elaborated in Chap. 6.3 and Table 6.3, suggesting the presence of immune dysregulation in a subset of MDS patients. This provides the rational for an immunosuppressivebased treatment approach (see MDS chapter). In the hypothesized immune mechanism of hypoplastic marrow failure syndromes, CD8þ T-cells are thought to expand in response to either a neoantigen, a quantitatively upregulated antigen, or an aberrantly expressed normal protein presented by MHC-I molecules of the dysplastic cells [7]. During the immune response against dysplastic cells, destruction of normal hematopoietic bystander cells occurs. This results in bone marrow failure and cytopenia mediated by cytokine-release from activated T-cells directed against the dysplastic clone (see also Fig. 6.1a, b on p. 156). However, the inciting antigenic peptide leading to CTL selection in MDS and other bone marrow failure syndromes is unknown, as in many other diseases classified as autoimmune diseases by the way. Nevertheless, an autoimmune-mediated reaction directed against hematopoietic stem cells with concurrent suppression of normal hematopoietic bystander cells as well as bone marrow stromal cells through release of myelosuppressive and apoptosis-inducing cytokines, is currently the commonly accepted working hypothesis in the etiopathogenesis of MDS- and MDS-overlap syndromes. Sufficient evidence has accumulated that supports a mainly autoimmune pathogene-

sis involving CTLs in aplastic anemia, hypoplastic MDS and also (but to a lesser extent) PNH. Finally, the efficacy of immunosuppressive therapeutic strategies targeting T-cells provides the strongest argument for the involvement of T-cells in the pathophysiology of hypoplastic MDS as well as MDS-overlap syndromes. Normalization of extensive Vb-skewing has been used to effectively monitor activity of the disease, treatment response as well as disease relapse [8]. It has been suggested that the high rate of emergence of PNH clones in bone marrow failure syndromes is related to a relative growth advantage conferred by disturbed immune function. In fact, certain GPI-anchored proteins function as receptors for growth inhibitory cytokines such as TGF-b, IFN-g, or TNF-a, which play well recognized pathophysiologic roles in AA as well as MDS (see Chaps. 10.3 and 6.3, respectively). Therefore GPI-anchor protein deficiency would confer a further indirect growth advantage, as growth inhibitory cytokines would no longer be able to exert their function in the absence of their GPI-linked receptor (summarized in [9]). Additionally, an elevated incidence of HLA-DR2 has been found in PNH, AA/PNH and MDS/PNH, and both the presence of HLA-DR2 and a PNH-clone has been identified as an independent predictor of response to immunosuppressive therapy [10]. This further solidifies the notion that clonal expansion of GPI-deficient cells is likely related to an immune mechanism.

10.2 MDS/PNH Overlap Syndromes While PNH can occur on its own (classic PNH), it can also precede, or evolve in the setting of, another bone marrow disorder. However, evidence is accumulating that there is always an underlying bone marrow disorder, which does not necessarily have to be clinically apparent, even in the case of classic PNH. Approximately 10 23% of MDS patients have erythrocytic and granulocytic PNH clones negative for decay accelerating factor (DAF, CD55) and/or CD59 [11]. Whereas exogeneic permissive factors are required for the dominance of the abnormal clone in PNH, which is basically a benign clonal myelopathy, MDS stem cells eventually undergo transformation steps resulting in growth and survival advantages. PNH may follow, or precede MDS. These far, the appearance of PNH clones per se has not been shown to increase the risk of transformation to AML. Thus the GPI-deficient phenotype does not seem to be leukemogenic in a myelodysplastic background. Many patients with MDS/PNH have more than one PNH clone with different types and seemingly random

Chap. 10 Clonal Bone Marrow Failure Overlap Syndromes

sites of mutations in the PIG-A gene, suggesting that PIGA is mutable in this subgroup, supporting the notion of genetic instability in MDS stem cells. Hemolysis is generally less severe in MDS/PNH as opposed to de novo PNH. It is important to incorporate flow-cytometric evaluation of PNH, due to the elevated risk of thrombosis. In particular, presence of PNH-clones in MDS patients should heighten the awareness and lower the threshold for diagnostic imaging of certain complaints (particularly abdominal bloating, discomfort or pain). Additionally, one should bear in mind that the presence of PIG-deficient clones in patients with MDS seems to predict responsiveness to immunosuppressive therapy. Dunn et al. reported that 89% of patients with MDS/PNH respond to immunosuppressive therapy, in comparison to 27% MDS patients without a PNH-clone [12]. The presence of PNH-clones in patients with aplastic anemia however, did not significantly change the response rates of these patients to the same immunosuppressive regimen [12]. Thus, screening for the presence of PNH clones should be incorporated into the routine workup of all diagnosed MDS-patients.

10.3 Aplastic Anemia (AA) and AA Overlap Syndromes 10.3.1 Aplastic Anemia Acquired aplastic anemia is characterized by pancytopenia and a hypocellular bone marrow, where normal hematopoietic marrow is replaced by fat cells. Currently aplastic anemia is considered to be an autoimmune disease in which hematopoietic stem cells (HSC) are the target of autoreactive T-lymphocytes [13]. It is generally accepted that aplastic anemia is caused by a toxic insult, e.g., viral infections, drugs, or irradiation, which leads to a temporary alteration of target proteins in hematopoietic stem cells. These abnormal self-proteins initiate an (auto)immune-mediated attack [14], which remains, even after the causative event has disappeared. This autoimmune attack is mediated by CTLs, which release TNF-a and IFN-g [14]. Certain single nucleotide polymorphisms linked to high production of IFN-g and TNF-a have been found in patients with aplastic anemia [15]. Importantly, suppression of the elevated IFN-g and TNF-a levels in aplastic anemia by immunosuppressive therapy with ATG seems to be associated with hematological remission [14]. Further evidence supporting the immune-mediated pathogenesis-theory is delivered by the detection of oligoclonal T-cell clones with T-cell repertoire skewing in patients with AA and PNH [3]. The bone marrow in

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AA (and PNH) shows quantitative and qualitative deficiency in CD34þ CD38 cells [16], which exhibit reduced clonogenic potential and abnormalities at different levels of maturation [17]. These inhibitory cytokines up-regulate Fas-expression on AA-CD34þ cells, which seems to be (at least partly) responsible for the increased apoptosis in this compartment [18, 19]. This is a trait which is generally recognized as a major feature in the pathophysiology of AA. In line with these data, TRAIL (tumor necrosis factor related apoptosis inducing ligand) has been proposed to play a relevant role in the apoptosis and pathogenesis of bone marrow failure syndromes [20, 21].

10.3.2 AA/PNH Overlap Syndromes Subclinical PNH occurs frequently in the setting of AA (60%) and sometimes actively participates in hematopoiesis, which is obviously beneficial for the patient, as the PIG-A deficient clones expand to fill the void left by the aplastic process, thereby alleviating cytopenias. Sensitive modern flow-cytometric techniques detect clones with PNH-phenotype in up to 89% of untreated aplastic anemia patients [22, 23]. This may indicate the presence of hypermutation in the PIG-A gene of aplastic anemia stem cells [24]. Conversely, it has long been recognized that de novo (i.e., classic) PNH can evolve to aplastic anemia with loss of the PNH clone as a late complication, when the PNH-clone becomes exhausted and is thus unable to sustain hematopoiesis. This overlap between AA and PNH was first reported in 1967 [25]. While it appears that AA predisposes to clonal hematopoietic disorders such as PNH, MDS and AML, the appearance of PNH clones per se does not seem to increase the risk of MDS or AML in the setting of AA. Thus, as was the case for MDS/PNH, the GPI-deficient phenotype is not leukemogenic in an aplastic background. Cumulating evidence suggests that the PIGmutation does not cause clonal expansion with an intrinsic growth advantage per se [26]. Rather, cytotoxic Tcells seem to be the predominant factor involved in the selection of PNH clones, which escape autoimmune destruction due to the lack of the PIG-anchor (for details, see Chap. 9.2). In majority of the patients with aplastic anemia, PNH cells are detected, often accompanied by improvements in peripheral cytopenias [27]. However, most of these PNH-cells are present at a subclinical level, whereas some aplastic anemia patients have clearly recognizable PNH clones, and only 10% of aplastic anemia patients eventually develop overt PNH [28]. Both aplastic anemia

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and PNH are strongly associated with the HLA haplotype DR2(DR15). Many aplastic anemia patients have minor GPI anchor deficient granulocytic clones, and to a lesser extent erythrocytic clones, at presentation. The large majority of patients have more than one PNH clone as determined by the presence of multiple mutations, suggesting genetic instability leading to hypermutation in the PIG-A gene in aplastic anemia stem cells [24]. Seemingly, PNH clones have a growth advantage over normal clones in the background of aplasia, and 10 15% of patients with aplastic anemia treated with iummunosuppressive therapy develop clinical evidence of PNH, which is a known late complication of treated aplastic anemia. This means that pre-existant PIG-A deficient stem cell clones expand sufficiently to become clinically apparent only in a minority of the patients.

a

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Fig. 10.2 Bone marrow histology of aplastic anemia. a Aplastic anemia with extreme hypoplasia/aplasia of the hematopoiesis and single residuals of a megaloblastic erythropoesis as well as residuals of lymphocytes and macrophages (NASD reaction, 100). b Enlargement of Fig. 10.2a, extreme hypoplasia/aplasia, with an almost empty marrow (NASD reaction, 400)

10.3.3 AA/MDS Overlap Syndromes Severe aplastic anemia has a hypocellular, fatty marrow (see Fig. 10.2a, b), in contrast to MDS, which is usually characterized by a hypercellular marrow. However, hypocellular forms of MDS with scattered foci of myelodysplasia exist, and can be misinterpreted as aplastic anemia if these foci are missed in the bone marrow biopsy. MRI can help distinguish hypoplastic marrow disorders due to differing proton relaxing properties of fatty and cellular tissues. Advantages of MR-imaging include the capacity to sample the bulk of active marrow, and non-invasiveness of the procedure, although it merely shows the gross anatomy of the marrow. Diffuse fatty replacement of bone marrow in severe AA bestows a typical appearance in MRI [29], whereas in MDS, typical patterns of small nodules superimposed on fatty background, inhomogenously distributed cellular regions, or diffuse cellularity are observed [30, 31]. One should bear in mind however, abnormal bone marrow patterns are not disease specific [30, 31]. Diffuse cellular patterns also occur in acute and chronic leukemias, inhomogenous patterns occur in multiple myeloma and lymphoma, and a speckled pattern can also be a sign of hematological recovery following bone marrow transplantation. Furthermore, the typical fatty appearance of aplastic anemia can also be altered by transfusion-related hemosiderosis or effective treatment due to appearance of normal foci of hematopoiesis [32 34]. However, when taking the individual disease, transfusion and treatment history into account, MRI has proven useful in (i) discriminating hypoplastic MDS from aplastic anemia, (ii) detection of treatment response, and (iii) detection of early clonal disease in patients with aplastic anemia who are at high risk for developing MDS and leukemia [35 38]. Long-term survivors of aplastic anemia have a 20 30% risk of clonal evolution to secondary hematological clonal disorders such as MDS (14% cumulative risk) and secondary AML (5 10%) or AA/PNH [39, 40]. A cumulative leukemic transformation risk of 40% has been observed in non-responders to immunosuppressive therapy due to remaining genetically unstable stem cell clones, compared to 10% cumulative risk in responders [41]. Currently it is uncertain whether AA should be viewed as a premalignant state, or whether secondary clonal evolution is therapy-related. In regard of the latter, a relationship between the number of days of G-CSF therapy and the development of MDS in non-responders to immunosuppressive therapy has been postulated, but not established, by several groups [42]. Alarming data reporting MDS/AML incidences in up to 45% of children treated with immunosuppressive drugs in combination


with G-CSF exists [43]. Although a retrospective survey could not confirm these unsettling data [44], hematologists should be aware, that adding G-CSF to immunosuppressive therapy is currently not standard for patients with AA [42]. G-CSF was found to particularly facilitate the growth of calls harboring monosomy 7, which is the most common cytogenetic characteristic in MDS/AML arising from aplastic anemia [41]. However, malignant transformation was recognized as a late hematological complication in aplastic anemia well before the availability of G-CSF.

10.4 T-cell Large Granular Lymphocyte Leukemia (T-LGL) and T-LGL Overlap Syndromes

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10.4.1 T-LGL T-cell large granular lymphocyte leukemia (T-LGL) is a rare clonal T-cell disorder comprising approximately 2 5% of all chronic lymphoid leukemias in the western world and up to 9% in the Chinese and Japanese population (summarized in [45]). T-LGL is characterized by an increased number of activated CD57 positive circulating effector/cytotoxic T-cells with abundant cytoplasm and azurophilic granules. Rare reports of familial variants also exist [46]. Bone marrow failure in T-LGL can be of comparable severity to that seen in MDS, and typically presents with neutropenia and/or anemia. In contrast to MDS however, the marrow is not dysplastic (see Fig. 10.3a, b) and the risk of transformation to AML is low [47, 48]. T-LGL can occur, and probably also plays an important pathogenetic role, in the setting of MDS [49], AA [50], pure red cell aplasia (PRCA) [45] or PNH [51] and represents the best example of lineage restricted cytopenia. It has been postulated that T-LGL clones observed in these bone marrow failure syndromes expand as a result of antigenic stimulation. Frequently however, bone marrow failure can be severe and typically presents with various degrees of neutropenia or, less commonly, red cell aplasia. T-LGL has even been implicated in the pathogenesis of adultonset, but not childhood onset, cyclic neutropenia, and treatment with steroids has been shown to result in decreased counts of clonogenic T-LGL cells and abrogation of neutrophil cycling [52, 53]. There is cumulating evidence that these cytopenias result from T-cell-mediated suppression of hematopietic stem cells, a key cellular pathomechanism which seems to be common in all bone marrow-failure syndromes. Interestingly, patients from western countries with

50 μm

Fig. 10.3 Bone marrow histology of T-LGL. a T LGL showing a low to moderate diffuse interstitial infiltration of middle to large sized T lymphocytes detected by immunohistochemistry (immunohistochemistry with CD3, 200). b T LGL showing a low to moderate diffuse interstitial infiltration of middle to large sized T lymphocytes detected by immunohistochemistry (immunohisto chemistry with CD57, 200)

T-LGL often suffer from rheumatoid arthritis and recurrent infections, whereas no such association is seen in Asian patients, in whom pure red cell aplasia seems to be a major cause of morbidity, occurring in up to 64% of T-LGL-patients [45]. In this respect, T-LGL cells have been shown to directly inhibit the growth of erythroid progenitors [54]. Quite possibly clinically manifest cases of T-LGL represent the extreme of clonal/oligoclonal T-cell proliferation, which can be observed to a lesser extent in other bone marrow-failure- and MDS-overlap-syndromes. T-LGL, like MDS, is associated with autoimmune diseases. It is important to keep in mind, that lymphocytosis may not be obvious in some patients with T-LGL, as the LGL-count is less than 1,000/ml in 8% of cases. Therefore, PCR

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assessment of TCR-clonality is essential in all patients in whom classic T-LGL or a T-LGL-overlap syndrome is a diagnostic possibility [47]. T-LGL is probably an underdiagnosed condition, and it may well be that a considerable portion of idiopathic bone marrow-failure syndromes are in fact secondary to T-LGL.

10.4.2 T-LGL/MDS Overlap Syndromes Several reports of coincident T-LGL and MDS exist. A recent study found characteristics of both T-LGL and MDS in 9/100 patients [49]. As T-LGL is a rare disorder, the frequent coincidence of T-LGL/MDS suggests a causal, or commen pathogenetic, relation. T-LGL clones may arise from MDS progenitor cells. Others think this unlikely and propose a non-malignant cause of T-LGL, whereby the occurrence of T-LGL clones is seen as a result of chronic immune stimulation by an antigenic abnormality in the (dysplastic) bone marrow. This is in accord with the increased numbers of activated T-helper cells commonly observed in T-LGL, MDS and MDS/ T-LGL.

10.4.3 T-LGL/PNH Overlap Syndromes Subclinical mimicry of T-LGL-disease with expansions of CD8þ T-cells with restricted TCR-b usage was demonstrated in 24/24 patients with PNH. This demonstrates that T-LGL-like expansions occur at an unexpected frequency in patients with PNH [3]. These observations confirm an earlier report [51]. It is generally accepted however, that the emergence of PNH in patients with (subclinical) T-LGL clones, which are thought to target an antigen on the surface of normal HSCs, is probably due to immune escape, and thus clonal selection for PNH stem cells.

10.4.4 T-LGL/AA and T-LGL/Pure Red Cell Aplasia (PRCA) Overlap Syndromes Aplastic anemia and pure red cell aplasia are two further types of immune-mediated clonal bone marrow failure syndromes that can be associated with T-LGL. In the setting of T-LGL, AA is found rarely, while PRCA is found more often [50, 55 59]. In fact, T-LGL is the disorder most commonly associated with PRCA [60] and clonal T-cells are found in up to 76% of patients with PRCA. Cooccurrence of T-LGL clones and PRCA coincides with a lower CD4/CD8 ratio, and the prevalence seems especially high in Chinese patients

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[45, 61]. As T-LGL is an underdiagnosed entity, it may be possible, that a significant proportion of the idiopathic forms AA and PRCA, may in fact be secondary to T-LGL [50]. In general, the clinical findings and disease outcome in AA/T-LGL and PRCA/T-LGL seem to be similar to the primary forms of AA and PRCA [50]. However, the association of PRCA with T-LGL seems to predict a superior response to immunosuppressive therapy [60]. The presence of clonal cytogenetic abnormalities predicts poor response to immunosuppressive therapy [60]. Good response rates have been achieved with cyclosporine alone or in combination with cyclophosphamide corticosteroids [45, 50, 55, 57 60, 62, 63]. Cyclosporine as well as cyclophosphamide need to be given continuously as maintenance therapy, as attempts to reduce the dosage or stop treatment led to relapses in nearly all patients [58, 59]. One report of successful treatment of PRCA associated with T-LGL with alemtuzumab exists [64].

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List of Contributors

Editors

Authors

Univ.-Prof. Dr. med. Richard Greil Universit€atsklinik f€ ur Innere Medizin III Paracelsus Medizinische Privatuniversit€at Salzburg, Austria E-mail: [email protected]

Prof. Dr. med. Justus Duyster III. Medizinische Klinik und Poliklinik Klinikum rechts der Isar Technische Universit€at M€unchen E-mail: [email protected]

Dr. med. Dipl. Ing. biomed. inf. Lisa Pleyer Universit€atsklinik f€ ur Innere Medizin III Paracelsus Medizinische Privatuniversit€at Salzburg, Austria E-mail: [email protected]

Dr. med. Thomas Melchardt Universit€atsklinik f€ur Innere Medizin III Paracelsus Medizinische Privatuniversit€at Salzburg, Austria E-mail: [email protected]

PD Dr. med. Daniel Neureiter, M.A. Universit€atsinstitut f€ ur Pathologie Paracelsus Medizinische Privatuniversit€at Salzburg, Austria E-mail: [email protected]

Doz. Dr. med. Nikolas von Bubnoff III. Medizinische Klinik und Poliklinik Klinikum rechts der Isar Technische Universit€at M€unchen E-mail: [email protected]

Dr. med. Victoria Faber Universit€atsklinik f€ ur Innere Medizin III Paracelsus Medizinische Privatuniversit€at Salzburg, Austria E-mail: [email protected]

Dr. med. Lukas Weiss Universit€atsklinik f€ur Innere Medizin III Paracelsus Medizinische Privatuniversit€at Salzburg, Austria E-mail: [email protected]

About the Editors

Univ. Prof. Dr. Richard Greil studied medicine at the Medical University of Innsbruck, where he also had his education for Internal Medicine. He became board certified for internal medicine in 1990, had his habilitation in 1992 with the specific topic of oncogenes and their role in normal and neoplastic B-cell development, board certified for hematology and medical oncology in 1995, was appointed associate professor and deputy chief of the Department of Hematology and Oncology of the Innsbruck Medical University in 1996. He was the founding member and medical director of the Tyrolean Cancer Research Institute in 2001. In 2004 Prof. Greil was appointed full professor and director of the IIIrd Medical Department with Hematology, Medical Oncology, Hemostaseology, Infectious Disease and Rheumatology at the Private Medical University Hospital in Salzburg and founded the Laboratory of Immunological and Molecular Cancer Research (LIMCR) in Salzburg which he is heading. He is the president of the AGMT (Arbeitsgemeinschaft Medikament€ ose Tumortherapie), vice president of the ABCSG (Austrian Breast and Colorectal Cancer Study Group) and panel member of the German Hodgkin Study Group (GHSG) and many other trial groups. Prof. Greil has a basic research focus on the molecular biology and immunology of leukemias and myelomas, he has authored or co-authored more than 150 publications in peer reviewed journals and many book chapters, and regularly served as reviewer for journals like J Exp Med, Blood, Leukemia, Ann Oncol, J Immunol, Int J Cancer, Oncogene. He serves as reviewer for granting

agencies like the MRC UK, the Italian Ministry of Research, the Mildreed Scheel Foundation and the European Commission. Prof. Greil is a member of many international research societies among them the American Society of Hematology (ASH), the American Association of Medical Onoclogy (ASCO), and the American Association of Cancer Research (AACR) as well as of the European Society of Medical Oncology (ESMO) where he is a regular reviewer and author of minimal recommendation guidelines on growth factors, leukemias and lymphomas. He is the member of the Hematology Maligancies Faculty of ESMO and the ESMO ethics committee.

Dr. Lisa Pleyer was born in Zell am See, Austria. She completed her M.D. at the Leopold Franzens University in Innsbruck and her D.I. at the Private University for Medical Informatics in Hall in Tirol. Dr. Pleyer then specialised in Haematology and Oncology under the direction of Professor Richard Greil. She works in the Haematology Outpatients Department at St. Johns Hospital, Salzburg, Austria and also provides clinical training and lectures in Haematology at the Paracelsus Medical University, Salzburg. Dr. Pleyers research interests focus on myeloproliferative diseases, including the underlying cellular and molecular biology, emerging therapies and the impact of concomitant infections. She has recently established a nationwide azacitidine registry with the aim of facilitating further scientific research.

292

Dr. Daniel Neureiter was born in J€ ulich, Germany and completed his M.D. as well as his consultant of pathology at the University of ErlangenNuernberg and Institute of Pathology (Prof. Dr. Th. Kirchner). Here, main research projects dealt with the association of chronic inflammatory diseases and the extracellular matrix components. After changing to the Institute of Pathology at Salzburg (Prof. Dr. O. Dietze), he was promoted to an assistant professor at the Paracelsus Private Medical University Salzburg and is now chief senior consultant with major responsibility for solid tumours and hematopathology. His main research interest is the morphological and molecular embryonic differentiation patterning (such as b-Catenin or Hedgehog pathway) during human tumorigenesis.

About the Editors

Dr. Victoria Faber was born in Waidhofen an der Ybbs, Austria. She completed her MTA diploma in 1972 in Innsbruck and became a specialist for blood and bone marrow cytology. Dr. Faber completed her M.D. at the University of Vienna in 1999. She then specialized in haematology and oncology under the direction of Professor Richard Greil. In 2002, Dr. Faber completed a diploma in palliative medicine, which is her main focus of interest. She is currently the head of the Routine Hematological Laboratory as well as of the Palliative Medical Unit of the 3rd Medical Department at the St. Johanns Hospital in Salzburg. Dr. Faber also lectures students at the Private Medical Paracelsus University in Salzburg, Austria.

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Chronic Myeloid Leukemia: Biology and Treatment

Chronic Myeloid Leukemia

Obesity: Epidemiology, Pathophysiology, and Prevention

Aplastic Anemia: Pathophysiology and Treatment

Narcolepsy: Pathophysiology, Diagnosis, and Treatment

Aplastic Anemia: Pathophysiology and Treatment

Myeloid Leukemia – Clinical Diagnosis and Treatment

Gastroparesis: Pathophysiology, Presentation and Treatment (Clinical Gastroenterology)

Compartment Syndromes: Diagnosis, Treatment, and Complications

Diagnosis and Treatment of Movement Impairment Syndromes

Male Reproductive Dysfunction. Pathophysiology and Treatment

Functional Somatic Syndromes: Etiology, Diagnosis and Treatment

Asymptomatic Atherosclerosis: Pathophysiology, Detection and Treatment

Pediatric Trauma: Pathophysiology, Diagnosis, and Treatment

Molecular Epidemiology of Chronic Diseases

Hyperuricemic Syndromes: Pathophysiology and Therapy (Contributions to Nephrology)

Options and Options Trading

Headache and Chronic Pain Syndromes (Current Clinical Practice)

Treatment of Stress Response Syndromes

Headache and Chronic Pain Syndromes (Current Clinical Practice)

Ovarian Hyperstimulation Syndrome: Epidemiology, Pathophysiology, Prevention and Management

Myeloid Leukemia: Methods and Protocols

Myeloid Leukemia: Methods and Protocols

Conduct and Oppositional Defiant Disorders: Epidemiology, Risk Factors, and Treatment

Conduct and Oppositional Defiant Disorders: Epidemiology, Risk Factors, and Treatment

Treatment Options in Urological Cancer

Heart Physiology and Pathophysiology

Cushing's Syndrome: Pathophysiology, Diagnosis and Treatment (Contemporary Endocrinology)

Chronic Myeloid Neoplasias and Clonal Overlap Syndromes: Epidemiology, Pathophysiology and Treatment Options

Chronic Myeloid Leukemia: Biology and Treatment

Chronic Myeloid Leukemia

Obesity: Epidemiology, Pathophysiology, and Prevention

Aplastic Anemia: Pathophysiology and Treatment

Narcolepsy: Pathophysiology, Diagnosis, and Treatment

Aplastic Anemia: Pathophysiology and Treatment

Myeloid Leukemia – Clinical Diagnosis and Treatment

Gastroparesis: Pathophysiology, Presentation and Treatment (Clinical Gastroenterology)

Compartment Syndromes: Diagnosis, Treatment, and Complications

Diagnosis and Treatment of Movement Impairment Syndromes

Male Reproductive Dysfunction. Pathophysiology and Treatment

Functional Somatic Syndromes: Etiology, Diagnosis and Treatment

Asymptomatic Atherosclerosis: Pathophysiology, Detection and Treatment

Pediatric Trauma: Pathophysiology, Diagnosis, and Treatment

Molecular Epidemiology of Chronic Diseases

Hyperuricemic Syndromes: Pathophysiology and Therapy (Contributions to Nephrology)

Options and Options Trading

Headache and Chronic Pain Syndromes (Current Clinical Practice)

Treatment of Stress Response Syndromes

Headache and Chronic Pain Syndromes (Current Clinical Practice)

Ovarian Hyperstimulation Syndrome: Epidemiology, Pathophysiology, Prevention and Management

Myeloid Leukemia: Methods and Protocols

Myeloid Leukemia: Methods and Protocols

Conduct and Oppositional Defiant Disorders: Epidemiology, Risk Factors, and Treatment

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Treatment Options in Urological Cancer

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Cushing's Syndrome: Pathophysiology, Diagnosis and Treatment (Contemporary Endocrinology)

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