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.

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L. Pleyer and R. Greil

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

Molecular and Cellular Biology of CMPD

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

L. Pleyer and R. Greil

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

Molecular and Cellular Biology of CMPD

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

L. Pleyer and R. Greil

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)

Molecular and Cellular Biology of CMPD

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

L. Pleyer and R. Greil

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

Molecular and Cellular Biology of CMPD

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

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[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

Molecular and Cellular Biology of CMPD

[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

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[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

Essential Thrombocythemia

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.

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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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Essential Thrombocythemia

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%)

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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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Essential Thrombocythemia

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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

Chap. 2

Essential Thrombocythemia

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

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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

Chap. 2

Essential Thrombocythemia

(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

28

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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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

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Essential Thrombocythemia

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-

38

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

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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

Essential Thrombocythemia

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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Essential Thrombocythemia

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[250] Paietta E, Rosen N, Roberts M, Papenhausen P, Wiernik PH (1987) Philadelphia chromosome positive essential throm bocythemia evolving into lymphoid blast crisis. Cancer Genet Cytogenet 25: 227 231 [251] Rice L, Popat U (2005) Every case of essential thrombo cythemia should be tested for the Philadelphia chromosome. Am J Hematol 78: 71 73 [252] Daly K, Nandula SV, Murty VV, Nichols G (2005) Variant translocation with a deletion of derivative (9q) in a case of Philadelphia chromosome positive (Phþ) essential thrombo cythemia (ET), a variant of chronic myelogenous leukemia (CML) with a poor prognosis. Leuk Lymphoma 46: 1801 1806 [253] Pajor L, Kereskai L, Zsdral K et al. (2003) Philadelphia chromosome and/or bcr abl mRNA positive primary thrombocytosis: morphometric evidence for the transition from essential thrombocythaemia to chronic myeloid leu kaemia type of myeloproliferation. Histopathology 42: 53 60 [254] Blickstein D, Aviram A, Luboshitz J et al. (1997) BCR ABL transcripts in bone marrow aspirates of Philadelphia negative essential thrombocytopenia patients: clinical presentation. Blood 90: 2768 2771 [255] Aviram A, Blickstein D, Stark P et al. (1999) Significance of BCR ABL transcripts in bone marrow aspirates of Philadelphia negative essential thrombocythemia patients. Leuk Lymphoma 33: 77 82 [256] Singer IO, Sproul A, Tait RC, Soutar R, Gibson B (1998) BCR ABL transcripts detectable in all myeloproliferative states. Blood 92: Abstract 427a [257] Heller P, Kornblihtt LI, Cuello MT, Larripa I, Najfeld V, Molinas FC (2001) BCR ABL transcripts may be detected in essential thrombocythemia but lack clinical significance. Blood 98: 1990 [258] Emilia G, Marasca R, Zucchini P et al. (2001) BCR ABL rearrangement is not detectable in essential thrombocythe mia. Blood 97: 2187 2189 [259] Hackwell S, Ross F, Cullis JO (1999) Patients with essential thrombocythemia do not express BCR ABL transcripts. Blood 93: 2420 2421 [260] Kwong YL, Chiu EK, Liang RH, Chan V, Chan TK (1996) Essential thrombocythemia with BCR/ABL rearrangement. Cancer Genet Cytogenet 89: 74 76 [261] Morris CM, Fitzgerald PH, Hollings PE et al. (1988) Essential thrombocythaemia and the Philadelphia chromosome. Br J Haematol 70: 13 19 [262] Cervantes F, Urbano Ispizua A, Villamor N et al. (1993) Ph positive chronic myeloid leukemia mimicking essential thrombocythemia and terminating into megakaryoblastic blast crisis: report of two cases with molecular studies. Leukemia 7: 327 330 [263] Marasca R, Luppi M, Zucchini P, Longo G, Torelli G, Emilia G (1998) Might essential thrombocythemia carry Ph ano maly? Blood 91: 3084 3085 [264] Damaj G, Delabesse E, Le Bihan C et al. (2002) Typical essential thrombocythaemia does not express bcr abelson fusion transcript. Br J Haematol 116: 812 816 [265] Mesa RA (2007) Navigating the evolving paradigms in the diagnosis and treatment of myeloproliferative disor ders. Hematol Am Soc Hematol Educ Program 2007: 355 362

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-

53

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-

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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

<93%

JAK2 mutation analysis

Positive

Normal red cell mass & normal plasma volume

Negative

Hypoxic erythrocytosis

Normal red cell mass & decreased plasma volume

Assess for: – Smoking habit – Androgen intake – Diuretic intake – Hypertension – Pheochromocytoma

Assess for: – Cardiac disorder – Pulmonary disorder

EPO

Normal/low Polycythemia vera EPO-R mutation (PFCP) Exon 12 mutation Sleep apnea Other secondary causes after compensation for hypoxia by polyglobulia

Elevated

Normal Exclusion of Epo producing tumor & renal disease (cysts, tumors, renal artery stenosis) VHL gene analysis PHD2 mutation

Hemoglobin oxygen affinity

Elevated Hemoglobin electrophoresis Globin gene analysis 2,3-BGP quantification

Fig. 3.6 Alternative algorithm for diagnostic workup for patients with suspected PV when leukocytosis, thrombocytosis or splenomegaly are not present (according to Spivak et al. [74, 88]). O2 Oxygen; EPO erythropoieitin; PFCP primary familial and congenital polycythemia; VHL von Hippel Lindau; PHD2 proline hydroxylase 2; BGP bisphosphoglycerate

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neither dependent on JAK2V617F, nor limited to PV, as this phenomenon also occurs in ET and PMF [84, 85]. As no additional discriminatory information is provided by this test, it is considered a research tool of historic interest by some [74]. Other laboratory abnormalities include elevated alkaline leukocyte phosphatase (ALP) levels, elevated vitamin B12 or unbound serum B12 binding capacity (Table 3.2), both of which are characteristic of PV when present, but neither sensitive nor specific enough to be included in the major or minor criteria in any of the recently published diagnostic algorithms of PV. In the absence of access to a nuclear medicine facility providing measurements of plasma volume and red blood cell mass, phlebotomy can be diagnostic as well as therapeutic. If reduction of hematocrit to the therapeutic values of H42% in females and H45% in males requires more than two phlebotomies, and the hematocrit increases byH10% within 3 months of this phlebotomy trial in the absence of iron deficiency, absolute erythrocytosis may be assumed [74]. The difficulty in correctly diagnosing PV is further implied by the amount of false negative and positive results obtained (see Table 3.7). In most cases however, a clear-cut diagnosis can be made easily. It is to be hoped and expected, that the plethora of currently existing diagnostic algorithms by the various study groups will be brought together in the near future.

Table 3.7: False positive and false negative results in diagnosing PV False positive results (0.5%) [73] * Patients with liver cirrhosis who smoke heavily Splenomegaly Elevated RCM due to high levels of carbonmonoxide and/or Development of COPD * ET or PMF patients with elevated hematocrit for other reasons Hypoxia Smoking False negative results (10%) [53] * In obese patients calculation of RCM may be falsely low * PV patients with significant GIT blood loss may present with a normal hematocrit (bled down PV) Iron deficiency with secondary thrombocytosis * PV patients who are hypoxic for other reasons Chronic lung disease Smoking Oxygen saturation G92% * Early PV (increased RCM may not be apparent) * PV patients with elevated plasma volume RCM Red cell mass; COPD chronic obstructive pulmonary dis ease; PMF primary myelofibrosis; GIT gastrointestinal

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3.8 Differential Diagnosis of Polycythemia Vera (see Table 3.8) The differential diagnosis against reactive conditions may be difficult, particularly in comorbid patients. A thorough workup of clinical findings, basic laboratory including ALP, vitamin B12 and oxygen saturation are mandatory. As mentioned above, an increase in hematocrit can either be caused by an absolute increase in erythroid cells in the presence of constant plasma volumes, as is the case in PV, or by a decrease in plasma volume in the absence of erythroid hyperproliferation, which is frequently observed under reactive conditions, relative/spurious polyglobulia or stress erythrocytosis (Gaissb€ ock syndrome). Differential diagnoses of PV and causes of secondary polyglobulia are presented in Table 3.8. An arterial oxygen saturation level of less than 92%, even if only intermittent in nature (such as in the obstructive sleep apnea syndrome (OSAS)) should be regarded as being indicative of a causal relationship with an absolute erythrocytosis and should lead to further cardiological and pulmological diagnostics (see also algorithm in Fig. 3.5).

3.8.1 Absolute Polycythemia/Erythrocytosis (see Summary Box 1) In absolute polycythemia there is a true increase of red cell mass outside the range of 2 standard deviations of the expected mean, while plasma volume is not lowered [78]. Absolute polycythemia includes primary and secondary polycythemia. Primary polycythemia is a condition in which the erythropoietic compartment is expanding independently of extrinsic influences, or by responding inadequately to them. Primary polycythemia may be congenital, e.g., due to the mutation of the erythropoietin receptor (EPO-R) gene with subsequent truncation of the EPO-R protein, or acquired (i.e., polycythemia vera). The congenital form is also known as primary familial and congenital polycythemia (PFCP). Due to physiologic negative feedback mechanisms, primary erythrocytosis is typically associated with a low erythropoietin serum level (for more details see the appropriate sections below). Secondary polycythemia is driven by hormonal factors extrinisic to the erythroid compartment and is usually accompanied by elevated erythropoietin levels. Patients with secondary erythrocytosis due to hypoxia-mediated dysregulated erythropoiesis may however have normal erythropoietin levels, once polyglobulia reaches a steady state level, in which it compensates for tissue hypoxia (e.g. [88]). Secondary erythrocytoses can be classified into congenital (e.g., hemoglobin variants with high

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Table 3.8: Differential diagnosis of PV Causes of secondary polyglobulia Causes of secondary polyglobulia with normal or high erythropoietin levels Smokers (combined) polycythemia * Increased red cell mass with reduced plasma volume * Elevated blood carboxy Hb concentration * Low oxygen saturation in the presence of high concentrations of arterial oxygen * Reduction of Hct of 4 percentage points within a few days of cessation of smoking Oxygen-sensitive Epo-response to hypoxia secondary to * Pulmonary disease (altered lung function, with ensuing symptoms) * High altitude abidance * Intracardiac right to left or intrapulmonary shunts * Obstructive sleep apnea * Massive obesity (Pickwickian syndrome) * Red cell defects: * Congenital methemoglobinemia * Chronic exposure to carbonmonoxidea Inappropriate, autonomous increases of Epo due to Epo-secreting tumors Paraneoplastic polycythemia (can also cause hyperglycemia and electrolyte disturbances such as hypokalemia):* * Hepatocellular carcinoma (23%), hamartoma or focal hyperplasia of the liver * Hemangioblastoma, especially cerebellar (15%) (possibly due to an underlying unrecognized VHL syndrome) * Myxomata of the atrium * Uterine fibroids/myomata/leiomyomas (0.02 0.5%) * Large abdominal mass may mechanically interfere with pulmonary ventilation or blood supply to the kidneys * Inappropriate erythropoietin production by smooth muscle cells * Absence of anemia despite significant menorrhagia Endocrine disorders with elevated levels of erythropoietin or volume regulating hormones leading to plasma volume dysregulation (atrial natriuretic peptide, renin, aldosterone, antidiuretic hormone)* * Pheochromocytoma * Aldosterone producine adenomas * Bartter syndrome * Dermoid cysts of the ovary * Cushing syndrome * Erythropoietic effect of androgens in syndromes with androgen overproduction * elevated erythropoietin levels * elevated levels of volume regulating hormones leading to plasma volume dysregulation: * Atrial natriuretic peptide * Renin * Aldosterone * Antidiuretic hormone Renal disease* * Renal cell carcinoma (1 5%) * Solitary large renal cysts * Polycystic kidney disease * Hydronephrosis * Wilms tumors * Metanephric adenomas * Stenosis of a/both renal artery(ies) * Arteriolosclerosis * Hypertensive damage to renal parenchyma Post-renal transplant erythrocytosis (5 10% of renal allograft recipients) * Epo producing areas of hypoxic kidneys via increased activity of the angiotensin II receptor pathway * Develops within 8 24 months post transplant and resolves spontaneously in 25% (Continued)

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65

Table 3.8: (Continued) Mutational causes: hemoglobins with certain amino acid substitutions lead to conformational changes resulting in altered hemoglobins with high affinity for, and reduced release of, oxygen, resulting in tissue hypoxia and compensatory erythrocytosis * High oxygen affinity hemoglobinopathies * Hereditary hemorrhagic teleangiectasia with pulmonary arteriovenous malformations * Activating mutations of the Epo receptor * Idiopathic familial polycythemia * Methemoglobinemia Certain deficiencies in red cell enzymes can also result in increased oxygen affinity, reduced deoxygenation, tissue hypoxia and compensatory polcythemia * Decrease/absence of 2,3 BPG (bis phospho glycerol) mutase * Decrease/absence of phosphofructokinase * Decrease/absence of methemoglobinreductase Von Hippel-Lindau (VHL) syndrome: * Autosomal dominant genetic abnormality affecting the post ranslational control of HIF 1a * Propensity for development of renal cell carcinomas, retinal hemangioblastomas, spinal and cerebellar hemangioblastomas, pancreatic cysts, pheochromocytomas due to an additional somatic mutation * Polycythemia is not part of VHL syndrome, but is sometimes associated with the tumors that often develop Congenital polycythemias * Chuvash polycythemia Endemic, autosomal recessive VHL 598 C ! T results in impaired interaction and thus degradation of pVHL with HIF 1 and leads to increased expression of target genes: inter alia Epo, transferrin, transferrin receptor and VEGF Abnormality in oxygen sensing pathway Survival beyond 60 years is uncommon (death due to thrombo/hemorrhagic complications) * Other congenital polycythemias characterized by VHL mutations VHL 598 C !T þ 562 C !G or 574 C !T or 388C !G or VHL376G !T * Congenital polycythemia resulting from altered oxygen sensing but without mutation of VHL Thalassemia minor * Elevated RBC, but normal or reduced Hct and Hb due to microcytic, hypochromic cells Doping * Administration of androgens, anabolic steroids, or diuretics Epo *Extirpation of the tumors often resolves polyglobulia. aAssociated with headache, dizziness, nausea, altered cognition; elevated blood carboxy Hb concentration and low oxygen saturation in the presence of high concentrations of arterial oxygen Hb Hemoglobin; Hct hematocrit; EPO erythropoietin; VHL von Hippel Lindau; VEGF vascular endothelial growth factor

oxygen affinity, 2,3-diphosphoglycerate deficiency, mutations in the VHL gene), autonomous (due to high erythropoietin production by tumors), and acquired forms (physiologic response to tissue hypoxemia, e.g., in renal disease). Causes of secondary polyglobulia are listed in Table 3.8 and Summary Box 1. In patients with erythrocytosis and inadequately normal or high erythropoietin levels, hemoglobin analysis should be performed to rule out hemoglobin mutants with high oxygen affinity and/or the even rarer 2,3-bispohosphoglycerate deficiency [88] (see algorithm in Fig. 3.6).

3.8.2 Relative and Spurious/Apparent Polyglobulia Relative erythrocytosis is characterized by reduction of plasma volume (e.g., due to diuretics or severe fluid loss

due to fever, vomiting or diarrhea), while the red cell mass remains within the normal range. Apparent/spurious erythrocytosis is characterized by the additive effects of increased normal red cell mass and a lower plasma volume within the respective normal ranges (e.g., smokers polycythemia). Diagnosis requires the measurement of a stable hematocrit by serial measurements and the exclusion of comorbid conditions known to be associated with secondary erythrocytosis. The most important and frequent differential diagnosis has to be made against a chronic smoking habit which is associated with a stress-induced catecholamine response and reduced plasma volume. As far as possible, nicotin abuse should be stopped and diagnostic evaluation be repeated within 14 days. Further important and common differential diagnoses are the use of diuretics resulting in plasma volume reduction, obstructive sleep apnea, which accounts for

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Summary Box 1: Classification of absolute erythrocythosis and proposed levels of Hct that should be aimed for (according to [3]) Primary erythrocytosis Congenital: Acquired:

(Low EPO levels) EPO-R mutations (PFCP) Polycythemia vera

Secondary erythrocytosis Congenital:

(Normal/high EPO levels) Affected oxygen affinity of Hb Hemoglobin with high oxygen affinity 2,3-bisphosphoglycerate deficiency Oxygen sensing pathway defect Chuvash polycythemia VHL mutation (sporadic or familial) PHD2 mutation HIF2A mutation Physiological EPO elevation due to: Cardiac Pulmonary Renal Hepatic diseases Autonomous EPO synthesis due to: Kidney (nephroblastoma, RCC) Liver (hepatome, HCC) CNS (hemangioblastoma) Endocrine tumor (phemochromocytoma) Uterus tumor

Acquired:

Risk of complications High (keep Hct G42%/45%)* High (keep Hct G42%/45%)*

Absent (keep Hct G45%/50%)* Absent (keep Hct G45%/50%)* High High High High

(keep (keep (keep (keep

Hct G42%/45%)* Hct G42%/45%)* Hct G42%/45%)* Hct G42%/45%)*

Absent Absent Absent Absent

(keep Hct G45%/50%)* (keep Hct G45%/50%)* (keep Hct G45%/50%)* (keep Hct G45%/50%)*

Absent Absent Absent Absent Absent

(keep Hct G45%/50%) (keep Hct G45%/50%) (keep Hct G45%/50%) (keep Hct G45%/50%) (keep Hct G45%/50%)

EPO Erythropoietin; EPO R erythropoietin receptor; PFCP primary familial and congenital polycythemia; VHL von Hippel Lindau; RCC renal cell carcinoma; HCC hepatocellular carcinoma; CNS central nervous system; Hct hematocrit; * female/male

25% of unexplained polycythemia [89]), and benign or malignant tumors producing either erythropoietin or plasma volume regulating hormones (see Table 3.8). Other important potentially confounding facts leading to apparent polycythemia include obesity, hypertension, cardiovascular disease, alcohol consumption and fluid loss, e.g., due to diarrhea, and are also summarized in Table 3.8.

3.8.3 Idiopathic Erythrocytosis (IE) Idiopathic erythrocytosis (IE) is a heterogenous collection of rare hematologic disorders that may be sporadic or familial in origin. Idiopathic erythrocytosis is characterized by an elevated red cell mass and an elevated hematocrit without an identifiable cause, as well as by the absence of splenomegaly and megakaryocytic or granulocytic hyperplasia [90]. The diagnosis of IE is based on the exclusion of PV, secondary acquired poly-

cythemias and various congenital primary and secondary polcythemias [91]. Furthermore, idiopathic erythrocytosis is associated with variable erythropoietin levels and patients typically lack JAK2V617F mutations [26, 34]. However, almost a third of patients initially classified as IE, were found to bear the JAK2 exon 12 mutation. These individuals all have EEC, and must be reclassified as having a variant of PV with isolated erythroid hyperplasia [34]. Differentiation is important, as IE is a stable disease with a low thrombotic risk and negligible, if any, tendency to progress to myelofibrosis or AML [91]. In contrast to PV, a beneficial effect of phlebotomy is controversial in IE and cytoreductive drugs should be avoided in the absence of anamenstic thromboembolic events [91]. It must be kept in mind however, that a significant proportion of IE may in fact comprise early PV, or unrecognized/unrecognizable congenital or secondary acquired erythrocytosis [92]. Differential diagnosis must also be made for other conditions leading to thrombocytosis (see Table 2.2 in Essential thrombocythemia chapter).

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3.9 Risk Stratiﬁcation of Patients with PV For a detailed discussion see Risk factors for thrombotic events in ET/PV and Risk factors for myeloid disease progression sections of Chapter on Essential thrombocythemia and Tables 3.9 and 3.10a, b. Multivariate analysis of data from 459 PV patients identified advanced age (H60 years), leukocytosis H15,000/ml and a history of thrombosis at diagnosis as independent predictors of inferior survival [93]. In the absence of the first two risk factors, median survival is 272 months, compared to 108 years in patients H60 years with H15,000 leukocytes at diagnosis [93]. The risk of major thrombotic episodes occurring in 1,638 PV patients over a median follow-up period of 2.8 years is also associated with age above 65 years, history of previous thrombosis as well as arterial hypertension and claudication [59].

Table 3.9: Risk stratification of patients with PV Age H60 years or history of thrombosis

Cardiovascular risk factors

Risk category

No No Yes

No Yes Yes or no

Low Intermediate High

Table 3.10a:

Furthermore, leukocytosis independently predicts leukemic transformation, and venous thrombosis during follow-up [93], as well as risk of disease evolution to post-PV myelofibrosis [94]. Others have also demonstrated a consistent association between age H65 years and risk of leukemia [4, 95], as well as between duration of the disease and risk of disease progression to myelofibrosis [4]. However, disease duration failed to reach statistical significance as an independent risk factor for leukemic transformation in a cohort of 1,638 PV patients [95]. Total mortality is additionally negatively influenced by diabetes, smoking and splenomegaly [59]. Other significant predictors of survival include a long duration of thrombocytosis (G1,500,000/ml) as well as the usual risk factors for cardiovascular morbidity (diabetes mellitus, hypertension, adipositas, hyperlipidemia, smoking, congestive heart failure). The at first thought seemingly paradox association of platelet counts H1,500,000/ml with bleeding complications is explained by an acquired von Willebrand syndrome type II (for details, see 2.8.). No clear association between single or multiple cytotoxic drug exposure and either leukemic or fibrotic transformation has been found [93]. The ECLAP study failed to demonstrate significant differences in terms of leukemic evolution between patients treated with hydroxy-

Staging of PV patients according to WHO/ECMP criteria with therapeutic implications (adapted from [71])

PV stage Clinical

0 Early PV

ALP score and/or PRV 1 Red cell mass Serum EPO WBC (109/l) PLT (109/l) Hb (g/dl) Hct (%) Ery (1012/l) BM cellularity (%) MF grading Spleen size (cm) EEC formation BFU E JAK2V617F Therapeutic implication

" N N/# G12 G400 G16 G51 G6 50 80 MF0 G12 þ þ/þþ þ ASA

1 Polycythemic PV "

2 Classic PV

3 Advanced PV

4 Post PV MF

"

"/""

Variable

5 Spent phase PV Variable

" # G12 G400 H16 H51 H6 50 80 MF0 12 15 þ þ/þþ þ Phlebotomy, ASA

" # N ! 12 H400 H16 H51 H6 80 100 MF0/1 12 15 þ þþ þ/þþ Phlebotomy, ASA

" # H15 H1,000 H16 H52 H6 80 100 MF1/2 12 20 þ þþ þ/þþ Phlebotomy, ASA, Cytoreduction

Variable Variable H20 Variable Variable Variable Variable Decreased MF3 H20 þ þþ þþ Cytoreduction

N/# N/# H20 Variable N/# N/# N/# Decreased MF3 H20 /þ þþ þþ BSC

þ heterozygous; þ þ homozygous (in this context); ALP Alkaline leukocyte phosphatase; PRV 1 polycythemia rubra vera 1; EPO erythropoietin; WBC white blood cell; PLT platelet; Hb hemoglobin; Hct hematocrit; Ery erythrocytes; BM bone marrow; EEC endogenous erythroid colony; BFU E burst forming units erythroid lineage; ASA acetylic salicylic acid, aspirin; N normal; WHO World Health Organization; ECMP European Clinical, Molecular and Pathological

a

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Table 3.10b:

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Risk stratification and possible guides for treatment of patients with PV (according to [97])

Score

Risk level

AR

Suggested treatment for PV

G1 1 3

Low Moderate

G1.5 1.5 3

3.1 5.5 H5.5

High Very high

3.1 6 6 10

Consider ASA (careful balancing of risk/benefit ratio) ASAa; consider cytoreductive therapy with hydroxyurea; for very young patients or pregnant women consider interferon alpha ASAa; hydroxyurea ASAa; hydroxyurea; consider more aggressive treatment

a

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

Table 3.10c:

Scoring system for Table 3.10b

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

urea versus those managed with phlebotomy only [96]. Exposure to radiophosphorus, busulphan and or pipobroman may produce an excess risk of progression to MDS/ AML compared to treatment with hydroxyurea, interferon-a (INF-a) or phlebotomy alone [95], although this is very controversially discussed (for details, see respective section in ET chapter (2.11.2.) and below).

3.10 Treatment of Polycythemia Vera (PV) Staging of PV patients according to WHO/ECMP criteria with proposed therapeutic implications are summarized in Table 3.10a.

females. Shortly after diagnosis, phlebotomy may be required as often as every second day during the first 2 weeks in symptomatic patients with extremely high hematocrit levels (provided this treatment is tolerated by the patient). Infusion of physiologic saline to compensate for volume loss is usually helpful in elderly patients. The frequency of phlebotomy generally recedes, and usually levels off to once every 2 or 3 months, even when cytoreductive therapy is not indicated. Venisection usually results in substantial iron deficiency, but iron should not be substituted unless there are clinically relevant symptoms like severe glossitis or cheilitis or massive effluvium, since the substitution with iron can dramatically stimulate the growth of the neoplastic clone. Treatment of blood hyperviscosity remains a primary objective in patients with PV. Recent findings, generated from the prospective follow-up of 1,638 PV patients suggest, that the target hematocrit values may be safely higher than those generally accepted [59]. In this trial, patients with hematocrit levels H45% had a comparable risk of death, major thrombosis or total thrombosis, when compared with those patients whose hematocrit was kept G45%. Until these preliminary data have been confirmed in prospective randomized trials however, it is prudent to recommend the widely acknowledged hematocrit target values of G45% for men, G42% for women and G36% in pregnancy [98]. Proposed levels of Hct that should be aimed for in primary and secondary erythrocytosis are summarized in Summary Box 1.

3.10.2 Antiaggregatory Therapy 3.10.1 Phlebotomy Treatment has to be tailored to the individual needs of the patients. Usually the hematocrit can be managed sufficiently by phlebotomy, which remains the cornerstone of treatment. The hematocrit level should be maintained below 45% in males and below 42% in

The antithrombotic efficacy of ASA has been demonstrated in PV subjects in the ECLAP trial [96]. The therapeutic trade-off between reducing thrombotic events and possibly increasing the risk of bleeding complications must be approached by patient risk stratification as can be taken from Tables 3.9 and

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3.10.3 Indications for Treatment and Choice of Cytoreductive Drugs in Patients with PV (see Fig. 3.7)

3.10b, c. The incidence of major bleedings was not increased in this double-blind placebo controlled clinical trial with 518 enrolled PV patients, and the risk of minor bleeding events was 7.9% versus 4.5% in the placebo group. Sceptics argue that this trial may underestimate the risk in the whole PV population, due to a selection bias, as a quarter of the screened PV patients were excluded from the trial because they were judged to have a contraindication for ASA due to gastrointestinal symptoms [96]. However, the general tendency to avoid aspirin due to feared gastrointestinal bleeding events is long outdated, as the trials that generated these data used inappropriately and unnecessarily high-dose aspirin (900 mg daily) [98]. For further details, see Sects. 2.11.1, 2.11.5, 2.11.6 and 2.11.7.

Due to the above-mentioned similarities between ET and PV, practically all of the treatment paradigms, apart from the additional necessity for adequate control of hematocrit levels in PV, are very similar for these two disease entities. Therefore, the reader is referred to the Indications for treatment and choice of drugs section in the Essential thrombocythemia chapter for details on antiaggregatory and cytoreductive treatment (2.11.). When adequate control of hematocrit levels with periodic phlebotomy is not possible and/or the patient is (a) highly symptomatic, (b) at risk for cardiovascular complication or stroke, (c) older than 65 years, (d) has a

Adult non-pregnant patients with PV

Phlebotomy if Hct > 45% in males or > 42% in females

Aspirin (stop when PLT > 1,000,000/μl) (see Tables 3.10 a–b for details)

Age < 60 years

Age > 60 years

Cytoreductive treatment

No history of thrombotic or hemorrhagic events

History of thrombotic or hemorrhagic events

Cytoreductive treatment

PLT < 1,000,000/μl

PLT > 1,000,000/μl

Cytoreductive treatment

Cardiovascular risk factors

Familial thrombophilia

- arterial hypertension - smoking habit - hypercholesterinemia - diabetes mellitus

- homocystinuria - familial dominant hypercholesterinemia

Microcirculatory symptoms - erythromelalgia - angina pectoris - transient ischemic attacks

Cytoreductive treatment - first line: - second line: - third line: - fourth line:

Hydroxyurea Anagrelide Pipobroman, Interferon-alpha Radiophosphorus, Busulfan, Platelet apharesis, etc.

Fig. 3.7 Algorithm for cytoreductive treatment indications for patients with PV (adapted from [97])

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history of a thromboembolic event, phlebotomy should be combined with medical suppression of erythropoiesis. An example of a reasonable treatment algorithm for patients with PV is given in Fig. 3.7.

3.10.3.1 Hydroxyurea Hydroxyurea (HU) is usually the drug of choice for patients with PV necessitating cytoreductive therapy (important details and side effects can be found in Chap. 2.11.2.1). Its short biologic and clinical half life make it a very convenient drug, since its effects can be monitored easily. HU is also indicated when both the elevation of the hematocrit and an increased platelet count require treatment. Importantly, the presence and the allele burden of JAK2V617F in PV patients predicts chemosensitivity to hydroxyurea and correlates inversely with the daily hydroxyurea dose required in responders [10]. Thus information on JAK2V617F allele burden may help in assigning the optimal starting and/or maintenance dose in individual patients with PV [10]. Hydroxyurea toxicity may comprise cutaneous disorders (acne), buccal disorders (aphthous ulcers), leg ulcers, as well as an increased risk for cutaneous malignancy [99, 100]. In some cases, excessive thrombocythemia is the main problem of PV patients, or remains the essential problem as soon as PV can successfully be controlled by phlebotomy. In this situation, the same indications for platelet reductive therapies apply as described for ET in the respective chapter. When selective platelet reduction is necessary, anagrelide is the additional treatment of choice. For further details, see Sects. 2.11.2.1 and 2.11.2.2.

3.10.3.2 Interferon-a Interferon-a was used before hydroxyurea became available for this indication. Although high molecular response rates have been achieved in PV with pegylated interferona with seemingly preferential reduction of the malignant JAK2V617F bearing clone [101], this drug has several caveats, which have already been listed in the chapter of ET. To briefly summarize the most important, depression, local irritations at the s.c. injection site, and cardiovascular problems must be taken into consideration. For further details, see Sect. 2.11.2.3.

3.10.3.3 Pipobroman Pipobroman, a piperazine derivative, is effective in PVand induces hematologic remissions in 92 100% of patients [99, 102]. Pipobroman is more effective than hydroxyurea with regard to control of megakaryopoiesis. The drug is

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generally well tolerated [103, 104] with mild dose-related side effects, mainly gastrointestinal in nature, which generally occur early during the initial phase of treatment [100]. A study carried out from 1970 1981 observed an actuarial leukemia rate of 6%, 9% and 10% at 5, 7 and 13 years [102]. However, more recent publications report a 10-year leukemia risk of 5% in PV, which is only slightly higher than that expected as the natural evolution of the disease [105]. Several other groups show how leukemic transformation rates for ET patients treated with pipobroman [106 109]. Interestingly, the rate of progression to myelofibrosis seems much lower for patients treated with pipobroman, than for those treated with hydroxyurea [99, 105, 110]. This is thought to be due to the better control of platelet count, and consequent lowering of the secretion of fibrogenic cytokines, such as platelet-derived growth factor [99]. In addition, the antiproliferative capacity of pipboroman on megakaryocytes may be of particular value in lowering the occurrence of post-PV myleofibrosis, which seems to be the lowest registered with available treatments [105]. Five cases of severe aplastic anemia related to pipobroman have been reported in the literature, with an immune-mediated suppression of hematopoiesis as the underlying mechanism (subsumed in [104]). As immunosuppressive treatment seems effective [104], it is important to be aware of this extremely rare, but severe complication. For further details, see Sect. 2.11.2.4.

3.10.3.4 Other Cytoreductive Agents only Rarely Used Nowadays The use of myleran, busulfan and radiophosphorus (32P) should be avoided whenever possible. Radiophosphorus (32P) is convenient and effective in preventing thrombotic and hemorrhagic complications in very old and symptomatic patients with a low compliance, since a single or few injections can control the hematocrit for sustained time periods [111 114]. 32P is almost always perfectly tolerated [115]. Oral administration of 32P is also effective, and has not been associated with shorter survival or higher incidence of AML [113]. However, the leukemogenic properties of the radionucleide become significant when hydroxyurea is used simultaneously or sequentially as maintenance, as has been demonstrated by the early trials of the PVSG [115]. In this setting, hydroxyurea reduced life expectancy by 15% due to an increased leukemia and cancer risk of 30% after 15 years, versus 15% in patients treated with 32P alone [115, 116]. Therefore, maintenance therapy with hydroxyurea, rather than the initial use of 32P, increases both the risk of leukemia and carcinoma, in comparison with 32P alone. However, in cases with rapid relapse, maintenance treatment with hydroxyurea re-

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duced the vascular risks and moderately prolonged survival [115]. For these reasons, the use of hydroxyurea in 32 P pretreated patients is only justified under these circumstances. Busulphan must also be handled with extreme caution as this substance has a very tight therapeutic range and takes weeks to act adequately, leading impatient physicians to increase the dosage early. Due to the long half life of the drug, this may cause prolonged and severe cytopenias, which cannot be corrected and may be life-threatening. Leukemogenic potency of busulphan is currently controversially discussed and it seems, that 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 ineffective, may have to be revised in the future (see respective chapter in section 2.11.2.5. of ET chapter) [117 119].

3.10.4 Allogeneic Bone Marrow Transplantation in PV Allogenic bone marrow transplantation has been performed on extremely rare occasions, but is not generally considered as a treatment option in patients with classical PV. The reported cases comprise patients who developed PV at a very young age or who rapidly progressed to intractable post-PV myelofibrosis (summarized in [120]).

3.10.5 Future Treatment Possibilities – JAK2 Inhibitors Future treatment possibilities may include JAK2-inhibitors. The reader is referred to the appropriate section in the Introduction to classic CMPDs chapter (1.3.) and the chapter on PMF (4.13.6.1.).

3.11 Polycythemia Vera in Pregnancy Pregnancies are pro-thrombotic states eo ipso and confer a greater risk for venous thromboembolism, especially in patients with an intrinsic propensity for such complications, such as women with PV. Reports on pregnancies in women with PVare a lot more sparse than in women with ET. Women with uncontrolled PV may appear infertile due to recurrent early spontaneous abortions, whereas adequate disease control leads to near normal fertility rates [121, 122]. Only a handful of well documented reports on overall less than 40 such pregnancies exist. Obstetric complications are substantial, whereas maternal

71

morbidity seems minor in comparison [121, 123, 124]. However, major maternal complications occur more frequently in women with PV (44.4%) compared with ET (7.7%) [125]. The rate of surviving neonates is 50% (19/ 38). In common with ET, first trimester loss is the most common complication (21 22%), followed by late pregnancy loss and intrauterine growth retardation (15%) as well as preterm delivery (13%) [125, 126]. Preconception planning should include cessation of possible teratogenic medications, advice on wash-out periods and precise control of hematocrit prior to conception. A comprehensive thrombophilia profile should be obtained in all women with PV desiring to get pregnant. As already elaborated on in detail in the Management of Pregnancy in ET section (2.12.), interferon-a is recommended as therapeutic agent of choice in women requiring cytoreductive therapy, based on reports on its successful use in pregnant women with ET [126]. Interferon-a appears to be the cytoreductive drug least harmful for the fetus as it is not teratogenic and does not cross the placental barrier [127]. PV pregnancies are to be considered at high risk of complication to the mother and/or fetus in the case of (a) previous venous arterial or venous thrombosis or hemorrhage attributed to myeloproliferative disease, (b) previous pregnancy complications, or (c) development of any such complication in the index pregnancy [126]. Pregnancy complications include (a) first trimester fetal losses or second or third trimester pregnancy loss, (b) birth weight G5th centile for gestation, (c) intrauterine death or stillbirth and (d) preeclampsia necessitating preterm delivery G37 weeks. In the largest report of a series of 18 pregnancies in high-risk women with PV, aggressive treatment with control of hematocrit by phlebotomy to levels G45%, 75 mg ASA and prophylactic low-molecular weight heparin for 6 weeks post-partum appeared to be associated with a significantly better outcome [126, 127]. There were live births in 10/11 patients, nine at term, and no intrauterine growth retardation in women treated with phlebotomy, 75 mg aspirin and post-partum low molecular weight heparin [126]. This is in stark contrast to 1/7 live births (at 34 weeks due to placental insufficiency), three first trimester miscarriages, three stillbirths, and one neonatal death in women whose underlying PV did not receive specific attention [126]. In highest risk pregnancies, the additional use of low molecular weight heparin [128] and/or interferon-a should be contemplated and discussed with the patient [125]. Uterine artery Doppler studies should be performed at regular intervals in order to assess placental function and fetal growth monitoring should be assessed at least every 4 weeks. In case of bilateral notching, the addition of

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antioxidant vitamins C (1,000 mg/d) and E (400 IU/d) as well as dose escalation of low molecular heparin has been used in individual cases [126], although currently no evidence-based recommendations exist.

3.12 Childhood Polycythemias/ Erythrocythosis Polycythemias or erythrocytosis in childhood and adolescence not secondary to other disorders such as cyanotic heart disease, are very rare. However, their actual prevalence may be underestimated since clinical problems frequently do not occur before adulthood [88]. Primary polcythemias are caused by intrinsic defects in the erythroid precursors, whereas secondary polcythemias are driven by factors (predominantly erythropoietin) extrinisic to the erythroid progenitor cells. Generally secondary erythrocytosis results from a physiologic response to tissue hypoxia or abnormally increased erythropoietin levels.

3.12.1 Primary Familial and Congenital Polycythemia (PFCP) Primary familial and congenital polycythemia (PFCP) is characterized by elevated red blood cell mass, low serum erythropoietin level, normal oxygen affinity hemoglobin and typically autosomal dominant inheritance [129, 130]. PFCP is a rare condition caused by mutations in the gene encoding the erythropoietin receptor (EPO-R). So far, at least 14 different mutations of the EPO-R gene have been found, 11 of which result in truncation of the binding site for SHP-1 phosphatase on the cytoplasmatic proportion of the EPO-R protein (reviewed in [88]). As SHP-1 negatively regulates EPO-R signaling by dephos-

phorylation of JAK2, truncations in this region result in lack of negative feedback regulation and hypersensitivity of erythrocyte progenitors to circulating erythropoietin [131]. EPO-R truncation may also negatively influence proteosomal degradation, leading to further prolongation of EPO-R activation [132, 133]. PFCP usually follows an autosomal dominant inheritance pattern, although incomplete clinical penetrance has been reported [134]. PFCP is currently the only molecularly characterized congenital type of primary erythrocythosis (for the classification of absolute erythrocythosis see Summary Box 1, p. 66). Clinical symptoms, if present, are usually mild and relieved upon phlebotomy, although severe and fatal clinical complications do occasionally occur at mainly in adult patients [88]. Congenital defects of downstream EPO signaling pathway members, or of proteins interfering with the angiotensin-receptor pathway have been suggested as further causes of primary erythrocytosis, but remain speculative to date [135].

3.12.2 Sporadic Pediatric Non-Familial PV Pediatric PV can also present as a sporadic disease [136 138]. The median age at onset is 12.5 years [138]. Among these children with non-familial PV, the incidence of the JAK2V617F mutation may be lower (37%) than in adults (H95%) [139], although these results have not been confirmed by others [138]. JAK2 exon 12 mutations have been described in children with PV [138]. This suggests, that other molecular defects, functionally similar to the JAK2V617F mutation, affect the JAK2 dependent signaling pathway in pediatric PV. As a consequence of these apparent genetic and molceularbiologic differences (see Table 3.11), the proposed

Table 3.11: Presence of myeloproliferative and genetic markers in sporadic PV, familial PV and childhood PV [141, 169 171] Marker

Adult PV

Childhood PV

JAK2V617F JAK2 Exon 12 TPO mutations MPL mutations EEC formation PRV-1 mRNA Monoclonal hematopoiesis Low serum EPO levels Elevated TPO levels EPO-R mutation

þþþ þ n.a. n.a. þþþ þþ(þ) þþþ þþþ þ

þ(þ)

n.a. Not assessed

n.a. n.a. n.a. n.a. þ(þ) n.a. n.a.

Familial PV

/þ /þ n.a. n.a. þ

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WHO diagnostic guidelines for adult PV cannot be directly applied to pediatric cases [140], which may require a set of specific diagnostic criteria [141]. Teofili et al. propose that these pediatric criteria include (a) exclusion of familial forms due to inherited molecular defects, and (b) the consideration that pathogenetic alterations (i.e., JAK2V617F, EEC-formation, elevated PRV-1) found in adult PV patients are detectable only in some children [141]. Although the incidence of thrombotic episodes has been reported to be lower in pediatric patients than in adults [139], these children are at risk of developing thrombohemorrhagic complications, with an especially high prevelance of Budd Chiari syndrome being suggested from the available literature [138]. Therefore, these children should be treated accordingly [137].

3.12.4 Congenital Secondary Erythrocytosis

3.12.3 Familial Polycythemia Vera

3.12.4.1 High Afﬁnity Hemoglobin Variants

Polycythemia vera is the only aquired form of primary erythrocythosis and is extremely rare in children and adolescents, with only 5% of PV patients presenting at an age of les than 40 years and 0.1% at less than 20 years [142, 143]. Rare reports of autosomal dominant inheritance of familial predisposition for PV exist [144 148]. In familial cases of PV, which usually manifests in elderly family members, a genetic predisposition for the disease is extremely likely. However, clonal hematopoiesis resulting in clinical PV necessitates a supplementary second-hit in the form of an aquired somatic mutation, in addition to the inherited germline mutation [145]. In familial PV, as in sporadic PV, the JAK2V617F mutation constitutes an acquired somatic secondary event. Still unknown primary genetic aberrations, convey a background of preexisting clonal hematopoiesis and an inherited predisposition for hematopoieitc cells to acquire the JAK2V617F mutation [149 152]. This is affirmed by the pattern of lineage involvement of the JAK2V617F mutation, which may differ among diseased family members [150, 152]. A 6-fold higher risk of developing PV among first degree relatives of PV patients further supports the existence of common shared susceptibility genes predisposing to PV [153]. To date only 30 children and adolescents diagnosed with PV have been reported in the literature. Severe thromboembolic complications, including Budd Chiari syndrome [144] have been reported in children with PV and it is currently recommended to treat these children necessitating cytoreductive therapy with interferon-a or anagrelide (reviewed in [88]).

More than 200 hemoglobin variants with increased oxygen affinity are known, approximately half of which are associated with secondary erythrocytosis [154]. All these hemoglobin variants are inherited in an autosomal dominant manner [155]. Multiple structural modifications caused by autosomal dominant mutations in aglobin or -globin genes lead to impaired formation of a stable tetramer state, direct modification of the oxygen binding site, as well as formation of novel hybrid tetramers with altered oxygen binding properties. All of these structural modifications ultimately result in enhanced oxygen binding affinity which leads to a left-shift in the oxygen dissociation curve and decreased oxygen delivery into peripheral tissues. This results in compensatory upregulation of erythropoietin and compensatory erythrocytosis [154]. Once adequate tissue oxygenation has been re-established by compensatory erythrocytosis, erythropoietin production levels off, and at this new steady state, erythropoietin levels are often normal [155]. In other cases, the increase in oxygen affinity is too mild, the amount of variant hemoglobin too low, or erythrocytosis may be masked by hemolysis if the abnormal hemoglobin is unstable, or the patient is additionally heterozygote for a thalassemic trait [156]. Assessment of oxygen dissociation curves, hemoglobin electrophoresis and globin gene sequencing in case of abnormal hemoglobin in electrophoresis should be performed in polycythemic patients with oxygen saturation levels above 92% and inadequately normal or elevated serum erythropoietin levels (see diagnostic algorithm in Fig. 3.6).

Congenital heart or cyanotic lung disorders which lead to tissue hypoxia and elevated erythropoietin levels, are the most common causes of congenital secondary polycythemia, and are characterized by a normal hemoglobin dissociation curve. It is important to distinguish secondary forms of erythrocytosis from primary forms, as phlebotomy treatment should be performed with caution in the former group, since erythrocytosis is a compensatory mechanism to avoid tissue hypoxia in these patients. Therefore, the aimed at hematocrit levels are higher than in PV, in order to avoid anaerobic metabolism. Generally hematocrit levels of G45% in females and G50% in males should be aimed at in order to prevent thrombosis (see also Summary Box 1, p. 66).

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Inherited methemoglobinemias also result in secondary congenital polyglobulia [155]. Importantly, polycythemic patients with high affinity hemoglobin variants are asymptomatic and have a normal life expectancy [155]. Therefore, their differentiation form PV is crucial in order to avoid unnecessary treatment.

3.12.4.2 Congenital 2,3-Bisphosphoglycerate (BPG) Deﬁciency 2,3-BPG deficiency is an extremely rare disease caused by congenital 2,3-BPG mutase deficiency. Similar to the above-mentioned hemoglobinopathies with enhanced oxygen affinity, 2,3-BPG deficiency results in a left-shifted oxygen dissociation curve, impaired oxygen delivery and compensatory secondary erythrocytosis, however from a structurally normal hemoglobin [156]. In polycythemic patients with oxygen saturation levels above 92% and inadequately normal or elevated serum erythropoietin levels, elevated hemoglobin oxygen affinity but normal hemoglobin electrophoresis, quantification of 2,3-BPG should be performed (see diagnostic algorithm in Fig. 3.6). 2,3-BPG deficiency also seems to be the origin of secondary erythrocytosis in patients with inherited pyruvate kinase abnormalities associated with low 2,3-BPG levels [157].

3.12.4.3 Polycythemias due to Abnormal Hypoxia Sensing The molecular network responsible for cell response to hypoxia can be simplified into three major constituents: (a) the propyl hydroxylase domain-containing proteins (PHD1 3), (b) the hypoxia inducible factor (HIF), and (c) the protein product of the von Hippel-Lindau (VHL) oncosuppresor gene (pVHL). Each of these three proteins contributes critically to the maintenance of erythropoietin levels and familial erythrocytosis can be caused by mutations in either of the genes encoding for them [158]. Under normal oxygen tension, PHDs hydroxylate the a-subunit of HIF and target HIF-a for degradation by the pVHL. Under hypoxic conditions, HIF-a subunit hydroxylation is hampered, HIF-a subunits accumulate, dimerize with HIF-b, migrate into the nucleus and induce the expression of erythropoietin, among other target genes (summarized in [159]). Importantly, mutations in these genes not only lead to secondary erythrocytosis, but also seem to regulate the organ systems upon which cellular oxygen delivery ultimately depends. In this regard, abnormal pulmonary function, elevated pulmonary vascular tone, altered ventilatory, pulmonary vasoconstrictive and/or heart rate

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responses to acute hypoxia, as well as congestive heart failure are thought to be direct or indirect consequences of mutations in HIF PHD VHL signaling pathway [160, 161]. VHL mutations Chuvash polycythemia. Chuvash polycythemia, a congenital form of secondary erythrocytosis, is currently the only known endemic form of an erythrocytosis, and was discovered in central Russia [162]. Chuvash polcythemia is caused by a homozygous mutation in the VHL gene, which usually targets HIF-1 for proteasomal degradation. So far eight mutations in the VHL gene have been described, resulting in defective oxygen sensing mechanisms. Patients with Chuvash polycythemia carry a high risk for thromboembolic complications as well as for bleeding episodes and the risk for cerebral infarction seems especially high in these patients, reaching up to 45% [163]. Estimated overall survival to age 65 is approximately half of the normal population of the same geographic area and complications and mortality do not seem to be influenced by the intensity of phlebotomy treatment in these patients [163]. Proline hydroxylase mutations and hypoxia inducible factor mutations. Mutations within the PHD2 gene, which result in defective HIF hydroxylation, leading to consequent familial erythrocytosis have been reported [164, 165]. Gain of function mutations in the HIF2A gene also cause hereditary erythrocytosis [158, 159, 166, 167]. Similarly to patients with VHL or PHD2 mutations, patients with HIF2A mutations seem to have an increased thromboembolic risk and require appropriate therapy [159].

References [1] Johansson P (2006) Epidemiology of the myeloproliferative disorders polycythemia vera and essential thrombocythemia. Semin Thromb Hemost 32: 171 173 [2] Ma X, Vanasse G, Cartmel B, Wang Y, Selinger HA (2008) Prevalence of polycythemia vera and essential thrombocythe mia. Am J Hematol 83: 359 362 [3] Passamonti F, Rumi E, Pungolino E et al. (2004) Life ex pectancy and prognostic factors for survival in patients with polycythemia vera and essential thrombocythemia. Am J Med 117: 755 761 [4] Marchioli R, Finazzi G, Landolfi R et al. (2005) Vascular and neoplastic risk in a large cohort of patients with polycythemia vera. J Clin Oncol 23: 2224 2232 [5] Passamonti F, Malabarba L, Orlandi E et al. (2003) Polycythemia vera in young patients: a study on the long term risk of thrombosis, myelofibrosis and leukemia. Haematologica 88: 13 18 [6] Passamonti F, Rumi E, Arcaini L et al. (2005) Leukemic transformation of polycythemia vera: a single center study of 23 patients. Cancer 104: 1032 1036

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Chap. 3

Polycythemia Vera

[154] Wajcman H, Galacteros F (2005) Hemoglobins with high oxygen affinity leading to erythrocytosis. New variants and new concepts. Hemoglobin 29: 91 106 [155] Agarwal N, Mojica Henshaw MP, Simmons ED, Hussey D, Ou CN, Prchal JT (2007) Familial polycythemia caused by a novel mutation in the beta globin gene: essential role of P50 in evaluation of familial polycythemia. Int J Med Sci 4: 232 236 [156] Cario H, Schwarz K, Debatin KM, Kohne E (2004) Congenital erythrocytosis and polycythemia vera in child hood and adolescence. Klin Padiatr 216: 157 162 [157] Rosa R, Max Audit I, Izrael V, Beuzard Y, Thillet J, Rosa J (1981) Hereditary pyruvate kinase abnormalities associated with erythrocytosis. Am J Hematol 10: 47 55 [158] Percy MJ, Furlow PW, Lucas GS et al. (2008) A gain of function mutation in the HIF2A gene in familial erythrocy tosis. N Engl J Med 358: 162 168 [159] Martini M, Teofili L, Cenci T et al. (2008) A novel heterozy gous HIF2AM535I mutation reinforces the role of oxygen sensing pathway disturbances in the pathogenesis of familial erythrocytosis. Haematologica 93: 1068 1071 [160] Smith TG, Brooks JT, Balanos GM et al. (2008) Mutation of the von Hippel Lindau gene alters human cardiopulmonary physiology. Adv Exp Med Biol 605: 51 56 [161] Minamishima YA, Moslehi J, Bardeesy N, Cullen D, Bronson RT, Kaelin WG Jr (2008) Somatic inactivation of the PHD2 prolyl hydroxylase causes polycythemia and congestive heart failure. Blood 111: 3236 3244 [162] Percy MJ, McMullin MF, Jowitt SN et al. (2003) Chuvash type congenital polycythemia in 4 families of Asian and Western European ancestry. Blood 102: 1097 1099 [163] Gordeuk VR, Sergueeva AI, Miasnikova GY et al. (2004) Congenital disorder of oxygen sensing: association of the homozygous Chuvash polycythemia VHL mutation with thrombosis and vascular abnormalities but not tumors. Blood 103: 3924 3932 [164] Percy MJ, Furlow PW, Beer PA, Lappin TR, McMullin MF, Lee FS (2007) A novel erythrocytosis associated PHD2 mu

79

[165]

[166]

[167]

[168]

[169]

[170]

[171]

[172]

[173]

[174]

tation suggests the location of a HIF binding groove. Blood 110: 2193 2196 Al Sheikh M, Mazurier E, Gardie B et al. (2008) A study of 36 unrelated cases with pure erythrocytosis revealed three new mutations in the erythropoietin receptor gene. Haematologica 93: 1072 1075 Percy MJ, Sanchez M, Swierczek S et al. (2007) Is congenital secondary erythrocytosis/polycythemia caused by activating mutations within the HIF 2 alpha iron responsive element? Blood 110: 2776 2777 Percy MJ, Beer PA, Campbell G et al. (2008) Novel exon 12 mutations in the HIF2A gene associated with erythrocytosis. Blood 111: 5400 5402 Levine RL, Pardanani A, Tefferi A, Gilliland DG (2007) Role of JAK2 in the pathogenesis and therapy of myeloprolifera tive disorders. Nat Rev Cancer 7: 673 683 Randi ML, Putti MC, Pacquola E, Luzzatto G, Zanesco L, Fabris F (2005) Normal thrombopoietin and its receptor (c mpl) genes in children with essential thrombocythemia. Pediatr Blood Cancer 44: 47 50 Randi ML, Putti MC, Scapin M et al. (2006) Pediatric patients with essential thrombocythemia are mostly polyclonal and V617FJAK2 negative. Blood 108: 3600 3602 Veselovska J, Pospisilova D, Pekova S et al. (2008) Most pediatric patients with essential thrombocythemia show hy persensitivity to erythropoietin in vitro, with rare JAK2 V617F positive erythroid colonies. Leuk Res 32: 369 377 Wiestner A, Schlemper RJ, van der Maas AP, Skoda RC (1998) An activating splice donor mutation in the thrombo poietin gene causes hereditary thrombocythaemia. Nat Genet 18: 49 52 Berk PD, Wasserman LR, Fruchtman SM et al. (1995) Treatment of polycythemia vera: a summary of clinical trials conducted by the polycythemia vera study group. In: Berlin NI (ed) Polycythemia vera and the myeloproliferative dis orders. Saunders WB, Philadelphia, p 166 Smith BD, La Celle PL (1982) Blood viscosity and thrombosis: clinical considerations. Prog Hemost Thromb 6: 179 201

4

Primary Myeloﬁbrosis (PMF) [Previously Chronic Idiopathic Myeloﬁbrosis (CIMF), Myeloﬁbrosis with Myeloid Metaplasia (MMM), Agnogenic Myeloid Metaplasia (AMM)] Lisa Pleyer, Victoria Faber, Daniel Neureiter, and Richard Greil

Contents 4.1 Introduction to PMF::::::::::::::::::::::::::::::::::::::::::::::::::: 82 4.2 Epidemiology of PMF ::::::::::::::::::::::::::::::::::::::::::::::::: 82 4.3 Pathophysiology and Molecular Biology of PMF :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 84 4.4 Cytogenetics in PMF::::::::::::::::::::::::::::::::::::::::::::::::::: 86 4.5 Clinical Features of PMF:::::::::::::::::::::::::::::::::::::::::::: 86 4.6 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 4.7 Cytological Findings in PMF :::::::::::::::::::::::::::::::::::::: 91 4.8 Histological Findings of Bone Marrow Biopsy Specimen in PMF:::::::::::::::::::::::::::::::::::::::::::::::::::::::: 91 4.9 Imaging in Patients with PMF :::::::::::::::::::::::::::::::::::: 91 4.10 Diagnosis of Primary Myelofibrosis:::::::::::::::::::::::::::: 91 4.11 Differential Diagnosis for Primary Myelofibrosis::::::: 93 4.12 Prognostic Scores and other Prognostic Factors in PMF ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 94 4.13 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

Tyrosine Kinase Inhibitors in PMF ::::::::::::::::: 4.13.6.1 Targeting Constitutively Activated JAK2 by Selective Tyrosine Kinase Inhibitors :::::::::::::::::::::::::::::::::::::::: 4.13.6.2 Imatinib Mesylate (STI571, Gleevec)::::::::::::::::::::::::::::::::::::::: 4.13.6.3 Farensyltransferase Inhibitors :::::::::: 4.13.6.4 Other Tyrosine Kinase Inhibitors that have been Used in PMF ::::::::::: 4.13.7 Indications for Splenectomy in PMF::::::::::::::: 4.13.8 Indications for Splenic Irradiation::::::::::::::::::: 4.13.9 Treatment of Other Foci of Extramedullary Hematopoiesis and Their Complications :::::::::::::::::::::::::::::::: 4.13.9.1 Irradiation of Tumor like Manifestations of Extramedullary Hematopoiesis :::::::::::::::::::::::::::::::: 4.14 Atypical Myelofibrosis Variants ::::::::::::::::::::::::::::::::: 4.14.1 Secondary Myelofibrosis, i.e., Post Polycythemia and Post Essential Thrombocythemia Myelofibrosis :::::::::::::::::::: 4.14.2 Primary Autoimmune Myelofibrosis (AIMF) :::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 4.14.2.1 Treatment of AIMF::::::::::::::::::::::::: 4.14.3 Familial Myelofibrosis ::::::::::::::::::::::::::::::::::: 4.14.4 Idiopathic Myelofibrosis in Childhood :::::::::::: 4.13.6

104

104 104 104 104 105 106

106

106 107

107 108 108 108 109

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4.1 Introduction to PMF

4.2 Epidemiology of PMF

Primary myelofibrosis is a disease that has been characterized by a number of terms in previous literature. The International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) convened in April and November 2006 to clarify definition, response criteria and nomenclature of this condition [1]. The term primary myelofibrosis (PMF) was chosen over several other designations including chronic idiopathic myelofibrosis (CIMF), agnogenic myeloid metaplasia (AMM), and myelofibrosis with myeloid metaplasia (MMM). Myelofibrosis that develops in the setting of either polycythemia vera (PV) or essential thrombocythemia (ET) is referred to as post-PV MF and post-ET MF, respectively, and leukemic transformation is now recognized as blast phase disease (PMF-BP, post-PV/ET MF in blast phase). However, the recent change in nomenclature from chronic idiopathic myelofibrosis to primary myelofibrosis [1] is of questionable benefit at best, as well as inexact and confusing, considering that biologically, there is no such thing as primary myelofibrosis [2]. As will be further delineated in the pathophysiology section below (see 4.3.), bone marrow fibrosis is reactive and reversible if the underlying cause can be eliminated [3], and not a disease per se. Primary myelofibrosis is a stem cell-derived clonal disease, characterized by an abnormal population of hematopoietic cells that inappropriately release fibrogenic cytokines in the bone marrow, resulting in bone marrow fibrosis and osteosclerosis. The disease involves the granulo-monopoietic, erythropoietic, megakaryopoietic and sometimes B lymphocytic lineage [4, 5]. Following an initial hyperproliferative phase with leukocytosis and thrombocytosis, the evolving marrow fibrosis leads to progressive anemia and thromobocytopenia. Marrow-repopulating hematopoietic CD34 þ stem cells start to egress the marrow and colonize extramedullary sites [5], causing marked hepatosplenomegaly and other signs of extramedullary hematopoiesis, which will be described in detail below (see 4.3.). Foci of extramedullary hematopoiesis may occur in any tissue including lymph nodes, meninges, lungs, myocard or endocard, to name but several sites. These foci may behave like tumors with sometimes severe disturbances of organ functions, in that they can cause symptoms of compression of other organs due to space-consuming infiltrative growth. In the terminal phase of the disease constitutional symptoms may be very severe including fever, nocturnal sweating, pruritus, bone pain, weight loss, and severe cachexia. Finally, leukemic transformation (blast phase myelofibrosis) may occur. The heterogeneity of the hematologic picture is distinctive. PMF is the least common of the CMPDs with the worst prognosis.

The incidence rate is 0.4 1.46 cases per 100,000 annually, with the median age at diagnosis being 54 67 years. Reported female preponderance (female-to-male ratio 1.6) seems to disappear after appropriate adjustments for age. With a median progression time of 7 months, 3-year survival of 52.4% and an 8-year survival of 33%, PMF is the CMPD with the poorest prognosis. Life expectancy is reduced by 31% compared with age and sex-matched controls [6]. The main causes of death are (a) leukemic transformation (5-year risk 16% [7]), (b) portal hypertension or hepatic failure due to hepatic/splenoportal vein thrombosis or myeloid metaplasia of the liver, (c) thrombosis in other territories, (d) infectious complications secondary to progressive bone marrow failure, (e) bleeding complications due to secondary von Willebrand syndrome type II or progressive bone marrow failure, and (f) heart failure. Low risk younger patients (G55 years), which constitute 22% of all cases, have a less aggressive course of disease, and a substantially better prognosis with a median survival of 13 years [8, 9]. Childhood cases, typically presenting with an aggressive and fatal course, minimal to no splenomegaly as well as extensive bone marrow fibrosis with dysplastic features, have been occasionally reported. However, it has to be stated that these so-called cases of acute osteomyelofibrosis show an overlap spectrum to, and may mimic, acute megakaryocytic leukemia (AMLM7). In these rare patients, the differentiation between primary myelofibrosis with acute onset and AML-M7 is especially important due to the resulting differing prognostic and therapeutic consequences. This delineation can be difficult, particularly because of disease mimicry and time delaying dry taps due to marrow fibrosis, which means waiting for histologic work up [10, 11]. Additionally, long-standing primary myelofibrosis may also transform into acute megakaryocytic leukemia in its terminal phase [12]. Familial presentation is exceptionally rare and is suggested to be of autosomal recessive inheritance [13]. However, in 1st degree relatives of patients with PMF and other classical chronic myeloproliferative disorders, the risk for the development of PMF, polycythemia or ET is increased by a factor of 3.53, 5.7 and 7.37, respectively [14], whereas the risk of developing, other chronic myeloid neoplasias such as chronic myeloid leukemia is merely borderline. Interestingly, the risk of development of MDS and/or acute myeloid leukemia does not seem to be increased in 1st degree relatives, whereas the risk for developing these diseases in patients with PMF is 0.2% and 2.5%, respectively.

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Emperiopolesis

Other nosogenic, profibrotic cytokines

Proliferation of fibroblasts & mediation of osteosclerosis

Fig. 4.1 Pathophysiology of PMF. Emperiopolesis of neutrophils by magakaryocytes (MKs) is enhanced by cross talk with mono cytes and leads to the lysis of neutrophilic a granules. The profi brotic and myelopoiesis enhancing cytokine content is released into the bone marrow microenvironment. As a result of progressive bone marrow fibrosis and sclerosis, CD34þ hematopoietic stem cells are forced to egress and leave the protective bone marrow environment in search of new niches in which to sustain hemato

poiesis. This results in foci of extramedullary hematopoiesis, which can occur in virtually any organ, predominantly however in the spleen, liver and lungs, with consecutive, hepatosplenome galy, enhanced pulmonary arterial pressure, and cytological signs of inefficient extramedullary hematopoiesis (erythroblasts, reti culocytes, chronic low level hemolysis with depletion of hapto globin and grossly elevated LDH, dacryocytes, left shift in granulopoiesis)

84

4.3 Pathophysiology and Molecular Biology of PMF (Fig. 4.1) Abnormalities in the expression of cytokines play an essential role not only in myeloproliferation but also in the generation of a disease-specific microenvironment characterized by myelofibrosis, angiogenesis and osteosclerosis, all of which typically characterize PMF. These changes are reactive, since bone marrow fibroblasts are polyclonal and show normal function and growth [15]. Nosogenic cytokines include plateletderived growth factor (PDGF), angiogenesis-inducing vascular endothelial growth factor (VEGF) [16], osteosclerosis mediating and osteoclastogenesis-inhibiting stromal cell-derived RANKL-binding osteoprotegerin, basic fibroblast growth factor (b-FGF), its type I and type II receptors and especially hematopietic cell-derived, collagen synthesis inducing transforming growth factor beta (TGF-b) and its receptors [17 19]. Enhanced angiogenesis coinciding with elevated levels of VEGF may be the predominant histomorphological manifestation in the early cellular phase of PMF, whereas other functionally pleiotropic cytokines such as TGF-b, bFGF and PDGF with fibro-osteogenic potential play a progressively dominant role with transition to advanced stages of the disease [16]. In fact, bone marrow microvascular density is significantly higher in PMF and post-ET/PV-MF than in ET or PV, correlates with JAK2V617F mutational burden as well as lactate dehydrogenase (LDH) levels, and may be a useful additional tool in the histopathological definition of the disease [20]. In murine models with thrombopoietin (TPO) overexpression, TGF-b produced by hematopoietic cells was shown to be pivotal for the pathogenesis of myelofibrosis [21]. The key role of TGF-b in this process is due to the upregulation of osteoprotegerin expression, which disrupts osteoclastogenesis, thereby promoting osteosclerosis [17, 22]. Furthermore, TGF-b significantly alters the balance in synthesis and degradation of matrix [23] and induces upregulated expression of bone morphogenetic proteins (BMP) and their receptors, resulting in aberrant bone marrow matrix homeostasis [24]. As TGF-b production by neoplastic megakaryocytes seems to be under the control of NFkB [25], the latter might qualify as a target for further therapeutic approaches. Some of these cytokines are released during emperiopolesis, i.e., the random passage of different cell types through the cytoplasm of megakaryocytes [26]. This process is typically increased in patients with extreme thrombocytosis, and has been attributed to the increased expression and altered P-selectin distribution

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observed in clonal megakaryocytes of patients with PMF [23, 27]. The release of enzymes from megakaryocyte-engulfed neutrophils causes lysis of cytokinecontaining a-granules and leakage of profibrotic and myeloproliferative a-granular proteins and growth factors into the bone marrow microenvironment (see Fig. 4.1). Additionally there seems to be an emperiopolesis potentiating cross-talk as a result of pathologic interaction of neutrophils with monocytes, and the cooperation of these cells seems to be required for induction of myelofibrosis [26 28] (Fig. 4.1). In line with the above, PMF patients have increased baseline platelet activation, as shown by significantly higher levels of soluble and platelet P-selectin expression, and also higher percentages of platelet monocyte complexes, the formation of which has been attributed to P-selectin mediated binding [28]. The numbers of clonogenic bone marrow megakaryocytes are typically increased in colony assays. This growth pattern is highly cytokine-independent, but may simultaneously be extremely stimulated by hematopoietic growth factors such as TPO and/or stem cell factor [29]. In addition, not only the number of megakaryocytes derived from CD34þ cells is increased, but also their expression level of Bcl-xL which results in the resistance of these megakaryocytes towards apoptosis [29]. Megakaryocytes in PMF produce higher levels of TGF-b and matrix metalloproteinase 9 (MMP9), both of which contribute to the development of many pathological phenomena associated with PMF, such as extracellular matrix deposition, angiogenesis [16] and bone marrow fibrosis [29]. MMP9 creates a proteolytic bone marrow environment which enables constitutive mobilization of CD34þ cells into the peripheral blood [30]. Intrinsically upregulated TGF-b and b-FGF are also essential for the pathologic orchestration of neoplastic hematopoiesis. TGF-b maintains myeloid progenitor cells in quiescence, while b-FGF abrogates this growth suppressive effect of TGF-b. Escape mechanisms from the negative regulation of TGF-b include decreases in TGF-b type II receptor expression or increases in b-FGF and/or its type I and II receptors [31]. In concordance, CD34 þ cells in PMF show significantly increased expression of both b-FGF and its type I and type II receptors, as well as decreased expression of the receptor for TGF-b. The malignant hematopoietic progenitor cell clone gives rise to myeloid as well as lymphoid cells [4, 5, 32], which further ascertains the clonal stem cell character of the disease. Although bone marrow fibrosis is the principle hallmark of myelofibrosis, this phenomenon is generally accepted as the consequence, rather than the

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cause of the neoplastic disease. This is demonstrated by several findings: (i)

(ii)

(iii)

Marrow fibrosis, cytopenias and splenomegaly all disappear after successful allogeneic stem cell transplantation, thus providing evidence that the fibrotic process is under the control of the malignant but not the normal hematopoietic stem/progenitor cells [33 35]. In a murine model of myelofibrosis, targeting of TGF-b, a key cytokine controlling marrow fibrosis, effectively inhibited development of bone marrow and spleen fibrosis. However, this blockage of TGF-b was unable to hinder the further evolution of progenitor stem cell mobilization or myeloproliferation, and was thus unable to inhibit an ultimately fatal outcome [36]. Therefore it seems that myeloproliferation, rather than bone marrow fibrosis, is responsible for the lethality of this disease, at least in mice. Myelofibrosis can be induced by several exogenic factors in mice (see Table 4.1).

As outlined earlier, the insight into the gain of function mutation of JAK2V617F has led to a significant improvement in the understanding of the pathogenesis of the disease. Up to 50% of PMF patients harbor this mutation [37, 38], whereas it can be detected in nearly all patients with postTable 4.1: Induction of myelofibrosis by exogenic factors in murine models Induction of myelofibrosis in mice by *

* *

* *

Chemicals Lead acetate Saponin Benzene High dose ionizing radiation Virus infections Rauscher rat leukemia virus S37 sarcoma virus Myeloproliferative leukemia virus High doses of estrogens Antimarrow serum (immunologic mediated hyperplasia of BM connective tissue)

PV-MF (Table 4.2). Homologous recombination causes homozygosity for the mutation in 13% of patients [37], and is often associated with additional, unfavorable cytogenetic alterations [39]. Patients bearing the JAK2V617F mutation have elevated leukocyte counts, increased neutrophil and monocyte activation markers, compared to those with wildtype JAK2 [28]. Furthermore, endothelial and coagulation activation, as demonstrated by increased plasma levels of thrombomodulin and prothrombin fragments are also significantly higher in JAK2V617F positive patients [28]. For more information on the vele of JAK2V617F see Chap. 1. These levels of platelet, leukocyte, endothelial and coagulation activation markers are similar to those observed in ET and PV, and may be expected to contribute to the elevated frequency of thrombotic events (12%) observed in PMF patients [28] (for more details, see respective section in Essential thrombocythemia (2.7.)). Mutations in the TPO-receptor (cMPL) are detected in 9% of JAK2V617F mutation negative patients, and occur either in codon MPLW515L or in codon MPLW515K [40]. In murine systems, transplantation of MPLW515L mutated stem cells causes a lethal myeloproliferative disorder characterized by all clinical features of PMF, including thrombocytosis. However, these mice lack the erythrocytosis typical of JAK2 mutations [40]. Patients with MPLW515L/K mutations differ from the respective wildtype patients by older age, more severe anemia, and a higher need for transfusions [41]. About 5% of patients with PMF carry a mutation within MPL [42]. These mutations may occur concurrently with JAK2V617F mutations [42, 43], suggesting that these alleles may be functionally complementary as well as a potent effect of MPLW515L/K in supporting megakaryocytic hyperplasia and thrombocytosis. The expression level of these mutated genes remains constant during the course of the disease [43]. As mentioned above, only 50% of PMF patients have detectable mutations within JAK2V617F and/or MPL. The other 50% lack these mutations, but nonetheless show clonal hematopoiesis [44] and identical clinical symptoms. Other genetic and/or epigenetic events are therefore likely to have an impact on the pathogenesis of the disease

Table 4.2: Frequency of JAK2V617F and MPLW515L/K mutations in CMPDs [39, 48, 184, 216, 242] ET (%)

Post-ET-MF (%)

PV (%)

Post-PV-MF (%)

PMF (%)

MF-BP (%)

JAK2V617F heterozygous homozygous

60 4

40 11

H95 21 35

91 18

45 50 2 23

n.a.

MPLW515L/K Other cytogenetic abnormalities (t8; t9; 20q )

1

n.a.

0

n.a.

5 10

n.a.

4 10

n.a.

14

78

45

100

n.a. Not assessed

86

(see below and [45]). Recently, defined abnormalities in the microRNA profiles of PMF granulocytes were found, which enabled to distinguish PMF granulocytes from those of normal subjects and, partially also from ET and PV patients [46]. This may not only be of diagnostic relevance in the future, but may also provide more insight into pathogenetic mechanisms of the disease.

4.4 Cytogenetics in PMF The presence of cytogenetic alterations indicates poor prognosis in several studies [47]. They occur in approximately 50% of chemotherapy na€ıve patients with PMF, 65% of which are constituted by del(20)(q11;q13), del (13)(q12;q22), which putatively involves RB1 and seemingly confers good prognosis, and partial trisomy 1q. Trisomy 8, trisomy 9, del(12)(p11;p13) and monosomy 7 or long arm deletions involving chromosome 7 account for most other mutations [48]. So far none of the aforementioned lesions have turned out to be specific for PMF. Allelic loss of heterozygosity (LOH) frequently occurs on 1q, 3p and 3q. Chromosome 3p24 houses the retinoicacid-receptor (RAR) tumor suppressor gene, which is considered important for myeloid differentiation. In good concordance, RAR2 gene expression is markedly increased in most patients, making a case for evaluating retinoic acid-based agents in this disease. Although chromosome 6 anomalies are rare in PMF with an incidence of 2.3 3.7%, breakpoint 6p21.3 seems specifically associated with PMF [49]. Der(6)t(1;6) (q21 23;p21 23) involving 6p21 accommodates the FKBP51 gene, overexpression of which displays an antiapoptotic effect and has been repeatedly reported in megakaryocytes of patients with primary myelofibrosis. At present, the cytogenetic alterations cannot clearly be associated with certain causal disturbances of gene functions and their particular contribution to the molecular pathology is ill-defined. Negative prognostic influence seems to be associated with trisomy 8, deletion of 12p [50], trisomy 13, del(1)t (1;9) and t(6;10)(q27;q11), all of which augur early blast transformation, while deletions of 13q and 20q do not seem to associated with shorter survival, and have been termed as favorable cytogenetic abnormalities [39]. Del 7q indicates reduced survival time [51]. This is in good correlation with the observation that the proportion of patients with this karyotypic abnormality increases from 6% at diagnosis to nearly 20% at leukemic transformation [52]. Unfavorable cytogenetic clones (i.e., all cytogenetic abnormalities excluding sole 13q or 20q) in patients with PMF seem to cluster with JAK2V617F homozygosity (23% vs. 9%) [39].

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Importantly, peripheral blood can be considered as a valid alternative to bone marrow for the investigation of cytogenetic and molecular lesions in myelofibrosis [48].

4.5 Clinical Features of PMF Approximately 15 30% of patients are asymptomatic at presentation and the disease is usually diagnosed due to randomly detected leukocytosis or asymptomatic splenomegaly [53]. Symptoms due to leukostasis are uncommon in PMF, and if present, then usually in the context of leukemic transformation. Severe fatigue is the most common presenting complaint (50 70%), but this depends on the phase of the disease at initial presentation. Splenomegaly represents a hallmark of the disease, is usually mild to moderate at the beginning of the disease particularly during the hyperproliferative phase where it is present in 90% of cases [54]. Palpable hepatomegaly is present in 40 70% of patients [55]. With progression of the disease to the fibrotic phase, changes in peripheral blood usually coincide with a rapid progression of splenomegaly which then becomes symptomatic. In up to 23% of cases the spleen may extend more than 16 cm below costal margins [56]. Severe complications of splenomegaly develop in 25 50% of cases, especially in patients with huge and excessive splenomegaly (Fig. 4.2a c). As a consequence, constitutional symptoms such as early satiety, diarrhea and/or obstipation due to obstruction of the left colon flexure and/or the gastroesophageal junction, as well as B-symptoms with energy- and quality of lifeconsuming nocturnal sweats, may develop. Subjective signs of a hypermetabolic state like weight loss, nocturnal sweats, and low-grade fever are seen in 5 20% of cases especially during the terminal phase of the disease. Additionally, splenic infarctions, pleuritic inflammation and pain in the left upper abdominal quadrant with extension into the left shoulder can be caused by grossly enlarged spleens in terminal PMF. Due to the immense enlargement, the spleen is no longer protected by the rib cage. Therefore even slight traumata can lead to splenic rupture (Fig. 4.3) with often life-threatening intraabdominal blood loss and nauseating pain due to acute strain on the organs capsule. It is therefore important to inform the patient of this fact, so that the appropriate precautions can be undertaken (e.g., during sportive activities such as cycling, sailing, skiing, etc.). It is recommended to advise patients with excessively enlarged spleens to abstain from such activities. As mentioned above, symptoms due to extramedullary myeloid metaplasia can occur in almost any organ. Potential complications of hepatosplenomegaly include portal hypertension with ensuing ascites and/or esophageal as

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a

b Fig. 4.3 Rupture of the spleen. When of normal size, this organ is protected, as it is covered by the rib cage. Trauma to massively enlarged spleens can easily leed to life threatening intraabdominal blood loss

c

Fig. 4.2a Clinical hepatosplenomegaly. The blue lines depict the easily palpable margins of spleen and liver. This patient experienced significant constitutive symptoms due to hepatosplenomegaly, with predominant debilitating asthenia, weight loss and nocturnal sweats. b Gigantic spleen as seen intraoperatively. This is a photo taken intraoperatively of the massively enlarged spleen of the same patient as seen in a. It is easily imaginable that such an enormous organ can lead to constitutive symptoms and symptoms resulting form com pression and misplacement of surrounding organs. c Massive splenomegaly as seen in CT-scan. Kissing phenomenon of greatly enlarged spleen and liver

well as gastral varices with the potential for gastrointestinal blood loss due to the vulnerable vessel walls of these varices, as well as hepatic encephalopathy. Severe disturbances of liver functions may arise due to infiltration with hematopoietic foci and may result in bleeding complications due to reduced synthesis of clotting factors. In fact, liver failure is one of the major causes of death in this disease. Portal vein thrombosis may proceed the diagnosis of primary myelofibrosis and these patients may carry a JAK2V617F mutation as the sole hint for the presence of the disease [9, 57 59]. Generalized lymphadenopathy, pleural, abdominal or pericardial effusions which may cause pericardial tamponade, as well as dysuria and/or hematuria as a result of involvement of the gastrointestinal or genitourinary tract, are examples of sequelae of extramedullary hematopoiesis. Extramedullary involvement of the central nervous system may be relevant and can occur in the brain as well as in the myelon, especially in the thoracic area. Involvement of the central nervous system can be associated with symptoms due to elevated intracranial pressure including severe headaches or seizures [60 62]. When motor or sensory impairment, loss of control over bladder or bowel functions, or other neurological symptoms including progressive paraparesis occur, spinal cord compression by extramedullary foci of hematopoiesis must be considered [63, 64]. In rare cases, these may even be the presenting symptoms [65, 66]. Lung disorders are multifactorial in cause, and present as respiratory distress, pulmonary hypertension and sometimes even pulmonary failure. Pulmonary arterial hypertension (PAH) occurs in up to 36 48% of patients

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with myeloproliferative diseases, PMF being at the fore [46]. The cause of pulmonary hypertension has not been fully elucidated. Diffuse pulmonary extramedullary hematopoiesis caused by the invasion of CD34 þ cells [67], recurrent microemboli consisting of megakaryocytes and thrombocytes, pulmonary parenchymal infiltration by hematopoietic cells and extramedullary hematopoiesis [68], as well as cytokine-induced smooth-muscle hyperplasia in pulmonary arteries [69 71] have been discussed. In particular, serotonin and PDGF released from platelets in the pulmonary circulation has been proposed to be one of the mechanisms involved. Enhanced circulating endothelial progenitor cells, elevated serum VEGF levels and increased bone marrow microvessel density have been found in PMF patients with pulmonary arterial hypertension (PAH), arguing in favor of an association of PAH with a pro-angiogenic status in PMF [46]. It is tempting to speculate that inefficient extramedullary hematopoiesis, resulting in a low but chronic degree of hemolysis and subsequent NO-consumption by free hemoglobin, may also contribute to the pathogenesis of enhanced pulmonary arterial pressure, in analogy to the pathomechanism involved in pulmonary hypertension in PNH (see respective section in PNH chapter (9.2.4.)). Considering the above, patients with myelofibrosis and dyspnea should have Doppler echocardiography to evaluate pulmonary artery pressures [72]. Despite the high prevalence, the clinical impact of PMF-associated PAH seems to be small [46]. However, symptoms of PAH may be underestimated, due to an overlap of general symptoms associated with anemia, such as dyspnea on exertion or fatigue. Skin symptoms due to foci of extramedullary hematopoiesis may also occur, albeit infrequently, and present as erythematous plaques, papular skin nodules, erythema, ulcers and/or bullae [73 76]. The strongly increased cell turnover of the clonal proliferation sometimes causes severe bone and joint pain, particularly in the lower extremities, and secondary gout attacks can be seen in any phase of the disease. The increased blood flow in the areas of neoangiogenesis in cortical areas of the bones causes warmth over tibiae and knees. This increased blood flow also results in the superscan phenomenon in bone scintigraphy [77]. As mentioned above, primary myelofibrosis involves disturbances of the monocyte/macrophage lineage and sometimes the B cell lineage, as well as hypocomplementemia. The immune response may be severely disturbed, leading to recurring infections of increased severity and duration and/or infections with unusual/ atypical pathogens. Autoimmune phenomena are also frequent as shown e.g., by elevated levels of ANA, rheumatoid factor and/or lupus type anticoagulant. Clinically this may result in

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Coombs positive autoimmune hemolytic anemia, nephrotic syndrome, cutaneous vasculitis and dermatitis resembling Sweets syndrome or pyoderma gangrenosum. Frequently associated coagulopathy due to prolongation of prothrombin and partial thromboplastin times and/ or decreased levels of factor V and VIII can be observed. This is likely due to be the result of compromised synthesis in the liver burdened by foci of extramedullary hematopoiesis.

4.6 Laboratory Findings in PMF 4.6.1 Abnormal Laboratory Tests Non-specific abnormal laboratory findings include elevated serum levels of indicators of increased cell turnover, ineffective hematopoiesis or increased neutrophil mass and are summarized in Table 4.3 (e.g., [53, 56]).

4.6.2 Blood Cell Anomalies Observed in the Hyperproliferative Phase In the hyperproliferative phase an increased white blood cell count due to neutrophilia, often with a moderate left Table 4.3: Laboratory findings in patients with PMF Laboratory findings typical of primary myelofibrosis Laboratory anomalies associated with increased cell turnover Increased levels of plasma alkaline phosphatase * Increased levels of LDH * Hyperuricemia * Hyperkalemia and other electrolyte fluctuations Laboratory anomalies associated with ineffective erythropoiesis * Increased levels of LDH * Decreased levels of haptoglobin * Increased levels of reticulocytes * Increased levels of erythroblasts * Increased levels of serum bilirubin Laboratory anomalies due to increased neutrophil mass * Elevated serum level of vitamin B12 binding protein * Elevated serum level of vitamin B12 Other laboratory anomalies * Decreased levels of serum albumin * Decreased serum levels of cholesterol * Increased serum levels of VEGF (most patients) * Elevated serum levels of TPO and IL 6 * Increased levels of markers of collagen and bone synthesis, e.g., BMPs [24] * Reduced MPL surface expression in platelets * Increased urinary excretion of calmodulin *

BMP Bone morphogenetic protein

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shift is frequently observed (50%). Approximately 11% of patients demonstrate significant leukocytosis with H30,000 106/ml [7, 56]. Both polyglobulia/erythrocytosis and thrombocytosis are observed in 15% of patients [7, 56], but are often underdiagnosed due to masking by splenomegaly-induced hemodilution. Reticulocyte counts and erythroblasts are elevated due to (inefficient) extramedullary hematopoiesis, but this may be further aggravated by hemolysis. Elevated numbers of circulating CD34 þ -HSC are typically observed in early hypercellular stages and can reach, levels up to 360 times higher than in the normal population and 18 30 times higher than in patients with other Ph CMPDs [78]. These hematopoietic stem cells (as determined by the phenotype CD34 þ , CD38low, Thy1 þ , c-Kit þ , and sometimes point mutation of SCF-receptor c-Kit) mobilize to the bloodstream and colonize the spleen and other organs (see Fig. 4.1). A small fraction of circulating myeloid blasts can also be seen in up to 30% at presentation or during the course of the disease and should not lead to the diagnosis of acute leukemia [78]. These blasts are the result of inefficient, left-shifted extramedullary hematopoiesis (due to the lack of bone marrow stroma) and/or represent CD34 þ hematopoietic stem cells that have been forced to egress the progressively fibrotic bone marrow, in the search of other extramedullary niches in which they can settle down and establish extramedullary hematopoiesis. An excessively increased number of blasts (H10%) however is usually indicative of pending transformation. Furthermore, a clear-cut differentiation against erythroblasts is necessary in order not to overestimate the actual blast count, or total white blood cell count, for that matter.

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a

b

c

4.6.3 Blood Cell Anomalies Observed During the Late-Stage Osteosclerotic Phase Leukopenia is typical for the later phases of the disease, and is present in only 8% of cases. At diagnosis, normocythemic anemia is present in 50 70% of patients, 25% of which have severe anemia (HbG8 g/dl) [7, 56]. This is the result of a reduction in medullary hematopoieses and inefficient extramedullary hematopoiesis, a premature destruction of red cells in the enlarged spleen, and a dilutional anemia due to pooling of erythrocytes in excessively enlarged spleens. In addition, autoimmune hemolytic syndromes may develop, and the increased risk for bruising and bleeding due to excessive thrombocytosis or secondary von Willebrand syndrome, as well as hepatic impairment of production of procoagulatory factors, may contribute to anemia. Furthermore, thrombocytopenia is

Fig. 4.4 Cytology of peripheral blood smear in myelofibrosis. a Anisocytosis of erythrocytes with tear drop cells (white arrow), target cells (black arrows) and reticulocyte (red arrow); blast (blue arrow), giant platelets (green arrows), neutrophil myelocyte (yellow arrow). b Micromegakaryocyte with membrane blebbing of thrombocytes. c Leukerythroblastosis (left shift with erythro blasts), erythroblast (white arrow), tear drop cell (dakryocyte) (green arrow), target cell (orange arrow) Howell jolly body (red arrow), giant platelet (blue arrow), immature granulocyte (yellow arrow)

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a

b

200 μm

c

d

e

f

Fig. 4.5 Bone marrow histology in myelofibrosis. a Prefibrotic myelofibrosis with granulocytic and megakaryocytic hyperplasia (HE staining, 50). b Typical morphological aspects of megakar yocytes with dense clusters of small to giant forms with hypolobu lated nuclei and asynchronous nuclear cytoplasm maturation (HE

staining, 400). c Low grade of reticulin fibrosis surrounding the clustered megakaryocytes (Gomori staining, 400). d and e Ad vanced fibro osteosclerotic changes of myelofibrosis. d Gomori staining, 200, e HE staining, 50. f Clusters of atypical mega karyocytes (immunohistochemistry with CD61, 200)

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frequently observed in the fibrotic phase of the disease (26 37%) [7, 56]. Circulating CD34 þ cell numbers are gradually reduced with progression of the disease to the overt fibrosclerotic stage, reflecting the decrease in bone marrow reserves.

4.7 Cytological Findings in PMF (See Fig. 4.4a c) The peripheral blood smear provides the first clue to the diagnosis of primary myelofibrosis and characteristically shows nucleated erythrocytes and granulocyte precursors as leukerythroblastic findings of myelophthisis. Additionally, anisocytosis, poikilocytosis, tear-drop shaped erythrocytes (dacryocytes), increased numbers of reticulocytes, normocytic anemia, hypersegmented neutrophils, variable degrees of polychromasia, abnormally large platelets with altered granulation, fragmented megakaryocytes and less than 5% myeloblasts are usually present. As mentioned above, platelet and white blood counts are variable in primary myelofibrosis, with thrombocytopenia becoming more common during disease progression (see Fig. 4.4a c). Bone marrow aspiration usually yields a dry tap in advanced stages of the disease. If successful, the results of aspiration alone are not diagnostic and at best indicative of primary myelofibrosis, usually in the hyperproliferative early phase. Hyperlobulated neutrophilic and megakaryocytic hyperplasia with both micro- and macro-megakaryocytes are usually seen, with normal or increased erythroid precursors.

4.8 Histological Findings of Bone Marrow Biopsy Specimen in PMF (Fig. 4.5a e) Despite impressive advances in the area of molecular biology, bone marrow morphology remains the diagnostic cornerstone to identify and differentiate various myeloid neoplasms, including PMF [79]. Bone marrow biopsy is essential in the differential diagnosis of infiltration by metastatic cancer or infectious granulomata. Hypercellular bone marrow with scant fibrosis defines the cellular phase of primary myelofibrosis. Prefibrotic and early stage primary myelofibrosis presenting with a high platelet count shows megakaryocytic and granulocytic proliferation with typical clustering of megakaryocytes, which vary considerably in size and show gross abnormalities and maturation defects including: plump lobulation of megakaryocytic nuclei revealing a

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dense, hyperchromatic, bulbous, cloud-like appearance, asynchronous nuclear cytoplasm maturation, and frequent occurrence of naked nuclei. Usually some degree offibrosis, with reticulin surrounding the abnormally clustered megakaryocytes, isseenalong witha thickening andshorteningof trabeculae (osteosclerosis) as the disease progresses. Bone marrow sinusoids are expanded and intravascular hematopoiesis is observed. Erythropoiesis ranges from just detectable to increased up to 10 times normal (see Fig. 4.5a e).

4.9 Imaging in Patients with PMF Osteosclerosis resulting in diffuse or patchy increase in bone density may lead to a mottled radiographic appearance of the bone, which must be differentiated from osteoblastic metastatis of solid tumors [80]. Bone marrow imaging with MRI can demonstrate conversion of fatty marrow to cellular or fibrotic marrow [81, 82]. MRI imaging of the whole spine is essential when considering splenectomy, in order to adequately predict whether sufficient hematopoietic capacity will remain after removing one of the potentially most important sites of hematopoiesis. In case of a completely sclerotic spine without sufficient hematopoietic reserves in the bone marrow, the consequences of splenectomy will almost certainly be lethal (see also 4.13.7.). Isotopic imaging [83, 84] with 52Fe, 59Fe or 111In can directly visualize erythropoietically active marrow and may be useful for patterns of marrow loss, extension of erythropoiesis into long bones and sites of extramedullary hematopoiesis. Technetium 99 m-sulphur colloid imaging can assist in the diagnosis of extramedullary hematopoietic foci. This may be important when considering splenectomy, or when there is need to differentiate between an extramedullary hematopoietic focus and another infectious or malignant process. However, there does not appear to be any advantage to uncovering such occult foci in the asymptomatic patient.

4.10 Diagnosis of Primary Myeloﬁbrosis PMF is the least common of the CMPDs and the hardest to define because of its phenotypic, molecular and biological mimicry of a wide variety of hematologic and non-hematologic diseases. The recent scientific insight into the pathogenesis of CMPDs, boosted by the detection of the JAK2V617F mutation and consequent research shedding light on the cell biological consequences thereof, has altered the general conception of this group of diseases. Accordingly, the diagnostic criteria of chronic myeloproliferative disorders, including primary myelofibrosis,

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Table 4.4: Revised WHO (World Health Organization) criteria for the diagnosis of primary myelofibrosis (according to [38]) Revised WHO criteria for the diagnosis of PMF. Diagnosis requires the presence of all three major and 2 minor criteria Major criteria a * Presence of megakaryocytic proliferation and atypia usually accompanied by either reticulin and/or collagen fibrosis, or, in the absence of significant reticulin fibrosis, the megakaryo cyte changes must be accompanied by an increased marrow cellularity characterized by granulocytic proliferation and often decreased erythropoiesis (i.e., prefibrotic cellular phase disease) b c d * Not meeting the WHO criteria for PV , CML , MDS or other myeloid neoplasms V617F * Demonstration of JAK2 or other clonal markers (e.g., MPL5151WHL/K), or in the absence of a clonal marker, no evidence of bone marrow fibrosis due to underlying inflammatory or other neoplastic diseases Minor criteriae * Leukoerythroblastosis * Increase in serum LDH * Anemia * Palpable splenomegaly a

Small to large megakaryocytes with an aberrant nuclear/cyto plasmic ratio and hyperchormatic nucleus or irregularly folded nuclei and dense clustering b Exclusion of PV is based on hematocrit and hemoglobin levels. Red cell mass measurement is not required. Requires the failure of iron replacement therapy to increase hemoglobin level to the polycythemia vera range in the presence of decreased serum ferritin c Requires the absence of Bcr Abl d Requires the absence of dyserythropoiesis and dysgranulopoiesis as well as absence of toxic or drug induced myelopathies, infec tions, autoimmune disorders or other chronic inflammatory con ditions; Hairy cell leukemia or other lymphoid neoplasms as well as other metastatic malignancies must be excluded. It should be noted that patients with conditions associated with secondary myelofibrosis may nevertheless have primary myelofibrosis and the diagnosis should be considered in such cases if other criteria are met e Degree of abnormality is variable and does not need to be marked

have been redefined. The diagnostic criteria for PMF according to the revised WHO classification are given in Table 4.4. In addition, the International Working Group on Myelofibrosis Research and treatment (IWG-MRT) has accordingly defined the criteria necessary for diagnosing post-PV/ET myelofibrosis (Tables 4.5a, b). However, in order to compare future data with previous laboratory and clinical findings as well as with treatment results, knowledge of the previous diagnostic criteria for chronic primary myelofibrosis according to the Italian study Group (Table 4.6) as well as the Cologne criteria for agnogenic myeloid metaplasia (Table 4.7) will still be required. Bone marrow biopsy and histology is essential for diagnosing PMF [2]. It is important to remember, that the mere presence of myelofibrosis is not tantamount to the diagnosis of PMF, as many other hematological dis-

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Table 4.5a: International Working Group for Myelofibrosis Re search and Treatment (IWG MRT) criteria for the diagnosis of post PV MF (according to [214]) IWG-MRT criteria for post-Polycythemia Vera myelofibrosis Required criteria * Documentation of a previously diagnosed polycythemia vera as defined by the WHO criteria [38] * Bone marrow fibrosis grade 2 3 (on 0 3 scale of the European classification) [243] or grade 3 4 (on 0 4 scale of the standard classification) [244] Additional criteria (two are required) * Anemia (defined as below the reference value for age, sex, gender and altitude consideration) * Sustained loss of requirement of phlebotomy (in the absence of cytoreductive therapy) * Sustained loss of requirement of cytoreductive treatment of erythrocytosis * Leukoerythroblastic peripheral blood picture a * Increasing splenomegaly * Development of 1 of three constitutional symptoms * H10% weight loss in 6 months * Night sweats * Unexplained fever H37.5 C Defined as either an increase in palpable splenomegaly 5 cm below costal margin or the appearance of a newly palpable splenomegaly

a

Table 4.5b: International Working Group for Myelofibrosis Re search and Treatment (IWG MRT) criteria for post ET MF (accord ing to [214]) IWG-MRT criteria for post-essential thrombocythemia myelofibrosis Required criteria Documentation of a previously diagnosed essential thrombocythemia as defined by the WHO criteria [38] * Bone marrow fibrosis grade 2 3 (on 0 3 scale of the European classification) [243] or grade 3 4 (on 0 4 scale of the standard classification) [244] Additional criteria (two are required) * Anemia and a 2 mg/ml decrease from baseline hemoglobin level * Leukoerythroblastic peripheral blood picture a * Increasing splenomegaly * Increased LDH level * Development of 1 of three constitutional symptoms * H10% weight loss in 6 months * Night sweats * Unexplained fever H37.5 C *

Defined as either an increase in palpable splenomegaly 5 cm below costal margin or the appearance of a newly palpable splenomegaly a

eases may also have varying degrees of bone marrow fibrosis (see Table 4.8). Typical laboratory features must also be taken into consideration (see Table 4.3). Splenomegaly is so typical for PMF, that absence thereof makes the diagnosis of PMF suspect. Consequently, it has been argued, that splenomegaly should be regarded as a major

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Table 4.6: Italian criteria for the diagnosis of myelofibrosis with myeloid metaplasia Necessary criteria Diffuse bone marrow fibrosis Absence of Ph chromosome or Bcr Abl rearrangement in PB cells Optional criteria Splenomegaly of any grade Anisopoikilocytosis with tear drop erythrocytes (dakryocytes) Presence of circulating immature myeloid cells Presence of circulating erythroblasts Presence of clusters of megakarioblasts and anomalous megakaryocytes in bone marrow sections Myeloid metaplasia Diagnosis of MMM is acceptable if the following combinations are present The two necessary criteria plus any other two optional criteria when splenomegaly is present The two necessary criteria plus any other four optional criteria when splenomegaly is absent

criterion [2] (currently it is a minor criterion, as defined by the WHO [1]). Clinical staging of myelofibrosis according to the European Clinical, Molecular and Pathological (ECMP) criteria can be taken from Tables 4.9a, b ([85]). A simplified diagnostic algorithm for the

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work-up of patients with suspected PMF is given in Fig. 4.6.

4.11 Differential Diagnosis for Primary Myeloﬁbrosis (Table 4.8) Differential diagnosis of PMF can be tricky, due to disease mimicry and considerable overlap between other myeloproliferative disorders. However, a variety of other neoplastic as well as non-neoplastic causes of myelofibrosis must also be taken into consideration. Non-neoplastic causes of myelofibrosis include welldefined autoimmune diseases, particularly systemic lupus erythematodes (SLE) [86 88], and rarely systemic sclerosis [89], polyarteritis nodosa [90], Hashimotos thyroiditis [91] or Sj€ogrens syndrome [91, 92] (see Table 4.8). Approximately 10% of patients with other CMPDs have substantial collagen deposition in the bone marrow. In addition, immature myeloid cells in the peripheral blood and myeloid metaplasia can occur during the course of any CMPD, further hampering differential diagnosis. Furthermore, absence of reticulin fibrosis in early stage PMF (CIMF-0) as well as the lack of characteristic lineage

Table 4.7: Cologne clinicopathologic criteria for the diagnosis and staging of chronic idiopathic myelofibrosis [245] Clinical criteria

Pathological criteria

A1 * No preceding or allied subtype of CMPD, CML or MDS

B1 * MK and granulocytic myeloproliferation * Relative reduction of erythroid precursors * Abnormal clustering and increase in atypical giant sized MK * Cloud like lobulated nuclei * Definitive maturation defects MF stage 0: prefibrotic stage CIMF (i.e., cellular phase or smoldering MMM) * No reticulin fibrosis

A2 early clinical stages Normal Hb or anemia grade I: Hb 12 g/dl * Slight to moderate splenomegaly on palpation or H11 cm on ultrasound scan or CT * PLT H400,000/ml A3 intermediate clinical stage * Anemia grade II: Hb 10 g/dl * Definitive leukoerythroblastic blood picture and/or tear drop erythrocytes * Splenomegaly a * No adverse signs A4 advanced clinical stage * Anemia grade III: Hb 10 g/dl a * 1 or more adverse signs *

MF stage 1: early CIMF * Slight reticulin fibrosis

MF stage 2: manifest CIMF * Marked increase in reticulin and/or collagen fibrosis

MF stage 3: overt CIMF * Advanced collagen fibrosis osteosclerosis * Endophytic bone formation The combination of A1 þ B1 establishes CIMF; any other criterion confirms CIMF a

Adverse signs: ageH79 years, HbG10 g/dl,H2% myeloblasts in PB,H2% erythroblasts in PB, leukocytosisH20109/l, PLTG300,000/ml, severe constitutional symptoms, massive splenomegaly, cytogenetic abnormalities MK Megakaryocyte; Hb hemoglobin; PB peripheral blood

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Table 4.8: Differential diagnosis for primary myelofibrosis MF and/or myelophthis may occur in Myeloid disorders Transitional myeloproliferative syndrome * MDS with myelofibrosis (dyserythropoiesis, absence of splenomegaly) [222] * CMML with myelofibrosis * Atypical myeloproliferative disorders * CML with myelofibrosis (Ph positive) * Mast cell disease with myelofibrosis * Malignant histocytosis with myelofibrosis * ET (lower degree of fibrosis, splenomegaly and absence of leukerythroblastic picture) * PV (increased RBC mass) * Acute myelofibrosis (occasionally seen in patients with acute megakaryoblastic leukemia (FAB M7), associated with fever, severe BM fibrosis, pancytopenia, tear drop erythrocytes and leukerythroblastic blood picture (no palpable splenomegaly, excess MK in BM) [223]) Lymphoid disorders * Lymphomas * Hairy cell leukemia * Multiple myeloma Non-hematologic disorders * Infectious granulomata * Lipid storage disease * Osteogenic/osteoblastic metastases [246] * Mottled radiographic appearance of bone in 25 66% of patients with PMF * Autoimmune disorders * SLE [86 88] * Systemic sclerosis [89] * Sj€ ogrens syndrome [91, 92] * Hashimotos thyroiditis [91] * Mixed connective tissue disease * Polyarteritis nodosa [90] * Polymyositis * Primary pulmonary hypertension * Secondary hyperparathyroidism with vitamin D deficiency or renal osteodystrophy Atypical myelofibrosis variants * Prefibrotic myelofibrosis * Myelofibrosis with fatty marrow Primary autoimmune myelofibrosis [221] *

BM Bone marrow; RBC red blood cell; MK megakaryocyte

Table 4.9a: European clinical, molecular and pathological (ECMP) criteria for the staging of chronic idiopathic myelofibrosis (CIMF) (according to [85]) Clinical staging of myelofibrosis (ECMP) Early clinical stage * Platelet count H400,000/ml, usually 1,000,000/ml * No leukerythroblastosis * No anemia * No or only slight splenomegaly (in sonography) * MF 0 or MF 1 (according to [243] and see Table 4.9b) Intermediate clinical stage * Definitive leukerythroblastosis * Anemia grade 1 (Hb G12 g/dl but H10 g/dl) * Splenomegaly on palpation * MF 1 or MF 2 (according to [243] and see Table 4.9b) Advanced clinical stage * Pronounced leukerythroblastosis * Anemia grade 2 (Hb G10 g/dl or H10 g/dl but with the presence of adverse signsa) * Pronounced splenomegaly * Leukocytosis or leukopenia * Normal or decreased platelet count * MF 2 and MF 3 (according to [243] and see Table 4.9b) Adverse signs: age H70 years, hemoglobin (Hb) G10 g/dl, myeloblasts in peripheral blood H2%, erythro normoblasts H2%, leukocytosis H20,000/ml, thrombocytopenia G300,000/ml, severe constitutional symptoms, massive splenomegaly, cyto genetic abnormalities a

proliferation difficulties.

in

PMF

can

lead

to

diagnostic

4.12 Prognostic Scores and other Prognostic Factors in PMF Biomarkers are essential tools in order to more precisely predict the spontaneous prognosis of the disease and/or the response to certain types of treatment. The establishment of risk scores is of utmost importance for an adequate tailoring of risk/benefit ratios for treatments

Table 4.9b: Grading of myelofibrosis (according to [243]) Grading of myelofibrosis MF 0

Prefibrotic MF

MF 1

Early fibrotic CIMF 1

MF 2

Fibrotic CIMF 2

MF 3

Classic CIMF 3

MFH3

Endstage MF

Scattered linear reticulin with no intersections (cross over) corresponding to normal bone marrow Loose network of reticulin with many intersections, especially in peripheral areas, no collagenization Diffuse and dense increase in reticulin with extensive intersections, occasionally with only focal bundles of collagen and/or focal osteosclerosis Diffuse and dense increase in reticulin with extensive intersections with only course bundles of collagen often associated with significant osteosclerosis Hypocellular bone marrow with extensive osteomyelosclerosis

Chap. 4

Primary Myeloﬁbrosis

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Leukerythroblastic blood picture and splenomegaly on ultrasound Bone marrow biopsy, reticulin stain, cytogenetic studies Increased marrow reticulin in the absence of an infiltrative or granulomatous process also known to be associated with marrow fibrosis (see Table 4.8)

Mutation screening for JAK2V617F (peripheral blood), screening for Bcr–Abl

Ph-chromosome positive CML

V617F-positive OR del(13q)

Other cytogenetic abnormalities

Normal cytogenetics AND V617F-negative

PMF likely BUT use histology to exclude other CMPD

Could be PMF OR MDS OR other myeloid neoplasm

Use histology for specific diagnosis

Fig. 4.6 Algorithm for diagnostic workup of patients with suspected PMF according to WHO diagnostic criteria (modified from [253])

such as e.g., allogeneic transplantation (see below). A multitude of risk scores have been devised for patients with PMF, comprising a multitude of different risk parameters (e.g., Tables 4.10a c and 4.11). However,

Table 4.10c: Cervantes scoring system for PMF [247] Scorea

Risk group

Median survival

0 1 2

Low High

98.8 months 20.6 months

The presence of either Hb G10 g/dl, age H64 years or constitu tional symptoms (night sweats, weight loss, fever) scores 1 point

a

Table 4.10a: Dupriez scoring system [7] Scorea

Risk group

prognostic classification

Median survival

0

Low

93 months

1 2

Intermediate High

26 months 13 months

Median survival with normal/ abnormal karyotype 112 months/ 50 months n.a. n.a.

n.a. Not assessed The presence of either hemoglobin G10 g/dl, leukocyte count G4,000/ml or H30,000/ml scores 1 point

Table 4.11: Cervantes scoring system for patients G55 years [8] Scorea

Risk group

Median survival

0 1 2

Low High

15 years (H176 months) G3 years (33 months)

a The presence of either hemoglobin G10 g/dl, presence of circu lating blast cells 1% in peripheral blood or constitutional symp toms (night sweats, weight loss, fever) scores 1 point

a

Table 4.10b: Mayo clinic prognostic scoring system for PMF [93] Scorea

Risk group

Median survival

0 1 2

Low Intermediate High

173 months 61 months 26 months

a The presence of either hemoglobin G10 g/dl, leukocyte count G4,000/ml or H30,000/ml, monocyte count H1,000/ml or platelet count G100,000/ml scores 1 point each

their prognostic value can probably be broken down to anemia as the common denominator [7]. The Dupriez score (Table 4.10a) [7] has widely been used as stratification system for prognosis. The Mayo clinic recently devised a prognostic score for PMF patients (Table 4.10b) [93], which has been independently validated in a separate population with a performance that may be superior to the Dupriez and Cervantes prognostic scoring systems (Table 4.10c) [94]. A scoring system more precisely separating the prognosis of younger patients (G55 years) has also been developed

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(Table 4.11) [8]. Such a differentiation seems necessary since patientsG55 years tend to have less severe anemia, higher incidence of splenomegaly, low rates of thrombocytopenia and a lower frequency of chromosomal abnormalities. Other adverse prognostic factors indicating disease progression and/or impending leukemic transformation include age at presentation, constitutional symptoms (fever, night sweats, weight loss), clinical picture of erythroid failure, splenomegaly, high proportion of circulating immature precursors and/or blood blasts, thrombocytopenia, increased angiogenesis/microvessel density, abnormal karyotype, as well as splenectomy, especially in patients with preoperative thrombocytopenia [95]. Regarding overall survival however, only anemia, leukocytosis or immature myeloid cell count sustain significance as independent prognostic indicators during multivariate analysis in a prospective trial [96], with the latter two emerging as the sole predictors of clinical progression and blastic transformation in this trial. Others have found increased neo-angiogenesis to be a significant and independent risk factor for overall survival [97, 98]. Smoldering myelofibrosis or prefibrotic myelofibrosis according to previous classifications, is a disease variant with slow evolution occurring in younger patients, where watchful waiting in lieu of immediate treatment is advisable. It is characterized by normal white blood cell counts, normal or slightly elevated platelet counts, no evidence of bone marrow fibrosis, but with biphasic cellular proliferation of megakaryocytes and neutrophils (chronic megakaryocytic granulocytic myelosis), low numbers of immature myeloid cells without blasts, absent or modest splenomegaly and a history of thromboembolic episodes. Risk factors for leukemic transformation have been discussed controversially in the literature. High peripheral CD34 þ counts (H300106/l CD34 þ ) have been linked with an increased probability of acute transformation (50% probability of transformation 11 months from diagnosis) [78], although this could not be confirmed by others [96]. Peripheral blood blast percentage 3% and/or platelet counts below 100,000/ml at the time of diagnosis are not only independent predictors of survival in patients with PMF as incorporated in the above-mentioned Mayo score, but were also found to be strong and independent predictors of leukemic transformation in one of the largest retrospective studies to date [99]. Importantly, an unsettling association between leukemic transformation and treatment with erythropoiesis stimulating agents such as erythropoietin and danazol was found [99]. Whether JAK2-status is associated with blastic phase transition or not is currently fiercely debat-

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ed. However, the majority of studies argue against such an association [99 101]. One group reported that JAK2V617F predicted the evolution toward massive splenomegaly, need of splenectomy and leukemic transformation [102]. However, as most of these patients were splenectomized prior to leukemic transformation, such a conclusion seems invalid due to the supposed elevated risk of blastic phase transition by the process of splenectomy per se, which interestingly was observed by the same group [103]. Patients with abnormal karyotypes may carry a high risk of progression to AML [7]. These results have not been confirmed by others, who only demonstrated shorter survival for patients with abnormal karyotype, but not increased risk of blastic phase transition [47]. The observation that JAK2 mutant CMPD patients have the potential to develop acute leukemia from a JAK2 wildtype clone [104 107] demonstrates, that the role of this mutation in disease progression is currently not clarified. Furthermore, low JAK2V617F allele burden has been linked with an aggressive disease phenotype, lower overall survival and leukemia free survival in PMF [107, 108], likely because patients bearing the mutation tend to have higher hemoglobin levels. It was concluded, that low JAK2V617F allele burden might indicate the presence of an overriding JAK2V617F negative clone with a higher propensity to undergo clonal evolution [108]. Thus, the potential long-term impact of JAK2 inhibition on disease progression must be carefully monitored and evaluated in the upcoming clinical trials.

4.13 Treatment of Patients with Myeloﬁbrosis (Fig. 4.7) The conventional therapy for patients with myelofibrosis is largely palliative, and no conventional drug has so far been shown to improve patients overall survival. Ablation of the hematopoietic clone with high dose chemotherapy and allogenic stem cell transplantation (allo-SCT) is currently the only treatment modality with a curative potential in myelofibrosis, but at the price of a high-risk of immediate death in select patients where a suitable donor is available. Alas, treatment with currently available drugs has not yet been shown to prolong life or to alter the natural history of the disease. Therefore, any treatment other than allogeneic stem cell transplantation must be considered as palliative and therapeutic choices must be made carefully. The first decision when considering treatment of a patient with PMF is whether he/she qualifies for allotransplantation as the only curative option. If the answer to this question is yes, it is crucial to be clear about the best time point in the course of

Chap. 4

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97

Curative treatment

Palliative treatment options

Allogeneic SCT

Hyperproliferation and/or constitutional symptoms

Cytopenias

(1) im/high risk score pts with: - available suitable donor - age < 60 years - low comorbidity - 2 adverse risk factors

- Thrombocytosis - Leukocytosis - Symptomatic splenomegaly - Severe constitutional symptoms

- Anemia - Leukopenia - Thrombocytopenia

Cytostatic therapy

Supportive measures

- Hydroxyurea (thrombocytosis + leukocytosis) - Anagrlide (isolated thrombocytosis) - Interferon-alpha (young age and/or pregnancy) - Cladribine (post-splenectomy thrombocytosis) - Melphalan - High dose CTX + auto-SCT (rapid progression & no suitable donor) - Investigational agents*

- RBC transfusions - PLT transfusions - G-CSF - Androgens - Vitamin D3 analogues - Thalidomide ± corticosteroids - Cyclosporine ± corticosteroids - Bisphosphonates - Investigational agents*

(2) low risk score pts with - acquisition of novel risk factors - very young age

Nonmyeloablative SCT (RIC) (1) high risk score pts with: - advanced age - low comorbidity

Splenectomy in case aleviation of splenomegaly associated symptoms doesn’t occur AND sufficient marrow reserve AND - mechanical symptoms - abdominal pain - dyspnea - altered digestive habits due to displacement of the bowel - hypercatabolic symptoms - recurrent splenic infarction - refractory hemolysis - refractory thrombocytopenia - portal hypertension

Splenic irradiation (only when splenectomy is contraindicated AND no significant anemia or thrombocytopenia)

*Investigational agents - Lenalidomide - Pomalidomide - Etanercept - 5-azacytidine - Decitabine - HDAC-inhibitors - TGF-beta inhibitors - JAK2-inhibitors - Imatinib - Tipifarnib - Erlotinib - SU5416 - Vatalanib

Fig. 4.7 Treatment algorithm current state of the art. im Intermediate; pts patients; SCT stem cell transplantation; CTX chemotherapy; RBC red blood cell; PLT platelet; RIC reduced intensity conditioning

disease in which the procedure should be carried out. Obviously, the top priority is primum non nocere. The benefit of potentially life-threatening therapeutic proce-

dures such as allo-SCT, but also splenectomy, must be carefully weighed against the individual prognosis of the patient on the one hand, and the consequences

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L. Pleyer et al.

of omission of the respective procedure on the other hand. Tragically, patients with leukemic transformation still have a very grim prognosis with a median survival

of 2.6 months from diagnosis. Aggressive AMLlike therapy does not seem to have a major impact on survival [52].

Table 4.12: Results with allogeneic stem cell transplantation in primary myelofibrosis Reference

n

European American 55 study [114]

Conditioning regimen, donor type, risk profile *

* *

Seattle group [113]

62b

* * *

IBMTR and National Donor Program [248]

320

[249]

56

*

Myeloablative, mostly TBI containing Matched donors 89% Im/high risk Lille score 76% Myeloablative Related donor 57% Unrelated donor 43% Myeloablative conditioning

TRM *

*

[34]

21

* *

[109]

21

* *

EBMT [250]

104

* * *

Genua, single center [251]

39

* *

*

47% 5a OS [low vs. im/high risk 85% vs. 30 45%]a

*

*

Acute grade III, IV: 33% Extensive chronic 35%

34% at 5 years

*

61% at 7 yearsc

n.a.

*

22% at 100 days (siblings) 27% alternative family donor 42% at 100 days MUD 32% Engraftment failure 10%

*

39% at 5ad

n.a.

*

31% at 5ad

10%

*

*

*

27% at 1 year

GvHD

*

*

*

OS

*

31% at 5ad 58% at 3ae

Myeloablative conditioning related donors 64% im/high risk 55%

*

RIC Im/high risk 100%

*

RIC Im/high risk 76%

*

16% at 1 year

*

84% at 2.2 years

RIC Im/high risk 77% related donor 32% RI Im/high risk 92%

*

19% at 1 year

*

70% at 3 yearsf

*

*

*

*

86% at 2.7 years

* *

* * * *

*

0% vs. 41% for 0 1 vs. 2 3 unfavorable factorsg

*

Acute: 21% grade III, IV Chronic: 59% at 2 years Acute II/ IV: 10% Extensive chronic: 44% Acute III/IV 19% Chronic 55% Acute III IV 7% Chronic 32%

50% after 823 days

RIC Reduced intensity conditioning; MUD matched unrelated donor; im intermediate; n.a. not assessed; TBI total body irradiation Factors predicting for impaired survival: Hb G10 g/dl, abnormal karyotype, high risk Lille score, osteosclerosis b 65 out of 104 patients included suffered from myelofibrosis c Improved outcome observed for busulphan/cyclophosphamide regimen, younger age, higher platelet count, low comorbidity score d Patients with favorable Karnovsky index H90% and absence of blasts in the peripheral blood had 81% survival at 5 years e Risk factors for overall survival were Dupriez score, cytogenetic abnormalities and degree of marrow fibrosis f 3a EFS 55%, risk factors for the 3 year OS were: age G50a (92% vs. 62%), low vs. im/high risk score (100% vs. 62%) g Univariate risk factors for survival: Karnovsky index 100% (93 vs. 37%), HLA identical donor (68 vs. 47%), diagnosisG1 year 85 vs. 55%, splenectomy 60 vs. 50%. Patients with 0 1 vs. 3 4 favorable risk factors had an OS of 40 vs. 83% a

Table 4.13: Risk factors for the outcome of allotransplantation after myeloablative chemotherapy in primary myelofibrosis (modified and adapted from [116]) Risk parameter

Deeg [249]

Kerbauy [113]

Conditioning regimen bu/cy vs. others Younger age High platelet count Low comorbidity index Low risk according to Dupriez score Normal karyotype Hemoglobin H10 g/dl No osteosclerosis Circulating blasts

Yes

Yes Yes Yes Yes

Bu/cy Busulfan/cyclophosphamide

Yes Yes

Guardiola [114]

Ballen [248]

Yes

Yes

Yes Yes Yes Yes Yes

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99

4.13.1 Curative Treatment Options – Allogeneic Stem Cell Transplantation Allogeneic stem cell transplantation is capable of replacing the neoplastic clone by normal myeloid progenitors, thereby reversing all features of the disease, including the regression of marrow fibrosis [109]. The capacity of donor lymphocyte infusions to eliminate residual JAK2 positive clones demonstrates the effectiveness of the graft vs. leukemia effect [110]. Simultaneously, a graft vs. myelofibrosis effect has consistently been demonstrated for donor lymphocyte infusions [111, 112], thus contradicting previous fears of failure of allogeneic approaches due to the presumed persistence of marrow fibrosis. Results with myeloablative conditioning regimens and allotransplantation are depicted in the upper half of Table 4.12. Overall, the one year non-relapse mortalities are still in the range of 20 48% and 5 year non-relapse mortality is approximately 34% [113]. Others however, have achieved 5 year overall survival rates of 47% and 67% [113, 114]. Different risk factors for the outcome of allotransplantation have been defined (Table 4.13). Of these, age may be the most important, as 62% of patients younger than 45 years survive, whereas only 14% of patients beyond this age limit are alive after the same procedure [114]. In order to reduce the transplant-related mortality and increase the age range for qualifying patients, reduced intensity conditioning (RIC) protocols have been introduced (lower half of Table 4.12). Non-relapse mortalities seem lower and 3 year overall survival appear promising with 84% alive at 3 years [109]. In a retrospective analysis of conventional and reduced intensity conditioning transplantation, the non-relapse mortality was 10% in the RIC cohort, compared to 30% in the myeloablative group, in spite of the fact that patients in the former were 14 years older (on average) than in the latter [115]. For an adequate risk/benefit estimation, the following aspects need to be considered: (i)

The success of allotransplantation decreases with increasing risk profiles, as defined by high Lilleand/or Cervantes-scores or by transformation into the leukemic phase or overt AML [116]. These patients suffer from higher relapse and treatment related mortality rates. In fact the outcome of patients with overt leukemic transformation is exceptionally dismal, as no complete remissions are obtained by AML-like therapy and the median overall survival is merely, 2.6 months [52]. Only 41% of all PMF patients are reverted into a short lasting chronic phase and the price to be paid for induction therapy is a 33% treatment related mor-

(ii)

tality. For patients qualifying for allogeneic stem cell transplantation it is therefore extremely important not to overlook the adequate time point for this procedure. The comorbidity index for allogeneic transplantation has to be strictly integrated into the decision algorithm [117].

Currently, no universally accepted algorithm has been defined for the indication of allogeneic transplantation in primary myelofibrosis. However, the following suggestions for patient selection may be reasonable: (i)

(ii)

(iii)

Patients at high risk according to the Cervantes score or intermediate/high risk according to the Dupriez score can be evaluated. Patients with low risk according to the Cervantes and Lille scores are characterized by lower transplant related mortalities. As life expectancy is significantly reduced in young patients with PMF, the risk-benefit ratio of allogeneic transplant seems acceptable, when a significant increase in disease dynamics is perceived. In this group of patients, one should considerproceedingto transplant when either severe constitutional symptoms occur, decreases in hemoglobin or increases in LDH are measured, or when rapid development of marrow fibrosis or hematopoietic failure is obvious [18, 116]. It must also be kept in mind, that unfavorable cytogenetics have a high impact on the outcome of the disease, but are usually not part of the scoring systems. In older patients, comorbidity index, age-adjusted life expectancy, risk score, type of available donor and the capacity to overcome the neoplasticclone by reduced intensity conditioning have to be carefully weighed. Usually, high risk patients with low comorbidity index may be considered eligible for RIC.

Preoperative splenectomy has been used for the reduction of graft failure and feared delayed engraftment [118, 119]. Patients allotransplanted after RIC without prior splenectomy demonstrated some delay in engraftment. This delay was most notable in patients with a splenic longitudinal diameter H30 cm [120]. However, progressive reduction of spleen size was observed in all patients, and this paralleled reduction of marrow fibrosis [120]. This suggests that splenectomy prior to allogeneic hematopoietic stem cell transplantation is not necessary, even in patients with excessive splenomegaly, especially since no clear benefit in terms of overall survival has become apparent for those patients who were splenectomized prior to allotransplantation [121]. The attitude towards splenectomy before allogeneic hematopoietic stem cell transplantation differs between transplant cen-

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ters, particularly since the substantial morbidity and 9% mortality associated with splenectomy is differently weighed. It may be fair to state that pretransplantation splenectomy is not universally considered standard and the approach should be individualized.

4.13.2 Treatment of Symptomatic Myeloproliferation as well as Constitutional Symptoms in the Early Hyperproliferative Phase or in Blast-Phase PMF in Patients Without Curative Options or Who are Refractory to the Above-Mentioned Treatment Options Patients not qualifying for allogeneic transplantation are treated in a palliative manner. Cytostatic therapy is indicated in patients with peripheral signs of hyperproliferation, in particular thrombocytosis and/or leukocytosis, and in order to relieve severe constitutional symptoms, such as nocturnal sweats and progressive, symptomatic splenomegaly. Hydroxyurea is the agent of choice and most commonly used, although it is discussed controversially due to a potential risk of leukemia (see respective section in the ET chapter (2.11.2.1.)). It is well suitable to control thrombocytosis and leukocytosis [122]. Reversal of marrow fibrosis has also been described [123]. As was the case in PV, JAK2V617F presence seems to identify those patients who are likely to respond to hydroxyurea therapy [124]. Note that the doses are lower than those used for PV and ET due to the presence of cytopenias in many patients. Anagrelide can be used to successfully decrease platelet counts, particularly after splenectomy (see respective section in chapter on ET for more details (2.11.2.2.)). Melphalan in reduced doses (2.5 mg p.o. 3 times/week increased to a maximum of 2.5 mg/d) has been successfully used [125] with 66% of patients responding within 7 months. Of these, 26% had a complete response with normalization of all clinical and hematological parameters and 40% demonstrated an improvement of H50% (partial responses) [125]. However, a substantial rate of leukemic transformation (26%) was reported in this trial. Busulfan (2 4 mg/d p.o.) and other alkylating agents have been used in the past, but are rarely used nowadays. This is due to the unusual sensitivity of PMF patients to these agents, which results in prolonged, severe cytopenias that can persist long after cessation of therapy. Understandably, this substance has not been further considered for treatment of patients with PMF. 2-Chlorodesoxyadenosine (0.05 0.1 mg/kg/d as continuous intravenous infusion for 7 days or 5 mg/ m2 as 2 h infusion for 5 days, 4 6 monthly cycles) has

L. Pleyer et al.

been used to control post-splenectomy thrombocytosis [126, 127]. Anemia, leukocytosis, thrombocytosis and hepatomegaly were shown to improve in up to 55% of patients. High dose chemotherapy with autologous stem cell transplantation has been carried out in very few patients, mostly with high dose treosulfan [128 130]. This may be an option for rapidly progressive, aggressive forms of PMF in the older patient without a suitable stem cell donor. Graft failures may be substantial and engraftment may be significantly postponed. Transplant related mortality was reported in a substantial amount of patients (14%). However, reduction of marrow fibrosis, improvement of cytopenia and reduction of spleen size was achieved in a significant proportion of patients.

4.13.3 Treatment of Cytopenias in Advanced Stage Myeloﬁbrosis Treatment of anemia with red blood cell transfusions remains the mainstay of palliative treatment. Long-term periodic red cell transfusions can be complicated by hemosiderosis, often requiring the use of chelation therapy. Some patients may require platelet transfusions.

4.13.3.1 Growth Factors Response rates of 33 45% have been reported in PMF patients following treatment with erythropoietin [131, 132] or darbopoietin [133], particularly in those with an inadequate Epo level. However, ESA application should be initiated with a restrictive attitude, as serum Epo-levels in PMF are usually adequate, responses are mostly only seen in non-transfusion dependent patients. Furthermore, aggravation of splenomegaly due to stimulation of extramedullary hematopoiesis can occur. In addition, in a recent retrospective search for factors associated with leukemic transformation, ESA were associated with a threefold increase in the leukemic transformation rate [134]. G-CSF may be used in patients with severe neutropenia (G500/ml) and/or recurrent neutropenic infections.

4.13.3.2 Androgens Androgens (e.g., nandrolone, oxymetholone or fluoxymesterolone (halotestin) 10 mg twice/day) with or without addition of corticosteroids (0.5 mg/kg/d) lead to responses rates of 50 60% after 3 months of treatment. Severely compromised hematopoiesis is indicative of poor response. Patients who fail to respond to one

Chap. 4

Primary Myeloﬁbrosis

androgenic preparation may however respond to another. Women should be notified of virilizing side effects and males should be screened for prostate cancer prior to therapy. Other side effects include fluid retention, increased libido, abnormal liver function tests and hepatic tumors, necessitating monitoring of liver function and periodic abdominal ultrasonographies. Androgens should be given for a minimum of 6 months. Once a response has been achieved, the dose should be titrated to find the lowest maintenance dose. Vitamin D3 analogues, such as danazol (600 800 mg/d), a synthetic attenuated androgen, have the added effect of correcting thrombocytopenia and reducing the degree of splenomegaly in some patients. Recently, however, treatment with danazol was associated with an increased risk of leukemic transformation [134].

4.13.3.3 Bisphosphonates The beneficial effects of bisphosphonate therapy may be due to decreased bone resorption, fibroblast inhibition with decreased osteosclerosis and/or an antitumor effect similar to that seen in myleoma or bone metastases of solid tumors [135]. There is now extensive in vitro and in vivo evidence indicating a direct cytostatic effect of bisphosphonates on tumor cells. Especially nonamino-bisphosphonates inhibit the release of inflammatory cytokines, thereby modulating the neoplastic cytokine microenvironment [136]. In fact, etidronate not only alleviated debilitating bone pain, but also led to a sustained hematological improvement in a patient with PMF, which was assigned to effects on bone marrow microenvironment [137]. Pamidronate also seems effective in reducing myelofibrosis pain [138]. Others report marked decrease in bone marrow fibrosis coinciding with normalization of blood counts and transfusion independence after 8 months of treatment with clodronate [139]. Zoledronate also resulted in recovery from pancytopenia and disappearance of leukemic infiltration in a patient with acute panmyelosis with myelofibrosis [140]. Bone pain may respond to bisphosphonates which may also display an antiproliferative effect on megakaryocytes, and cause a reduction of fibrosis and hematological improvement [137].

4.13.3.4 Cyclosporine A Several reports suggest a role for immunologic mechanisms of anemia in PMF, as abnormal immune responses are frequently associated with PMF. Immunosuppression with cyclosporine A has proven to be

101

effective in improving anemia in autoimmune disorders. It has been employed in primary myelofibrosis patients with immune defects that do not respond to corticosteroids at a dosage of 5 mg/kg twice per day p.o. and has been shown to ameliorate anemia and leed to prolonged transfusion independence in individual cases [141 143].

4.13.4 Targeting and Modulating the Bone Marrow Microenvironment in Myeloﬁbrosis with Off-Label Drug Use According to pathogenic mechanisms mentioned above, future treatment options should focus on specific targeting not only of the neoplastic clone, but also on the tumor microenvironment. A reduction in the bulk of neoplastic megakaryocytes may be achieved by, e.g., IFN-a. Antiangiogenic properties may result from the direct inhibition of PDGF (e.g., by imatinib), VEGF (e.g., farensyltransferase inhibitors (R115777), SU-5416, SU6668, PTK 787). Targeting TNF-a (e.g., thalidomide, lenalidomide, etanercept) or TGF-b mediated fibroblast proliferation and collagen synthesis (e.g., suramin, pirfenidone), may address biologic processes central for symptom development and marrow fibrosis.

4.13.4.1 Thalidomide Thalidomide is not only the cheapest antiangiogenic drug available, but is also endowed with strong cytokine-modulating properties (VEGF, bFGF and TNF-a in particular) [144]. The above-mentioned (see Pathophysiology section (4.3.)) involvement of diverse cytokines (creating a pro-angiogenic microenvironment) in the pathogenesis of the disease, provides the theoretical basis for using thalidomide in patients with PMF. Beneficial responses with improvement of anemia (in up to 43%), platelet count increases (in up to 80%) and splenic size reductions (in up to 63%) have been achieved in patients treated with thalidomide (50 800 mg/d p.o.) [144 147]. In 20% of patients spleen size decreased by more than 50% of the initial size and significant reduction of fatigue is observed in 1/3 of patients [148]. While PMF patients treated in early stage disease obtain significant benefits from treatment with thalidomide, this substance seems to be ineffective in patients with advanced stage and patients with secondary post-ET or post-PV myelofibrosis [149 151]. However conflicting reports exist [152]. A placebo-controlled randomized phase IIb study could not demonstrate a benefit for anemic patients treated within the thalidomide 400 mg/d arm, when analyzed on an intention to treat basis [159]. Standard-dose-thalidomide is burdened

102

with high rates of toxicity (fatigue (50%), drowsiness/ sedation (35%), constipation (48%), rash or pruritus (37%), paresthesias, neutropenia, peripheral neuropathy (22%), peripheral edema (29%) [153]). Peripheral neuropathy can lead to chronic debilitating pain which persist of up to years after cessation of thalidomile. This may lead to discontinuation of therapy in 50% of patients [146]. However, low starting doses of 50 mg/d with gradual dose escalation seem to be both more tolerable and more effective, especially when combined with prednisone (0.5 mg/(kg day 1 ) p.o. and slowly tapered over 3 months) [145, 154]. Although reductions of karyotypic alterations in the marrow were not observed, one-third of patients responded in terms of spleen size, two-third in terms of anemia and three quarters in terms of thrombocytopenia [154]. Undesired increases in white blood cell counts and platelet counts as a consequence of treatment with thalidomide or its analogues are observed in 23% and 38% of patients, respectively [146]. Flares, characterized by extreme thrombocytosis with consecutive thrombotic complications and/or leukocytosis may also occur, and may lead to relevant complications such as pericardial effusion secondary to extramedullary hematopoiesis [147, 155]. Serious myeloproliferative reactions may be more frequent in patients with post-thrombocytemic MF [147]. Some authors have combined thalidomide with erythropoietin in the hope of achieving a synergistic effect [156, 157]. However, we advise extreme caution and do not recommend the combined use of these substances, as both are thrombogenic and the corresponding adverse effects with sometimes fatal complications have been observed [158].

4.13.4.2 Thalidomide Analogues Lenalidomide (CC-5013) has been shown to be substantially active in PMF. Response rates were 25% for anemia, 33% for splenomegaly and 50% for thrombocytopenia. In addition, bone marrow histology was improved in responders [160]. Similar to MDS patients with 5q syndrome, patients with PMF bearing deletions in chromosome 5 seem particularly sensitive to this drug [161, 162]. Flare reactions that lead to significant leukocytosis, and may sometimes resemble leukemic transformation due to a massive left shift with stark increases of blast counts, have been observed. In our hands, these flare reactions were reversible after cessation of lenalidomide. Pomalidomide (CC-4047) is 20,000 times more active in inhibiting TNF-a than thalidomide and is currently being tested in a phase II clinical trial (ClinicalTrials.gov Identifier: NCT00463385).

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4.13.4.3 Targeting TNF-a with Etanercept As briefly mentioned above, TNFa is one of the cytokines known to be involved in the pathophysiology of bone marrow fibrosis by stimulation of fibroblast proliferation [163] (see 4.3.). Furthermore, TNF-a also directly inhibits hematopoiesis [164, 165], negatively regulates hematopoietic stem cell fate, by promoting myeloid differentiation instead of self-renewal [166], and contributes to hypercatabolic symptoms such as fever and cachexia [167], explaining the rational for targeting this cytokine in patients with PMF. Etanercept is a soluble TNF-receptor which binds TNF-a and thus functions as a TNF-a antagonist. Early clinical trials show promising preliminary results for etanercept (25 mg s.c. 2 times/ week). Constitutional symptoms are alleviated in 60% of patients, with seemingly low toxicity. An objective response rate was observed in 20% of patients, with mitigation of cytopenia or reduction in spleen size [168]. Consequently, this drug may be beneficial for those patients experiencing severe constitutional symptoms. However, the extremely high costs of such a treatment may inhibit its administration and further development.

4.13.4.4 Interferons Interferon-a-2b (starting dose 5 610 IU s.c. 3 5 times/ week; lower doses for maintenance) not only reduces proliferation of fibroblasts but also inhibits fibrogenic cytokines in experimental models of myelofibrosis, thereby reducing collagen production. Furthermore IFN-a has an antiproliferative effect on hematopoietic progenitor cells from patients with myelofibrosis, and preferentially inhibits megakaryopoiesis. Despite initial hopes, the drug was unable to alter angiogenesis, marrow fibrosis or osteosclerosis [169]. Response rates of up to 50% in patients with hyperproliferation, with control of thrombocythemia, hyperleukocytosis and/or reduction in spleen size, have been shown. In patients with more advanced stage PMF however, this drug has failed to show significant activity. In addition, IFN-a is generally poorly tolerated and response rates are hampered by high drop-out rates due to side effects and inconvenient dosing schedules. Similarly, unsatisfying results have been reported for the long-acting pegylated interferon-a-2b [170] which merely yielded a response in 1/11 patients, but demonstrated similar toxicities to the un-pegylated form. Interferon-g does not seem effective either [171]. On the other hand, Interferon-a-2a has yielded impressive results in polycythemia vera patients, with a favorable toxicity profile [172]. In this study, molecular responses with reduction of cells bearing JAK2V617F were obtained in 89% [172]. Further improvement of response rates with

Chap. 4

Primary Myeloﬁbrosis

pegylated interferon-a-2a, compared to interferon-a-2b have also been observed in other myeloproliferative diseases [173]. Therefore, clinical trials with Interferon-a-2a seem warranted in patients with PMF.

4.13.4.5 Targeting TGF-b As already mentioned in 4.3., an important role for TGF-b in the disease pathogenesis of PMF has been established and explained by the following aspects: (i)

(ii)

(iii)

The potent inhibitory effect of TGF-b on proliferation are central to its tumor-suppressive effect on hematopoietic stem cells. Resistance to these effects, which is achieved by downregulation of Smad4 or TGF-bRII in ET, PV, primary myelofibrosis and CML, seems to provide a proliferative advantage to clonal myeloid stem cells of CMPDs. TGF-b has also been repeatedly designated as the critical cytokine mediating reactive myelofibrosis in myeloproliferative diseases and hairy cell leukemia via augmentation of collagen synthesis. Additionally, secreted TGF-b may be essential for evasion of immunosurveillance of cancer cells.

Therefore, in accordance with the dual role of TGF-b in tumorigenesis, the rational for targeting of the TGF-b pathway in PMF is twofold: (i)

(ii)

One would aim to reconfer sensitivity of tumor cells to TGF-b, thus breaching one of the pathogenetic mechanisms sustaining abnormal proliferation. This could be achieved by re-expression of Smad4 and/or TGF-b-receptors (as has been proposed as one of the mechanisms of action of decitabine and bortezomib), or by degradation of PML-RARa with, e.g., ATRA. One could also aim to eliminate evasion of immunosurveillance and thus disrupt the pathomechanism underlying increased myelofibrosis by decreasing TGF-b activity. This may be accomplished with large-molecule TGF-b signaling inhibitors (neutralizing TGF-b antibodies, antisense strategies, oligonucleotides) or small molecule inhibitors of TGF-bRI kinase activity, a plethora of which are currently being evaluated in clinical trials (for more details, see e.g., [174, 175]).

4.13.5 A Possible Role for Epigenetic Therapy in PMF? As mentioned above, PMF is characterized by constitutive mobilization of primitive hematogenic progeni-

103

tor cells and CD34 þ hematopoietic stem cells, the quantity of which is related to disease progression and may serve as biomarker of disease activity [78]. These circulating CD34 þ cells also encompass bone marrow-repopulating cells belonging to the malignant clone [5]. This increased trafficking of CD34 þ cells is largely dependent on the interaction between integrins and chemokine receptors expressed by hematopoietic stem and progenitor cells with a variety of matrix proteins and chemokines, respectively produced by marrow fibroblasts, osteoblasts or endothelial cells [176]. Of these, the CXCR4/SDF-1 interaction plays a particularly important role [176]. The sustained stem cell mobilization in PMF has been attributed to a proteolytic environment [30] on the one hand and down-regulated expression of the chemokine receptor CXCR4 on the other hand [177]. Hypermethylation and thus inactivation of several critical genes including CXCR4 has been shown to be associated with the malignant phenotype and possibly disease progression in PMF (e.g., [178, 179]), thus providing the theoretical background for a phase II clinical trial with 5-azacytidine (Vidaza) for patients with PMF. However, clinical improvement in a mere 21% after a median of 5 months with a median response duration of 4 months, paid for by 29% grade 3 4 neutropenia, is not particularly impressive [180]. Currently several other phase II clinical trials with demethylating substances are underway, including 5-aza-20 deoxycytidine (decitabine Dacogen) (ClinicalTrials.gov Identifier: NCT00095784 and NCT00630994) and 5-azacytidine (ClinicalTrials.gov Identifier: NCT00381693). In vitro data is accumulating, that sequential application of DNA-methyltransferase inhibitors (leading to demethylation) followed by histone deacetlase (HDAC) inhibitors is necessary, in order to achieve re-expression of silenced genes (e.g., [181, 182]). Accordingly, sequential in vitro application of decitabine and the HDAC-inhibitor trichostatin-A led to a quantitative reduction of JAK2V617F bearing cells and resulted in an upregulation of the expression of CXCR4 in CD34 þ cells from patients with PMF [183]. The authors interpret this as a positive phenomenon, which should be further examined in clinical trials. However, one must consider, that the egression of CD34 þ cells in PMF is a consequence of progressive marrow fibrosis/sclerosis, and without this egression, resulting in extramedullary hematopoiesis, patients would not be able to sustain even an inefficient form of hematopoiesis. Robbing the CD34 þ cells of the possibility to establish hematopoiesis in extramedullary sites may therefore have deleterious effects.

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4.13.6 Tyrosine Kinase Inhibitors in PMF 4.13.6.1 Targeting Constitutively Activated JAK2 by Selective Tyrosine Kinase Inhibitors Table 1.4 in the Introduction to the CMPDs chapter summarizes phase I/II clinical trials with JAK2 inhibitors. However promising this targeted approach may seem, one must bear in mind, that the leukemic phase of PMF is characterized by a JAK2V617F negative status. This supports fears that specific suppression of the mutated clones might foster the outgrowth and direct transformation of the common JAK2 mutation negative precursor cells, possibly resulting in frank leukemia. However, the prognosis of this disease is so dismal in many patients, and their symptoms so difficult to treat, that the benefit/risk ratio seems in favor of the novel drugs. The general prognosis of patients with ET or PV is significantly better, and overall survival often not much reduced when compared to the normal population, once platelet numbers and hematocrit are sufficiently controlled. Therefore, PMF is the ideal subset of chronic myeloproliferative disorders in which to study the potential impact of inhibitors of JAK2 specific tyrosine kinase inhibitors (TKI). Promising preclinical data have been documented for various JAK2 specific inhibitors [184, 185]. Phase I/II trials have been initiated (ClinicalTrials.gov Identifier NCT00509899), (ClinicalTrials.gov Identifier NCT00631462), (ClinicalTrials. gov Identifier NCT00522574) for the JAK2 inhibitors INCB018424, TG101348 and XL019, respectively. Early results were reported at the annual ASH meeting 2007 [186]. The orally available JAK2 inhibitor INCB018424 achieved dramatic reductions in spleen size, e.g., from 25 cm to 8 cm and 22 cm to 10 cm below costal margin within 1 month of treatment in patients with PMF or post-PCV/ET MF patients. Furthermore, the number of JAK2 mutated clones was significantly reduced in some patients within this time frame. These promising drugs are now under rapid development (see recent review [187]). Of interest, preclinical evidence exists, that JAK2 selective antagonists may also be effective in patients with myeloproliferative disorders that do not bear JAK2 or MPL mutations [188].

4.13.6.2 Imatinib Mesylate (STI571, Gleevec) Imatinib is a selective tyrosine kinase inhibitor with significant activity against c-Abl, Bcr Abl, ARG-kinase, c-Kit (CD117), as well as PDGF receptor. c-Kit is highly expressed on CD34 þ cells of patients with PMF and PDGF also plays a role in the pathogenesis of PMF. Although imatinib is most effective in CML, several other

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myeloproliferative diseases with activated imatinib sensitive tyrosine kinases also respond [184]. This has been attributed to modulation of the overexpression of PDGFR, VEGF and TGF-bR. Furthermore, imatinib has demonstrated an antifibrogenic effect on bone marrow fibrosis in CML, which was accompanied by normalization of megakaryocyte numbers [189]. Therefore one might expect a similar effect on bone marrow fibrosis in PMF. However, highly variable results have been produced in clinical studies conducted in patients with PMF (e.g., [190, 191]). Most patients were unable to tolerate the normal dosage of 400 mg once daily due to myelosuppression [192]. In others, undesired increases in white blood cell or platelet counts [193] in the absence of any discernible clinical benefit were observed. In addition, the emergence of a JAK2-mutated clone with features of myelofibrosis has been reported in a CML patient under imatinib treatment, supporting the low efficacy of the drug against JAK2- mutated clones [194]. Nontheless, several clinical trials implementing imatinib in patients with PMF are underway (ClinicalTrials.gov Identifier NCT00245128 and NCT00039416).

4.13.6.3 Farensyltransferase Inhibitors (R115777 Tipifarnib (Zarnestra)) In vitro, tipifarnib reduces the growth of myeloid colonies derived from PMF patients in drug concentrations achievable in vivo [195]. The orally bio-available tipifarnib has shown promising results in elderly patients with AML [196]. In a phase II trial including 34 symptomatic patients with PMF (n ¼ 28) or post-PV/ET MF (n ¼ 6), 33% and 38% of patients responded in terms of reduction of hepatosplenomegaly or transfusion dependence, respectively [197]. Although the growth of myeloid colonies was substantially suppressed, decreases of the JAK2V617F burden, angiogenesis or marrow fibrosis were not observed, and cytogenetic status was not affected. Nearly half the patients terminated the trial early due to progression of the disease or unsustainable side effects. It remains unclear at present, whether tipifarnib is capable of postponing or preventing leukemic transformation in patients with PMF. The drug remains experimental in this indication.

4.13.6.4 Other Tyrosine Kinase Inhibitors that have been Used in PMF Erlotinib (Tarceva), an inhibitor of the tyrosine kinase domain of the EGFR, has recently been shown to be a potent inhibitor of JAK2V617F activity [198]. This substance effectively suppressed growth and expansion of JAK2V617F PV hematopoietic progenitor cells while hav-

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ing little effect on normal cells, suggesting that erlotinib may be useful in the treatment of JAK2V617F positive CMPDs, including PMF. As increased bone marrow angiogenesis [97], as well as increased splenic neoangiogenesis [199] have been documented in patients with PMF, inhibition of angiogenesis has been attempted with a number of small molecules. SU5416, is a synthetic RTK-inhibitor of VEGFR-2, c-Kit and FLT3, and may directly target bone marrow angiogenesis through inhibition of VEGF-dependent endothelial cell proliferation. Furthermore, SU5416 may target blast cell proliferation via inhibition of FLT3 and c-Kit. Given at a dosage of 145 mg/m2 twice a week, only modest responses have been reported in PMF. Toxicities include mild to moderate gastrointestinal side effects, headache, fatigue, dyspnea, and catheter site reactions [200]. Currently MD Anderson has an ongoing study with SU5416 (ClinicalTrials.gov Identifier NCT00387426). Vatalanib (PTK787/ZK222584), an oral inhibitor of the VEGFR1, demonstrated merely minimal clinical activity [201]. The effect of thalidomide and lenalidomide in patients with PMF (see above) can therefore most probably not be attributed to the inhibition of angiogenesis.

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Table 4.15: Potential complications of splenectomy in patients with primary myelofibrosis Short-term complications * 9% mortality of operative procedure, rising to 26% after 3 months * 31 46% morbidity * Intra abdominal bleeding * Subphrenic abscess, sepsis * Acute liver failure due to massive compensatory exacerbation of EMH, may result in death Long-term complications (not predictable) * Post surgical thrombocytosis (H600,000 and H1,000,000/ml in 22% and 6%, respectively) is significantly associated with postoperative thrombotic, hemorrhagic events and decreased survival, may necessitate platelet apharesis and cytoreductive therapy post splenectomya * Significant compensatory increase in liver size due to myeloid metaplasia in 24% (elevation of alkaline phosphatase and other liver enzymes, elevation of bilirubin) * Marked increase of EMH sites and pulmonary hypertension * Independent risk for blast transformation through acceleration of pre existing myeloproliferation (cumulative risk 55% vs. 27% in non splenectomized patients) EMH Extramedullary hematopoiesis HU (500 mg 3 times/d p.o.) or 2 Cda (chlorodeoxyadenosine) (0.05 0.1 mg/(kg day1 ) for 7 days or 0.14 mg/kg for 5 days) can be used in patients with progressive accelerated hepatomegaly or symptomatic, extreme thrombocytosis and leukocytosis after ther apeutic splenectomy (response rates: 78%). The latter can also be controlled by anagrelide

a

4.13.7 Indications for Splenectomy in PMF Splenomegaly is an important problem of patients with PMF. Substantial symptoms arise particularly during the progression of the disease from the hyperproliferative to the sclerosing phase, when an increase in spleen size may be rapid. In this phase of the disease the pros (Table 4.14) and cons (Table 4.15) of splenectomy must be carefully discussed. The overall median survival is 2.0 years from the time of splenectomy, and there is no evidence for an improvement in overall survival by this

Table 4.14: Indications for, and beneficial effects of, splenectomy Alleviation of the following symptoms *

* * *

*

*

Mechanical and hypercatabolic, constitutional symptoms due to symptomatic splenomegaly (alleviation in 100% and 80%, respectively) Recurrent episodes of splenic infarction Refractory hemolysis Transfusion dependent anemia (substantial improvements in anemia are seen in 45% and 52% of patients at 3 months and 1 year post splenectomy, respectively, and a 25 50% reduction in transfusion dependency can be expected) Portal hypertension (improvement in 50 83% at 1 year) due to splenomegaly (not due to intrahepatic obstruction; requires porto systemic shunt: TIPS) Refractory thrombocytopenia (improvement in 56%)

maneuver [202]. At this stage of the disease, the spleen becomes the predominant organ of residual hematopoiesis in an often cytopenic patient. Simultaneously the large spleen further augments anemia and thrombocytopenia due to a massive pooling effect (Fig. 4.2a c). The potential benefit from eliminating the pooling compartment and the site of pain, e.g., after splenic infarction, must carefully be balanced against the removal of the major site of hematopoiesis, which may lead to longlasting profound cytopenias and a propensity for severe infections. An especially feared complication of splenectory is acute liver failure due to compensatory massive hepatic infiltration of hematopoietic stem cells. This happens when the bone marrow does not have sufficient residual hematopoietic capacity, which is why an MRI of the spine as well as the femura to evaluate the aforementioned is prudent prior to splenectomy. In addition, bone marrow biopsy may be helpful in determining marrow reserve. Chronic transfusion requirement exceeding 3 units of red cells every 2 weeks in a non-bleeding patient lacking autoimmune hemolytic anemia, is indicative of negligible splenic red cell production. These patients derive little benefit from preservation of a potential hematopoietic reserve compartment in their spleen. The serious problem of post-operative hepatomegaly may be controlled by low-dose cladribine (0.1 mg/kg/d for

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5 days per cycle), at least in the experience of the Mayo clinic [126]. However, patients in this state are often spontaneously granulopenic, thrombopenic and suffer from infections, thus making myelosuppressive therapy a difficult task. The release of the previously pooled platelets into the circulation can cause post-splenectomy thrombocytosis H600,000/ml in 20% of patients. This is associated with according thromboembolic consequences and reduced survival [103, 203, 204]. This complication may successfully be prevented by preoperative prophylactic platelet reduction in patients with platelets H200,000/ml with hydroxyurea or anagrelide (second choice) to achieve a platelet count in the lower normal range. Not surprisingly the acute and long-term complications of splenectomy are extremely high (see Table 4.15), as is mortality (56% in 1937 but still 9 11% in the year 2000) [202]. One has to keep in mind that the mortality of splenectomy in patients with PMF is in the range of allotransplantation with reduced intensity conditioning. Presplenectomy thrombocytopenia (G50,000 70,000/ml) and bone marrow hypocellularity seem to be associated with reduced perioperative and overall survival, but this may merely reflect a more advanced stage of disease. This is supported by the negative impact of spleen weights H2,000 mg and leukocytesG10 106/ml. However, when performed at the right time and in the right patients at the right stage of the disease, splenectomy is efficient in reducing complaints associated with splenomegaly (Table 4.14). Especially B symptoms, which are often experienced as particularly cumbersome and exhausting by the patients, can be substantially alleviated in many. Furthermore, gastrointestinal symptoms caused by mechanical obstruction, cytopenias and transfusion requirements, as well as portal hypertension and its sequelae improve in a substantial proportion of patients and obviously, the risk of splenic infarction and splenic rupture is banned (see Table 4.14). Taken together, the adequate patient selection is difficult for splenectomy. (i) Patients should be symptomatic with failure of conventional medical treatments to control symptoms. (ii) The general condition should be acceptable. (iii) Patients should be at the transition from the hyperproliferative to the sclerotic phase, since severe cytopenias and excessive spleen size are associated with an unfavorable outcome. Patients with myelofibrosis should be carefully followed during their course of the disease in order not to overlook indications for splenectomy or transplantation if possible. In the rare cases of patients presenting with excessive spleen size, overt pancytopenia and exhaustive constitutional symptoms, splenectomy must be considered a highest risk, but sometimes inevitable, intervention requiring detailed patient

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consent and perfect cooperation between hematologists and surgeons.

4.13.8 Indications for Splenic Irradiation Low-dose splenic irradiation is an alternative in patients with overt contraindications for splenectomy but symptoms of splenic mass and who do not have significant anemia or thrombocytopenia (for review see [202]). Effective palliation of splenic pain and reduction in spleen size is achieved in 94% of patients, with the median duration of response being 6 months [205]. However, complications may be relevant and include significant cytopenias in 45% of patients. In fact, unpredictable lifethreatening pancytopenia may occur after a single course in 26%, resulting in fatal sepsis or hemorrhage in 13%. It must be considered, that especially high post-splenectomy mortality rates have been observed in patients who have received splenic irradiation prior to splenectomy. Therefore, the procedure of splenic irradiation should be reserved for patients who cannot qualify for splenectomy.

4.13.9 Treatment of Other Foci of Extramedullary Hematopoiesis and Their Complications Extramedullary hematopoiesis in liver and spleen occurs (to some degree) in 90 100% [206] and frequently causes portal hypertension with subsequent visceral bleedings and ascites refractory to medical intervention [207]. Transjugular intrahepatic porto-systemic shunt systems (TIPS), an invasive radiologic procedure that creates a side-to-side porto-caval shunt, have been used for the palliation of such a type of portal hypertension [208].

4.13.9.1 Irradiation of Tumor-like Manifestations of Extramedullary Hematopoiesis The diffuse infiltrative nature of extramedullary hematopoiesis generally limits surgical resection of foci. However, as myeloid progenitors are exquisitely radiosensitive, involved field irradiation of symptomatic sites generally achieves excellent results in palliation of symptoms [202], but does not induce disease remissions or prolong survival. Typical dosages delivered within 5 10 fractions and with high success rates are 100 1,000 cGy for intraspinal/paraspinal EMH [209], 30 Gy for whole brain irradiation of intracranial myelopoiesis, and 100 125 cGy for whole lung treatment for pulmonary hypertension [209, 210]. Sildenafil has successfully been applied for many conditions associated with pulmonary

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hypertension with satisfying long-term results [211]. However, little is known concerning its efficacy in pulmonary hypertension associated with myelofibrosis. As the onset of pulmonary hypertension in patients with PMF is associated with a dismal prognosis, with an overall survival of 18 months [212], it seems worth a try. Hepatosplenomegaly with ascites has been treated with radiotherapy of the upper or the whole abdomen in a small series. The resolution of palpable organs and ascites did not correlate with the particular field sizes. Substantial reduction of symptoms, ascites and hepatomegaly was achieved, but anemia and thrombocytopenia worsened in about 60% of cases [209]. In addition 21% of all patients died. Therefore this method has not achieved wide acceptance and should be handled with extreme caution.

4.14 Atypical Myeloﬁbrosis Variants 4.14.1 Secondary Myeloﬁbrosis, i.e., PostPolycythemia and Post-Essential Thrombocythemia Myeloﬁbrosis Secondary myelofibrosis accounts for approximately 10% of all cases of myelofibrosis. In PV the risk for disease progression to secondary myelofibrosis is estimated to be 6% at 15 years after diagnosis [213]. The criteria for the diagnosis of post-PV and post-ET myelofibrosis have been redefined because of the rapid development of research in the field of CMPDs in general and the emerging role of JAK2V617F mutations in particular. Secondary myelofibrosis therefore requires the demonstration of the pre-existence of PVor ET as defined by the recent WHO criteria [38], the presence of marrow fibrosis and the presence of at least two out of 4 (PV) or two out of 5 (ET) additional criteria [214] (see Tables 4.5a b). The necessity for the pre-existence of WHO-criteria defined ET or PV is particularly important because of the difficulties in differentiating ET from prefibrotic primary myelofibrosis. The survival of patients with post-PV myelofibrosis is similar to that of patients with primary myelofibrosis (mortality 11.1 vs. 10.1 per 100 person years [215]). In contrast, the evolution from PV into myelofibrois significantly worsens the prognosis as compared to PV patients without such an event. Several prognostic factors have been defined for post-ET and post-PV myelofibrosis, and just recently a prognostic score to predict survival in postPV-MF has been defined (Table 4.16) [215]. Whereas cytogenetic results do not seem to predict evolution of PV to post-PV-MF [216], the presence of unfavorable cytogenetics, i.e., aberrations other than 13q or 20q, remained the only unfavorable prognostic

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Table 4.16: Prognostic risk score for post PV MF [215] Scorea 0 1 2 3

b

Estimated median survival H108 months 52 months 16 months 5 months

The score predicts a 4.2 fold worsening of survival for each risk factor acquired at any time during follow up of post PV MF a The presence of either hemoglobin G10 g/dl, leukocyte count H30,000/ml or platelet count G100,000/ml scores 1 point b Patients who never acquire risk factors during follow up

parameter in a multivariate analysis [217]. In a very recent analysis of 68 post-PV-MF patients, the risk of leukemic transformation in post-PV-MF patients was 50/1,000 person years, yielding a leukemia free survival rate of 82% at 3 years [215]. The only parameter predicting the leukemic transformation was the circulating CD34 þ cell count in this analysis. In a more dynamic model, which allows for the prediction of outcome not only at diagnosis but also at any time of post-PV myelofibrosis, hemoglobin G10 g/dl, platelets G100,000/ml, and leukocytes H30,000/ml retained significance. The risk for death increases 4.2-fold for the acquisition of any risk factor during the time of post-PV myelofibrosis [215]. Figure 4.8 demonstrates the dramatic difference in the mortality risk for patients with different risk profiles. Based on hemoglobin levels with a cut-off level of 10 g/dl alone, a median survival of 6.6 years for patients with H10 g/dl vs. 1.9 years for those with G10 g/dl has been recorded [215].

Fig. 4.8 Risk factors in secondary myelofibrosis. In a dynamic model, Hb G10 g/dl, platelet counts below 100,000/ml and leuko cytesH30,000/ml turned out to define risk factors. The model allows the estimation of survival at any point in time during post PV myelofibrosis. Acquisition of any additional risk factor leads to a 4 fold decrease in survival [215]

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4.14.2 Primary Autoimmune Myeloﬁbrosis (AIMF) Primary AIMF was first described in 1987 [218], and is seen as a distinct clinicopathologic syndrome by several authors [91, 219 221], although it has not been taken up in the WHO classification as yet. However, since the prognosis and therapy of PMF with autoimmune manifestations and primary AIMF differ drastically, it is mandatory to make as clear a distinction as possible. Pullarkat and colleagues present an expedient guideline in which several criteria are necessary for the diagnosis of AIMF [221] (see Table 4.17). MDS with myelofibrosis [222, 223] and acute myelofibrosis [223] are important differential diagnoses of primary AIMF, as these disorders also present with pancytopenia and lack of significant splenomegaly or poikilocytosis and dacryocytosis. However, (multilineage) dysplasia and/or elevated blast counts in the bone marrow aspirate generally enable differentiation. Most patients with acute myelofibrosis classify as acute megakaryocytic leukemia or other forms of AML with destructive fibrosis and elevated blast counts. Lymphoproliferative disorders causing fibrosis can easily be distinguished by immunohistochemistry, if the classical morphological features (such as i.e., hairy cells) are not sufficient. Early stage primary myelofibrosis is more tricky to separate from AIMF, especially as prominent hepatosplenomegaly may Table 4.17: Criteria for the diagnosis of primary autoimmune myelofibrosis AIMF (according to Pullarkat et al. [221]) Criteria for the diagnosis of primary AIMF * *

* * * *

*

* *

* *

*

*

Grades 3 or 4 reticulin fibrosis of the bone marrow Lack of clustered or atypical megakaryocytes in the bone marrow Lack of myeloid or erythroid dysplasia in peripheral blood Lack of eosinophila or basophila in peripheral blood Peripheral blood cytopenias Interstitial lymphocyte infiltration of the bone marrow or lymphoid aggregates which release cytokines, such as TGF b and substance P [252], which are known inducers of myelofibrosis Elevated levels of TGF b and substance P have been found in patients with AIMF. Cumulating evidence etiologically implicates a role for these substances in the pathophysiology of AIMF [252]. Lack of osteosclerosis Absent or mild splenomegaly (due to lack of splenic hematopoiesis) Presence of autoantibodies Absence of another disorder known to be associated with myelofibrosis Rare nucleated red blood cells or immature myeloid cells due to the lack of extramedullary hematopoiesis Absence of significant amounts of dacryocytes or leukerythroblastosis in peripheral blood smears

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be missing at such early disease stages, and as autoimmune phenomena also occur in patients with PMF. In doubtful cases, a trial with corticosteroids should be implemented.

4.14.2.1 Treatment of AIMF Corticosteroids are the mainstay therapy of AIMF. Results are typically less impressive in secondary AIMF, i.e., myelofibrosis associated with a distinct autoimmune disease, such as SLE or others mentioned in the chapter on differential diagnosis of primary myelofibrosis and in Table 4.8. Response to corticosteroids (1 mg/kg tapered over 3 months) resulted in complete normalization of peripheral blood counts in 6/7 patients in one series [221], and has also been observed by others [224]. Cases with partial response to corticosteroids may profit from addition of another immunosuppressive agent [220].

4.14.3 Familial Myeloﬁbrosis Familial presentation of myelofibrosis is exceptionally rare and only few reports of well-documented families with first-degree relatives with myelofibrosis exist [13, 225 231]. Familial myelofibrosis has been suggested to be of autosomal recessive inheritance [13, 232], although the mode of inheritance often remains illusive [229]. Familial myelofibrosis may be indistinguishable from its sporadic form. However, any combination of CMPDs may exist [231]. Usually the disease is diagnosed in infants or children, may take on a fulminant, often lethal character [229, 230], and must be differentiated from idiopathic myelofibrosis of infancy (see below). In the few known families with documented familial myelofibrosis, cosegregation analysis and study of candidate genes have thus far failed to reliably identify predisposing factors. In this context, it was recently investigated whether the somatic JAK2V617F mutation present in many of the sporadic PMF cases, may also occur as a germ-line mutation and predispose to the development of familial myelofibrosis. However, 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 [225]. It has however recently been shown that first-degree relatives of CMPD patients have a 5 7-fold enhanced risk of developing a CMPD, supporting the hypothesis that there are common, strong susceptibility genes predisposing to ET, PV and PMF [233].

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Primary Myeloﬁbrosis

4.14.4 Idiopathic Myeloﬁbrosis in Childhood Infantile myelofibrosis is exotically rare, except in Down syndrome [234, 235]. In pediatric cases without hereditary character, variable outcomes with either fulminant courses [236, 237] or relatively indolent courses [238, 239], sometimes even with spontaneous remissions, have been described. The etiology is unclear and its natural history seems different from the adult variant. A thorough search for an underlying disease should be made when myeloid metaplasia is first diagnosed in a child, in order to exclude secondary myelofibrosis of childhood [240]. Fulminant courses of childhood myelofibrosis may present with or without hepatosplenomegaly and with or without myeloid metaplasia [241] and seem to undergo rapid progression to an overt leukemic phase, at which point the only hope of effective treatment lies in allogeneic bone marrow transplantation. Indolent courses on the other hand, have been reported to respond to corticosteroid treatment [236, 240].

References [1] Mesa RA, Verstovsek S, Cervantes F et al. (2007) Primary myelofibrosis (PMF), post polycythemia vera myelofibrosis (post PV MF), post essential thrombocythemia myelofibrosis (post ET MF), blast phase PMF (PMF BP): consensus on terminology by the international working group for myelofi brosis research and treatment (IWG MRT). Leuk Res 31: 737 740 [2] Spivak JL, Silver RT (2008) The revised World Health Organization diagnostic criteria for polycythemia vera, es sential thrombocytosis and primary myelofibrosis: an alter native proposal. Blood 112(2): 231 239 [3] Thiele J, Kvasnicka HM, Dietrich H et al. (2005) Dynamics of bone marrow changes in patients with chronic idiopathic myelofibrosis following allogeneic stem cell transplantation. Histol Histopathol 20: 879 889 [4] Delhommeau F, Dupont S, Tonetti C et al. (2007) Evidence that the JAK2 G1849T (V617F) mutation occurs in a lym phomyeloid progenitor in polycythemia vera and idiopathic myelofibrosis. Blood 109: 71 77 [5] Xu M, Bruno E, Chao J et al. (2005) The constitutive mobilization of bone marrow repopulating cells into the peripheral blood in idiopathic myelofibrosis. Blood 105: 1699 1705 [6] Mesa RA, Silverstein MN, Jacobsen SJ, Wollan PC, Tefferi A (1999) Population based incidence and survival figures in essential thrombocythemia and agnogenic myeloid metapla sia: an Olmsted County Study, 1976 1995. Am J Hematol 61: 10 15 [7] Dupriez B, Morel P, Demory JL et al. (1996) Prognostic factors in agnogenic myeloid metaplasia: a report on 195 cases with a new scoring system. Blood 88: 1013 1018 [8] Cervantes F, Barosi G, Demory JL et al. (1998) Myelofibrosis with myeloid metaplasia in young individuals: disease char acteristics, prognostic factors and identification of risk groups. Br J Haematol 102: 684 690

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[9] Cervantes F, Barosi G, Hernandez Boluda JC, Marchetti M, Montserrat E (2001) Myelofibrosis with myeloid meta plasia in adult individuals 30 years old or younger: present ing features, evolution and survival. Eur J Haematol 66: 324 327 [10] Orazi A, OMalley DP, Jiang J et al. (2005) Acute panmye losis with myelofibrosis: an entity distinct from acute mega karyoblastic leukemia. Mod Pathol 18: 603 614 [11] Paredes Aguilera R, Romero Guzman L, Lopez Santiago N, Trejo RA (2003) Biology, clinical, and hematologic features of acute megakaryoblastic leukemia in children. Am J Hematol 73: 71 80 [12] Amin MB, Maeda K, Carey JL, Babu RV, Raman BK (1992) Megakaryoblastic termination of myeloproliferative disor ders. Henry Ford Hosp Med J 40: 122 126 [13] Rossbach HC (2006) Familial infantile myelofibrosis as an autosomal recessive disorder: preponderance among children from Saudi Arabia. Pediatr Hematol Oncol 23: 453 454 [14] Landgren O, Goldin LR, Kristinsson SY, Samuelsson J, Bjorkholm M (2007) Increased Risks of Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Myelofibrosis (MF) among 24577 First Degree Relatives of 11039 Patients with Chronic Myeloproliferative Disorders (MPD) in Sweden. Blood 110(11) [15] Tefferi A (2005) Pathogenesis of myelofibrosis with myeloid metaplasia. J Clin Oncol 23: 8520 8530 [16] Ho CL, Arora B, Hoyer JD, Wellik LE, Mesa RA, Tefferi A (2005) Bone marrow expression of vascular endothelial growth factor in myelofibrosis with myeloid metaplasia. Eur J Haematol 74: 35 39 [17] Chagraoui H, Tulliez M, Smayra Tet al. (2003) Stimulation of osteoprotegerin production is responsible for osteosclerosis in mice overexpressing TPO. Blood 101: 2983 2989 [18] Hoffman R, Rondelli D (2007) Biology and treatment of primary myelofibrosis. Hematol Am Soc Hematol Educ Program 2007: 346 354 [19] Wang JC, Chang TH, Goldberg A, Novetsky AD, Lichter S, Lipton J (2006) Quantitative analysis of growth factor pro duction in the mechanism of fibrosis in agnogenic myeloid metaplasia. Exp Hematol 34: 1617 1623 [20] Boveri E, Passamonti F, Rumi E et al. (2008) Bone marrow microvessel density in chronic myeloproliferative disorders: a study of 115 patients with clinicopathological and molecu lar correlations. Br J Haematol 140: 162 168 [21] Chagraoui H, Komura E, Tulliez M, Giraudier S, Vainchenker W, Wendling F (2002) Prominent role of TGF beta 1 in thrombopoietin induced myelofibrosis in mice. Blood 100: 3495 3503 [22] Dong M, Blobe GC (2006) Role of transforming growth factor beta in hematologic malignancies. Blood 107: 4589 4596 [23] Rameshwar P, Oh HS, Yook C, Gascon P, Chang VT (2003) Substance p fibronectin cytokine interactions in myelopro liferative disorders with bone marrow fibrosis. Acta Haema tol 109: 1 10 [24] Bock O, Hoftmann J, Theophile K et al. (2008) Bone mor phogenetic proteins are overexpressed in the bone marrow of primary myelofibrosis and are apparently induced by fibro genic cytokines. Am J Pathol 172: 951 960 [25] Komura E, Tonetti C, Penard Lacronique Vet al. (2005) Role for the nuclear factor kappaB pathway in transforming growth factor beta1 production in idiopathic myelofibrosis: possible relationship with FK506 binding protein 51 overexpression. Cancer Res 65: 3281 3289 [26] Centurione L, Di Baldassarre A, Zingariello M et al. (2004) Increased and pathologic emperipolesis of neutrophils within

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Chap. 4

Primary Myeloﬁbrosis

[198] Li Z, Xu M, Xing S et al. (2007) Erlotinib effectively inhibits JAK2V617F activity and polycythemia vera cell growth. J Biol Chem 282: 3428 3432 [199] Barosi G, Rosti V, Massa M et al. (2004) Spleen neoangio genesis in patients with myelofibrosis with myeloid metapla sia. Br J Haematol 124: 618 625 [200] Giles FJ, Cooper MA, Silverman L et al. (2003) Phase II study of SU5416 a small molecule, vascular endothelial growth factor tyrosine kinase receptor inhibitor in patients with refractory myeloproliferative diseases. Cancer 97: 1920 1928 [201] Giles FJ, List AF, Carroll M et al. (2007) PTK787/ZK 222584, a small molecule tyrosine kinase receptor inhibitor of vascular endothelial growth factor (VEGF), has modest activity in myelofibrosis with myeloid metaplasia. Leuk Res 31: 891 897 [202] Mesa RA, Tefferi A (2005) Surgical and radiotherapeutic approaches for myelofibrosis with myeloid metaplasia. Semin Oncol 32: 403 413 [203] Jarvinen H, Kivilaakso E, Ikkala E, Vuopio P, Hastbacka J (1982) Splenectomy for myelofibrosis. Ann Clin Res 14: 66 71 [204] Lafaye F, Rain JD, Clot P, Najean Y (1994) Risks and benefits of splenectomy in myelofibrosis: an analysis of 39 cases. Nouv Rev Fr Hematol 36: 359 362 [205] Elliott MA, Chen MG, Silverstein MN, Tefferi A (1998) Splenic irradiation for symptomatic splenomegaly associated with myelofibrosis with myeloid metaplasia. Br J Haematol 103: 505 511 [206] Pereira A, Bruguera M, Cervantes F, Rozman C (1988) Liver involvement at diagnosis of primary myelofibrosis: a clini copathological study of twenty two cases. Eur J Haematol 40: 355 361 [207] Shaldon S, Sherlock S (1962) Portal hypertension in the myeloproliferative syndrome and the reticuloses. Am J Med 32: 758 764 [208] Angermayr B, Cejna M, Schoder M et al. (2002) Transjugular intrahepatic portosystemic shunt for treatment of portal hypertension due to extramedullary hematopoiesis in idio pathic myelofibrosis. Blood 99: 4246 4247 [209] Koch CA, Li CY, Mesa RA, Tefferi A (2003) Nonhepatos plenic extramedullary hematopoiesis: associated diseases, pathology, clinical course, and treatment. Mayo Clin Proc 78: 1223 1233 [210] Steensma DP, Hook CC, Stafford SL, Tefferi A (2002) Low dose, single fraction, whole lung radiotherapy for pulmonary hypertension associated with myelofibrosis with myeloid metaplasia. Br J Haematol 118: 813 816 [211] Barnett CF, Machado RF (2006) Sildenafil in the treatment of pulmonary hypertension. Vasc Health Risk Manag 2: 411 422 [212] Dingli D, Utz JP, Krowka MJ, Oberg AL, Tefferi A (2001) Unexplained pulmonary hypertension in chronic myelopro liferative disorders. Chest 120: 801 808 [213] Passamonti F, Rumi E, Pungolino E et al. (2004) Life expectancy and prognostic factors for survival in patients with polycythemia vera and essential thrombocythemia. Am J Med 117: 755 761 [214] Barosi G, Mesa RA, Thiele J et al. (2008) Proposed criteria for the diagnosis of post polycythemia vera and post essen tial thrombocythemia myelofibrosis: a consensus statement from the International Working Group for Myelofibrosis Research and Treatment. Leukemia 22: 437 438 [215] Passamonti F, Rumi E, Caramella M et al. (2008) A dynamic prognostic model to predict survival in post polycythemia vera myelofibrosis. Blood 111: 3383 3387

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[216] Diez Martin JL, Graham DL, Petitt RM, Dewald GW (1991) Chromosome studies in 104 patients with polycythemia vera. Mayo Clin Proc 66: 287 299 [217] Dingli D, Schwager SM, Mesa RA, Li CY, Dewald GW, Tefferi A (2006) Presence of unfavorable cytogenetic abnor malities is the strongest predictor of poor survival in second ary myelofibrosis. Cancer 106: 1985 1989 [218] Hasselbalch H, Jans H, Nielsen PL (1987) A distinct subtype of idiopathic myelofibrosis with bone marrow features mim icking hairy cell leukemia: evidence of an autoimmune pathogenesis. Am J Hematol 25: 225 229 [219] Bass RD, Pullarkat V, Feinstein DI, Kaul A, Winberg CD, Brynes RK (2001) Pathology of autoimmune myelofibrosis. A report of three cases and a review of the literature. Am J Clin Pathol 116: 211 216 [220] Gruson B, Brevet M, Vaida I, Sid IS, Damaj G (2006) Myelofibrosis and cytopenia are not always malignant. Eur J Intern Med 17: 136 137 [221] Pullarkat V, Bass RD, Gong JZ, Feinstein DI, Brynes RK (2003) Primary autoimmune myelofibrosis: definition of a distinct clinicopathologic syndrome. Am J Hematol 72: 8 12 [222] 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 [223] Imbert M, Nguyen D, Sultan C (1992) Myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) with myelofibrosis. Leuk Res 16: 51 54 [224] Hattori N, Nakashima H, Usui T et al. (2007) Successful treatment with prednisolone for autoimmune myelofibrosis accompanied with Sjogren syndrome. Rinsho Ketsueki 48: 1539 1543 [225] 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 [226] Bonduel M, Sciuccati G, Torres AF, Pierini A, Gallo G (1998) Familial idiopathic myelofibrosis and multiple hemangio mas. Am J Hematol 59: 175 177 [227] Kaufman S, Briere J, Bernard J (1978) Familial myelopro liferative syndromes. Study of 6 families and review of literature. Nouv Rev Fr Hematol 20: 1 15 [228] Rossbach HC (2007) Hereditary and familial syndromes of bone and blood. Genetic pathways, diagnostic pitfalls. Fetal Pediatr Pathol 26: 1 16 [229] Sheikha A (2004) Fatal familial infantile myelofibrosis. J Pediatr Hematol Oncol 26: 164 168 [230] Sieff CA, Malleson P (1980) Familial myelofibrosis. Arch Dis Child 55: 888 893 [231] Skoda R, Prchal JT (2005) Lessons from familial myelopro liferative disorders. Semin Hematol 42: 266 273 [232] Mallouh AA, Sadi AR (1992) Agnogenic myeloid metapla sia in children. Am J Dis Child 146: 965 967 [233] Landgren O, Goldin LR, Kristinsson SY, Helgadottir EA, Samuelsson J, Bjorkholm M (2008) Increased risks of poly cythemia vera, essential thrombocythemia, and myelofibrosis among 24577 first degree relatives of 11039 patients with myeloproliferative neoplasms in Sweden. Blood 110(11): Abstract 680 [234] Ueda K, Kawaguchi Y, Kodama M, Tanaka Y, Usui T, Kamada N (1981) Primary myelofibrosis with myeloid meta plasia and cytogenetically abnormal clones in 2 children with Downs syndrome. Scand J Haematol 27: 152 158 [235] Lau SO, Ramsay NK, Smith CM, McKenna R, Kersey JH (1981) Spontaneous resolution of severe childhood myelofi brosis. J Pediatr 98: 585 588

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[236] Wang JK, Lin DT, Hsieh HC, Chuu WM, Wang CH, Lin KS (1990) Primary myelofibrosis in children: report of 4 cases. J Formos Med Assoc 89: 719 723 [237] Walia M, Mehta R, Paul P, Saluja S, Kapoor S, Sharma M (2005) Idiopathic myelofibrosis with generalized periostitis in a 4 year old girl. J Pediatr Hematol Oncol 27: 278 282 [238] Sekhar M, Prentice HG, Popat U et al. (1996) Idiopathic myelofibrosis in children. Br J Haematol 93: 394 397 [239] Boxer LA, Camitta BM, Berenberg W, Fanning JP (1975) Myelofibrosis myeloid metaplasia in childhood. Pediatrics 55: 861 865 [240] Naithani R, Tyagi S, Choudhry VP (2008) Secondary myelo fibrosis in children. J Pediatr Hematol Oncol 30: 196 198 [241] Tebbi K, Zarkowsky HS, Siegel BA, McAlister WH (1974) Childhood myelofibrosis and osteosclerosis without myeloid metaplasia. J Pediatr 84: 860 862 [242] Vannucchi AM, Barbui T (2007) Thrombocytosis and throm bosis. Hematology Am Soc Hematol Educ Program 2007: 363 370 [243] Thiele J, Kvasnicka HM, Facchetti F, Franco V, van der WJ, Orazi A (2005) European consensus on grading bone marrow fibrosis and assessment of cellularity. Haematologica 90: 1128 1132 [244] Manoharan A, Islam A (1979) Acute megakaryoblastic leu kemia or acute myelofibrosis? Br J Haematol 43: 157 158 [245] Barosi G (1999) Myelofibrosis with myeloid metaplasia: diagnostic definition and prognostic classification for clini cal studies and treatment guidelines. J Clin Oncol 17: 2954 2970 [246] Pleyer L, Went P, Russ G et al. (2007) Massive infiltra tion of bone marrow in colon carcinoma after treatment with activated protein C. Wien Klin Wochenschr 119: 254 258

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5

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

Contents Introduction ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Epidemiology ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Course of Disease ::::::::::::::::::::::::::::::::::::::::::::::::::::::: Etiology and Pathogenesis of CML:::::::::::::::::::::::::::: Classification of CML :::::::::::::::::::::::::::::::::::::::::::::::: Clinical Features and Disease Complications in CML :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 5.7 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 :::

5.1 5.2 5.3 5.4 5.5 5.6

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

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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:::::::::::::::::::::::::::::::: 5.9 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) :::::::::::::::::::::::::::::::::::::::

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5.1 Introduction Chronic myelogenous leukemia (CML) is a chronic myeloproliferative disorder with an uncontrolled production of maturing neutrophils being the predominant laboratory feature. The presence of translocation t(9;22), the so-called Philadelphia chromosome, is commonly acknowledged as the disease initiating event, and as such, is essential for the diagnosis of CML. This chapter addresses the pathophysiology of CML, explains diagnostic procedures, summarizes current treatment options including clinical results with imatinib in the treatment of CML, reviews current recommendations for monitoring and management of treatment failure, surveys results with approved second generation Abl kinase inhibitors after imatinib failure, and finally gives an outlook for promising treatment strategies examined in current and future clinical trials.

5.2 Epidemiology Chronic myelogenous leukemia (CML) occurs at an annual incidence of 1 2/100,000, with a slight male predominance. CML accounts for 15 20% of leukemias in adults, and as such is one of the most common leukemias, surpassed only by chronic lymphocytic leukemia (CLL). The peak incidence is in the middle age between 50 and 70, but CML can occur in every age, even in children. Exposure to ionizing radiation is the only known risk factor [1].

5.3 Course of Disease

adequate treatment. The chronic phase is characterized by predominantly mature neutrophilia, with a lower percentage of maturing neutrophils leading to a characteristic left shift in the differential blood count. Basophilia and sometimes eosinophilia are also common and thrombocytosis can reach exorbitantly high levels, sometimes mimicking essential thrombocytosis. Inevitable progression to blast crisis used to occur after a median of 3 5 years prior to the development of Bcr Abl-targeted therapies. In this preimatinib era, busulfan, hydroxyurea or interferon-a represented the (palliative) treatment options. Suddenonset blast crisis is a seldom, but well-acknowledged phenomenon, which occurs within 3 months of a documented hematologic response to treatment. Whilst various definitions for the accelerated phase exist, blast crisis is defined by the presence of 20% or more peripheral blood or bone marrow blasts, and/or the presence of extramedullary blastic infiltrates (myeloid sarcoma, chloroma). The median survival with CML without therapy is about 2 3 years, with conventional chemotherapy such as Hydroxyurea about 4 years and with interferon therapy about 6 years. Currently, the only curative therapy for CML is an allogenic stem cell or bone marrow transplantation with a 10 year survival between 10% and 70%, depending on disease stage and transplant constellation. Today the tyrosine kinase inhibitor (TKI) imatinib is considered to be the gold standard in the first line therapy of CML in CP. Data with a follow-up of over 7 years are now available for this compound. The rate of complete hematologic responses is above 90% and the median overall survival is not reached yet. Thus, responses to TKI therapy in CML seem to be very durable and it may be possible that in the future the majority of CML patients will not die from their leukemia.

CML has a tri-phasic clinical course: (1)

(2)

(3)

The chronic phase (CP) is characterized by an indolent course with only few symptoms and lasts for 3 5 years on average, if left untreated. The accelerated phase (AP) is characterized by poor response to therapy and worsening of the hematological parameters. The blastic phase (BP) (myeloid or lymphatic) defines the transformation of CML in an acute leukemia and is difficult to treat. Myeloid transformation of CML is more common (70%) than lymphoid blastic crisis (30%).

Whilst the duration of these phases is variable and dependent on individual disease biology, the progression to blast crisis, which requires the acquisition of additional chromosomal changes, is ultimately inevitable without

5.4 Etiology and Pathogenesis of CML CML was the first leukemia for which the underlying cause could be successfully elucidated on the molecular level. Already in 1960 Nowell and Hungerford from the University in Philadelphia could demonstrate a specific, small abnormal chromosome in CML patients. They named this chromosome after the city of discovery the Philadelphia chromosome (Phþ) [2]. The Ph-chromosome is the product of a reciprocal translocation which places parts of the long arm of chromosome 9 to chromosome 22 [t(9q;22q)]. This results in the expression of a 210-kDa large fusion protein, Bcr Abl, which consists of the first 927 or 902 amino acids of Bcr and the carboxyterminal 1,097 amino acids of the tyrosine kinase c-Abl (see Fig. 5.1).

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Chromosome 22 c-Bcr

Exon 1

Exon 2

Exon 3

Exon 4

Exon 5

Chromosome 9 Exon 2–11

Exon 1

Exon 1

Exon 1

Exon 2

± Exon 3

c-Abl

p210 Bcr-Abl

Exon 2–11

p185 Bcr-Abl

Exon 2–11

Breakpoints in Ph + ALL Breakpoints in Ph+ CML Intron (= non-coding region)

Fig. 5.1 Bcr-gene, Abl-gene and the most common Bcr Abl fusion products. Top left: Bcr gene on chromosome 22, Top right: Abl gene on chromosome 9; Bottom: The two most common Bcr Abl fusion genes resulting in the respective fusion proteins p210 Bcr Abl, which is more common in chronic phase CML, and p185 Bcr Abl, which is more common in Ph+ ALL

Induction of transcriptin

Induction of transcriptin via autocrine loop triggered by membrane receptors

Fig. 5.2 Important signaling pathways in CML

In Phþ ALL a smaller 185 kDa protein is expressed, which only contains the first 426 amino acids of Bcr (see Fig. 5.1). Bcr contains an oligomerization domain in the first 63 amino acids which leads to the dimeriza-

tion and tetramerization of Bcr Abl with subsequent activation of the Abl tyrosine kinase activity [3]. Thus, the fusion of Bcr to the Abl tyrosine kinase leads to the constitutive activation of this hybrid protein. Bcr Abl

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Table 5.1:

N. von Bubnoff et al.

Epigenetic events during disease progression in CML

Epigenetic event

Gene [chromosome]

(Putative) association

CP

AP

BP

Reference

De novo hypermethylation De novo hypermethylation De novo methylation Loss of imprinting (LOI) De novo methylation

p15 [9p21]

Disease progression

2/17

2/9

4/8

[256]

Calcitonin [11p]

Disease progression

2/31

5/8

11/12

[257, 258]

Histone H3

Prognosis of reponse to treatment and risk of relapse Disease progression

"

""

"""

[259]

1/6

3/3

3/3

[260]

[261]

Disease progression (reversal in remission)

51/112

75/112

[262, 263]

[264]

IGF2 [11p15] SOCS1

elicits strong promitogenic and antiapoptotic signals when expressed in cells such as activation of Ras, PI3-kinase, Rac, and Stats (see Fig. 5.2). Activation of these signaling cascades is crucial for the oncogenecity of Bcr Abl [3]. Expression of Bcr Abl leads to the transformation of myeloid cells. In a syngenic retroviral murine transplantation model, expression of Bcr Abl in murine bone marrow cells results in a CML like disease in mice, with high penetrance and short latency [4, 5]. Therefore, it is believed that Bcr Abl is the sole causing oncogene for the development of CML. Hereby, the constitutive tyrosine kinase activity of Bcr Abl is the driving force of leukemogenesis [6]. These discoveries led to the development of specific Bcr Abl tyrosine kinase inhibitors such as imatinib. However, Bcr Abl fusion genes have been found in healthy individuals using extremely sensitive two-step RP-PCR assays [7]. This may be explained by expression of Bcr Abl in determined progenitors without self-renewing capacity. CML is a clonal stem cell disease, i.e., all of the leukemic cells originate from a single transformed stem cell. This was demonstrated by the analysis of the expression pattern of glucose-6-phosphatdehydrogenase isoforms on the x-chromosome of Phþ cells. Therefore, the Philadelphia chromosome is not only in myeloid cells, but also in erythrocytes, megakaryocytes, B-lymphocytes and possibly in certain T-lymphocytes [3]. Progression of the disease to accelerated and blastic phase is often accompanied by the occurrence of additional clonal chromosomal abnormalities (ACAs or CCAs) in Ph+ cells as well as by epigenetic alterations (see Table 5.1).

5.5 Classiﬁcation of CML CML in the majority of cases is defined by the typical Philadelphia chromosome, which can be detected reli-

Conflicting reference

ably by classical cytogenetic analysis. The Ph-chromosome is found in approximately 95% of CML cases (Phþ -CML). In about 5% of cases it is not possible to demonstrate the existence of a Ph-chromosome by conventional cytogenetics. Here, variant translocations with involvement of additional chromosomes or cryptic translocations lead to the expression of Bcr Abl. By classical karyotype analysis cryptic translocations cannot be detected. Therefore, when CML is suspected but no Phchromosome can be detected by classical cytogenetics, FISH or PCR analysis for the detection of a Bcr Abl fusion is obligatory.

5.6 Clinical Features and Disease Complications in CML [8] The majority of CML patients are diagnosed in the chronic phase of the disease. In about 50% of cases the diagnosis of CML is made by chance on the occasion of a routine blood control. In the other cases, usually rather mild and nonspecific symptoms are present. Many of these patients are asymptomatic at diagnosis (50%), with elevated white blood cell counts detected in routine blood tests raising first suspicions of the disease. In the other cases, usually rather mild and nonspecific symptoms are present. The excess of neutrophils is often accompanied by splenomegaly (50 75%) which sometimes leads to abdominal fullness or discomfort, early satiety, upper left quadrant pain (sometimes perceived as pain in the left shoulder) with or without perisplenitis or splenic infarctions. Splenic rupture is an extremely rare event [9, 10]. Hepatomegaly or lymphadenopathy only occur occasionally. With medical care being sought earlier, increased sensitivity of laboratory tests, as well as heightened awareness due to better physician education, however, the percentage of asymptomatic CML patients without splenomegaly at diagnosis is rising. When symptoms do occur, they are mostly vague, non-specific and

Chap. 5

Chronic Myeloid Leukemia (CML)

gradual in onset. Among symptomatic patients fatigue and reduced intolerance to exercise and physical stress, with or without anemia, predominate. Signs of excessive hypermetabolism (B-symptoms), characterized by the well-known trials of excessive weight loss (H10% of body weight in 6 months), debilitating fatigue and nocturnal sweats, are uncommon presenting symptoms. Gouty arthritis may also be present as a consequence of elevated cell turn-over. In patients with massive leukocytosis (WBC H300,000/ml) the intravascular blood flow may be impeded, resulting in clinical signs of leukostasis. Impaired circulation of the lungs, retinal veins, the penis or the central nervous system, resulting in dyspnea and cyanosis, retinal hemorrhages, retinal vein distension or papilledema with visual blurring and/or diplopia, priapism, renal insufficiency, tinnitus, impaired hearing, dizziness and rarely stupor, respectively, may occur [11]. In such cases, it is imperative to perform leukapharesis as soon as possible, combined with cytoreduction by hydroxyurea. Care must be taken to prevent tumor lysis syndrome. Thrombohemorrhagic complications as a result of thrombocytosis, secondary von Willebrand syndrome or thrombocyte dysfunctions are infrequent. Some patients experience tenderness over the lower sternum due to bone marrow expansion. Rarely observed CML-associated clinical features include Sweets syndrome [12], autoimmune hemolytic anemia [13] and paraneoplastic digital necrosis [14]. Hyperhistaminemia resulting in acne urticaria [15] or diabetes insipidus [16, 17] have also been described. In accelerated phase and in blast crisis patients, additional symptoms may occur, which are similar to those seen in AML patients. Increasing bone marrow insufficiency can lead to bleeding, infectious complications or anemia. Massive splenomegaly can lead to very painful splenic infarctions. Furthermore, extramedullary disease or chloromas at any site can be found in advanced CML. Lymphatic blast crisis may be associated with meningeosis leucaemica with symptoms such as severe head ache, nausea, emesis, vertigo or other neurologic impairments. CML may be associated with lymphoproliferative diseases in several ways. Firstly, CML may occur after radiation therapy of patients with Hodgkin- or NonHodgkin Lymphoma. Secondly, blastic phase CML in approximately one third of cases features a lymphoid phenotype. Furthermore, simultaneous or sequential occurrence of T-cell lymphoma [18 20], M. Waldenstr€ om [21], multiple myeloma [22 24], mantle cell lymphoma [25] or CLL [26 29] have been reported.

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5.7 Diagnosis of CML The diagnosis of CML is primarily based on the examination of the peripheral blood smear (see Fig. 5.3) and a bone marrow specimen (see Figs. 5.4 5.6). Diagnosis must be confirmed by documentation of Bcr Abl expression by karyotype analysis and molecular diagnostics. Patients with CML typically present with a highly characteristic white blood cell differential blood count with peaks in the percent myelocytes and segmented neutrophils. Absolute basophilia and eosinophilia are characteristic findings (see Fig. 5.3). Thrombocytosis is another characteristic feature found in many CML patients. The detection of the Philadelphia chromosome or

Fig. 5.3 CML Cytology of peripheral blood. Leukerythro blastosis (erythroblast (blue arrow)); left shift (promyelocyte (black arrow); metamyelocyte (green arrow, blast (white arrow)); anisocytosis of the erythrocytes (tear drop cells (red arrow)); megakaryocytic nuclei with ruptured cytoplasm; anisocytosis of platelets

Fig. 5.4 CML-chronic phase bone marrow aspirate. Hyper plastic bone marrow with preponderance of granulocytes in various degrees of maturation, sporadic erythroblasts (red arrows)

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its transcriptional or translational products is a requirement sine qua non for the diagnosis of classic CML. The occurrence of philadelphia chromosome negative, Bcr Abl positive disease variants has been repeatedly reported [30], and probably reflects the lack of sensitivity of conventional cytogenetics. Thus, a diagnostic workup should include RT-PCR or nested PCR of the Bcr Abl gene transcript.

5.7.1 Baseline Diagnostics

Fig. 5.5 CML accelerated phase cytology of bone marrow aspirate. Hyperplasia, with prominent left shift and predominant promyelocytes, reduced fatty marrow

a

Baseline diagnostics in CML include a physical examination with recording of the spleen size, liver size, lymph node status and search for chloroma. Alkaline leukocytes phosphatase (ALP) is no longer considered to be an important parameter for the diagnoses of

b

100 μm

50 μm

d

c

50 μm

Fig. 5.6 CML bone marrow histology. a Marked granulocyte proliferation with reduced age dependant adipose tissue by neutro philic granulopoiesis (NASD reaction, 100); marked hyperplasia of megakaryocytes. b Enhanced megakaryopoiesis with micro

50 μm

megakaryocytes (NASD reaction, 200). c Hyperplasia of mega karyocyte microforms with hypolobulated, dense nuclei (immunohistochemistry with CD61, 200). d Slightly enhanced reticulin fibrosis (Gomori, 200)

Chap. 5

Table 5.2: * * * *

* *

Chronic Myeloid Leukemia (CML)

Typical differential white blood cell count in CML

Blasts (0 9% of WBC) Promyelocytes (4% of WBC) Myelocytes, metamyelocytes (40% of WBC) Mature segmented and hypersegmented neutrophils (35% of WBC) Basophilia (10 15% of WBC) Eosinophilia

CML, since molecular markers are by far more specific.

5.7.2 Cytology of Peripheral Blood in CML (Fig. 5.3)

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ase-granules [34] and hyperbasophilia in CML is only rarely associated with hyperhistaminemia [35], in contrast to mastocytosis. Monocytosis is found predominantly in patients with p190 Bcr Abl fusion protein [36, 37].

5.7.2.2 Changes in the Lymphoid Compartment Absolute lymphocytosis due to increases in CD4 and CD8-cells may be found in CML patients, whereas Blymphocytes are not increased [38]. Furthermore number and activity of circulating natural killer cells (NKC) is typically decreased, with further decreases occurring during disease progression [39, 40].

5.7.2.3 Changes in the Platelet Compartment Leukocytosis with white blood cell counts (WBC) greater than 50,000/ml with a continuous left shift up to promyelocytes and myeloblasts is characteristic (see Fig. 5.3). The number of myeloblasts and basophils is diagnostically relevant. The findings of a typical differential WBC are summarized in Table 5.2. Per definition, myeloblasts must be G10% and basophils G20%, in chronic phase CML.

Thrombocytosis is present in approximately half of all newly diagnosed CML patients, with platelet counts between 600,000 and 700,000/ml (15 34%), and H1,000,00/ml not being unusual [41]. Thrombocytopenia due to splenic pooling in case of massive splenomegaly or massive bone marrow infiltration is rare and usually portends pending progression to the accelerated phase.

5.7.2.1 Changes in the Myeloid Compartment Leukocytosis with elevated white blood cell counts (WBC) of H25,000/ml is found in many newly diagnosed patients, and in some patients present with WBC of H100,000/ml or even H300,000/ml. Absolute neutrophilia, with the presence of cells of the neutrophilic lineage at all stages of development, down to the level of myeloblasts, is present. The presence of a greater percentage of myelocytes, as compared to the more mature metamyelocytes, defined as a hiatus leukemicus, is pathognomonious for patients with CML. Interestingly, significant periodic variations in leukocyte (and erythrocyte) counts with cycle intervals of approximately 60 days have been documented in some patients [31, 32]. Functional abnormalities of neutrophils are usually mild and compensated for by high neutrophil concentrations, so that CML patients in chronic phase do not have an increased liability for infections, be they common or opportunistic. Although relative eosinophilia and basophilia are almost universal findings, absolute eosinophilia/basophilia is less common. However, rare cases of prominent eosinophilia/basophilia may occur and reach such an extent, that they dominate the granulocytic cells, leading to the terminus Ph-positive eosinophilic/basophilic CML [33]. Interestingly, CML-basophils contain trypt-

5.7.2.4 Changes in the Erythroid Compartment Anemia of varying degrees due to splenic pooling in case of massive splenomegaly or massive bone marrow infiltration may be present in up to 50% of patients. Red cell membrane porosity and loss of biconcave shape can typically be observed by electron microscopy.

5.7.3 Bone Marrow Cytology in CML (Figs. 5.4 and 5.5) Hyperplastic bone marrow with preponderance of granulocytes in various degrees of maturation is typically found in bone marrow aspirates of chronic phase CML patients (see Fig. 5.4). The granulopoiesis/erythropoiesis ratio is shifted in favor of the marked granulopoiesis, but the maturation of granulopoiesis and erythropoiesis appears normal without major myelodysplastic stigmata. A predominant left shift with preponderance of promyelocytes and only occasional erythroblasts is observed in the accelerated phase (see Fig. 5.5). Blasts usually do not exceed 5% of all nucleated cells. Blast counts of 10% or more of all nucleated cells defines acceleration of the disease, whereas blast counts H20% define blastic transformation of CML (see Summary Box 5.1).

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Summary Box 5.1

N. von Bubnoff et al.

Table 5.3:

Diseases with abnormal ALP levels [44, 265, 266]

Elevated ALP

Acceleration criteria: Rising leukocytes/platelets counts or increasing splenomegaly under adequate therapy; thrombopenia G100,000/ml, which is not induced by therapy; additional chromosomal aberrations; basophils 20% or more in the peripheral blood; 10 19% blasts in the peripheral blood or bone marrow.

* * * * *

Decreased ALP *

Blast crisis criteria: Blasts 20% or more in the peripheral blood or bone marrow; extramedullary blast proliferation (chloroma). Immunophenotyping of the blasts: Lymphatic (30%) or myeloid (70%) blast crisis. In lymphatic blast crisis CNS involvement is possible.

5.7.4 Bone Marrow Histology in CML (Fig. 5.6a–c) Similar findings as discussed in the bone marrow cytology section are found, with marked increased cellularity and predominance of granulopoiesis. Hypercellular bone marrow with marked granulocytic proliferation and reduced age-dependant adipose tissue are characteristically found in chronic phase CML (see Fig. 5.6a and b). Marked hyperplasia of megakaryopoiesis (see Fig. 5.6a c) with sometimes typical small hypolobulated micromegakaryocytes (see Fig. 5.6b and c) is also common. Approximately 30% of the patients shows marrow fibrosis (see Fig. 5.6d). These patients tend to present with thrombocytosis, distinct splenomegaly and are reported to have a worse prognosis.

Inflammatory disorders Infections Pregnancy PV CIMF (25%)

* * * *

CML PNH Hypophosphatemia CIMF (25%) Androgen abuse

Elevated serum levels of Vitamin B12 and Vitamin B12-binding proteins are common in most myeloproliferative disorders, and particularly so in CML. This is easily explained, as mature neutrophils are one of the main sources of Cobalamin-binding proteins [46, 47]. Pseudohyperkalemia may be present in patients with extreme leukocytosis as a consequence of potassium release from white blood cells during clotting [48]. Furthermore, decreased levels of cholesterol are often detected in patients with CML, and lower levels have been implicated to have a negative prognostic value [49].

5.7.6 Molecular Diagnostics in CML Presence of Bcr Abl can be confirmed at the DNA-level (conventional cytogenetics, FISH, Southern Blot), at the

5.7.5 Laboratory Findings in CML Hyperuricemia, hyperuricosuria as well as urate stones in the urinary tract are a common consequence of enhanced cell turnover in patients with CML, and may result in gouty arthritis or uric acid nephropathy [42, 43]. Serum lactic acid dehydrogenase (LDH) may also be elevated, and relates to increased cell turnover. Low to absent alkaline leukocyte phosphatase (ALP) in the circulating neutrophils [44] is found in H90% of patients. Low ALP-levels were classically used to exclude PV or ET, disorders in which ALP-levels are typically increased (see Table 5.3). Furthermore, ALP-levels are normal or increased in reactive leukocytosis due to infections, a feature which used to be important in the differential diagnosis (see Table 5.2). Effective treatment of CML results in a return of ALP-levels to normal levels [45].

Fig. 5.7 Philadelphia chromosome t(9;22). Fluorescence in situ hybridization (FISH): Reziproke translocation with co localization of Bcr (chromosome 9q34: spectrum orange) and Abl (chromosome 22q11.2: spectrum green) and 2 normal chromosomes without translocations

Chap. 5

Chronic Myeloid Leukemia (CML)

RNA-level (PCR) or at the protein level (Western Blot). For the routine diagnostic approach karyotyping via conventional cytogenetics is the most important technique and is considered to be the gold standard in the diagnosis and monitoring of CML patients. Quantitative PCR is mandatory for the detection of minimal residual disease and should be performed regularly to monitor patients under TKI therapy or following allogenic transplant. PCR and FISH (see Fig. 5.7) should be performed in any case of suspected Ph-negative Bcr Abl positive CML.

5.7.6.1 Conventional Cytogenetics in CML Karyotyping requires cells to be in the metaphase of the cell cycle. Therefore it is necessary to cultivate and sychronize cells prior to cytogenetic analysis. Usually a bone marrow biopsy is required, since cells from the peripheral blood cannot be synchronized in metaphase. Only if significant numbers of myeloblasts are present in the peripheral blood (typically in the accelerated or blastic phase), or in cases of excessive leukocytosis, can karyotyping be performed with peripheral blood cells. The Philadelphia chromosome can be easily identified by this method. Moreover, additional chromosomal aberrations can be diagnosed. While the prognostic significance of additional aberrations at the time of diagnosis is unclear, additional characteristic aberrations appearing in the course of the disease are associated with a bad prognosis. Examples are isochromosome 17 (i(17q)), trisomy 8 or 19, a double Ph chromosome or loss of the Y chromosome. A conventional cytogenetic analysis requires 2 4 working days and detection of a Philadelphia chromosome substantiates the diagnosis of CML. In about 5% of CML patients no Philadelphia chromosome is found by conventional karyotyping. Here alternative molecular testing such as FISH or PCR must be performed.

Fluorescence In-Situ Hybridization (FISH) (Fig. 5.7) FISH can be used to detect the Bcr Abl gene rearrangements in metaphase or in interphase cells (Fig. 5.7). Thus, in contrast to karyotyping, a bone marrow biopsy is not absolutely required. FISH can also prove atypical gene rearrangements, which are missed by conventional cytogenetics. With FISH generally more cells are analyzed compared to karyotyping (usually H100 cells with FISH; H20 cells with karyotyping), so that the method has a greater sensitivity. FISH thus is a suitable method both for diagnosis and for monitoring and is used in many hematology centers as a standard diagnostic method in CML, together with cytogenetics and quantitative PCR (qPCR).

125

However, in contrast to conventional karyotyping FISH has not been prognostically validated for monitoring CML patients treated with imatinib. Real-time Polymerase Chain Reaction (RT-PCR) The Bcr Abl fusion product at the mRNA level is detected by RT-PCR. PCR products are subsequently separated by electrophoresis. Both existence of a translocation as well as the location of the breakpoint can be determined. The breakpoint occurs within the M-Bcr-(M ¼ Major)-gene locus in about 98% and in the m-Bcr-(m ¼ minor)-gene in less than 2% of CML cases. In contrast, in Phþ ALL rearrangements are in 50% within the M-Bcr- and in 50% within the m-Bcr-gene locus (see Fig. 5.1). The advantage of PCR compared to cytogenetic analysis is the substantially higher sensitivity. Additionally, PCR analysis can be performed with cells from the peripheral blood since no cell division is required. Thus, no bone marrow biopsy is needed. Therefore it is the method of choice for a close molecular monitoring during therapy. Today PCR is performed predominantly as a quantitative PCR (real time PCR) test. Here, incorporation of fluorescent dyes during the PCR reaction permits the quantification of the transcript levels in a given sample. The high sensitivity of quantitative PCR (qPCR) allows the early detection of a potential therapy failure and enables timely initiation of alternative therapy. Tight quality controls of the PCR procedure are mandatory for this very sensitive and error-prone method, in order to receive reliable and comparable results. Thus, diagnostic PCR analysis should only be performed in laboratories with a high volume of PCR samples and close quality controls.

5.7.7 Differential Diagnosis of CML Chronic myeloid leukemia has to be delineated from non-clonal, reactive leukocytosis occurring during acute or chronic inflammation or after G-CSF treatment (medical history, bone marrow diagnostics, Bcr Ablrearrangement, acute phase parameters). Nowadays, other myeloproliferative/myelodysplastic diseases such as PV, ET, CMML or PMF can easily be differentiated by bone marrow analysis, cytogenetics and molecular testing.

5.8 Treatment of Patients with CML Chronic myelogenous leukemia (CML) is caused by the constitutively activated kinase Bcr Abl and thereby con-

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stitutes a disease, which is amenable to treatment using specifically acting kinase inhibitors. The Abl tyrosine kinase inhibitor imatinib (Glivec) induces durable responses in a high proportion of patients with chronic phase chronic myeloid leukemia and therefore represents the current standard first line treatment. However, especially in advanced phase CML a proportion of patients display clinical resistance to imatinib. The availability of approved second line therapies and the need for specialized diagnostic tools makes the clinical management of CML more complex.

5.8.1 Treatment in Chronic Phase CML For the treatment of CML in chronic phase, available options include the Abl kinase inhibitor imatinib, interferon alpha (IFN, optionally in combination with lowdose cytarabine), the cytostatic drugs busulphan and hydroxyurea, and allogeneic blood stem cell transplantation.

5.8.1.1 Hydroxyurea, Busulphan and Alpha Interferon Hydroxyurea and busulfan can induce hematologic but however, no cytogenetic responses in chronic phase CML [50, 51]. A meta-analysis demonstrated a survival benefit at 4 years for hydroxyurea compared to busulphan (53.6% versus 45.1%) [52]. In contrast, treatment with alpha interferon (IFN) alone or in combination with low-dose cytarabine induces hematologic responses in 70 80% of cases and durable complete cytogenetic responses (CCyR; for definition see Sect. 5.8.3.4 and Table 5.4) in 5 15% of cases, with higher rates of

cytogenetic responses achieved by IFN in combination with low dose cytarabine [53 56]. A survival advantage of IFN-based therapy to hydroxyurea or busulphan was demonstrated in a meta-analysis (5-year-survival 57% versus 42%) [57], and survival at 10 years was 72% with IFN-based therapy when a CCyR was attained [56]. Thus, IFN in combination with low-dose cytarabine in the 1990s of the 20th century was regarded as treatment standard in chronic phase CML, and the use of hydroxyurea should be restricted to palliative or initial cytoreduction.

5.8.1.2 Imatinib in the Treatment of CML The phenylaminopyrimidine CGP57148B (Imatinibmesylate) in the 1990s was developed by Ciba-Geigy (now Novartis) as ATP-competitive tyrosine kinase inhibitor of PDGFR, cKit and Abl. Preclinical studies demonstrated activity in Bcr Abl positive cell lines and in animal models [58 60]. Based on these observations, clinical trials in Bcr Abl positive CML were initiated in 1998. Phase 2 clinical trials demonstrated activity of imatinib in chronic phase as well as in accelerated phase and blast crisis CML [61 63] and lead to the approval of imatinib for the treatment of CML in 2002. Imatinib After Interferon Failure in Chronic Phase A phase 2 clinical trial examining the activity of imatinib 400 mg daily in patients with chronic phase CML and IFN resistance (65% of the patients) or intolerance (35% of the patients), a complete hematologic response (CHR; for definition see Sect. 5.8.3.4 and Table 5.4) was achieved in 96% and a CCyR in 57% of the patients [64]. Progression-

Table 5.4: Definitions for remissions and current recommendations for monitoring imatinib therapy in chronic phase CML. Based on recommendations by the European Leukemia Net (ELN) [72, 249, 252] Definition CHR (complete hematologic response)

* * *

*

Cytogenetic response (at least 25 BM metaphases counted)

Molecular response (Bcr Abl transcripts PB)

WBC count G10 G/L Platelet count G450 G/L Differential without immature myeloid cells and G5% basophils Nonpalpable spleen

Complete (CCyR) 0% Phþ Partial (PCyR) 1 35% Phþ Minor CyR 36 65% Phþ Minimal CyR 66 95% Phþ No CyR H95% Phþ Complete (CMR) Not detectable Major (MMR) Bcr Abl/control gene 0.10

Monitoring (CML CP, Treatment with Imatinib) Every 2 weeks until CHR, then every 3 months

At 3 and 6 months, then every 6 months until CCyR achieved and confirmed

Every 3 months, at CCyR þ MMR every 6 months

Chap. 5

Chronic Myeloid Leukemia (CML)

free survival at 6 years was 61%, overall survival was 76%. Imatinib First Line in Chronic Phase A phase 3 clinical trial (IRIS trial; International Randomized Study of Interferon and STI571) compared first-line imatinib 400 mg daily with IFN in combination with low-dose cytarabine in 1,106 patients with newly diagnosed, untreated chronic phase CML. The superiority of imatinib was already apparent after 19 months with a CCyR rate of 76% with imatinib compared to 15% with IFN and cytarabine [65, 66]. After 12 months, major molecular responses (MMR; for definition see Sect. 5.8.3.4 and Table 5.4) were seen in 40% of cases with imatinib compared to 2% with IFN and cytarabine [66]. After 6 years, the rate of CCyR attained at any time (cumulative best CCyR rate) was 82% with imatinib. Despite a crossover rate of 65% from IFN to imatinib (see below), superiority of imatinib was also shown with respect to overall survival [67, 68]. Analysis of primary endpoints after 7 years documented stable and ongoing responses in the majority of cases with a cumulative CCyR rate of 82% with imatinib (456 out of 553 patients) [69]. A loss of a previously achieved CCyR was observed in 79 out of these 456 patients (17%). Progression to accelerated or blast phase was reported in 15 patients who previously had achieved CCyR (3%) and in 7% of all patients randomized to imatinib treatment [69]. At 7

Fig. 5.8 Imatinib-induced periorbital edema

127

years, 60% of patients randomized to receive imatinib were still on study medication with 57% still in CCyR, and the overall survival rate for patients randomized to imatinib (intention to treat) was 86% or 94% when only CML-related deaths were considered [69]. Imatinib side effects were mainly considered as mild (grade 1) or moderate (grade 2) and included superficial edema, especially periorbital edema (see Fig. 5.8), which is a characteristic side effect, nausea, muscle cramps, musculoskeletal pain, rash, fatigue and headache [65, 70]. Grade 3 or 4 side effects were mainly limited to neutropenia (17%), thrombocytopenia (9%), anemia (4%) and elevated serum alanine or aspartate amino-transferase (5%) and predominantly were observed during the first and second year of treatment. After 19 months, 58% of patients allocated to study treatment with IFN and cytarabine crossed over to imatinib, 43% of these due to side effects including fatigue, depression, myalgia, joint pain, neutropenia and thrombocytopenia [65]. In contrast, a discontinuation of imatinib study medication after 6 years was reported in 5% of patients [67]. These results were reproduced outside the setting of a clinical trial [71], and established imatinib 400 mg daily as standard treatment for patients with CML in chronic Phase [72, 73].

5.8.2 Treatment of Accelerated and Blast Phase Treatment options for advanced phase CML include imatinib, chemotherapy, and allogeneic hematopoietic stem cell transplantation. In phase 2 trials in patients with CML in accelerated phase (AP) and blast crisis (BC), response rates induced by imatinib were inferior to response rates achievable in chronic phase, but superior to chemotherapy [62, 63, 74 76]. With imatinib 400 600 mg daily, overall survival in phase 2 trials was 53% at 4 years and 43% at 7 years in accelerated phase [74, 75], and it was 11% at 3 years in blast crisis CML [76]. For patients with advanced phase CML, the recommended imatinib dose is 600 mg daily. If feasible, patients in BC should proceed to allogeneic hematopoietic stem cell transplantation as soon as a hematologic response has been achieved [72]. For patients in AP, the achievement of a cytogenetic response at 3 months or CCyR at any time receiving imatinib treatment is predictive for more sustained responses and prolonged overall survival [74, 75]. These patients should be closely monitored, and in case of a loss of hematologic or cytogenetic response, should be submitted to stem cell transplantation. After progression during imatinib, options to regain a response before allogeneic transplant

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include second-generation Abl kinase inhibitors like dasatinib or nilotinib (see Sect. 5.8.4.2) and chemotherapy.

5.8.3 Response Criteria in CML

Bcr-Abl ratio

Determining the response to treatment is the prerequisite for the recognition of a treatment failure. According to the techniques used for monitoring, three levels of response can be discriminated (Fig. 5.9, Table 5.4) [72, 77]. With decreasing leukemic burdon, the primary finding will be the normalization of blood cell counts (hematologic response). Later on, the decrease of Philadelphia-positive metaphases in the bone marrow indicates cytogenetic response. Molecular response is reflected by a decrease of Bcr Abl transcripts in peripheral blood or bone marrow using quantitative real-time PCR (qRT-PCR). The magnitude of molecular response is expressed as ratio of Bcr Abl to a control gene or as log reduction compared to either the pretreatment value or a standard [72]. Efforts of harmonizing the methodology for detection of Bcr Abl transcripts and expressing results have lead to the calculation of lab-specific correction factors allowing

Fig. 5.9 Relatedness of leukemic burdon, response and amount of Bcr Abl transcripts in CML in the course of treatment (adapted from ref. [249]). With decreasing leukemic burdon, normalization of blood counts will be achieved at first (hematologic response). Cytogenetic response will document the decline of Philadelphia positive bone marrow metaphases. Molecular response reflects the regression of Bcr Abl transcripts in peripheral blood. Once a complete cytogenetic response (CCyR) is attained, a further decrease of leukemic burdon only can be monitored using quantitative real time PCR (qRT PCR). Molecular response is expressed as Bcr Abl to control gene ratio or log decrease compared to a standard. A complete molecular response (CMR; Bcr able transcripts not detected, i.e., qRT PCR and nested PCR negative) depends on the lab specific test sensitivity [14]. Thus, achieving a CMR mirrors not only leuke mic burdon, but also the threshold of detection of the assay and is not equivalent to eradication of the disease or cure

conversion of PCR results into the internationally uniform and comparable international scale [72, 78]. The high sensitivity of PCR allows monitoring of disease activity and treatment response below the level of CCyR (Fig. 5.9). In case of a negative test result for quantitative PCR, nested PCR allows a qualitative detection of Bcr Abl transcripts with a one log higher sensitivity compared to qRT-PCR [72]. However, the presence of a complete molecular response (CMR; negative results for qRT-PCR and nested PCR) is also dependent on test sensitivity which might vary between different assays and labs [72].

5.8.4 Monitoring Response in CML Regular monitoring of imatinib treatment in CML is indispensable to confirm adequate response and to identify patients with suboptimal response or treatment failure early enough to make appropriate treatment changes. An international expert panel as well as consensus statements of the Deutsche Gesellschaft f€ ur H€amatologie und Onkologie (DGHO) and the National Comprehensive Cancer Network (NCCN) include recommendations for monitoring CML patients receiving imatinib (Table 5.4) [72, 73, 79]. Hematologic and cytogenetic response to first-line imatinib at 3, 6 and 12 months in patients with chronic phase CML determines progression-free and overall survival. Patients who are continued with imatinib despite a lack of cytogenetic response face the risk of progression to accelerated and blast crisis. In contrast, achieving a CCyR is associated with excellent progression-free survival, provided that imatinib is continued without dose reduction or interruptions [68, 70, 80, 81]. Looking at the IRIS study, achieving a major cytogenetic response (MCyR 35% Phþ; see also Table 5.4) at 12 months indicated superior progression-free and overall survival [68]. This finding was confirmed and extended with respect to the notion that attaining CHR at 3 months or PCyR at 6 months was associated with a significant improvement of 5 year overall survival [80, 81]. These findings underscore the importance of a regular monitoring particularly in the first year of treatment (Table 5.4). Achieving a major molecular response (MMR; Bcr Abl ratio 0.1) at 12 or 18 months correlates with stability of a previously achieved CCyR, and therefore can be regarded as save haven [82], but is not predictive for progression-free or overall survival [70, 80, 81, 83, 84]. A sequencing analysis for the presence of Bcr Abl mutations that mediate inhibitor resistance (see Sect. 5.8.5.2) should be initiated in case of treatment failure and repeated qRT-PCR results demonstrating an

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129

Table 5.5: Definitions for optimal response, suboptimal response and failure in chronic phase CML receiving imatinib. Based on recommendations by the European Leukemia Net (ELN) [72]. Definitions for hematologic (CHR), cytogenetic (PCyR, CCyR) and molecular response (MMR) see Table B1. Bcr Abl mutation: Bcr able gene mutation mediating imatinib resistance. The resulting amino acid exchange affects binding of the kinase inhibitor. ACA: additional chromosomal aberrations in Bcr Abl positive (Phþ) cells as revealed by cytogenetic analysis Time

Failure

Suboptimal response

Optimal response

3 months 6 months

No CHR No cytogenetic response (Phþ H95%) No PCyR (Phþ H35%) NoCCyR (Phþ 1%) Loss of CHR loss of CCyR Bcr Abl mutation, ACA in Phþ cells

No cytogenetic response (Phþ H95%) No PCyR (Phþ H35%)

CHR, minor CyR (Phþ 65%) PCyR (Phþ S35%)

NoCCyR (Phþ 1%) No MMR (Bcr Abl/control gene H0.10) Loss of MMR

CCyR (Phþ 0%) MMR

12 months 18 months Anytime

increase of Bcr Abl transcripts [72]. The factor increase that should trigger mutation analysis is not yet established [72, 85, 86].

5.8.5 Resistance to Imatinib in CML 5.8.5.1 Deﬁnition and Incidence of Suboptimal Response and Treatment Failure In patients with chronic phase CML treated with imatinib 400 mg daily, a lack of hematologic or cytogenetic response at specific time points indicates suboptimal response or treatment failure (Table 5.5) and determines an adverse outcome with respect to overall survival, progression-free survival, gain of a CCyR, or loss of a previously achieved CCyR. Lack of a hematologic response at 3 months, a CHR at 6 months and lack of a cytogenetic response at 6 months, a PCyR at 12 months, and a CCyR at 18 months indicate primary hematologic or cytogenetic failure (primary resistance) and are associated with inferior progression-free survival [68, 70, 80, 81]. One study performed by the Hammersmith group shows that treatment failure at these time points also affects overall survival and, moreover demonstrates that not only failures, but also suboptimal responders either at 6 months (less than PCyR) or 12 months (less than CCyR) had a significantly poorer progression-free survival, a lower probability of CCyR, and in the case of 12 months suboptimal responders also worse survival [81]. Hematologic imatinib failures in early chronic phase are rare (G5% of cases) [65, 81]. In contrast, primary cytogenetic failures are more prevalent and were reported to occur in 3 18% at 6 months [67, 81], 15 27% at 12 months [70, 81], and 23 49% at 18 months [68, 81]. Acquired treatment failure (secondary resistance) denotes loss of a previously achieved hematologic or cytogenetic response or progression to

AP or BC despite continued imatinib treatment. In early chronic phase CML patients receiving imatinib treatment, the annual rates of secondary resistance or death in the IRIS study continuously decreased from the second (7.5%) to the sixth year (0.4%) [67]. In advanced phase CML, primary hematologic failure occurs more frequently and in phase 2 clinical trials was reported in 18 30% of patients with AP and in 60% of patients with BC. After 4 years, resistance to imatinib had emerged in 45 70% (AP), and 90% (BC), respectively [74, 75, 87 90].

5.8.5.2 Mechanisms of Resistance to Imatinib in CML The mechanisms of imatinib resistance were first studied in cell culture-based systems. Bcr Abl positive cells which were kept at suboptimal concentrations of imatinib for prolonged periods of time developed a moderate imatinib resistance up to concentrations of about 1,000 nM [91 93]. Further investigations revealed molecular changes underlying resistance in these cell lines. During the imatinib selection process, these cells acquired amplification of the Bcr Abl gene or overexpression of MDR-1, which codes for a multidrug-resistance membrane associated transporter protein [91 93]. Clinical resistance to imatinib, primarily occurring in advanced-phase CML and Phþ ALL gave rise to intensive studies elucidating the mechanisms underlying resistance to imatinib in the clinic. In 2001 it was shown that 3 out of 11 imatinib resistant patients with advanced CML or Phþ ALL displayed amplification of the Bcr Abl gene [94]. Furthermore, in six out of nine imatinib resistant patients a mutation in the Bcr Abl kinase domain was discovered, which leads to an amino acid exchange at position 315 from threonine to isoleucine [94]. This amino acid exchange interferes with imatinib binding [95] and results in a significant resistance against the drug (IC50 H10,000 nM) [94].

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Abl1 Tyrosine kinase region Gate-keepers

ATP-binding

P-loop

SH3-SH2

Catalytic domain

G

Activation loop

A-loop

C

57

224 M244V L248V Bold italics: G250E/A E255K/V 85% of resistance Q252H/E assoc. mutations Y253H/F

Destabilize inactive conformation imatinib binding

489 V289A

F317L

F311L/I

M343T

E355G/D

M351T/V

V379I

L387M/F F382L H396R/P

F359V A380T

T315I

DFG-motif: P open Controls catalytic activity by switching between different states (P-dependent)

Drug contact site: direct inhibition of imatinib binding

Fig. 5.10 Location of Bcr Abl point mutations

Subsequently, a large number of additional Bcr Abl mutations could be identified causing a variable degree of imatinib resistance [96 101]. The location of the most common Bcr Abl point mutations is depicted in Fig. 5.10. Furthermore, in a study of 36 imatinib resistant patients, cytogenetic abnormalities in addition to the philadelphia chromosome were noted, indicating that clonal evolution in an imatinib resistant leukemic clone had occurred [101]. Epigenetic aberrations occurring during disease evolution to accelerated and blastic phase are summarized in Table 5.1. Mechanisms of imatinib resistance observed both in cell lines or in clinical

a

b

c

samples are summarized in Fig. 5.11. Proposed mechanisms for genetic instability that might contribute to the generation of secondary Bcr Abl resistance mutations are summarized and depicted in Fig. 5.12. Bcr–Abl-dependent resistance Mutations

The first imatinib resistance mutation which was discovered leads to an exchange of threonine at position 315 to isoleucine [94]. Interestingly, a crystal structure analysis of the Abl kinase domain published in 2000 already

Bcr-Abl Imatinib Mutant Bcr-Abl

d

e

f

Additional genomic alterations Alternative signaling pathways Membrane transporters

Fig. 5.11 Mechanisms of resistance towards imatinib (adapted from ref. [250]). a Imatinib is available within the cell at a sufficient quantity for inhibition of all Bcr Abl mole cules. b Overexpression of Bcr Abl allows the leukemic cell to maintain a baseline level of signaling that is sufficient for cell survival even in the presence of imatinib. c Specific mutations within the Abl kinase domain prevent binding of imatinib but

still allow binding of ATP thus retaining Bcr Abl kinase active. d Secondary genetic alterations or e the activation of alternative signaling pathways contribute to the Bcr Abl independent growth and/or survival of the malignant clone. f Expression of membrane bound transport proteins leads to diminished concen trations of inhibitor available within the cell by increased eflux or decreased influx

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Chronic Myeloid Leukemia (CML)

131

Protein tyrosine phosphatases

ROS

PI3K/mTOR ඎchanges in mt. e-transport & glucose metabolism

Oxidized nucleic acids

DNA-incorporation by unfaithful polymerases

Bcr-Abl

Bcr-Abl remains in active conformation

Imatinib resistance

Kintics of DNA-repair & aberrant DNA-repair & p53 accumulation

Unfaithful/no repair

Bcr-Abl mutations

genomic instability ?

Oxidative DNA damage normal

centrosome alt. (9–20%) spindle defects (6–53%) chr. aberrations (20–45%)

Mitotic spindle & centromer defects

Imatinib, Nilotinib, Dasatinib

Fig. 5.12 Reasons for the frequent occurrence of Bcr Abl point mutations in CML

Fig. 5.13 Activity-/resistance profiles of imatinib (left), dasatinib (middle), and nilotinib (right). Representation of the Abl kinase domain [150, 228, 251], with C helix in green, P loop in magenta,

A loop in blue. Spheres indicate sites of resistance mutations, color represents the amount of resistance, with green: sensitive, yellow: intermediate, and red: resistant. See also Table 6

predicted the threonine 315 to be a critical position required for imatinib binding to Abl (see Fig. 5.13) [95]. Indeed, the T315I mutation leads to a complete biochemical imatinib resistance even at high imatinib concentrations while the kinase activity is preserved

[94]. Today, more than 40 different imatinib resistance mutations have been described (see Fig. 5.13, Table 5.6) [94, 96 100, 102, 103]. In CML CP and AP, mutations are more abundant in the setting of imatinib resistance (40 59%) than in cases with intolerance to the drug

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Table 5.6: Cellular IC50 values for Bcr Abl gene mutations associated with resistance to imatinib, nilotinib and dasatinib. IC50 values indicate inhibitor concentrations needed for half maximal suppression of proliferation in cell lines expressing Bcr Abl harboring specific mutations [11, 29, 39, 93, 120, 211, 251, 252, 285, 288, 291, 293, 294]. Imatinib: sensitive (G1.2 mM), intermediate (1.2 2.5 mM), resistant (H2.5 mM). Nilotinib: sensitive (G150 nM), intermediate (150 750 nM), resistant (H750 nM). Dasatinib: sensitive (G6 nM), intermediate (10 60 nM), resistant (H60 nM) Imatinib IC50 [mM] Wild type P-loop M244V L248V G250A G250E Q252H Y253F Y253H E255K E255V C-helix D276G SH3-contact V299L F311L T315A T315I T315S F317L F317V SH2-contact D325N S348L M351T E355G F359C F359V A-loop L387F L387M H396P H396R

Nilotinib Factor IC50 wt

0.4

Dasatinib

IC50 [nM]

Factor IC50 wt

25

IC50 [nM] 1

2.3 1.5 1.3 3.9 1.2 9 H10 10 H10

5.8 3.8 3.3 7.5 3 22.5 H25 25 H25

67 102 65 145 67 125 700 566 681

2.7 4.2 2.6 5.8 2.7 5 28 23 27

1.3 NR NR 1.8 3.4 1.4 1.3 5.6 11

1.5

3.8

69

2.8

NR

0.54 1.3 0.97 H10 3.8 1.5 0.5

1.35 3.25 2.43 H25 9.5 3.8 1.3

NR 23 61 H10,000 NR 80 NR

1.5 0.7 1.3 2.3 1.2 1.2

3.8 1.4 3.25 5.75 3 3

25 26 33 47 291 161

1 1 1.3 1.9 12 6.4

NR NR 1.1 NR NR 2.2

1.1 1 2.5 1.75

2.8 2.5 6.25 4.4

39 49 41 41

1.6 2 1.6 1.6

NR 2 0.6 1.3

(8 15%) [104 108]. However, in primary resistant CML CP, Bcr Abl mutations are less common and were found in 24% [108, 109], whereas the majority of patients with CML BC and Phþ ALL and imatinib resistance display resistance mutations [110, 111]. All mutations identified in imatinib resistant patients so far are located within the Abl kinase domain. They lead to structural changes so that imatinib is no longer able to displace ATP and preserve kinase activity. Imatinib resistance mutations can be divided into two distinct groups (see also Fig. 5.13, Table 5.6):

Factor IC50 wt

1 2.4 H400 3.2

1.

2.

18 1.3 125 H5,000 NR 7.4 53

1.3

1.8 3.4 1.4 1.3 5.6 11

18 1.3 125 H5,000 7.4 53

1.1

2.2

2 1 1.3

Imatinib-contact positions such as Y253, T315 and F317. This class of mutations affects amino acids, which are directly involved in binding of the drug. Mutations at these positions thus directly impede drug binding. Mutations that destabilize the inactive conformation of Bcr Abl. These may include exchanges at positions located within the activation loop, such as H396 and M388, or mutations of SH2-contact positions, such as S348 or M351. This second class of mutations probably shifts the equilibrium of

Chap. 5

Chronic Myeloid Leukemia (CML)

inactive to active state of Bcr Abl kinase towards the active state. Since imatinib can only bind to an inactivated Abl kinase domain with the activation loop in a closed conformation [95], access of imatinib to the ATP-binding site is impaired [96, 100, 112]. Mutations directly affecting imatinib binding typically lead to strong imatinib resistance. Examples are T315I, Y253H and E255V [94, 96]. In contrast, mutations, which stabilize the inactive conformation such as H396P, often only lead to moderate resistance [96, 100, 112]. Thus, the type of mutation affects therapeutic management in case of imatinib resistance. Increasing the dose of imatinib may be sufficient to block moderately resistant Bcr Abl mutants. However, in the event of a strong imatinib resistance mutation, increasing the dose will not achieve plasma concentrations that are sufficient to effectively block Bcr Abl kinase activity. Table 5.6 shows the degree of imatinib resistance for frequently observed mutations. Resistance mutations can also be categorized based upon the affected region of the kinase domain which is affected by these mutations. Hot spots not only include single positions such as the gatekeeper position T315, which holds an interaction with imatinib and therefore is vulnerable to an exchange, but also functional domains, such as the activation loop (A-loop, blue in Fig. 5.13), the ATP phosphate-binding loop (P-loop, magenta in Fig. 5.13), and C-Helix (green in Fig. 5.13). However, the position of an exchange does not allow to estimate the degree of resistance. As an example, the class of P-loop mutations includes moderate (G250A, Q252H, E255D) as well as strong imatinib resistance mutations (G250E, Y253H, E255V) [113]. Meanwhile, mutations conferring clinical resistance to therapeutically used kinase inhibitors were also identified in several other target kinases in various malignant diseases. Imatinib resistance mutations were identified in FIP1L1-PDGFRalpha in patients with hypereosinophilic syndrome [114, 115], and in cKit in patients with gastrointestinal stromal tumors (GIST) [116, 117]. In addition, a resistance mutation in the kinase domain of FLT3-ITD in a patient with acute myeloid leukemia (AML) treated with the kinase inhibitor PKC412 has been described [118]. Similarly, in patients with non small cell lung cancer (NSCL) treated with the kinase inhibitor gefitinib, an exchange of threonine at position 790 to methionine in the epidermal growth factor receptor (EGFR) was described [119, 120]. This mutation together with the imatinib resistant mutations cKit/T670I and FIP1L1-PDFGRalpha/T674I are homologous to the position T315 in the Abl kinase domain. Thus, mutations in kinase domains seem to be a

133

general mechanism of resistance against tyrosine kinase inhibitors. Gene Ampliﬁcation and Protein Overexpression

Bcr Abl gene amplification and protein over expression as cause of imatinib resistance were both identified in vitro [91 93] as well as in CML patient samples [94, 121]. Amplification of a kinase inhibitor target could also be demonstrated in imatinib resistant GIST patients [122]. Amplification of the target gene results in a shift of the inhibitor/target ratio towards a surplus of the target protein. Consequently, the amount of inhibitor available within the cell is not sufficient to effectively block all target proteins. In imatinib resistant CML, Bcr Abl overexpression allows for residual Bcr Abl activity even in the presence of imatinib, which enables the leukemic clone to survive, and can be found in 10 15% of cases [94, 103]. Bcr–Abl-independent Resistance Phamacokinetic Mechanisms

Alterations in the pharmacokinetic of imatinib also can be a cause for imatinib resistance. The intracellular concentration of imatinib is determined by membrane-bound, active import and export pumps and by its binding to plasma proteins. It could be shown that imatinib is bound to plasma proteins such as a-acid-glycoprotein (a-1-GP) [123]. It has been proposed that increased plasma a-1-GP levels might reduce the plasma concentration of free and unbound imatinib available for inhibition of Bcr Abl [124]. Indeed, the tumor burdon in a mouse model [124] and the CML disease stage in patients [125] correlated with plasma a-1-GP levels, and a priori elevated a1-GP levels led to a less rapid response to imatinib in CML patients [125]. However, elevated a-1-GP plasma levels in patients were reversible in the course of treatment [125], and a-1-GP did not alter the efficacy of the drug in vitro [125, 126]. Therefore, it is currently unclear whether plasma proteins such as a-1-GP contribute to imatinib resistance. Imatinib is a substrate of the multidrugresistanceassociated membrane transporter MDR-1 and thus can be actively pumped out of the cell [127, 128]. Over expression of MDR-1 was found in imatinib resistant cell lines, and by inhibition of MDR-1 imatinib resistance was partially reverted [93]. Increased expression of MDR-1 was also demonstrated on progenitor cells of patients with CML in myeloid blast crisis when compared to healthy controls. However, the level of expression did not predict response to imatinib [129]. Therefore, multidrugresistance-associated membrane transporters such as MDR1 may be of potential importance for the survival of CML cells in the presence of imatinib.

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Table 5.7: FDA/EMEA approved drugs: pharmacological mediators of resistance

Imatinib Nilotinib Dasatinib

P-Gp MDR1 ABCB1 þ þ

substrates

BCRP-1 ABCG2

Oct-1

AAG

Reference

þ þ

þ

þ ?

?

?

?

[132, 267] [132, 268, 269] [270]

for

Recently, a membrane transporter was identified (human organic cation transporter-1; hOCT-1), which is involved in the active transport of imatinib into the cell (import) [130]. Two studies demonstrated a correlation of hOCT-1 expression and response to imatinib [131, 132]. Also, it has been demonstrated that low hOCT-1 activity can be overcome by increasing the imatinib dose [132]. In addition, pharmacokinetic mechanisms may be important for a cell in the course of acquiring secondary resistance due to mutations or secondary genetic alterations in the presence of imatinib. Currently there is insufficient data available, as to whether the 2nd generation TKI dasatinib and nilotinib are substrates for these pharmacological mediators of resistance (Table 5.7). Secondary Genetic Alterations, Alternative Pathways

In a subset of imatinib resistant patients, it can be shown that Bcr Abl kinase activity still is effectively blocked by the drug. This indicates that the leukemia has become at least partially Bcr Abl independent by secondary genetic hits. Secondary genetic alterations can lead to a clonal selection of cytogentically abnormal imatinib resistant leukemic clones under imatinib treatment. Such a clonal evolution in CML is frequently associated with a progression of the disease [101, 133, 134]. In imatinib resistant CML, a cytogenetically detectable clonal evolution can be observed in 30 50% of cases [101, 134, 135]. However, the molecular mechanisms by which specific chromosomal alterations lead to progression of the disease or resistance are not understood. Two frequently described cytogenetic abnormalities are the loss of chromosome 17p, leading to inactivation of p53 [136, 137], and trisomy 8, resulting in amplification and overexpression of c-Myc [138]. Both events may contribute to disease progression and imatinib resistance [138 140]. In one imatinib resistant CML patient an inversion of chromosome 11 (inv(11)(p15q22)) led to the expression of a NUP98/DDX10 fusion protein [141]. Interestingly, NUP98/DDX10 is also associated with AML and myelodysplastic syndrome and thus may play a functional important role for disease progression and

imatinib resistance [142]. In addition, the finding of LYN kinase overexpression in bone marrow samples derived from imatinib resistant CML patients suggests that activation of Src-family kinases may bypass the dependence of the leukemic cell on active Bcr Abl, and thus contribute to imatinib-resistance [143]. Nevertheless, no clear-cut genes could be identified yet, which are directly responsible for imatinib resistance in a significant number of cases. Different molecular mechanisms of imatinib resistance can act together in a synergistic manner [113, 144, 145]. In single CML patients with imatinib resistance, Bcr Abl mutations were identified in combination with clonal cytogenetic evolution [144], or in combination with Bcr Abl gene amplification [145]. Compliance

Patient compliance is an issue with drugs that have to be administered continuously. Over the years compliance usually declines. Poor compliance in the case of imatinib may lead to resistance because suboptimal drug concentrations may facilitate the selection of resistant leukemia clones. Patients with a good response particularly need to be reminded that suboptimal dosage or interruptions can lead to relapse or development of resistance. In case of a suboptimal response or treatment failure, a lack of compliance to the drug has to be excluded prior to change of therapy.

Management of Suboptimal Response and Treatment Failure In chronic phase CML, suboptimal response or treatment failure at 3, 6 or 12 months of imatinib indicates inferior progression-free and overall survival [68, 70, 80, 81] and thus should trigger a change of treatment. Increasing the imatinib dose from 400 mg to 600 or 800 mg daily can improve the quality of response and can induce sustained responses in a subset of patients [146 148]. Patients harboring Bcr Abl resistance mutations may also benefit from increasing the imatinib dose. However, only few patients with the frequently observed P-loop mutations responded to a dose increase, and patients exhibiting the abundant T315I exchange will not profit from a higher imatinib dose [149]. A suboptimal response to imatinib or treatment failure should trigger a Bcr Abl mutation analysis. Increasing the imatinib dose may be considered in case of a weak resistance mutation. In the presence of a strong imatinib resistance mutation, a switch to dasatinib or nilotinib should be initiated, provided that activity against the individual mutation is documented. The extent of resistance to imatinib, nilotinib and dasatinib

Chap. 5

Chronic Myeloid Leukemia (CML)

is known from in vitro studies for the majority of mutations and can guide selection of the appropriate kinase inhibitor (see Table 5.6) but however, does not allow prediction of actual response to the selected compound in any case. If an increased imatinib dose of 600 800 mg daily turns out to be ineffective, a switch to one of the approved second generation Abl kinase inhibitors (dasatinib, nilotinib; see Sect. 5.8.4) should be considered [72, 73]. Alternative options include allogeneic hematopoietic stem cell transplantation (see Sect. 5.8.6) or investigational treatments (see Sect. 5.8.5.1) [68, 72, 73, 81]. In case of progression to accelerated or blast phase, responses with nilotinib or dasatinib are generally short lived and thus allogeneic hematopoietic stem cell transplantation should be instituted, if feasible.

5.8.6 Novel Abl Kinase Inhibitors 5.8.6.1 Preclinical Data The finding of clinical resistance to imatinib triggered the development of novel Abl kinase inhibitors. Preclinical models revealed a higher inhibitory activity of these drugs against wild-type Bcr Abl in cell lines and animal models, and also demonstrated activity of these novel compounds against many of the known imatinib resistant Bcr Abl exchanges. Examples include nilotinib (AMN107, Tasigna) [150], and dasatinib (BMS354825, Sprycel) [151], which both already have been approved for the treatment of imatinib resistant or intolerant CML (see below), and other compounds, such as Bosutinib (SKI-606) [152] and INNO-406 (NS-187) [153], which are currently investigated in clinical trials. The abundant T315I gatekeeper exchange displays full and complete resistance against imatinib, nilotinib, dasatinib, bosutinib and INNO-406 (see also Table 5.6). It was therefore particularly demanding to identify compounds demonstrating inhibitory activity against Bcr Abl/T315I. Compounds with documented T315I activity in vitro include MK-0457 (VX-680) [112, 154], ON012380 [155], SGX-70430 [156], TG101223 [157], VE-465 [158], AP24534 [159], PHA-739358 [160], AS703569 [161], XL228 [162] and DCC-2036 [163]. When novel ATP competitor-type Abl kinase inhibitors entered the clinic, it was not known whether these compounds again would be vulnerable to secondary mutations in the Bcr Abl kinase domain mediating resistance. However, in vitro methods were developed that allow prediction of specific profiles of Bcr Abl resistance mutations for different Abl inhibitors [164]. Using these strategies, specific in vitro resistance profiles were generated for

135

imatinib, nilotinib, and dasatinib. For imatinib, these in vitro profiles match to Bcr Abl resistance mutations detected in clinical isolates [165, 166]. Nilotinib and dasatinib in vitro generated resistant clones at a lower frequency, and displayed narrowed, albeit overlapping resistance profiles when compared to imatinib [166 169]. More recently, resistance mutations in patients treated with nilotinib and dasatinib were reported, and it became evident that the already known in vitro profiles very well recovered Bcr Abl resistance mutations emerging in the clinic in patients developing resistance to nilotinib or dasatinib [105, 165, 166, 170]. As stated above, the activity profiles of imatinib, nilotinib and dasatinib are known from in vitro studies for the majority of mutations. Both specific resistance profiles, and activity profiles for each inhibitor derived from in vitro studies can guide selection of the appropriate kinase inhibitor in the clinic with respect to sequential inhibitor treatment and also can be used to generate combination strategies.

5.8.6.2 Approved 2nd Generation Kinase Inhibitors in Imatinib Resistant or Intolerant CML The increased inhibitory activity of nilotinib and dasatinib against wild-type and mutant forms of Bcr Abl in preclinical models gave reason to clinical trials examining the activity of these two compounds in imatinib resistant and intolerant CML. Mature phase 2 data are available for both nilotinib and dasatinib and demonstrate clinical activity of both drugs in imatinib resistant and intolerant CML [171 181]. Dasatinib is approved for the treatment of CML in all phases and Phþ acute lymphoblastic leukemia (ALL) and resistance or intolerance to imatinib. Nilotinib has been approved for imatinib resistant and intolerant CML in chronic or accelerated phase.

Dasatinib Dasatinib Chronic Phase

A phase 2 clinical trial examined dasatinib in chronic phase CML with imatinib resistance or intolerance. At a dose of 70 mg two times daily (BID), high rates of hematologic and cytogenetic responses were observed (CHR 91%; CCyR imatinib resistance 44%, intolerance 78%) [172]. Survival at 15 months was 96%, and progression-free survival (PFS) was 90%. Forty-nine and forty-eight per cent of the patients experienced grade 3/4 neutropenia and thrombocytopenia, respectively. Nonhematological toxicities included dyspnea (5%) and pleural effusions (6%). In a randomized phase 2 study, dasatinib 70 mg BID was superior to an increased imatinib dose of 400 mg BID in patients with imatinib resistant

Study title

START-C [172]

START-R [252]

START-A [174]

START-B [175] START-L [175]

Disease phase

CML CP

CML CP

CML AP

CML BC Myeloid Lymphoid 12 (minimal) 12 (minimal)

14 (median)

24 (minimal)

15 (median)

Follow-up (months)

109 48

174

150

387

Number of patients

27 29

D 93 res. 45

I 82 intol. 46 33 52

intol. 80 I 33 intol. 39

CCyR

26 46

res. 40 D 44 res. 31

intol. 75 I 18 intol. 39

D 86 66 (@12 mos)

96 (@15 mos)

PFS

res. 52 D 53 intol. 39

MCyR

CHR 91

Survival (%)

Response rate (%)

6.7 mos 3 mos

I 65

11.8 mos 5.3 mos

82 (@12 mos)

NR

90 (@15 mos)

OS

Table 5.8: Efficacy data of clinical phase 2 trials with dasatinib in imatinib resistant or intolerant CML [172, 174, 175, 252]. All trials are single-arm except START-R, which randomized dasatinib 70 mg two times daily in a 2:1 ratio to imatinib 400 mg two times daily. Res. (resistant), intol. (intolerant), PFS (progression-free survival), OS (overall survival), NR (not reported), mos (months)

136 N. von Bubnoff et al.

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137

chronic phase CML (PFS at 2 years was 86% versus 65%) [173]. Grade 3/4 neutropenia/thrombocytopenia more frequently occurred with dasatinib. A phase 3 trial demonstrated comparable efficacy and better tolerability of dasatinib 100 mg once daily (OD) compared to 70 mg BID [182]. Thus, the recommended dasatinib dose in chronic phase CML is 100 mg OD. Data from clinical trials on the efficacy of dasatinib in chronic phase CML are summarized in Table 5.8. Dasatinib Accelerated and Blast Phase

In imatinib resistant or intolerant accelerated phase CML, the rates for CHR and CCyR with dasatinib 70 mg BID at 14 months were 45% and 32% [174], survival at 12 months was 82%, and PFS 66%. In a phase 3 trial, pleural effusions were less common with dasatinib at 140 mg OD verses 70 mg BID, so that the recommended dose in imatinib resistant or intolerant accelerated phase CML is 140 mg OD [183]. Hematologic and cytogenetic responses with dasatinib 70 mg BID were also seen in blast crisis CML with imatinib resistance or intolerance but however, PFS was below 6 months and survival less than one year [175]. Clinical data on the efficacy of dasatinib in advanced phase CML are summarized in Table 5.8. Nilotinib

temia (16%). Nilotinib efficacy data in chronic phase CML are summarized in Table 5.9. Nilotinib Accelerated and Blast Phase

Patients with accelerated phase CML and imatinib resistance or intolerance in a phase 2 trial displayed a CHR in 31% and CCyR in 20% of cases at 6 months nilotinib 400 mg BID [179, 180]. PFS at 1 year was 57%, survival at 2 years 67%. Hematologic and cytogenetic responses to nilotinib 400 mg BID were also seen in blast crisis CML after imatinib failure [181]. In this trial, survival at 12 months was 42%. Nilotinib efficacy data in advanced phase CML are summarized in Table 5.9. In summary, both dasatinib and nilotinib induce high rates of hematologic and cytogenetic responses in chronic phase CML after imatinib failure. Longer follow-up will show whether these responses are durable. In advanced phase CML, responses to dasatinib or nilotinib after imatinib failure are short-lived but may open the opportunity to subject these patients to allogeneic hematopoietic stem cell transplantation [72, 73, 79, 184 187]. Both compounds are active against frequently observed imatinib resistant Bcr Abl mutations but however do not display activity against Bcr Abl/T315I (Table 5.6). Selection of the appropriate 2nd generation TKI should take into account the sensitivity of existing mutations (if present) (see Table 5.6) and the comorbidity profile of the patient.

Nilotinib Chronic Phase

In chronic phase CML resistant or intolerant to imatinib, nilotinib 400 mg BID in a phase 2 trial resulted in high rates of hematologic and cytogenetic responses (CHR 94%; CCyR imatinib resistance 41%; intolerance 51%) [177, 178]. PFS at 2 years was 64%, survival was 88%. Grade 3/4 neutropenia and thrombocytopenia each were evident in 31% of patients, and grade 3/4 non-hematologic toxicity mainly consisted of increases of bilirubin (8%), lipase (17%), hyperglycemia (12%), and hypophospha-

5.8.7 Outlook – Promising Strategies in Current and Future Clinical Trials 5.8.7.1 Novel Compounds in Clinical Trials In addition to the already approved compounds imatinib, dasatinib and nilotinib, other Abl kinase inhibitors currently are investigated in clinical trials. These include INNO-406 and Bosutinib (SKI-606). Both drugs display

Table 5.9: Efficacy data of clinical phase 2 trials with nilotinib in imatinib resistant or intolerant CML [177, 179 181, 252]. Res. Resistant; intol. intolerant; PFS progression free survival; OS overall survival; NR not reported; mos months Disease phase

Follow-up (months)

Number of patients

Response rate (%) CHR

MCyR

CCyR

PFS (%)

OS (%)

res. 56 res. 30

res. 41 res. 18

64 (@24 mos)

88 (@24 mos)

57% (@12 mos)

67 (@24 mos)

NR NR NR

42% (@12 mos) NR NR

CML CP [177, 252]

19 (minimal)

321

94

CML AP [179, 180]

6 (minimal)

137

31

CML BC [181] Myeloid Lymphoid

6 (minimal) 105 31

11 13

38 48

intol. 65 intol. 41

29 32

intol. 51 intol. 30

p38 [4 mM] FLT3, KIT Abl [1–170 nM] Src [0.8 nM] KIT [50 nM]

Pyrido-pyrimidineDerivate

Quniazolone Derivate

4-Anilino-3-quinlinecarbonitrile

Synthetic Myriocin Analogon

3-Benzamid-Derivate

BIRB-796 Doramapimod

SU-11248

PD180970 PD173955

MLN-518

SKI-606 (Bosutinib)

FTY720 (Fingolimod)

NS-187 (INNO 406)

Abl [26 nM] Lyn [11 nM] KIT [51 nM]

KIT [170 nM] PDGFR [200 nM] Flt3 [200 nM] Fgr [174 pM] Lyn [850 pM], Hck Abl [1–2.4 nM] PP2a-Aktivator [5–18 mM] Immunmodulator (hold up CD4trafficking)

[199, 273–275]

[112, 154, 190, 276–278]

In vitro

Phase I/II (CML/ Phþ-ALL/solid tumors)

Apoptosis is markedly enhanced in tumor cell lines with p53 deletion All other mutations

[154, 279–282]

In vitro

Phase I (AML)

(Abl) T315I (KIT) T607I, D816V –

Apoptosis is enhanced with coadministration of CyA (P-gp-inhibitor) or ABT-737 (Bcl2inhibitor) (Abl) T315I

(Abl) T315I

(Abl) E255K, E255V, Q252H, M351T, Q253H, Y253F, M244V, G250E, F317L, E355G, F359V, H396P, F486S (KIT) V560G

–

[152, 284–287]

[288]

[153, 289, 291–294]

Phase I/II (CML)

Phase III (MS, renal transplant)

In vitro murine

[283]

[154]

–

Phase II/III (RA, M. Crohn) In vitro

[154]

[199, 253, 271, 272]

T315I, F317C/V/L/I

Reference

In vitro murine

Development phase (Tumor cell type)

Mutations with known resistance against the substance T315I, F317C/V/L

(Abl) Y253F, E255K, D276G

(KIT) V559D, T670I, N822K (KIT) (Abl) Q252H, Y253F, E255K, M351T, F359V, H396P, Q252H (KIT) N822K, V559D (KIT) N822K, V559D, D816V

(Abl) T315I

Aurora-Kinasen A-C, Flt3, JAK2 Abl [10–30 nM]

Src, Abl [300 pM–5.4 nM]

VX-680 (MK-0457)

Pyridopyrimidine

PD166326 SKI DV-M016

Mutations with known sensitivity for the substance (KIT) D816Y, D816V, D816F (Abl) Q252H, Y235F/H, E255K, M351T, H369P, L248R, G250E (Abl) E255K, Y253H, E255K/V, L248 R, G250E, M351T, H396P, A269V (Abl) T315I, Q252H, Y253F, E255K, M351T, F359V, H396P, V299L

ATP-based 2,6,9-trisubstituted Purine

AP23848 AP23464

[IC50] for various kinases

KIT [3–20 nM] Src, Abl [10–110 nM]

Chemical group

Substances

Table 5.10: Novel tyrosine kinase inhibitors and other substances with (preclinical) efficacy against T315I

138 N. von Bubnoff et al.

HDAC-inhibitor

LBH589

Farensyltransferase Modulator subzellul€arer Ras-Lokalisation P-gp-Hemmer

Abl [50–200 ng]

Cephalotaxine esther

Homoharring-tonine

SCH66336 (Lonafarnib)

Abl [9 nM] PDGFRa, Lyn, Fyn

ON012380

Table 5.10: (Continued)

(Abl) T315I

(Abl) T315I, F317L, H396R, M351T, H396P, Y253H, M244V, E355G, F359V, G250E, Y253F, F311 L, E255V, Q252H, L387M, E255K (Abl) T315I Synergism with imatinib in vitro Additive effect with nilotinib in vitro Synergism with imatinib reduction ofquiescentCD34þ CML stem cells, and (re)sensitization for imatinib

Not found Synergism with imatinib

[295, 296]

[297–303] [201, 202, 304]

[197, 305–308]

In vitro murine

Phase II Phase I (Hemat. N)

Phase I–III (CML, solid tumors, MDS, CMML)

Chap. 5 Chronic Myeloid Leukemia (CML) 139

140

activity in CML and Phþ ALL after failure of imatinib or other Abl TKIs [177, 188, 189]. However, neither imatinib, nilotinib and dasatinib, nor INNO-406 and bosutinib exhibit T315I activity. Patients with this mutation should be treated in clinical trials investigating compounds with documented T315I activity and should be submitted to allogeneic stem cell transplantation, if feasible. Clinical activity against T315I positive disease was reported for the Aurora kinase inhibitors MK-0457 [190], PHA-739358 [191], AS703569 [161] and XL228 [182] and for the protein translation inhibitor Homoharringtonin [192, 193]. A number of novel compounds are under preclinical development [155 163, 194 198], including substances with T315I activity [155 159, 163] (see Table 5.10). Combinations of different Abl kinase inhibitors might constitute a promising approach in advanced and imatinib resistant CML, since combinations might narrow the profile of possible resistance mutations [165, 167, 199]. This situation is reminiscent of antibiotic or antiretroviral combination treatment. In addition to ATP-competitors, preclinical development includes allosteric Bcr Abl inhibitors such as ON012380 or GNF-2 [155, 200], which are thought to act as substrate- rather than as ATP-competitors, histone deacetylase (HDAC) inhibitors such as LBH589 [201, 202], and farnesyltransferase-inhibitors (FTI), which block Ras activation [194]. In phase 1 clinical trials, the FTIs lonarfanib (SCH66336) and tipifarnib (R115777) induced hematologic and cytogenetic responses in patients with CML [195 197], and combination treatment with imatinib induced responses in imatinib resistant CML [198].

5.8.7.2 Second Generation Abl Kinase Inhibitors for 1st Line Treatment of Chronic Phase CML Ongoing clinical trials examine dasatinib (100 mg OD or 50 mg BID) and nilotinib (400 mg BID) as first-line treatment of chronic phase CML and in comparison to historic controls with imatinib 400 800 mg daily suggest more rapid cytogenetic responses with dasatinib or nilotinib [80, 203, 204]. A phase 3 clinical trial currently compares bosutinib (SKI-606) to imatinib 400 and 600 mg first-line in patients with chronic phase CML. It remains to be shown whether the use of newer Abl kinase inhibitors in early chronic phase will further improve long term outcome.

5.8.7.3 Can Tyrosine Kinase Inhibitors Cure CML? At present, tyrosine kinase inhibitor-based treatment in CML is considered non curative. Although presence of

N. von Bubnoff et al.

CMR indicates low disease activity, a CMR does not accord with eradication of the disease or cure. Results of several trials indicate that stopping imatinib in patients with sustained CMR will lead to a molecular relapse in approximately 50% of cases [205 211]. It will be interesting to see whether some of the remaining patients will be free of molecular, cytogenetic or hematologic relapse after a longer follow-up. Also, it is presently not clear whether in patients remaining free of measurable disease, an equilibrium of residual CML and host mechanisms is established or whether residual leukemic stem cells can be eradicated by TKIs in some patients. A mathematical model suggested that the CML stem cell compartment during TKI-based treatment might slowly decrease over time [212]. However, a different mathematical approach concluded that TKIs in CML suppress the production of differentiated leukemic cells, but do not deplete leukemic stem cells [213].

5.8.7.4 Immunotherapy of CML Several years ago, imatinib has displaced Interferon-alpha (IFN) as first-line treatment of chronic phase CML [65]. Currently active and future clinical trials will evaluate whether concurrent treatment with imatinib and IFN will increase response rates and whether maintenance treatment with IFN might allow control of residual disease. These trials will be of particular interest since it was recently shown that IFN can activate dormant hematopoietic stem cells [214], thereby possibly making them more susceptible to inhibition by imatinib. A different immunologic strategy that might improve response to TKIbased therapy and also might allow control of residual disease makes use of the disease-specific antigenic repertoire present in CML cells. Preliminary studies with vaccines that target the Bcr Abl-derived p210 fusion protein suggest that vaccination in combination with imatinib may further reduce residual disease in CML [215 217]. A recent report demonstrated that in addition to Bcr Abl itself, Bcr Abl-regulated antigens can elicit Tcell responses and thus might be employed for the development of immunotherapeutic approaches for the treatment of CML patients with residual disease following therapy with Bcr Abl kinase inhibitors [218].

5.8.8 Allogeneic Stem Cell Transplantation According to the state of knowledge, only allogeneic stem cell transplantation (SCT) can cure CML [219 222]. In early chronic phase, 5-year OS after allogeneic SCT are between 25% and 70%, depending on the presence of

Chap. 5

Chronic Myeloid Leukemia (CML)

individual risk factors [72, 223 225]. On the other hand, 6-year OS with imatinib in early chronic phase is 88% [67]. There are no trials comparing allogeneic SCT to first-line imatinib in chronic phase CML. Taking into account transplantation-associated morbidity and mortality on the one side and efficacy and tolerability of imatinib on the other side, current guidelines consistently recommend imatinib for first-line treatment of chronic phase CML [72, 73, 79]. Allogeneic SCT should be considered in patients failing imatinib and newer Abl kinase inhibitors (dasatinib or nilotinib) and patients progressing to accelerated phase or blast crisis.

5.8.9 Prognostic Scores in CML Response to imatinib determines PFS and OS (see Sect. 5.8.4). In addition, pre-treatment factors have been demonstrated to affect PFS and also OS. These include additional chromosomal aberrations in Phþ cells (clonal evolution), Sokal score (which integrates age, spleen size, platelet count and peripheral blood blast count [226]). Both factors have been demonstrated to affect PFS and OS in patients with CML treated with imatinib [65, 81, 134, 135, 227 229].

5.9 CML Variants In general, the absence of cytogenetic or molecular evidence for Bcr Abl rearrangement or transcriptional or translational products thereof virtually eliminates classic CML as a consideration in the differential diagnosis of conditions coinciding with neutrophilia (inflammatory diseases, infections, paraneoplastic leukocytosis). The following entities are not only extremely rare, but it is also highly questionable whether they truly exist or are merely misdiagnosed forms of classic CML.

5.9.1 Philadelphia Chromosome Negative CML (Formerly Atypical CML) The presence of the Bcr Abl fusion gene and/or its protein product is necessary to establish the diagnosis of CML. In fact, many hematologists question whether the disease entity Philadelphia chromosome negative CML truly exists, or whether the presence of a translocation t(9;22) is merely masked or not detected due to alternative breakpoints or additional mutations, e.g., leading to changes in the PCR-primer annealing sites. The following data are in accordance with this line of argumentation:

141

*

*

Approximately 50% of patients with clinical features of CML, but lacking the cytogenetic Philadelphia chromosome by cytogenetic analysis have complex genetic aberrations masking the t(9;22) translocation [8]. Another group of patients thought to have philadelphia chromosome negative CML were Ph-negative by karyotype, but had evidence of Bcr Abl gene fusion by metaphase or interphase FISH or RT-PCR [8].

Therefore, the terminus Philadelphia chromosome negative CML should be avoided, unless the characteristic clinical features of classic CML are present and a masked Ph-chromosome or Bcr Abl rearrangement are not found. However, many cases of Philadelphia chromosome negative CML seem to fulfill the criteria for CMML or other MDS subtypes. In fact, a good part of patients diagnosed with Ph-negative CML, have probably been misdiagnosed, and should be grouped within the myelodysplastic syndromes. In accordance with this hypothesis, patients respond poorly to CML therapy, which correlates with short survival [230]. Therefore, the world health organization (WHO) created the category of myeloproliferative/myelodysplastic disorders for those patients with granulocytic dysplasia lacking the Bcr Abl fusion. Many of these patients (30%) have an additional chromosome 8, another feature typical for myeloproliferative/myelodysplastic disorders. Thus, in the new WHO classification Bcr Abl negative atypical CML no longer belongs to the myeloproliferative neoplasms, but instead constitutes an independent entity of the myelodysplastic/myeloproliferative neoplasms. According to the WHO, Bcr Abl negative CML is characterized both by myeloproliferative and myelodysplastic features. Affected patients are usually older than CML patients and often present with very high leukocyte counts with dysgranulopoiesis. This group of diseases is often associated with thrombocytopenia, lack of basophilia and disease progression is associated with leukocytosis, organomegaly, extramedullary infiltrates and bone marrow failure. The prognosis is worse compared to Bcr Abl þ CML with a medium survival of only 24 months.

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) For cases of thrombocythemia with the Philadelphia chromosome and/or Bcr Abl rearrangement [30, 231,

142

232] the terminus Bcr Abl positive essential thrombocythemia has been used. In reality, these entities most likely reflect CML with an initial thrombocythemic pre-phase (for more details see Sects. 2.15.1 and 2.15.2 in the chapter on Essential Thrombocythemia). The same probably holds true for Bcr Abl positive polycythemia vera [233 235] and Bcr Abl positive idiopathic myelofibrosis [236, 237], which are likely to represent CML with a polycythemic prephase and CML with marrow fibrosis, respectively. In line with this theory, the overwhelming majority of patients with Phþ-CML, so far investigated for the JAK2-V617F mutation, have been negative [238, 239]. The same holds true for those CML patients with a thrombocythemic prophase [239]. Recently, a single case of coexisting JAK2-V617F mutation in PhþCML evolving to myelofibrosis during imatinib treatment has been reported [240]. In this case, however, the authors come to the conclusion that these mutations do not coexist within one stem cell clone, i.e., within the same cells. Rather, their evidence argues in favor of the existence of two independent stem cell clones within this patient. CML with thrombocythemic onset has been proposed to be a CML subtype with distinct hematological (marked platelet elevation, extreme megakaryocytic hyperplasia, low levels of leukocytosis) molecular and clinical features [241].

5.9.3 Other Ph+ Entities Furthermore, 20 30% of adult ALL and 5 10% of childhood ALL are Ph positive. 1% of adult AML has also been reported to be Ph positive [242]. These patients are divided into those with prior CML presenting in blast phase and those with de novo Ph+-acute leukemia. The Philadelphia chromosome can rarely be detected in tumor cells (plasma cells or lymphocytes) of other malignancies, including multiple myeloma and B-NHL [243, 244]. In these cases, it is thought, that dedifferentiation of the original CML clone into one that supports lymphoid proliferation occurs.

N. von Bubnoff et al.

CML demonstrate an alternative breakpoint in the Bcrregion resulting in a larger 230 kDa variant fusion protein compared to the 210 kDa or 185/190 kDa fusion proteins in classic CML [245]. As the Philadelphia chromosome and/or Bcr Abl rearrangement has been found in several patients diagnosed with chronic neutrophilic leukemia [246, 247], one might speculate, whether this disease entity simply represents CML with atypical breakpoints and an indolent clinical course, which is in accordance with a report by Verstovsek [248].

Abbreviations Abl

AP BC Bcr Abl CCyR CHR CML CMR CP IFN MCyR MMR PCyR PDGFR PFS Phþ ALL qRT PCR

Abelson kinase (chromosome 9), component of the Philadelphia translocation t(9;22) and thera peutic target of imatinib and other tyrosine kinase inhibitors Accelerated phase CML Blast crisis CML Fusion protein translated from the Philadelphia translocation t(9;22) Complete cytogenetic response (definition see Table 5.4) Complete hematologic response (definition see Table 5.4) Chronic myelogenous leukemia Complete molecular response (definition see Table 5.4) Chronic phase CML Interferon alpha Major cytogenetic response (definition see Table 5.4) Major molecular response (definition see Table 5.4) Partial cytogenetic response (definition see Table 5.4) Platelet derived growth factor receptor Progression free survival Philadelphia chromosome positive acute lymphoblastic leukemia Quantitative real time polymerase chain reaction

References 5.9.4 CML with Atypical Breakpoints and an Indolent Clinical Course (Formerly Neutrophilic CML) Neutrophilic CML is a rare variant of Bcr Abl positive CML, in which the elevated WBC is comprised mainly of mature neutrophils. Lack of myeloid immaturity, splenomagelie or basophilia, as well as normal ALP-levels further contribute to the differentiation from typical CML. Importantly, most patients with neutrophilic

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Chap. 5

Chronic Myeloid Leukemia (CML)

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6

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

Contents 6.1 Introduction ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 154 6.2 Epidemiology of MDS :::::::::::::::::::::::::::::::::::::::::::::: 155 6.3 Pathophysiology and Molecular Biology of MDS :::: 156 6.3.1 Disturbances in Apoptosis :::::::::::::::::::::::::::::: 156 6.3.2 Alterations in T Cell Functions and Cytokines :::::::::::::::::::::::::::::::::::::::::::::::: 158 6.3.3 Microenvironment in MDS::::::::::::::::::::::::::::: 160 6.3.4 The Role of Tumor Suppressor Genes and Oncogenes in MDS Disease Initiation/ Perpetuation::::::::::::::::::::::::::::::::::::::::::::::::::: 160 6.3.4.1 Somatically Acquired Mutations of the AML 1 Gene in MDS::::::::::::: 161 6.3.4.2 Overexpression of EVI 1 in MDS ::::: 161 6.3.4.3 Oncogenic Fusion Products in MDS:::::::::::::::::::::::::::::::::::::::::::: 161 6.3.4.4 Mutation of the Ras Protooncogene in MDS:::::::::::::::::::::::::::::::::::::::::::: 162 6.3.4.5 The Role of Interferon Regulatory Factor 1 (IRF 1) in MDS:::::::::::::::::: 162 6.4 Clinical Features in MDS::::::::::::::::::::::::::::::::::::::::: 162 6.4.1 Infectious Complications in MDS::::::::::::::::::: 162 6.5 Laboratory Features in MDS::::::::::::::::::::::::::::::::::: 166 6.6 Typical Bone Marrow Findings in MDS :::::::::::::::::: 167 6.7 Diagnosis and Classification of MDS ::::::::::::::::::::::: 167 6.8 Prognostic and Predictive Parameters in MDS ::::::: 172 6.8.1 Cytogenetics in MDS ::::::::::::::::::::::::::::::::::::: 172 6.8.1.1 Frequency of Cytogenetic Aberrations in MDS:::::::::::::::::::::::::::::::::::::::::::: 172 6.8.1.2 Clinical and Prognostic Features of Patients with Particular Cytogenetic Aberrations in MDS ::::::::::::::::::::::::: 173 6.8.2 Molecular Factors Associated with Progression of the Disease :::::::::::::::::::::: 174 6.8.3 Prognostic Scoring Systems in MDS::::::::::::::: 175 6.8.4 Other Prognostic Markers in MDS:::::::::::::::::: 178 6.9 Best Supportive Care (BSC) of Patients with MDS ::::::::::::::::::::::::::::::::::::::::::::::: 178 6.9.1 Transfusion of Red Blood Cells and/or Platelets :::::::::::::::::::::::::::::::::::::::::::::: 178 6.9.2 Erythropoietin (EPO) ::::::::::::::::::::::::::::::::::::: 178 6.9.3 G CSF and Combination Treatment of EPO with G CSF ::::::::::::::::::::::::::::::::::::::: 179 6.9.4 Thrombopoietin (TPO) and TPO Mimetics ::::: 180 6.9.4.1 PEG rHuMGDF ::::::::::::::::::::::::::::::: 181 6.9.4.2 Recombinant Human TPO (rHuTPO) :::::::::::::::::::::::::::::::::::::::: 181 6.9.4.3 Romiplostim (AMG531, Nplate)::::: 181

6.9.4.4 Oral TPO Mimetics Eltrombopag and AKR 501 (YM477):::::::::::::::::::: 181 6.9.5 Other Drugs for Palliative Amelioration of Cytopenia :::::::::::::::::::::::::::::::::::::::::::::::::: 182 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 6.9.6.6.1 Deferiprone (Ferriprox)::: 184 6.9.6.6.2 Deferoxamine (Desferal):::::::::::::::::::::: 184 6.9.6.6.3 Deferasirox (Exjade) ::::: 184 6.10 Low-Dose Palliative Chemotherapy in MDS:::::::::::: 185 6.10.1 Low Dose Melphalan ::::::::::::::::::::::::::::::::::: 185 6.10.2 Low Dose Cytosine arabinoside (Ara C)::::::: 185 6.11 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 6.12 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

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Immunosuppressive Treatment in MDS ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.13.1 Treatment with Anti thymocyte Globulin (ATG) :::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.13.2 Immunosuppressive Treatment with Cyclosporin A (CyA):::::::::::::::::::::::::::: 6.13.3 Treatment of MDS Associated Autoimmune Manifestations:::::::::::::::::::::::::::::::::::::::::::::: Targeting Bone Marrow Microenvironment in MDS ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.14.1 Thalidomide::::::::::::::::::::::::::::::::::::::::::::::::: 6.14.2 Lenalidomide (Revlimid):::::::::::::::::::::::::::: 6.14.3 Direct Targeting of TNF a: Infliximab and Ethanercept:::::::::::::::::::::::::::::::::::::::::::::::::: 6.14.4 Antiangiogenetic Therapies :::::::::::::::::::::::::: Induction of Differentiation Retinoic Acids :::::::::: Molecular Therapies Using Kinase-Inhibitors ::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.16.1 Farensyltransferase Inhibitors (FTIs): Tipifarnib (Zarnestra) and Lonafarnib (Sarasar) :::::::::::::::::::::::::::::::::::::::::::::::::::: 6.16.2 FLT3 Antagonist Tandutinib (MLN518/CT53518) :::::::::::::::::::::::::::::::::::: Targeting NF-kB :::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.17.1 Bortezomib (Velcade)::::::::::::::::::::::::::::::::: 6.17.2 Arsenic Trioxide (Arsenox) :::::::::::::::::::::::: Modulation of Pro-Apoptotic Cytokines with Pentoxiphylline, Dexamethasone and Ciprofloxacine :::::::::::::::::::::::::::::::::::::::::::::::::::::::::: MDS Subtypes Associated with Certain Cytogenetic Features::::::::::::::::::::::::::: 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 :::::::::::::::::::::::::::::::::::::: 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 :::::::::::::::::::::::::::::::::::::::::::::: Simplified Treatment Algorithm for MDS :::::::::::::::

193 193 194 195 195 195 195 196 197 197 197

197 198 198 198 198

199 200 200 201 201 201 202 202 202 202 205 205 205 206

6.1 Introduction Primary myelodysplastic syndromes (MDS), the most common hematologic malignancy to affect the elderly, are clonal disorders of hematopoietic stem cells. MDS is characterized by an increased but ineffective and dysplastic hematopoiesis as well as peripheral cytopenias. The abnormal hematopoietic clone partly gives rise to mature, but functionally and morphologically abnormal blood cells, and, at least in some cases, is capable of both myeloid and lymphoid differentiation ([1] and reviewed in [2]). The term Myelodysplastic Syndromes encompasses a heterogenous group of diseases that can form a continuum from relatively indolent clonally derived refractory anemias with or without ringsideroblasts (RARS, RA) or unilineage thrombocytopenias, to clonal multilineage dysplasias (RCMD) and refractory anemia with excess blasts (RAEB), sometimes also termed oligoblastic leukemia (see currently valid WHO-classification of MDS, Table 6.1). Approximately 1/3 of all MDS patients, more so patients with advanced stage MDS such as RAEB-I and RAEB-II, eventually progress to overt acute myeloid leukemia (AML). One might argue, that the terminus myelodysplasia was ill-chosen to denote a clonal neoplastic stem cell disorder, as it implicates a non-neoplastic reactive process, but it nevertheless reflects the most prominent cytological and histological findings required for the diagnosis. In early stages of MDS, peripheral cytopenia is due to increased intramedullary apoptosis which aborts the differentiation products of potentially malignant, mutated stem cells, leading to inefficient erythropoiesis, granulopoiesis or thrombopoiesis (see Sect. 6.3 of this chapter as well as Table 6.2 and Figs. 6.1a and 6.2). Infections due to neutropenia and granulocyte dysfunction represent the main cause of death. Petechiae (Fig. 6.3a c) or hemorrhages are frequent consequences of severe thrombocytopenia (see Sect. 6.4 and Table 6.3 as well as Fig. 6.4a, b). In contrast, the reduction of apoptotic capacity and increased expansion of the immature blast compartment cause marrow failure and cytopenia in advanced stages (see Fig. 6.1b). This increased proliferative capacity of the malignant clone ultimately leads to the evolution of acute myeloid leukemia in 30 45% of patients. This is why MDS is considered to be a preleukemic condition. Autoimmune syndromes are common in MDS (see Table 6.3) and may also be the primary cause of death in some patients. Furthermore, patients with MDS have a high prevalence of autoantibodies against red blood cells, without clinical or laboratory signs of hemolysis [3, 4].

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Table 6.1: WHO Classification of the Myelodysplastic Syndromes (adapted from [492]) Type of MDS

Peripheral blood anomalies

Refractory anemia (RA); Refractory cytopenias with unilinege dysplasia (RCUD); Refractory neutropenia (RN); Refractory thrombopenia (RT) Refractory anemia with ring sideroblasts (RARS)

*

Refractory cytopenia with multilineage dysplasia (RCMD)

*

*

* *

*

* * * * *

Refractory anemia with excess blasts I (RAEB I)

* * * *

Refractory anemia with excess blasts II (RAEB II)

* * * *

MDS unclassified (MDS U)

* * *

MDS with isolated del(5q)

Unicytopenia or bicytopenia No or rare blasts

*

*

Refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD RS)

Bone marrow anomalies *

* * *

Anemia No blasts Bi or pancytopenia No or rare blasts No Auer rods Monocytes G1,000/ml Bi or pancytopenia No or rare blasts No Auer rods Monocytes G1,000/ml Cytopenias G5% blasts No Auer rods Monocytes G1,000/ml Cytopenias 5 19% blasts Auer rods Monocytes G1,000/ml Cytopenias No or rare blasts ( 5%) No Auer rods Anemia G5% blasts PLT normal or "

Unilinege dysplasia: 10% of the cells in one myeloid linege G5% blasts, G15% ringed sideroblasts

Erythroid dysplasia only G5% blasts, 15% ringed sideroblasts * Dysplasia in 10% of cells in 2 myeloid cell lines * No Auer rods * G15% ringed sideroblasts * G5% blasts * Dysplasia in 10% of cells in 2 myeloid cell lines * No Auer rods * H15% ringed sideroblasts * G5% blasts Unilineage or multlineage dysplasia * 5 9% blasts * No Auer rods * *

* * *

* * * * * * *

Unilineage or multlineage dysplasia 10 19% blasts Auer rods þ= Unilineage dysplasia in granulocytes or MKs No Auer rods G5% blasts Normal to increased MKs with hypolobulated nuclei No Auer rods G5% blasts Isolated del(5q)

PLT Platelets; MK megakaryocyte Table 6.2: Potential pathophysiological mechanisms involved in the ethiopathogenesis of MDS Potential mechanisms involved in the pathogenesis of MDS Acquired stem cell lesion * Inherent growth/survival advantage Clonal evolution * Additional somatic mutations required Initial CTL-mediated immune attack directed against * Neoplastic MDS stem cells * T against the clone scenario * Collateral damage of bystanding normal stem cells & Due to insufficient specificity or & Paracrine effect of released cytotoxic substances * Normal stem cells * Selection pressure for mutant stem cell clones with the capacity for immune escape * Shown for trisomy 8, or PNH (paroxysmal nocturnal hemoglobinuria) CTL CytotoxicT lymphocytes

6.2 Epidemiology of MDS The precise incidence of MDS is currently unknown, but is estimated to be 4.56 and 2.81 per 100,000 for males and females, respectively, for the European standard population [5]. The risk of developing MDS is strongly associated with increasing age, with the annual incidence ranging from 0.07 to 45.43 per 100,000 for patients G25 and H85 years, respectively [6 8]. A recent analysis from the North American Association of Central Cancer Registries (NAACCR), based on more than 40,000 observations, revealed an annual age-adjusted incidence rate of 3.3 per 100,000 in the years 2001 2003. However, the authors believe this number to be an underestimation due to lack of reporting [9]. The median age at diagnosis is 77 years. Diagnosis onset before the age of 50 is rare, except for treatment-induced MDS (see Sect. 6.20.1). Although rare, MDS also occurs in children at a median age of 6 years, with predominance of juvenile

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Scenario A: “T against the clone” CTL attack of “abnormal” MDS stem cells CTLs attack MDS-HSC aberrant antigens collateral damage of ‘‘bystanding” normal stem cells Release of cytokines, upregulation of Fas proapoptotic mileu

Scenario B: “autoimmune attack” CTL attack of normal HSC Functional T-reg defects promote autoreactive CD8+ clonogenic CTLs Selection pressure for MDS HSC

HSC apoptosis

Apoptosis of BM stroma normal HSC support

Acquisition of additional mutations

a

b

Normal HSC normal HSC lose contact with stroma loss of survival signals

Apoptosis of normal HSC

Increased T-reg numbers suppress anti-MDS immunity immune escape predominant malignant hematopoiesis neoangiogenesis

Apoptosis of normal BM stroma

CTL

MDS HSC capacity for immune escape maintain contact with viable stroma maintain contact with each other

CTL

T-reg

T-reg

Apoptotic body

Apoptotic BM stromal cell

Apoptotic MDS blasts / normal myeloid progenitors

CTL (cytotoxic T lymphocyte)

Viable MDS blasts / normal myeloid progenitors

Lytic granules & cytokines secreted by CTL

Viable BM stromal cell

T-reg

Cytokines and chemokines secreted by T-reg Neovasculature Fatty marrow

Fig. 6.1a Pathophysiology of early stage MDS RA/RARS. Proapoptotic cytokines are released from CD8þ clonogenic CTLs (irrespective of whether the original target of the immune attack was the normal or the neoplastic stem cell compartment). These cyto kines promote apoptosis of hematopoietic stem cells (HSC) either directly and/or via upregulation of Fas, as well as by apoptosis induction of the stem cell supporting bone marrow (BM) stromal cells. As is schematically depicted, the normal stem cells loose supportive contact to the stroma cells (viable stroma cells depicted in grey) once these become apoptotic (dark stromal cells represent apoptotic stromal cells), and thus loose essential prosurvival signals as well as protective effects mediated by intact stroma. Ultimately, normal HSC undergo apoptosis (grey) and dysplastic stem cells remain viable (various shades of pink) and maintain contact with each other, as well as with supportive viable stroma cells. This further enhances their survival and growth advantage over normal

HSC, which ultimately leads to their predominance and progression to RAEB and AML (see b). b Pathophysiology of late stage MDS RAEB-II/AML. In advanced stage MDS the neoplastic dysplastic clone has already become predominant over normal hematopoiesis, due to the acquisition of additional mutations which confer further survival/growth advantages, or which further enable immune sur veillance escape. As is schematically depicted, the bone marrow is hypercellular and packed with dysplastic viable blasts (various shades of pink), with barely normal hematopoiesis remaining. Due to secretion of neovasculature promoting cytokines, enhanced microvascular density which further promotes survival and growth of the neoplastic clone, is readily visible. The dysplastic stem cells dominating the bone marrow have managed to escape immune surveillance mechanisms. Therefore, cytotoxic lymphocytes, al though they may still be present to some extent, are no longer able to control noplastic growth

myelomonocytic leukemia (JMML) and the monosomy 7 syndrome [8]. The former is distinguished by elevation in hemoglobin F and progressive marrow failure, whereas excessive susceptibility to infection, a familial tendency and frequent transformation to acute leukemia are hallmarks of the latter.

6.3 Pathophysiology and Molecular Biology of MDS 6.3.1 Disturbances in Apoptosis As briefly mentioned above, MDS is typically characterized by increased clonal proliferation on the one hand, and

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d

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c

Fig. 6.2 Balance of apoptosis vs. proliferation during different stages of MDS. a In early phase MDS (RA/RARS/RCMD), apo ptotic normal hematogenic progenitor cells predominate the bone marrow picture. b In advanced stage MDS (RAEB I), apotosis of normal progenitors and proliferation of dysplastic MDS progenitors/

blasts remains balanced. In contrast, proliferation of neoplastic, dysplastic MDS blasts predominates during late stage MDS (RAEB II) c and especially so during progression to AML. This inversion of the ratio of apoptosis to proliferation is a companied by several genetic hits d

extensive apoptosis (particularly pronounced in early stages of MDS) resulting in ineffective hematopoiesis and reduced differentiation, on the other. This leads to peripheral cytopenia(s) despite bone marrow hypercellularity. So far however, it remains unclear whether increased apoptosis results from a desperate cellular immune reaction directed against a rapidly proliferating clone and/or antigens expressed by the aberrant cells, or whether apoptosis represents an integral part of the pathophysiology of MDS (see Table 6.2). However, increasing evidence favors the hypothesis that in early disease, excessive apoptosis represents a pathophysiological mechanism resulting from genetic aberrations within the malignant clone, rather than the result of a compensatory specific immune reaction directed against the dysplastic clone(s) (see e.g., [10]). Failure of differentiation and increased rates of apoptosis are responsible for the bone marrow failure in these early stages of MDS [11]. Phenotypically silent genomic events conferring survival and/or growth advantages are thought to forestall stem cell exhaustion and bone marrow hypocellularity, which would otherwise be the inevitable consequence of enhanced apoptosis (see Fig. 6.1a and b). During progression to secondary AML, increased rates of apoptosis disappear and the failure of differentiation is

replaced by a block in differentiation and increased rates of proliferation, as is observed in primary AML (see Fig. 6.2). Whereas apoptosis is significantly increased and always exceeds proliferation in refractory anemia (RA) and refractory anemia with ring sideroblasts (RARS), this ratio is equalized in refractory anemia with excess of blasts (RAEB), and inversed in refractory anemia with excess of blasts in transformation (RAEBt)/MDS-AML (see Fig. 6.2) (see e.g., [10, 12]). In fact, the disturbed balance between apoptosis and proliferation changes continuously during progression of MDS to more advanced stages. The extent of apoptosis is therefore also inversely correlated with the International Prognosis Scoring System (IPSS). This is paralleled by an identical run of the curve for the ratio between pro-apoptotic and anti-apoptotic cytokines (Bax/Bad:Bcl-2/Bcl-X) [12]. This means, that disease evolution to AML is accompanied by significantly increased expression of antiapoptotic Bcl-2 [13]. This reduction of apoptosis in advanced stages may further promote the acquisition of additional genetic alterations, which furthermore the evolution to AML [10]. Not surprisingly, steadily increasing levels of Bcl-2 have also been linked with resistance to chemotherapy and typically portend poor prognosis with short survival [12].

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Table 6.3: Incidence of typical clinical features in MDS Presenting clinical features of patients with MDS

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Fig. 6.3 Complications due to inefficient thrombopoiesis and thrombopenia: a Petechiae (typically localized on the lower extrem ities), b Petechiae close up, c Intra oral petechiae/hemorrhages

6.3.2 Alterations in T-Cell Functions and Cytokines Disturbances of the immune system in MDS are important and may represent a double edged sword. Activation of autoreactive T-cells leading to autoimmunity and a pathologic T-cell response against normal hematopoietic stem or progenitor cells has been shown to occur. In addition, suppression of antitumoral cytotoxic effector cells has also been reported. In this regard, CD4þ CD25þ Foxp3þ regulatory T-cells (T-regs) seem to be of particular importance and exert a master function in

Symptoms resulting from anemia (60%) * Pallor, fatigue, weakness, exercise intolerance * Angina, dizziness, cognitive impairment, altered sense of well being Symptoms due to inefficient thrombopoiesis (26%) * Easy bruising, petechiae, purpura due to thrombocytopenia * Other bleeding manifestations due to thrombocytopenia * Dysfunctional and/or morphologically abnormal platelets Symptoms due to inefficient granulopoiesis * Infections due to neutropenia and granulocyte dysfunctions * Predominance of bacterial infections * Skin as the most common site of infection * Infections may be occult, respond poorly to antibiotics, and typically resolve slowly Systemic symptoms * Fever * Weight loss * Nocturnal sweats * Generally occur late in the course of the disease Autoimmune manifestations (14%) (female preponderance) (e.g., [4, 493]) * Acute systemic vasculitic syndrome characterized by: * Cutaneous vasculitis, fever, arthritis * Peripheral neuropathy * Non infectious pulmonary infiltrates, pleural effusions and peripheral edema * Chronic autoimmune disorders such as: * Cutaneous vasculitis, monoarticular arthritis * Pericarditis, pleural effusions * Skin ulcerations, pyoderma gangrenosum * Iritis, myositis, inflammatory bowel disease, glomerulonephritis * Peripheral neuropathy * Idiopathic thrombocytopenia * Classical connective tissue disorders, especially: * Recurring polychondritis * Systemic lupus erythematodes (SLE) * Sj€ ogrens syndrome * Raynauds phenomenon * Vasculitic overlap syndromes, most commonly: * Leukocytoclastic vasculitis * Asymptomatic immunologic abnormalities: * Polyclonal hypergammaglobulinemia (39 54%) * Monoclonal gammopathy (12%) * Hypogammaglobulinemia (8%) * CD4/CD8 ratio G1 (20%) * Decreased NKC activity * ANA (30%), RA factor (14%), anti DNA antibodies (7%), positive direct antiglobulin test (12%), autoantibodies (34%), cold agglutinin (62%), AMA, anti TPO and anti TG antibodies NKC Natural killer cell; ANA anti nuclear antibodies; RA rheuma toid arthritis; AMA antimitochondrial antibodies; TPO thyroid peroxidase; TG thyroglobulin

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a

b

Fig. 6.4a Ecchymosis of buttocks, b Ecchymosis of the thigh and peri-inguinal region

both processes [14]. T-regs play an important role in regulating self-tolerance. Overt autoimmune disorders have been shown to occur in a substantial number of MDS patients [15] (see Table 6.3), although the pathophysiologic link with the immune response against the MDS clone is currently unclear as most of the target tissues are of non-hematopoietic origin. In fact, the occurrence of autoimmune manifestations in MDS is so high, that the possibility of an underlying MDS should be considered in patients with diseases of the rheumatic sphere and concomitant cytopenias [16 18]. In early stage MDS dysfunctional T-regs with defective suppressor function and bone marrow homing due to CXCR4 down-regulation are thought to be involved in autoimmune processes leading to disease initiation, as well as autoimmune corollary phenomena mentioned above [19]. In this regard, low numbers of T-regs in early stages of disease may de-repress the emergence of autoreactive T-cell clones, thereby facilitating the outgrowth of autoimmune phenomena often associated with MDS. The disturbed balance of T-cells between adequate tumor control and suppression of autoimmunity may also contribute to the increased rates of apoptosis in normal hematopoietic cells in early MDS stages. In late stage MDS, the same T-reg population can incur detrimental effects by modulating overall immune re-

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sponses against tumor cells. Whereas defective T-reg function has been demonstrated in low-risk MDS, increased numbers and activity of T-regs occur in high-risk MDS. These T-regs suppress anti-neoplastic immune responses specifically targeted against neoplastic MDS clones. This tumor specific immune response is mediated by CD8þ CTLs. It is thus hypothesized that increased Treg activity in late stage MDS favors leukemic progression via suppression of CD8þ T-cells through Th1-mediated excretion of immunosuppressive cytokines [14, 19, 20]. This decrease in CD8þ cells is thought to contribute to the progression of low-risk MDS to more aggressive subtypes [21]. In fact, an expansion of polyclonal T-regs in higher MDS stages has been demonstrated and associated with worse prognosis and progression of the disease [22]. In addition, antitumoral functions of natural killer cells (NKC) are reduced during evolution of MDS to AML [23] and decreased NK functions are correlated with unfavorable prognostic scores, cytogenetics and high blast counts. This sets the rational for the therapeutic use of 2nd and 3rd generation immunomodulatory drugs (imids) such as lenalidomide and pomalidomide, both of which inhibit the proliferation and function of T-regs [24]. Immune elimination of stem cells by cytotoxic T-cells seems to play an important role in at least some MDS subgroups. This immune attack may be part of a physiological antitumor surveillance response to abnormal or dysplastic cells, (T against the clone scenario) (see Fig. 6.1a). In cases of insufficient specificity, this would result in collateral damage of bystanding normal hematopoietic cells, leading to generalized bone marrow suppression and pancytopenia. Such bystander-killing may be mediated by cytokines released by CTLs in early stage of MDS. In fact, it has been hypothesized that HLADR15/DR2 typing may identify a subset of MDS with immune mediated marrow failure [33]. HLA-DR2 is associated with increased release of TNF-a, overexpression of which has been linked to the ethiology of cytopenia [34]. These cases of MDS are associated with rheumatoid arthritis and a more favorable prognosis as well as with a higher probability to respond to immunosuppressive therapy with antithymocyte globulin (ATG) or cyclosporin [33, 35, 36]. Conversely (and more likely), however, the initial CTL-attack may be directed against normal stem cells, resulting in selection pressure and outgrowth of mutant hematopoietic clones capable of immune escape, as has been recently shown to be the case in trisomy 8 MDS [25]. Clonally expanded CD8þ CTLs play a dominant role in the hypothesized (auto)immune mechanism of MDS ethiopathology. It is thought, that CD8 þ T-cells of a specific Vbsubfamily expand in response to a neoantigen (such as a virus) or a quantitatively upregulated or aberrantly ex-

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pressed normal protein, which could be presented through MHC-I molecules of MDS-blasts. PR-1 (proteinase) and WT-1 (Wilms tumor) have been proposed as examples of such overexpressed self-proteins. In fact, these proteins are overexpressed in patients with MDS, with levels correlating positively with advanced MDS stages [26 28] and negatively with overall survival in patients with overt AML [29]. Furthermore, circulating PR-1 and WT-1 specific CTLs have been demonstrated in these patients [30]. As further proof of principle, an immunologic response can therapeutically be raised against the malignant clone by vaccination protocols using the simultaneous application of PR-1 and WT-1 antigens as vaccines. This was not only well tolerated in a phase-I clinical setting, but also led to an increase in PR1/WT-1-specific CD8 þ T-cells with concomitant significant reduction in leukemic blasts in patients with MDS or AML [30], as well as in a patient with CMML [31], thus confirming prior results [32].

6.3.3 Microenvironment in MDS The microenvironment of the bone marrow is severely disturbed in MDS patients. Stroma cells are important for the outgrowth of normal stem cells and also give relevant signals for hematopoietic differentiation. In patients with MDS however, mesenchymal stem cells are prone to apoptosis and display a decreased capacity to support normal hematopoiesis (see Fig. 6.1a and b) [37]. This may at least in part be caused by abnormally produced cytokines from autoreactive CTLs. Although the interaction of stroma and matrix components with the clonal myelodysplastic cells is functionally abnormal [38] and seems to play an important role in the pathophysiology and progression of the disease [39], the stromal component of the bone marrow microenvironment probably does not derive from the malignant clone [40, 41], but conflicting data exist [42]. The activated T-cell system as well as the aberrant expression and regulation of the cell death receptor CD95/ Fas on hematopoietic precursor cells [43] and the specific cytokine milieu create an apoptotic environment, which delays leukemic evolution on the one hand, but drives bone marrow failure on the other [44]. In good concordance with such an hypothesis, increased levels of (proapoptotic) levels of cytokines such as tumor necrosis factor a (TNF-a), tumor derived growth factor b (TGF-b) and IL-1b in the microenvironment, as well as upregulation of Fas-antigen expression on the CD34 þ cells, have been shown to be fundamental events leading to apoptotic death of bone marrow cells in the earliest stages of MDS [39]. This is further underlined by clinical responses of MDS and other bone marrow failure syndromes to targeted suppression of TNF-a by pentoxifyl-

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line or anti-TNF-a antibodies [45] (see respective section in this Chapter 6.18). Neoangiogenesis and microvascular density are increased in MDS and this process is under the control of proangiogenic cytokines like vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), TNF-a and TGF-b [39, 46]. High affinity type III receptor tyrosine kinases for VEGF (VEGFR-1 (Flt-1) and VEGFR-2 (Flk/Kdr)) are also overexpressed in MDS [46, 47]. In addition to its role in inducing neoangiogenesis, VEGF has also been implicated in the mitogenesis of myeloblasts in primary MDS [47]. Higher levels of VEGF are found in the FAB subtypes RAEB, RAEB-t and CMML, compared to patients with RA or RARS or normal bone marrow [48]. Thus, the amount of expressed VEGF is related to the percentage of immature myeloid cells (blasts and monocytic precursors), and correlates with FAB category [13, 48]. Intense coexpression of VEGF with one or both of its receptors was found in loci of ALIPs (abnormally localized immature precursors), suggesting autocrine disease promoting and perpetuating interactions [47]. Neutralization of VEGF activity suppressed MDS-cell growth, whilst promoting the formation of BFU-E in vitro, suggesting a contribution of VEGF to leukemia progenitor self-renewal [47].

6.3.4 The Role of Tumor Suppressor Genes and Oncogenes in MDS Disease Initiation/Perpetuation In de novo AML the molecular pathogenesis is supposed to rely on a two hit model. Alterations in class I genes, like FLTD3-ITD, N- or K-Ras mutations seem to confer a proliferative advantage while class II hits such as AML-1/ETO, PML/RAR or MLL-related fusion genes more predominantly seem to interfere with the normal hematopoietic differentiation capacity [49, 50]. In this concept, isolated class I or class II mutations may be associated with myeloproliferative disorders, while the development of overt leukemia requires the cooperation between class I and class II gene alterations [50, 51]. This model may also be important for MDS, given its high probability to finally transform into AML. However, this model is probably oversimplifying the exact biological role of each individual gene since e.g., Ras-mutations are of particular importance for disturbances in erythropoietic differentiation [52]. In addition, models are required which recapitulate the characteristic feature of initial cytopenias and the subsequent development of a frank leukemic course which is typical of MDS. In this regard, it is important to consider the particular difficulties of

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human xenograft models in MDS patients [11]. While clonogenic cells from AML patients can easily be xenotransplanted, clonogenic cells of MDS patients are poorly clonogenic in NOD SCID mice [53, 54]. Even in improved models such as NOD/SCID b2microglobulin = mice, cytogenetically abnormal clones persist for only a short period of time before they are outcompeted by karytotypically normal cells [55]. In a further step to better understand the molecular biology of MDS, gene alterations observed in human MDS patients have been transgenically overexpressed within murine hematopoeitic stem cells and used in bone marrow transplantation systems (for review see [11]).

6.3.4.1 Somatically Acquired Mutations of the AML-1 Gene in MDS Somatically acquired mutations of the AML-1 gene have recently been identified in MDS (almost exclusively in RAEB and RAEB-t) and AML [56 59]. This gene is indispensable for the establishment of definitive hematopoiesis [60, 61] and its mutation is thought to be one of the major driving forces for the transformation of MDS into AML by altering the DNA binding potential of the encoded transcription factor (core binding factor beta). Normalizing AML-1 function would thus provide an important target for therapeutic intervention. Mutations in AML-1 are frequently observed in patients with poorly differentiated AML (M0) (12 33%) [57], after prior exposition to radiation (46% of late-onset MDS among survivors of the atomic bomb explosion in Hiroshima) [59], to local radiotherapy or to alkylating agents (38 42% of therapy-related AML/MDS) [59]. In contrast, only 2.7% of de novo MDS- and G5% of de novo AML-patients bear this mutation [56, 59]. Recent evidence suggests that point mutations of AML-1 are related to low-dose irradiation or alkylating agents and long latency periods of transformation from MDS to AML, whereas translocations involving the AML-1 gene site occur as a result of sublethal irradiation or topoisomerase-II inhibitors and tend to be associated with short latency periods. AML1 mutations are also observed in patients with AML secondary to essential thrombocythemia, polycythemia, chronic idiopathic myelofibrosis or paroxysmal nocturnal hemoglobinuria, and seem to sequentially occur and cooperate with the JAK2 mutation in CMPDs (e.g., [62]). Germline mutations of AML-1 are also prominent in FDP/AML (familial platelet disorder with predisposition to AML) [63]. The prognosis of patients with AML-1 mutations is significantly worse than for patients without AML-1 mutations [57]. In further proof of the above mentioned data implicat-

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ing that loss of AML-1 function is critically involved in the pathogenesis of MDS/AML, AML1 mutations were shown to induce MDS and MDS/AML in a mouse bone marrow transplant model [50].

6.3.4.2 Overexpression of EVI-1 in MDS Overexpression of EVI-1 is commonly associated with structural abnormalities involving chromosome 3q26 and del(7). It is found in up to 29% of patients with MDS (excluding CMML) and 9% of patients with AML, and has been shown to be an independent poor prognostic factor [64]. The EVI-1 gene product is a transcription factor, normally not found in cells of the peripheral blood or bone marrow, that blocks erythroid differentiation via inhibition of GATA-1 induced transcription [64, 65]. EVI-1 also suppresses terminal myeloid cell differentiation through an as yet unidentified mechanism, and inhibits TGFb-mediated apoptosis [66]. Consequently, overexpression of EVI-1 is thought to play an important role in the pathogenesis or progression of several myeloid malignancies [64, 67, 68]. Furthermore, leukemic transformation in MDS, as well as disease progression from chrone phase CML to accelerated and blast phase CML, have also been associated with inappropriate expression of EVI-1 from fusion genes such as RPN1-EVI-1, GR6EVI-1, TEL-MDS-1-EVI-1 or AML1-MDS-1-EVI-1 [65 67]. Retroviral insertion and overexpression of EVI-1 in murine hematopoietic stem cells is associated with erythroid dysplasia, megakaryocytic and erythroid hyperplasia with simultaneous downregulation of erythropoietin- and thrombopoietin-receptors, finally resulting in lethal pancytopenia [69]. In addition, cooperation with AML-1 mutations has been demonstrated (see above).

6.3.4.3 Oncogenic Fusion Products in MDS Apart from the above mentioned fusion genes with EVI, oncogenic fusion products also result from the translocation (5;12)(q33;p13), which is a rare cytogenetic anomaly. The translocation of the tel- and the PDGFb genes is mainly seen in a recently recognized subgroup of CMML, and coincides with monocytosis and eosinophilia [70, 71] (see Chapter 7.4). The translocation and fusion of the NPM-MLF-1 (myelodysplasia/myeloid factor 1) genes alters DNA replication and cell growth in MDS cases with t(3;5)(q25q34), another occasionally observed translocation which seems to be associated with multilineage dysplasia and progression to AML [72 76]. In addition, the NPM1 gene is frequently

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deleted in primary and secondary MDS, and NPM1 þ = mice develop erythoid hyperplasia [77]. Murine models have also demonstrated that NUP98-HOXD13 fusion genes and heterozygosity within the Pten þ = / Ship1 = loci play relevant roles in the development of MDS [78, 79].

6.3.4.4 Mutation of the Ras-Protooncogene in MDS Mutation of the RAS-protooncogene is the most common molecular abnormality in MDS [80]. Retroviral insertion and overexpression leads to ultimate failure of the erythroid differentiation program at the late-erythroblast stage of development with an increased propensity for apoptosis. This provides providing a causative link between mutational activation of N-RAS and the pathogenesis of preleukemia [52, 81]. Insertion of K-RASG12D has been shown to block differentiation at the proerythroblast stage [82], a finding which provides the basis for the treatment of MDS patients with farensyltransferase inhibitors which inactivate oncogenic RAS (see Sect. 6.16.1). Furthermore, the incidence of activated ras oncogenes increases from 6% in MDS to 12% in secondary AML, emphasizing the value of this mutation as a marker of progressive disease and impending malignant transformation [83].

6.3.4.5 The Role of Interferon-Regulatory Factor-1 (IRF-1) in MDS Interferon regulatory factor-1 (IRF-1) is a transcriptional activator of type I interferons and interferon-induced genes, which play important roles in inflammation, autoimmune diseases [84] and tumor suppression [85]. These effects are thought to be in part mediated by induction of nitric oxide synthase (NOS) and consequent elevation of NO-levels, which is known to have activities against tumors and microorganisms and possibly also plays a role in autoimmunity. Independent of its immunomodulatory function, IRF-1 is critically involved in the terminal myeloid differentiation mediated by IL-6 and leukemia-inhibitory factor [86]. Furthermore, IRF1 is involved in growth suppression and thus serves as a tumor suppressor gene in MDS, which seems ascertained by its ability to suppress transformation in vitro [85]. Loss of this transcription factor seems to link tumorigenesis with aberrant immune responses and has been shown to be involved in the pathogenesis of autoimmune manifestations in MDS [87]. Increased levels of IRF-1 reflect overactive interferon signaling and induce IL-12 production, mat-

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uration of natural killer cells (NKC) and Th1 polarization, thereby promoting inflammatory processes and autoimmunity. In turn, the downregulation or loss of functional IRF-1 has been correlated with a dramatically reduced incidence of, and seems to protect against, paraneoplastic autoimmune phenomena in MDS patients [87]. Loss of function of the IRF-1 gene through deletions, somatic mutations or alternative splicing seems to be a critical oncogenic event in MDS patients without autoimmune manifestations [87, 88]. Absence of IRF-1 gene expression is associated with more severe/aggressive disease and higher IPSS risk groups, further supporting a pathogenetic role of this gene in MDS [88]. Interestingly, IRF-1 has been mapped to 5q31.1, and is thus consequently deleted in MDS patients with loss of or aberrations at this locus [89].

6.4 Clinical Features in MDS Patients with MDS do not have pathognomonic clinical features as such. Rather, they present with unspecific symptoms resulting from their cytopenias (summarized in Table 6.3), such as anemia related lethargia, pallor and weakness, and/or bruises, petechiae (Fig. 6.3a c) or ecchymoses (Fig. 6.4a and b) resulting from thrombocytopenia and dysfunctional thrombocytes. Petechia typically first occur in the lower extremities as small, 1.3 mm in diameter measuring lesions, most often in the skin covering the shinbone (Fig. 6.3a and b). Intraoral petechia (Fig. 6.3c) or ecchymoses (Fig. 6.4a and b) are often a sign of extreme thrombocytopenia, i.e., lower than 10,000 platelets/ml.

6.4.1 Infectious Complications in MDS Patients with severe decreases of white blood cells display a tendency for sometimes dramatic infectious complications with atypical pathogens due to neutropenia and inadequate function of granulocytes (Figs. 6.5 6.12). Reactivation of herpes virus infections, such as herpes zoster (Fig. 6.5a) or oral herpes simplex virus (HSV) infections can be more severe and of longer duration than in non-MDS patients. Complications such as herpes simplex keratitis (Fig. 6.5b) must be looked out for, as recurrent ocular herpes virus infections may lead to scar formation which impairs eyesight (Fig. 6.5c). Abscess formations (Fig. 6.6a) may sometimes spread significantly (Figs. 6.6b and 6.7) and thus lead to compression of blood vessels, or other vital organs as for example the

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Fig. 6.5 Infectious complications resulting from neutropenia, dys functional neutrophils, as well as additional immune defects. Reactivation of viral infections, a exulcerating dermal Herpes Zoster. Characteristic spread of the lesions according to nerval innervation of dermatomes. b Herpes simplex keratitis. Typical dendritica figure, Flurekain stain. c Recurrent ocular HSV infec tions. Corneal scarring and vascularization after recurrent ocular simplex virus infections

Fig. 6.6a Cervical abscess. b Cervical abscess of patient demarked by the red arrows in the CT scan. c Cervical abscess of patient in b necessitating incision and drainage

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Fig. 6.7 Gall bladder abscess, arrow demarks air inclusions

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Fig. 6.9 Pneumocystis carinii pneumonia with cysts. Large and multiple cysts in both lungs resulting from pneumocytis carinii in a patient with MDS

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Fig. 6.10 Gas gangrene by clostridium perfringens. These anaer obic bacteriae enter the muscle through a wound or a breakdown of barriers, and proliferate producing toxins which destroy the sur rounding tissue, thereby generating gas

Fig. 6.8a Periorbital phelgmone. b Phlegmone with deep ulceration in calf, necessitating plastic surgery

trachea. These complications are observed rather frequently in severely neutropenic patients, especially so in those with pending transformation to AML. These abscesses often necessitate a surgical intervention in order for the puss to drain sufficiently (e.g., Fig. 6.6c). Some neutropenic patients develop a tendency towards

phlegmone (Fig. 6.8a) which may ultimately lead to central necrosis or deep ulcerations which can take months to heal, and sometimes require surgical resection and/or plastic surgery (Fig. 6.8b). Patients with MDS often experience not only infections with an unusually severe course, but also have a propensity for infections with atypical pathogens, that are non-pathogenic for the immunocompetent host (e.g., infections with pneumocystis carinii (see Fig. 6.9) clostridium perfringens, see Fig. 6.10). Especially pa-

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a

c

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d

Fig. 6.11a CMV-Pneumonia before antiviral therapy. Marked interstitial pneumonia in both lungs. b CMV-pneumonia after antiviral therapy. Dramatic, almost complete restitutio ad integrum after adequate antiviral therapy. c Stenotrophomonas pneumonia

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Fig. 6.12a CMV-Encephalitis, CMV encephalitis in an MDS/ AML patient during chemotherapy induced aplasia: red arrows demark significant changes in MR signals in the following regions: caput nuclei caudate; insular region, temporopolar;

before antibiotic therapy. Marked pneumonic infiltration. d Stenotrophomonas pneumonia after antibiotic therapy. Near complete resolution after adequate antimicrobial therapy (right)

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frontoparietal and cerebellum. b CMV-Myelitis. Patient with ascending weakness and ultimately hemiplagia due to CMV myelitis and polyradikulitis (red arrows demark significant changes in MR signals)

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tients with advanced disease (RAEB-II) and those in transition to overt AML may present with, or develop pneumonic complications such as pneumocystis carinii pneumonia with cysts (Fig. 6.10), stenotrophomonas pneumonia (Fig. 6.11c) or life-threatening cytomegaly virus (CMV) reactivations including CMV-pneumonia (Fig. 6.11a), CMV-encephalitis (Fig. 6.12a) and/or CMV-myelitis (Fig. 6.12b). If detected early enough, the correct therapy can lead to significant recovery (Fig. 6.11b and d). As mentioned before autoimmune phenomena occur frequently in MDS, but mostly remain a subclinical phenomenon. Hepatosplenomegaly and lymphadenopathy are uncommon ( 5%), with the exception of CMML, where splenomegaly is massive in 25%, and often accompanied by nodular cutaneous leukemic infiltrates and/or pleural/pericardial effusions and ascites (see respective chapter on CMML). Diabetes insipidus, clinically characterized by polydypsia, a dry mouth and polyuria, is sometimes associated with pending leukemic transformation, and seems to occur more often in patients lacking the long arm of chromosome 7q [90]. Sweets syndrome (acute neutrophilic dermatosis), an acute febrile state with erythematous patches that progress to painful brown patches which may ulcerate, has also been associated with MDS and MDS/AML [91]. Furthermore, the incidence of solid tumors appears elevated in patients with MDS [92, 93], whereas the incidence of B-cell neoplasms does not seem to be raised [94].

6.5 Laboratory Features in MDS As described in detail above, the myelodysplastic syndromes are characterized by inefficient hematopoiesis, resulting in peripheral cytopenias (for incidences see Table 6.4), abnormal cell morphology (e.g., abnormally large platelets or acquired Pelger-Huet anomaly (see Fig. 6.13)) and dysfunctioning hematopoietic cells. At diagnosis, anemia, neutropenia and thrombocytopenia are present in 85%, 50%, 25% of patients, respectively [95 97]. Reticulocyte counts are usually low for the degree of anemia. The proportion of monocytes is often increased. Lymphopenia may be observed, with an inversion of the CD4 to CD8 ratio, which has been inversely related to the number of transfusions received [98]. This inversion of CD4/CD8 ratio is thought to be induced by a Th1/Th2 imbalance [21]. Quantitative decreases in natural killer cells are also seen [99]. Immunoglobulin production may be variably affected, with hypogammaglobulinemia, polyclonal hypergammaglobulinemia, and monoclonal gammopathy reported in 13%, 30%, and 12% of patients, respectively [100] (also see Table 6.3).

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Table 6.4: Laboratory findings in patients with MDS [95 97] Laboratory findings in patients with MDS Alterations in red blood cells * Anemia (85%) * Inappropriately low reticulocyte response * The presence of reticulocytosis is indicative of superimposed AIHA * Elevation in hemoglobin F * Abnormalities of erythrocytic glycolytic enzymes * Increased sensitivity to complement mediated lysis Alterations in platelets * Thrombocytopenia (25%) can also rarely represent an isolated early manifestation (3%) * Thrombocytosis (in association with 5q syndrome) Alterations in neutrophils * Absolute neutropenia resulting in leukopenia (50% at diagnosis) * Pancytopenia (50% at diagnosis) * G5% present with isolated neutropenia, thrombocytopenia or monocytosis in the absence of anemia Alterations in lymphocytes and cytokines * Reduction of IFN g producing CD4þ Th1 cells with inversion of CD4þ /CD8þ ratio * Elevation of serum levels of INF g and TNF a * Skewing of TCR Vb repertoire Other laboratory findings * Diminished activities of myeloperoxidase and alkaline phosphatase * Reduced expression of CD11/CD18 resulting in: * Dysfunctional granulocytes * Predisposition to bacterial infections * Thought to be associated with monosomy 7 or 7q * Increased serum muraminidase/lysozyme activity and/or lysozymeuria * Elevated monocytopoiesis and cell turnover * Urinary potassium wasting and hypokalemia TCR T cell receptor; AIHA: autoimmune hemolytic anemia; IFNg interferen g; TNFa tumor necrosis factor a

Erythropoietin may or may not be elevated. Serum ferritin levels are often elevated. Lactate dehydrogenase, lysozyme and uric acid may be elevated due to enhanced cell turnover and/or cell death in the bone marrow. As mentioned above, serologic (auto)immunologic abnormalities are common in MDS with a tendency for female predominance [101] (see Table 6.3). Elevated levels of hemoglobin H with red cell morphologic features reminiscent of a-thalassemia, are found in approximately 8% of MDS patients. This has been termed acquired alpha thalassemia myelodysplastic syndrome (ATMDS) [102]. These and other laboratory findings often found in patients with MDS are outlined in Table 6.4.

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Table 6.5: Cytological findings in the peripheral blood smear of patients with MDS Cytological findings in the PB-smear of patients with MDS

Fig. 6.13 MDS Cytology of surrounded. MDS blood smear: Pseudo pelger cell

peripheral

Table 6.5 summarizes typical cytological findings in the peripheral blood smear of patients with MDS, and details dyserythropoietic and dysgranulopoietic changes as well as morphologic anomalies of platelets.

6.6 Typical Bone Marrow Findings in MDS Cytological findings of the bone marrow aspirate, as well as typical histological findings are summarized in Tables 6.6 and 6.7 and presented in Figs. 6.14a d, 6.16, 6.17a f and 6.19a d. Dysplastic features of all cell lineages may be observed. Dysplastic megakaryocytes (Figs. 6.14a and 6.17c f), and macroblastic erythropoietic changes (Figs. 6.14b, c and 6.17a) are characteristic. Ring sideroblasts are eponymous for RARS and RCMD-RS (Fig. 6.14d), whereas patchy distribution of hemosiderin may be observed histologically (Fig. 6.18). Bone marrow cellularity is usually increased (see Fig. 6.17a), represented by erythroid hyperplasia with elevated proerythroblasts/megaloblasts and/or granulocytic hyperplasia with often elevated marrow monocytes. Abnormal cytoplasmicto-nuclear ratio, nuclear fragmentation, hyper- or hyposegmentation, hypogranulation, cluster formation, as well as abnormal localization (Fig. 6.17b) of all cell forms may be found. Especially the presence of bone marrow basophilia, eosinophilia or ALIPs should warn the physician of potentially pending hematologic transformation to AML [103 107]. Elevated blast counts are observed in RAEB-I (Fig. 6.19a and b) and RAEB-II (Figs. 6.15 and 6.19c). Auer rods are very characteristic for imminent transformation to, or full blown AML (Fig. 6.16).

Erythrocytes * Macrocytosis (in the absence of vitamin B12 or folate deficiency) or macrocytic anemia * Normocytic anemia * Ovalomacrocytosis * Elliptocytes, teardrops, stomatocytes and acanthocytes (reflect intrinsic alterations in cytoskeletal proteins) * Ringed sideroblasts (see Fig. 6.14d) * H5 iron granules/cell occupying H1/3 of the nuclear rim * Usually found in hypochromic, microcytic cells * Basophilic stipling, Howell Jolly bodies, megaloblastoid nucleated RBCs * Acquired a thalassemia is occasionally associated with MDS: * Microcytosis, hypochromia, haemoglobin H containing cells Granulocytes * Pseudo Pelger Huet anomaly (see Fig. 6.13) * Due to abnormal myeloid maturation * Reduced granulocytic segmentation with a band form nucleus * Reduced or absent granulation * Ring shaped nuclei * Representing a transitional stage to the band form (acquired Pelger Huet formation) * Nuclear sticks (Auer rods) are typically seen in therapy related MDS (see Fig. 6.16) * Clumped chromatin pattern creating the appearance of nuclear fragmentation * Pseudo Chediak Higashi anomaly * Myelokathexis like features * Circulating myelocytes and/or myeloblasts * Auer rods (see Fig. 6.16) within leukemic blasts are rare but identify RAEB t, and should lead to the suspicion that the patient has already transformed to AML * Borderline or relative elevations of monocytes are common (the occurrence of absolute monocytosis (H1,000/ml) defines CMML) Platelets * Giant platelets and/or circulating megakaryocytic fragments * Hypogranular platelet forms * Dwarf or micro megakracyocytes (associated with increased bleeding tendency despite the presence of apparently adequate platelet numbers)

Only occasionally is bone marrow cellularity decreased, in case of hypoplastic MDS.

6.7 Diagnosis and Classiﬁcation of MDS The diagnosis of MDS should be considered in any patient, especially the elderly, with unexplained cytopenia(s) or monocytosis. Typical laboratory findings are summarized in Table 6.4. Bone marrow micro-

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d

Fig. 6.14a MDS RA/RARS cytomorphology of bone marrow aspirate. Dysplastic megakaryocyte with three nuclei (demarked by red arrow). b Strongly dysplastic macroblastic, nearly megalo blastic changes in erythropoiesis. White arrow demarks a mitosis.

c Dysplastic erythropoiesis. Red arrow demarks a dysplastic mega karyocyte with five nuclei. d RARS. Ringsideroblasts: iron laden mitochondria surround 2/3 of the circumference of the nucleus

Fig. 6.15 MDS RAEB-II: Cytology of bone marrow aspirate. Refractory anemia with excess of blasts characterized by 10 20% blasts and dysplastic features

Fig. 6.16 MDS/AML Auer rods. Arrows demark multiple Auer rods, typical for pending transformation to, or full blown AML

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b

a

20 μm

20 μm

d

c

10 μm

e

10 μm

f

10 μm

20 μm

Fig. 6.17 MDS-RA: Bone marrow histology. a Refractory anemia with disordered and makroblastic hyperplasia of erythropoesis (NASD reaction, 400). b Peritrabecular association of erythropoesis (immunohistochemistry with glycophorine, 400). c Typical morphological dysplastic features of megakaryocytes with separated nuclei (HE staining, 630). d Typical morphological dysplastic features of megakaryocytes: micromegakaryocyte with a single, monolubulated round nucleus (green arrow) (HE staining, 630). e Typical morphological dysplastic features of megakaryocytes with peritrabecular association (HE staining, 630). f Typical morphological dysplastic features of megakaryocytes with micromegakaryocytes. Emphasized using immunohistochemistry (immu nohistochemistry with CD61, 400)

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10 μm

Fig. 6.18 MDS-RA: Bone marrow histology. Patchy distribution of hemosiderin (Prussion blue staining, 630)

megakaryocytes and pseudo-Pelger-Huet anomaly (i.e., nuclear hyposegmentation of granulocytes (see Fig. 6.13)) in the peripheral blood smear represent the most specific features of MDS. Other relevant morphological characteristics of myelodysplasia typically found in the peripheral blood, the bone marrow aspirate or bone marrow histological sections are collected in Tables 6.5 6.7. As cytological and histological findings are not absolutely diagnostic of MDS, the detection of cytogenetic anomalies typically associated with MDS can provide crucial ancillary information. This is especially true in cases in which morphologic findings are compatible with other causes, such as e.g., severe sepsis. However, these can often be distinguished by elevated acute phase proteins, clinical signs of infection, as well as a marked left shift and toxic granulation of neutrophils (see also: Differential

b

a

10 μm

20 μm

c

d

10 μm

20 μm

Fig. 6.19a RAEB-I: Bone marrow histology. Refractory anemia with excess of blasts characterized by 5 10% blasts and the progressive loss of normal hematopoletic architecture (NASD reaction, 400). b Abnormally localized immature precursors (ALIPs) as adverse prognestic feature (NASD reaction, 630). c Refractory anemia with excess of blasts characterized by 5 10% blasts visualized by immunohistochemistry (immunohistochemistry with CD34, 630). d RAEB-II: Characterized by 10 20% blasts visualized by immunohistochemistry (immunohistochemistry with CD34, 400)

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Table 6.6: Cytological findings in the bone marrow aspirate of patients with MDS (see also Figs. 6.14a d, 6.15 and 6.16)

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Table 6.7: Bone marrow histological findings in MDS (see also Figs. 6.17a f, 6.18 and 6.19a c)

Cytological findings in the bone marrow aspirate of patients with MDS

Histological findings in the bone marrow of patients with MDS

Erythropoiesis * Dyserythropoietic features characterized by: * Delayed/distorted nuclear and cytoplasmatic maturation * Erythroid hyperplasia with megaloblastoid features * Nuclear budding, multinucleation, karyorrhexis and cytoplasmatic vacuolization * Erythroid hyperplasia (in conjunction with ineffective erythropoiesis) often represents the predominant finding * Rarely red cell aplasia or hypoplasia occurs (in hypoplastic MDS) * Intranuclear bridging due to chromatin threads tethering dissociated nuclei, reflects impaired mitosis * Red cell membrane porosity (especially large pores in MDS/ AML) and loss of biconcave shape (observed by electron microscopy * Presence of acanthocytes and echinocytes (appearance of thorn and horn like structures) Myelopoiesis * ALIPS (atypical localization of immature precursors): * Granulocytic precursors with an apparent growth arrest at the myelocyte stage * Increased and abnormally located (centrally in the marrow space rather than along the endosteal surface) * Maturation of the cytoplasm progresses more rapidly than the nucleus * Increases in mast cells and/or plasma cells may be present * Bone marrow basophilia H1% (in 12%) * Bone marrow eosinophilia H5% (in 12%) * Basophilia þ Eosinophilia (in 4%) Megakaryopoiesis * Megakaryocytes (MKs) numerically normal or increased * Occasionally grouped in clusters * Morphological abnormalities: * Micro MKs * Hypogranular MKs * MKs with multiple dispersed nuclei (pawn balls) * Nonlobulated, large mononuclear forms (especially in association with 5q syndrome)

*

diagnosis of MDS: Table 6.8a and b). Furthermore, cytogenetics may be of prognostic impact and also guide treatment choices in the era of molecular targeted therapies. The classification of MDS has frequently been adapted to clinical experience and the influence of cytogenetics in diagnosis and prognosis. Table 6.1 characterizes the currently valid WHO classification of MDS page 172 (adapted from [8]), which has replaced the FAB (French American British) classification (see Table 6.9 page 172) [108]. In this context, it is important to keep in mind that the diagnostic distinction between RA/RARS/RCMD (see Figs. 6.14a d and 6.17a f), RAEB-I (see Fig. 6.19a and

* *

*

*

*

*

*

Hypercellular bone marrow accompanied by single or multilineage dysplasia is common Hypocellularity of the bone marrow in hypoplastic MDS is rare vWF staining: * vWF is synthesized by megakaryocytes (MKs) and stored in a granules of the platelets * Is the most reliable marker for identifying abnormal MKs in myeloproliferative diseases * Especially in MDS with del(5q) and myelofibrosis [494] * Abnormal multimeric vWF patterns are a marker for abnormal function in MK dysplasia [495] * CD61 to assess megakaryocytic patterning Iron stains: * Distinction of ring sideroblasts Glycophorin A and PAS stains: * To asses erythropoiesis with hyperplasia and/or peritrabecular location Peroxidase, sudan black, CD13, CD14 and CD33 stains: * Essential for quantifying myeloid progenitors ALIPs: * Granulocyte precursors displaced * From the normal paratrabecular location to more central marrow spaces * Commonly observed in advanced MDS subtypes Mild to moderate myelofibrosis ( 5%) and marked fibrosis (10 15%) * Detected by silver impregnation stains of reticulin fibers * Irrespective of subtype * Mature collagen (measured by trichrome stain) however, is uncommon in MDS

vWF von Willebrand Factor; ALIPs Abnormal localization of immature precursors

b), RAEB-II (see Figs. 6.15 and 6.19c) and AML (see Fig. 6.16) by G5%, 5 10%, 10 20% and H20% bone marrow blasts, respectively, is not based on pathobiological criteria. Therefore this distinction is arbitrary to a certain extent and not always consistent with biologic behavior. On rare occasions, patients with secondary AML, developed from a prior MDS, have smouldering leukemias with H50% bone marrow blasts, with stable disease for more than 2 years. These patients are best left untreated for as long as possible, and use of growth factors should be contemplated with caution, as any exogenous manipulation can disrupt and tip the balance of this extremely fragile homeostasis between host and leukemia. However, these are rare exceptions, and watching and waiting for too long, will have fatal consequences for most patients. An overview of diagnostic principles of MDS is given in Summary Box 1 (p. 177).

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Table 6.8a: Differential diagnosis of myelodysplasia

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Table 6.8b: Bone marrow changes associated with intake of certain drugs

Differential diagnosis of myelodysplastic syndromes Dysplastic changes due to deficiencies in nutrients or exposure to exogenic factors * Megaloblastic anemia due to nutritional disorders: * Vitamin B12 and/or folate deficiency result in: * Increased neutrophil granulation (rather than reduced lob ulation characteristic of MDS) * Exposure to toxic substances leading to dysplastic changes of the marrow * Alcohol (often also associated with vitamin B12 or folate deficiency) * Heavy metals * Dysplastic changes after radiation therapy Differential diagnosis to other BM diseases resulting in mono- or multilineage cytopenias * Hereditary causes of bone marrow failure * Autosomal recessive Fanconi Anemia (which can sometimes present in early adulthood) * Congenital dyserythropoietic anemia * Hereditary sideroblastic anemia * Other bone marrow failure syndromes (see Chapter 10) * Aplastic anemia * Pure red cell aplasia * PNH (see Chapter 9) * Hairy cell leukemia (CD103 positive cells with typical morphology, bone marrow fibrosis) * Cyclic neutropenia * Congenital neutropenia * Autoimmune neutropenia * LGL (often severe anemia and/or neutropenia and/or thrombocytopenia but without dysplastic changes in the bone marrow; H15% CD3 þ/CD57 þ lymphocytes in the peripheral blood, clonal TCR gene pattern as determined by PCR) * Overlap syndromes * The distinction of RAEB II from early evolving AML can only reliably be made after an observation period of at least 30 days * Atypical CML and JMML are often difficult to distinguish from CMML. In fact, many cases of Philadelphia chromosome negative CML seem to fulfil the criteria for CMML or other MDS subtypes Dysplastic hematopoiesis and (pan)cytopenia due to infections * HIV * Hypercellularity, myelodysplasia, megaloblastic hematopoiesis * Fibrosis, plasmocytosis, lymphatic aggregates and granulomas * These dysplastic features may result from: & Medications & Opportunistic infections & A direct cytopathic effect of the HIV virus * Parvovirus B19 * Non A, non B and non C hepatitis * Any severe infection

Transient dysplastic BM changes with cytopenias associated with intake of certain drugs Reversible dysplastic changes Macrocytosis * Reduced neutrophil lobulation and/or neutropenia * Thrombocytopenia * Dysplastic bone marrow changes in all three cell lines Drugs known to cause transient BM toxicty * Ganciclovir * Valproic acid * Mycophenolate * Alemtuzumab * Chemotherapeutics * Many antibiotics or antimycotics * NSAR * Anticonvulsants * Anti thyroid medications may all result in * *

BM Bone marrow; PNH paroxysmal nocturnal hemoglobinuria; LGL large granular lymphocytic leukemia; TCR T cell receptor; RAEB refractory anemia with excess blasts; PCR polymerase chain reaction; CML chronic myeloid leukemia; JMML juvenile mye lomonocytic leukemia; CMML chronic myelomonocytic leuke mia; HIV human immunodeficiency virus; NSAR non steroidal antirheumatic drugs Table 6.9: FAB classification of MDS (according to [108]) MDS BM subtypes blasts (%) RA RARS

G5 G5

RAEB RAEB t CMML

5 20 21 30 20

PB Additional blasts (%) features 1 or 1 G5 5 G5

Ringed sideroblasts H15% of nucleated erythroid cells Auer rods Monocytes H1,000/ml

BM Bone marrow; PB peripheral blood

6.8 Prognostic and Predictive Parameters in MDS 6.8.1 Cytogenetics in MDS 6.8.1.1 Frequency of Cytogenetic Aberrations in MDS Cytogenetic abnormalities are found in 60% of de novo MDS and 90% of therapy-related MDS (see Table 6.10), with deletion of 5q/monosomy 5 being the most frequent single chromosomal abnormality (16 28%) (see Fig. 6.20a), followed by del(7q)/monosomy 7 (see Fig. 6.20b) and trisomy 8 (10% with a male

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Table 6.10: Occurrence of chromosomal abnormalities according to FAB subgroup (adapted from [110] and [109]) Chromosomal abnormality

* * * * * * * * * * * *

3q21q26 del(5q) 7 del(7q) þ8 del(11q)/t(11q) del(12p) i(17q) del(17p) del(20q) þ21 Y

Incidence (%)

Median survival (years)

2.6 8.7 2.7 4.1 4.5 1.0 3.4

2.6 4.3 1.2 1.3 2.1 3.8/2.2 NR 0.9 NR 5.8 1.2 4.9

1.2 3.0 1.0

1-Year cumulative risk of transformation (%) 75 15.5 47 30 24.5 16/40 12.5 100 0 20 25 6

5-Year cumulative risk of transformation (%) 100 35.5 59 30 62.4 50/92 12.5 0 20 50 14.3

RA (%)

RARS (%)

RAEB (%)

0.8 9 2 0.8 6 1.5 1 0.4 0.8 1.5

0.6 3 0.6 0.6 5.6 2.5 1

0.4 8 5 2 5 1.5 2.5 2

1 4 6 4 5 1

2

4

0.7 0.4 0.4

1 01 1

1 3

RAEB-t (%)

3

CMML (%)

0.5 0.5 1 1 6 0.6 0.6 1 0.6 3.5 2

Note that the FAB classification has meanwhile been replaced by the WHO classification

predominance) (see Fig. 6.20c) [109]. Aberrations of chromosomes 5 and 7 have been significantly associated with environmental/occupational exposure to mutagens, as well as constitutional predisposition to myeloid malignancies. Monosomy Y (10%), del(11q) (5%), del(12) (p13) (10% of CMML, 5% of RAEB-I II), del(20q) (5%), and loss of 17p (4%) are also frequently found (see Table 6.10 and e.g., [109, 110]). Complex abnormalities are seen in 10 20% of de novo MDS, compared with H90% of therapy-related MDS. Using conventional metaphase cytogenetics, lesions can be found in only approximately 50% of MDS patients [111]. The advance of newer technologies such as comparative genomic hybridization or SNParray based karyotyping, allows the identification of cryptic lesions. With these methods LOH (loss of heterogeneity) resulting from uniparental disomy has been shown to occur frequently in MDS [111].

6.8.1.2 Clinical and Prognostic Features of Patients with Particular Cytogenetic Aberrations in MDS Deletions of 5q (see Fig. 6.20a) are significantly associated with longer overall survival and progression-free intervals (e.g., [109]; for more details see Sect. 6.19.1). Isolated del (5)(q13q31) separates a subtype of MDS with significantly better prognosis with lower progression rates to AML [112]. However, when additional cytogenetic abnormalities are present, the favorable prognostic value of isolated 5q deletion is lost, and an aggressive clinical course with considerably poorer survival can be expected [113]. In contrast to earlier beliefs, the clinical outcome of 5q

patients does not seem to be determined by the different breakpoints of interstitial deletion, but rather by the percentage of bone marrow blasts [109, 114]. Overall survivial for untreated MDS patients with complex chromosomal aberrations is 8 months and leukemic transformation occurs after a median of 0.9 years, compared to an overall survival of 40 months and leukemic transformation at 5.6 years for patients with one or two cytogenetic abnormalities [115]. Others have reported similar numbers, with median survival being 53.4 months for patients with normal karyotype, compared to 8.7 months for those with complex anomalies [116]. Estimated 3-year survival rates of patients with RA and 5q syndrome are 78% and 77%, respectively, whereas patients with additional 5q aberrations or other cytogenetic abnormalities have a 3-year OS rate of merely 25 31% [117]. Interestingly, patients with deletions of 5q are relatively insensitive to erythropoietins, resulting in clearly reduced responsiveness to erythropoietin substitution (response rates of 5 16% versus 20 37% in the general MDS population) [118, 119]. Monosomy 7, del(7)(q31q35) (Fig. 6.20b), i(17q) and 3q rearrangements are indicative of a very aggressive disease course and have been associated with significantly shorter overall survival [109]. 1q-rearrangements, translocations of 11q, del(12p), trisomy 8 and 9 as well as del (17p), have been segregated into a cytogenetic category portending intermediate prognosis [110], although contradictory results have been published [114]. Normal karyotype, loss of chromosome Y, del(11)(q14q23), del (20q) do not seem to negatively impact on survival and were therefore placed into the good prognostic cytogenetic category [110] (see also 6.8.3. and Table 6.13).

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a

Serial cytogenetic evaluation may be informative, as leukemic transformation is preceded by karyotypic evolution in 60% of cases. Overwhelming evidence exists for the power of karyotyping in predicting the response to chemotherapy and conventional cytogenetics should be complemented with molecular cytogenetic techniques such as multiplex FISH analyses, in order to detect complex chromosomal aberrations more precisely [120].

6.8.2 Molecular Factors Associated with Progression of the Disease b

Several gene alterations important for the development of AML have been implicated in the transformation process of MDS to AML: (i)

(ii)

c

(iii)

FLT3-length mutations and FLT3-tyrosine kinase muations are frequent in primary AML (35% and 6%, respectively), but rare in MDS [83]. However, when present at MDS diagnosis they predict unfavorable prognosis due to imminent leukemic transformation [121]. In line with the above, FLT3 mutations more frequently occur with transformation to secondary AML [83]. N-Ras mutations frequently occur in the absence of cytogenetic aberrations and are then thought to represent initial events in the MDS process, requiring further events for leukemic transformation [83, 122]. In contrast, an increase of N-Ras mutations with progression through the MDS stages has been shown [83, 123, 124]. These data might indicate a dual role of the Ras mutation, which may be either an initiation or progression event, depending on genetic background and/or other factors present in individual patients. Bm-1, a transcriptional repressor gene, has important functions in regulating self renewing capacity

3 Fig. 6.20a MDS del (5q) (associated with a better prognosis when not accompanied by other chromosomal aberrations). Fluorescence in situ hybridization (FISH) of MDS cells from bone marrow aspirate: on the left hand side is a cell with two copies of the long arm (Spectrum orange 5q31) as well as the short arm of chromosome 5 (Spectrum green 5p15.2), whereas only one copy of 5q (Spectrum orange) is visible in the cell on the right. b MDS del (7q) (associated with worse prognosis). Fluorescence in situ hybridization (FISH) of MDS cells from bone marrow aspirate: clearly, there is only one signal for the long arm of chromosome 7 (Spectrum orange 7q31), whereas there are two signals for the centromeric region of chromosome 7 (Spectrum green CEP 7). c Trisomy 8 (often associated with a tendency for better prognosis and/or hypoplastic MDS). Fluorescence in situ hybridization (FISH) of MDS cells from bone marrow aspirate: three fluorescent signals for the marker of the centromeric region of chromosome 8 (Spectrum aqua CEP 8)

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in hematopoietic and leukemic stem cells ([125] and reviewed in [126]) and has been implicated in progression to advanced stage MDS and secondary AML [127]. Hypermethylation of the cak N2B gene, which encodes for pINK4b, is not only associated with MDS pathogenesis, but also with poor prognosis and disease progression to AML [306, 307] (for details see Sect. 6.12.1).

Given the complex pattern of cytogenetic and molecular aberrations described in MDS, it remains difficult to describe common pathways of cooperation utilized for the development and progression of MDS along the two hit hypothesis. However, at least two pathways seem to emerge: (i)

(ii)

A pathway in which AML-1 point mutations are an initial event causing the failure of hematopoietic differentiation and a rather long latency period for transformation [128]. In this pathway, Ras mutations, NF-1, PTPN-11 or FLT3 mutations might be cooperative and the frequent association with deletion in chromosome 7 be important. In a second pathway, the initial events remain elusive. Hematopoietic stem cells are wild type for AML-1, but frequently develop p53 mutations and 5q deletions [128]. Haploinsufficiency for nucleophosmin, which maps to chromosome 5q35, has been supposed to have a role as master gene in this regard [77, 128].

6.8.3 Prognostic Scoring Systems in MDS Apparently all classification systems used in the past were capable of providing prognostic information used for treatment decisions. As with many classification systems in hematology and oncology, novel classification systems are suitable for a clearer separation of entities, higher reproducibility of diagnosis and probably better prediction of the course of the disease.

However, for an adequate interpretation of clinical trial results in the past, the previously used FAB (French American British) classification (Table 6.9) will have to be kept in mind for many further years. Tables 6.11 and 6.12 report the prognostic impact of the FAB and the currently valid WHO (World Health Organization) classification (Table 6.1), which has been accepted as the standard classification for MDS [129]. More recently, individual parameters distinct from the WHO classification system have been used for the establishment of prognostic scores. This attempt is based on the understanding, that parameters which optimally discriminate the diagnostic entities might not be identical with the parameters optimally predicting the course of the disease. Tables 6.13 and 6.14 depict two important scoring systems of prognosis in MDS, i.e., the International Prognostic Scoring System (IPSS) [115] and the scoring system of the Grupo Cooperativo Espanol de Citogenetica Hematologica (GCECGH) [110]. The IPSS stratifies patients into four risk groups with different risks of death and leukemic transformation, according to the number of peripheral cytopenias, cytogenetics and percentage of bone marrow blasts [115]. This scoring system has been validated multifold, and was considered the gold-standard in prognosis prediction in MDS by many [130 133]. The prognostic stratification of both the WHO-classification and the IPSS can be improved on when revised cytogenetic categories are used [109]. These revised IPSS cytogenetic categories were more effective for predicting the risk of evolution to AML than the standard IPSS categories [109]. A retrospective study of the Spanish cooperative group for cytogenetics in hematology (GCECGH) analyzed 968 MDS patients and further refined the IPSS score. In particular, the GCEGH system uses four instead of three cytogenetic categories thereby leading to a better segregation of several specific chromosomal abnormalities assigned to the intermediate prognostic subgroup in the IPSS. In direct comparison with the IPSS, the GCECGH scoring system better discriminated for the risk of leukemic transformation and overall survival in cases in which cytogenetic information was available [110] (see Fig. 6.21).

Table 6.11: Prognosis of patients with MDS according to the FAB classification (adapted and modified from [5]) MDS subtypes

Median OS (years)

1-Year survival (%)

3-Year survival (%)

Incidence/100,000 for ESP (m/f)

RA RARS RAEB RAEB t MDS NOS 5q syndrome MDS all subtypes

3.3 H3.8 1.2 0.6 2.5 12.2 2.7

76 77 53 39 66

54 58 21 29 44 78 45

1.75/1.14 0.46/0.32 0.80/0.3 0.40/0.23

67

4.56/2.81

FAB French American British; ESP European standard population; NOS not otherwise specified; OS overall survival

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Table 6.12: Prognosis of patients with MDS according to WHO subtypes (adapted from [110]) MDS subtypes

Median overall survival (years)

RA RARS RCMD RCMD-RS RAEB-I RAEB-II del(5q) MDS-U

2.3 4.8 2.3 2.7 0.9 0.6 2.4 5.2

1 year cumulative risk of transformation (%) 12.8 5.9 15.5 7.7 38.0 45.2 20.5 17

5 year cumulative risk of transformation (%) 28.8 5.9 29.3 7.7 71.9 20.5 17

WHO World Health Organization

In line with the above, a recent external validation of the IPSS could not confirm prognostic significance of the IPSS [134]. Recently, a newer prognostic model, the World Classification-based Prognostic Scoring System (WPSS) has been designed (Table 6.15). This score stratifies patients into five subgroups and is based on the variables of the WHO classification, cytogenetics and importantly, transfusion requirements (not present in the IPSS) [135]. The WPSS predicts survival and leukemic evolution at any time-point during follow-up, compared to the IPSS, which is effective in predicting the outcome of treatment after diagnosis, but was not designed to provide prognostic information at any time after diagnosis, irrespective of

Table 6.13: IPSS (international prognostic scoring system) Parameters involved in the prognostic system

Score Score points points 0 0.5

Score Score points points 1.0 1.5

BM blasts Karyotypea

G5% Good

Poor

Cytopeniasb

0/1

5 10% Interme diate 2/3

Score points 2.0

11 20% 21 30%

a

Karyotype definitions: * Good: normal; Y, del(5q), del(20q) * Poor: abnormalities of chromosome 7, complex (3) aberrations * Intermediate: all other abnormalities b Cytopenia definitions: * RBCs: HbG10 g/dl * WBCs: ANCG1,800/ml * Platelets: PLTG100,000/ml IPSS scores for risk groups: * Low: 0 points * Intermediate 1: 0.5 1.0 points * Intermediate 2: 1.5 2.0 points * High: 2.5 3.5 points

whether the patients are treated or newly diagnosed [135, 136]. The WPSS, a novel dynamic time-dependent prognostic scoring system, has been externally validated and compared with the IPSS, and seems to provide more reliable and powerful stratification of patients than the latter [134, 136]. Especially very low risk patients with an excellent long-term survival and rare progression to overt leukemia could be singled out by the WPSS, but not

Table 6.14: GCECGH (Grupo Cooperativo Espanol de Citogenetica Hematologica) scoring system (adapted from [110]) Points Overal survival Age (years) Hb (g/dl) Cytopenias FAB subtype GCECGH cytogenetic subgroupa Leukemic transformation Hb (g/dl) FAB subtype GCECGH cytogenetic subgroupa a

0

0.5

1

1.5

60 10.0 0 1 RA, RARS, CMML Good

H60 G10.0 2 3 RAEB, RAEB t Intermediate

Unknown

Poor

10.0 RARS Good

G 10.0 RA, CMML Intermediate

Unknown

RAEB, RAEB t Poor

Karyotype definitions: * Good: normal; Y, del(5q), del(20q), del(12p), del(11q) * Intermediate: trisomy 8, rearrangements of 3q21q26, translocations of 11q, del(17p), trisomy 18, trisomy 19 * Poor: monosomy 7, del(7q), i(17q), complex (3) aberrations * Unknown prognosis: all remaining cases with single or double abnormalities SPS (Spanish prognostic score) scores for risk groups: * Low: 0 0.5 * Intermediate: 1 1.5 * High: 2.0 2.5 * Very high: 3

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a

GCECGH score IPSS score

Summary Box 1: Bone marrow diagnostic principles – things to be considered in MDS diagnosis (modified according to [129, 512]) *

*

low/good

intermed

high/poor

very high

GCECGH score IPSS score

*

*

b

*

*

*

low/good

intermed

high/poor

very high *

Fig. 6.21a The comparison of prognostic scores (i.e., IPSS and GCECGH) in terms of leukemic transformation (adapted from [110]). All numbers depicted represent % leukemic transformation in the respective risk categories, according to the GCEGH (brown) or IPSS (beige) score. GCECGH: Grupo Cooperativo Espanol de Citogenetica Hematologica; IPSS: international prognostic scoring system. b The comparison of prognostic scores (i.e., IPSS and GCECGH) in terms of overall survival (adapted from [110]). All numbers depicted represent years of overall survival in the respec tive risk categories

by the IPSS [134]. Furthermore, the WHO classification and the WPSS are capable of predicting post-allogeneic stem cell transplantation outcome of MDS patients [137].

*

*

Although dysplasia is the hallmark of myelodysplasia, the interobserver reproducibility of dyserythropoiesis (r ¼ 0.27) and dysgranuluopoiesis (r ¼ 0.45) is extremely poor [513]. A May-Gruenwald or Wright-Giemsa stain is mandatory to adequately detect cytosplasmic granules. Blasts, monocytes, and ring sideroblasts must be quantified. Myeloblasts, monoblasts and megakaryoblasts are counted as blasts whereas erythroblasts are not (except in the rare erythroblastic leukemias). In myelomonocytic leukemias, promonocytes are considered blast equivalents. Not all blasts express CD34, which underestimates the blast count, but immunophenotyping may be helpful in diagnosing MDS. A small number of erythroid, megakaryocytic or granulocytic cells can be dysplastic in healthy people [514]. Consider a 10% cut-off value per lineage to diagnose dysplasia. All biopsies should be core biopsies. Do not rely on material from the immediate subcortical area, which is often hypoplastic. There is no single cytogenetic aberration typical for MDS, but the presence of chromosomal aberrations may be supportive of diagnosis. Abnormal karyotypes may occur in patients with unexplained cytopenia(s) without dysplasia and are sometimes considered formes frustes of MDS, which often persist and may lateron transform into overt MDS [451].

Table 6.15: WHO Classification Based Prognostic Scoring System (WPSS) (adapted from [134]) Parameters involved in the prognostic system

Score points 0

Score points 1

Score points 2

Score points 3

WHO category Cytogenetic riska Transfusion requirement

RA/RARS/5q Good No

RCMD/RCMD RS Intermediate Yes

RAEB I Poor

RAEB II

a

Karyotype definitions: * Good: normal; Y, del(5q), del(20q) * Poor: abnormalities of chromosome 7, complex (3) aberrations * Intermediate: all other abnormalities WPSS scores for risk groups: * Very low: 0 points * Low: 1 points * Intermediate: 2 points * High: 3 4 points * Very high: 5 6 points

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6.8.4 Other Prognostic Markers in MDS Bone marrow histology and assessment of ALIPs (abnormally localized immature precursors) and CD34 immunoreactivity have been proposed to further improve the IPSS prognostic subcategorization of MDS patients [138]. Flow-cytometric analysis of MDS bone marrow aspirate demonstrates hyperexpression of CD13 on mature neutrophils as an independent risk factor [139]. Additional flow-cytometric aberrancies in the cell surface marker composition of the myelomonocytic lineage, which may distinguish MDS with pending transformation, are discussed by van de Loosdrecht et al. [140]. Other important variables found to be independent prognostic factors on multivariate analysis, include age and LDH levels [134, 136]. Not surprisingly, a progressive dependence on transfusions and refractoriness to hematopoietic growth factors is associated with an inferior overall outcome. The entity MDS-unclassified lacks specific prognostic markers, but these patients generally tend to have a poor prognosis [141]. As mentioned in Sect. 6.3.2, patients with MDS suffer from abnormal immune function of T-lymphocytes associated with the imbalance between Th1 and Th2 cells. A decreasing number of Th1 cells has been demonstrated in high-risk MDS patients with RAEB or RAEB-t, and the loss of the surface activation markers CD69 and HLA-DR [142] was indicative of disease progression to leukemia [143]. The survival of patients without immunological abnormalities and a CD4 þ/CD8 þ ratioH1 is significantly improved over those with an inverse scenario [21, 22]. Furthermore, immunologic abnormalities seem to be associated with disease progression and/or opportunistic infections [144].

6.9 Best Supportive Care (BSC) of Patients with MDS 6.9.1 Transfusion of Red Blood Cells and/or Platelets Supportive care, including red blood cell- and platelettransfusions, is still the mainstay of MDS treatment. It is important to keep hemoglobin levels high enough to avoid symptoms of anemia, and not to wait until patients become symptomatic again in the transfusion intervals. Generally the initiation of transfusion therapy should be based on individual anemia- and/or thrombocytopenia related symptoms, and not on predefined hemoglobin or thrombocyte levels. In clinical practice, hemoglobin levels of 8 9 g/dl usually trigger initiation of red blood cell

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transfusions, and platelet transfusions should be considered in patients with less than 20,000 platelets/ml, and definitively given in patients with less than 15,000 platelets/ml and in all patients with bleeding symptoms, irrespective of platelet counts. Sudden decreases in hemoglobin levels however, must be evaluated accordingly, irrespective of the fact that the patient has MDS. Gastrointestinal blood loss and other reasons for sudden aggravation of anemia such as autoimmune hemolytic anemia for example, must be excluded. Platelet transfusion therapy may be associated with several limitations, including refractoriness, alloimmunization, transmission of HIV or hepatitis C and transfusion reactions. Furthermore, the availability of blood products can also become problematic in some instances. Unexpected high transfusion requirement may result from the development of alloantibodies, splenomegaly, or gastrointestinal hemorrhages due to other neoplasias or angiodysplasias. Some patients develop allergic reactions to transfusions and necessitate prior application of steroids (75 250 mg, depending on severity of reaction) and/or antihistamins. Thus patients must carefully be followed for degree and reasons for transfusion requirement and number of red cell packages and intervals must carefully be individualized. Improvement of chronic anemia and the reduction of transfusion dependency significantly improves health-related quality of life (e.g., [145 148]). Transfusion dependency has a significant impact on overall survival and risk of leukemic transformation [149], which is the reason why this factor was included in the newest risk score, WPSS (see Table 6.15) [135]. This may be due to advanced or more aggressive disease, but secondary effects of chronic anemia eo ipso, such as cardiac remodeling (see Sect. 6.9.6), or transfusion-related iron overload (see Sect. 6.9.6) are likely to play an additional role. Anemia leads to increased cardiac output and gradual left ventricular hypertrophy associated with increased cardiac morbidity and mortality. The risk of cardiac remodeling is in part triggered by hemodynamic compensation of anemia and is reduced by a sustained elevation in hemoglobin [150]. Therefore patients with cardiac disease and advanced NYHA stages should be transfused more generously, and hemoglobin levels should be kept between approximately 9 and 10 g/dl in these patients.

6.9.2 Erythropoietin (EPO) Recent reports of adverse effects of erythropoietin (EPO) substitution in patients with solid tumors have caused considerable discussion of the use of EPO with or without G-CSF for the treatment of anemia in MDS patients. After considerable debate and review of the literature however,

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the current American Society of Clinical Oncology/ American Society of Hematology (ASCO/ASH) guideline still recommends the use of these growth factors for patients with low risk MDS [151]. The risks of thromboembolism must be considered and weighed, as randomized clinical trials demonstrate an increased risk of thromboembolism in patients receiving erythropoietin stimulating agents, especially in combination with other thrombogenic drugs such as thalidomide [152 154]. Erythropoietins may be successfully used in a proportion of patients with MDS. However, doses higher than those needed for the treatment of chemotherapy-induced anemia are required to induce responses in MDS, with doses up to 60,000 U/week [155]. It is currently unclear whether response to erythropoietin results from stimulation of normal residual hematopoiesis, differentiation of the abnormal clone, or a combination of both [147]. A meta-analysis of 17 studies reported response rates of 15 37% for recombinant human erythropoietin as monotherapy in MDS patients [118]. Interestingly, whilst 20 37% of unselected MDS patients respond to erythropoietin therapy, only 6 14% of patients with 5q deletions do so [118, 119]. More recently, 59 studies were reviewed in a meta-analysis. This meta-analysis revealed efficacy of all studied erythropoietin stimulating agents, with slightly higher response rates in darbopoietin patients than in epoietin patients, but similar safety profiles [156]. Hb response was 48% in single arm darbopoietin studies, 32% in epoietin single-arm studies and 27% in epoietin versus control trial [156]. However, this seems to be an artefact caused by the use of different response criteria, and could not be confirmed by others [157]. Response rates were shown to be similar for both substances (57.6% vs. 59.4%) in more recent studies that primarily utilized the international working group (IWG) criteria for evaluating response [157], and the Update Committee for the ASCO/ASH guidelines on use of erythropoiesis stimulating agents considers these agents equivalent with respect to effectiveness and safety [151]. Hematologic erythroid improvement is defined as an hemoglobin increase by 1.5 g/dl and/or a relevant reduction of transfused red blood cell units by an absolute number of at least 4 RBC transfusions per 8 weeks, compared with the pre-treatment transfusion number in the previous 8 weeks [158]. Several parameters predictive of erythropoietin response have been defined in the past. Low risk IPSS, endogenous serum erythropoietin levels less than 500 U/ L, absent or low transfusion requirements (G2 U/month), the presence of RARS and a hypocellular marrow predict a favorable erythropoietin response [147, 159, 160]. Generally most patients are treated for 12 16 weeks before erythropoietin substitution is deemed inefficient,

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although prolonged administration of up to 6 months has been reported to increase erythroid response rates [161]. We use a period of 12 weeks to evaluate efficacy, and when a patient does not respond within this time period, we add G-CSF for a period of several weeks (if the patient doesnt respond with hyperleukocytosis) (see below in Sect. 6.9.3).

6.9.3 G-CSF and Combination Treatment of EPO with G-CSF G-CSF (granulocytic colony stimulating factor) not only blocks apoptosis of neutrophilic granulocytes, but also of erythroid progenitors, via suppression of both spontaneous and Fas-mediated apoptosis [162]. These effects seem especially predominant in low risk MDS patients, and are most prominent in RARS [162]. It is thought that the potent antiapoptotic effect of G-CSF may allow erythroid progenitors to survive until they become erythropoietin responsive [163]. In vitro experiments show synergistic effects of erythropoietin and G-CSF on inhibition of apoptosis of myelodysplastic erythroid precursors via blockage of constitutive cytochrome-C release [162, 164]. Furthermore, these growth factors preferentially promote the outgrowth and survival of cytogenetically normal cells from 5q patients [164]. In fact, a dramatic reduction of 5q clones from a median of 97% to 35% was observed [164]. Notably, this in vitro synergistic effect of erythropoietin and G-CSF on erythroid response is also seen in vivo, predominantly in RA and RARS patients with higher erythroid response rates (40 50%) for patients receiving the combination (see also Table 6.16). This has been shown by many phase II and

Table 6.16: Response rates and duration in patients treated with Epo and G CSF (adapted from [160]) Prognostic subgroups of MDS * * *

*

*

IPSS low/intermediate 1 IPSS intermediate 2/high Good predictive group for responsea Intermediate predictive group for responseb Poor predictive group for responsec

RR (%)

CER (%)

Median duration of response (months) 25 7 24

46 27 60

27 12 36

18

9

23

6

0

3

CER Complete erythrocytic response; RR overall response rate a Serum EPO levels G500 U/l and no transfusion requirement b Serum EPO H500 U/l or transfusion requirement c Serum EPO H500 U/l and transfusion requirement

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III clinical trials (e.g., [165 169]). Long-term observations of 129 MDS patients treated with G-CSF and erythropoietin simultaneously, show long-lasting responses (median response duration 23 months) and transfusion independence in 40% of patients [160]. Patients with low or intermediate-1 IPSS scores had longer response durations than patients with higher risk scores (25 vs. 7 months), with no signs of increased blast proliferation after treatment and no influence on leukemic transformation or negative effect on overall survival in comparison with supportive care alone [160] (see also Table 6.16). This has recently been confirmed by others who demonstrated that treatment of MDS patients with erythropoietin and G-CSF was not linked to the rate of progression to acute myeloid leukemia in any defined subgroup, including patients with RAEB-1, RAEB-II or an unfavorable karyotype [170, 171]. In contrast, multivariate analysis revealed that combined growth factor treatment was associated with improved overall survival for low risk patients[170,171].Thispositiveeffectonoverall surviv-al in low risk groups is attributed to several factors: (i)

(ii) (iii)

Correction of anemia per se, with decreased incidences of cardiac failure due to anemia induced cardiac remodeling (see above). Prevention of iron overload and secondary consequences (see Sect. 6.9.6). Potential antitumoral effects due to immunomodulatory properties of erythropoietin on MDS T-cells, as well as induction of increased tumor antigen presentation capacity of hematopoietic precursor cells, as shown in murine experiments [170, 172].

However, it is still necessary to document disease stability and it is recommended to control the percentage of blasts in blood and marrow. Factors predicting response to the combination therapy of G-CSF and EPO include presence of WHO subcategory RA or RARS, serum erythropoietin levels of less than 500 U/l, transfusion requirement of less than 2 red blood cell units per month and reticulocyte response till day 7 [159, 173]. Patients without transfusion requirements and low serum-erythropoietin have 74% probability of erythroid response, whereas patients with one or both of these unfavorable factors have response rates of 23% and 7%, respectively.

6.9.4 Thrombopoietin (TPO) and TPO Mimetics Thrombopoietin (TPO), also known as megakaryocyte growth and development factor (MGDF), was first

purified in 1994. Since then several hematopoietic growth factors with thrombopoietic activity have been identified, i.e., GM-CSF, cKIT ligand, IL-1, IL-3, IL-6, and IL-11. Whereas TPO is relatively lineage specific, the afore mentioned cytokines have pleiotropic effects (reviewed in [174]). TPO is the primary regulator of both the proliferation and maturation of megakaryocytes into platelet producing cells. Furthermore, TPO induces proliferation of early myeloid and erythroid progenitors [175] and has a synergistic effect on erythro/myelopoiesis when combined with other hematopoietic growth factors such as erythropoietin or stem cell factor [176 178]. Several recombinant forms of TPO or TPO-mimetics have been developed in the past two decades, although none of them are currently approved for the treatment of MDS by the FDA or EMEA: (i)

(ii) (iii)

(iv)

Polyethylene glycol conjugated truncated nonglycosylated recombinant MGDF (PEGrHuMGDF), which has been withdrawn from clinical trials due to induction of neutralizing antibodies. Recombinant full length human TPO (rHuTPO). AMG-531 (romiplostim, Nplate), a subcutaneously applied Fc-peptide fusion protein (peptibody), represents recombinant TPO with a peptide fragment that shares no sequence homology with endogenous TPO. Romiplostim was recently approved by the FDA for the treatment of autoimmune thrombocytopenia. Oral non-peptide TPO mimetics eltrombopag and AKR-501 (YM477), are currently in clinical trials.

There is a typical lag-time of 4 5 days before the platelet count rises in response to TPO and TPOmimetics, reflecting the fact that TPO acts primarily on early and not late precursor cells [174]. In patients with low risk MDS, the endogenous TPO level is often elevated [179], and especially so in refractory anemia (MDS-RA) patients. Interestingly, in patients with RAEB or RAEB-t, there is no such inverse correlation between platelet and megakaryocyte counts and serum TPO level [180]. It has been suggested that TPO, and thus possibly also TPO-mimetics, may induce blast proliferation in some cases. In line with this hypothesis, MDS blast cells from patients with RAEB or RAEB-t have been shown to express TPO-receptor mRNA, whereas CD34þ cells from MDS-RA patients seem to have 50% downregulated TPO-receptor proteins [180]. Furthermore, TPO was shown to stimulate in vitro proliferation and increase blast numbers in 9/ 16 high risk MDS primary bone marrow cultures, but in none of the 11 cases with low risk MDS [181]. This effect on blast cell growth is not only restricted

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to endogenous TPO, but also applies to recombinant variants, as e.g., PEG-rHuMGDF also stimulated growth of MDS blast cells in vitro [182]. In addition, it must be kept in mind, that both rhTPO and PEGrHuMGDF have been successfully used for mobilizing and increasing the harvest of CD34þ cells used for stem cell transplantation (reviewed in [174]), as well as for the ex vivo expansion of primitive stem cells [183]. Therefore TPO-mimetics must be applied with extreme caution in high-risk MDS patients, especially so in those with high risk scores for transformation to AML.

6.9.4.1 PEG-rHuMGDF Several years ago small clinical trials were reported in which various intravenous doses of PEG-rHuMGDF were given to MDS-RA and RARS patients. Responses were observed in approximately one third of patients, and multilineage effects (e.g., elevation of hemoglobin levels) were recorded in a few [184]. However, administration of more than one dose of PEG-rHuMGDF resulted in unacceptable rates of development of neutralizing antibodies which not only abrogated the effects of PEG-rHuMGDF, but also of endogenous TPO via cross-reactions (e.g., [185, 186]). Thrombocytopenia occurred in approximately 2/210 (1.2%) and 11/124 (8.9%) of healthy volunteers who received two or three doses, respectively [185, 187]. In rare cases, thrombocytopenia caused by formation of an IgG antibody to PEG-rHuMGDF that cross-reacted with endogenous TPO coincided with development of neutropenia and anemia, suggesting an effect on the stem cell population [185, 186]. As a consequence of these results, PEG-rHuMGDF was withdrawn from clinical trials in 1998 by Amgen.

6.9.4.2 Recombinant Human TPO (rHuTPO) Recombinant human TPO (rHuTPO) represents the fulllength gylcosylated protein, which is identical to endogenous TPO. Due to the cross-reactivity of neutralizing antibodies induced by PEG-rHuMGDF, the clinical development of rHuTPO was put on hold. In the meantime several Chinese groups have conducted clinical trials and rHuTPO has been tested and deemed safe and efficacious in the treatment of patients with solid tumors with chemotherapy induced thrombocytopenia [188, 189]. Intravenous rhTPO (PN-152,243)/(PN-196,444) is currently being evaluated in phase III clinical trials in the prevention of thrombocytopenia in patients receiving myelosuppressive treatment regimens requiring platelet transfusion support (e.g., ClinicalTrials.gov Identifier NCT00037791).

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6.9.4.3 Romiplostim (AMG531, Nplate) Romiplostim has recently been approved by the FDA for the treatment of thrombocytopenia in patients with chronic idiopathic thrombocytopenic purpura (ITP) who have had an insufficient response to corticosteroids, immunoglobulins or splenectomy. Romiplastim activates transcriptional intracellular pathways via the TPOreceptor (c-mpl) and thus stimulates platelet production by a mechanism similar to endogenous thrombopoietin. The fact that romiplostim shares no sequence homology with the endogenous TPO prevents the production of neutralizing cross-reactive antibodies. Recent studies have shown its efficacy (at a dosage of 1 10 mg/kg weekly given subcutaneously), tolerability and longterm safety in patients with ITP (reviewed in [190]). In an extensive immunogenicity assessment, 25/236 (10.5%) subjects with ITP developed binding antibodies against romiplostim and 12/236 (5.1%) developed binding antibodies against TPO, but no clinical sequelae were observed in the presence of these binding antibodies, as they did not have neutralizing capacity [191]. Importantly, the incidence of anti-romiplostim neutralizing antibodies was only 1/236 (0.4%), and no anti-TPO neutralizing antibodies could be detected in these patients [191]. Several clinical trials testing romiplostim, in MDS patients are completed or currently recruiting (ClinicalTrials identifiers: NCT00472290, NCT00614523, NCT00303472). Preliminary data of an ongoing phase 2 multicenter, randomized, double-blind, placebo-controlled study evaluating the effect of romiplostim on the incidence of clinically significant thrombocytopenia in patients with low or intermediate risk MDS receiving azacytidine, were presented at ASH 2008 [192]. In this patient group, romiplostim was well tolerated in combination with azacytidine and reduced azacytidine induced thrombocytopenia as well as bleeding complications resulting thereof [192]. However, romiplostim still not approved by the authorities in the US and/or Europe for the treatment of MDS. Romiplostim may increase bone marrow fibrosis, and this question will be answered by several clinical trials that are investigating this hypothesis.

6.9.4.4 Oral TPO Mimetics Eltrombopag and AKR-501 (YM477) Results from 197 ITP patients included in the RAISE trial, a randomized double-blind placebo-controlled phase III study, demonstrating the efficacy, safety and tolerability of longterm oral treatment with eltrombopag (50 75 mg/ day p.o), were very recently published [193, 194]. Platelet

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increase becomes significant after 7 10 days and peaks between 10 14 days [195]. Although a plethora of clinical trials testing the efficacy of eltrombopag in a variety of conditions with thrombocytopenia are underway, there are currently only 2 trials for MDS patients listed at clinicaltrials gov (time of writing 02/10) (ClinicalTrails. gov identifier NCT00903422, NCT00961064). AKR-501 (YM477) is an orally active small molecule TPO-receptor agonist that was shown to elevate platelet counts in human platelet producing non-obese diabetic/ severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver CD34þ cells [196]. AKR-501 was well tolerated in multiple dose studies and elevated the platelet count in healthy volunteers [197]. Currently several clinical trials are open for oral administration of AKR-501 in patients with ITP. Studies in the setting of MDS are eagerly awaited. Although the response rate for patients treated with romiplostim or eltrombopag is above 70 80% in multiply pretreated and splenectomized patients with ITP, the response is almost invariably lost after several months of successful administration [195]. Thus, it remains to be seen, whether longterm beneficial effects on thrombopoiesis will occur in MDS patients receiving TPO mimetics.

6.9.5 Other Drugs for Palliative Amelioration of Cytopenia Other drugs that have been used in MDS for alleviation of anemia and/or thrombocytopenia include danazol, partly in combination with retinoic acid and/or corticosteroids [198 205] (see also respective sections in the chapter on Primary Myelofibrosis (Chapter 4)).

6.9.6 Iron Chelation Therapy (ICT) 6.9.6.1 Deleterious Sequelae of Iron Overload in MDS Patients Widespread (sub)clinical organ dysfunction results from transfusional iron overload in MDS patients and the pattern of organ involvement resembles that of idiopathic hemochromatosis. Excessive accumulation of iron leads to transferrin saturation and circulation of non-transferrin-bound iron, which leads to the formation of labile plasma iron. The latter is deposited in the parenchymal cells of liver heart, pancreas, brain and joints, where it generates reactive oxygen species that damage cell membranes, proteins and DNA [206]. Consequences are fibrogenesis and lipid peroxidation, resulting in congestive

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heart failure, arrythmias, hepatic cirrhosis, hepatocellular carcinoma, insulin resistance, diabetes mellitus, arthritis and an increased rate of infections [207]. Iron overload secondary to transfusion dependency represents a significant problem in all MDS patients requiring red blood cell transfusions at regular intervals. Development of secondary iron overload significantly affects the survival of transfusion-dependent low-risk MDS patients, and a 40% increase in the risk of death has been reported for every rise in 500 ng/ml of ferritin [149]. 10/16 patients with MDS-RA or aplastic anemia who had received a mean of 120 RBC units typically displayed focal portal fibrosis and had a 26-fold elevation of liver iron deposition compared to non-transfused patients [208]. All patients had glucose intolerance associated with significantly reduced insulin output, compared with controls. Pituitary reserve of adrenocorticotropin was limited in 10/12 patients, and that of gonadotropin in 5/ 13 patients [208]. Cardiac iron overload, grossly visible in autopsy, led to measurably reduced left ventricular cardiac output in the most heavily transfused patients or in those with coexisting coronary-artery disease cardiac iron overload is associated with chronic cardiac failure [208, 209]. Secondary iron overload is associated with abnormal liver (85%) and cardiac function (22%) [207]. A retrospective study of 840 MDS patients confirmed that transfusion dependent patients had a significantly higher risk of heart failure (28% vs. 18%) and cardiac death (69% vs. 18%) [211]. It has frequently been shown that once MDS patients develop red blood cell (RBC) transfusion dependence (e.g., [210]), their death hazard ratio increases, which is why this factor was included in the newest prediction score, the WPSS (see Table 6.15). As already discussed above, the following factors are thought to contribute to the reduced overall survival of transfusion dependent patients: (i) disease progression, (ii) cardiac remodeling due to chronically low hemoglobin levels and (iii) consequences of secondary iron overload. Furthermore, non-transferrin bound iron and labile plasma iron are taken up by tissues, where the production of reactive oxygen species, which are one of the major determinants of iron toxicity, is catalyzed. This effect cannot be measured by MRI, which merely visualizes storage iron [66]. Cardiac damage due to secondary hemosiderosis may therefore be underestimated. Most studies concerning iron chelation therapy have been conducted in patients with thalassemia major requiring multiple blood transfusions. In these patients, adequate iron chelation with deferoxamine clearly demonstrated a survival benefit by preventing the complications of iron overload, albeit after a long follow-up of ten years or until death [212]. MDS patients are generally much older than thalassemia major patients, and thus

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have a significantly higher frequency of cardiac and other comorbidities. It may thus be expected, that MDS patients are more vulnerable to the toxic effects of secondary iron overload resulting from multiple transfusions of packed red blood cells. Keeping this in mind, survival benefits may be seen after a much shorter time period of iron chelation in MDS patients.

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liver toxicity [221 224]. Future trials examining expected benefits of pre-transplant iron chelation therapy in this setting are awaited. Thus, in total one third to 55% of all MDS patients are expected to benefit from ICT [215].

6.9.6.4 When Should ICT Be Initiated and for How Long? 6.9.6.2 What are the Goals of ICT? The major goal of ICT in MDS patients is to avoid sequelae of chronic iron overload. Long-term chelation can reverse functional complications such as liver fibrosis, arrhythmia and echocardiographic abnormalities, but not complications due to extensive tissue alterations, such as frank diabetes, hypothyroidism and myocardiosclerosis [213]. Especially myocardial iron accumulation is a feared long-term consequence of secondary hemosiderosis. As many MDS patients are elderly and have preexisting cardiac disease, further cardiac damage due to iron overload is one of the primary goals of long-term ICT. Cardiac iron overload seems to be dependent on transfusion intensity and not on total transfused blood volume per se [214], but at least 75 RBC-units seem to be required to render the majority of patients positive for cardiac iron overload [209]. However, ICT is expected to be beneficial prior to the development of gross tissue iron deposition [215]. The reduction in iron overload was not supposed to modify the course of MDS, although improvements in myelopoiesis have been reported [216].

6.9.6.3 In Whom Should ICT Be Considered? Several consensus statements on diagnosis and treatment of iron overload in MDS have been published in the last years (e.g., [215, 217 220] and NCCN practice guideline: Myelodysplastic Syndromes version 2.2008). The bottom line of all these recommendations can be summarized as follows: Iron chelation treatment of MDS patients should be considered primarily in low risk MDS patients, i.e., patients with low- or intermediate-1 risk IPSS scores, WHO subtypes RA, RARS, RCMD, RCMD-RS, 5q -syndrome or documented stable disease. ICT should be offered to patients with a life expectancy of at least one year and in the absence of comorbidities severely limiting prognosis. Furthermore, all patients eligible for allogeneic transplantation should receive ICT, due to the accumulating evidence of the negative prognostic impact of elevated pretransplantation ferritin levels on TRM, mainly due to an increased rate of infectious complications and

ICT should be initiated when serum ferritin levels rise above approximately 1,000 mg/l and/or once a patient has received 20 25 RBC transfusions, or has a transfusion frequency of 2 U/month for at least 1 year. A minority of groups recommend more conservative ferritin levels as high as 2,500 mg/l. However, such high ferritin levels have been shown to be associated with significantly worsened survival (e.g., [149] and NCCN practice guideline: Myelodysplastic Syndromes version 2.2008). Therefore, we recommend the use of cutoff for initiation of ICT (in the absence of active inflammation), as do the latest recommendations from the biannually held MDS Consensus Meeting in Florence in 2007 published in 2008 [217]. Earlier chelation therapy may however be considered in patients with compromised organ function. The latest recommendations from the MDS Consensus Meeting in Florence in 2007 (published in 2008) leave the choice of chelating agent (i.e., deferasirox, deferoxamine or defriprone) at the discretion of the treating hematologist, and recommend that ICT be continued as long as the patient requires RBC transfusions and as long as iron overload remains clinically relevant [217]. The advantages and potential disadvantages of iron chelators mentioned below has been summarized by Maggio [225].

6.9.6.5 Monitoring of Body Iron Stores in MDS Body iron stores should be assessed at MDS diagnosis and at regular intervals thereafter. Once transfusion dependency occurs, serum ferritin levels should be monitored in 3-monthly intervals. Iron overload should be monitored using serum ferritin and transferrin saturation at least every 3 months during therapy. In those patients with chronic inflammatory diseases, in whom serum ferrtin level is not expected to reflect the true iron burden, additional imaging studies such as liver- and/or cardiac-MRI should be performed. In MDS patients liver biopsy is not only not necessary, as liver MRI can accurately estimate liver iron content, but is also potentially dangerous, due to bleeding complications in the often severely thrombocytopenic patients.

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6.9.6.6 Currently Available Iron Chelators 6.9.6.6.1 Deferiprone (Ferriprox) The oral iron chelator deferiprone has been applied to MDS patients over a period of 3 11 months and led to increased secretion of urinary iron and stabilization of serum ferritin levels [226]. Erythropoietin may potentiate mobilization of storage iron in MDS patients receiving ICT with deferiprone. MDS patients receiving deferiprone combined with recombinant erythropoietin therapy for at least 26 months demonstrated a significant increase in iron excretion, compared to patients receiving deferiprone as a single agent [227]. Deferiprone leads to reversible neutropenia in 5 10% of patients, which is a major reason, why its use should be very carefully weighed in MDS patients who often have pre-existing neutropenia and/or impaired neutrophil function. Generally deferiprone is viewed as third-line ICT choice for MDS patients. In patients experiencing agranulocytosis or neutropenic infections after initiation of ICT with deferiprone, the drug should be discontinued. 6.9.6.6.2 Deferoxamine (Desferal) Before the advent of oral iron chelators, daily (5 7 days/week) subcutaneous deferoxamine transfusions overnight (12 h) were the standard treatment of transfusional iron overload in anemic patients who could not be treated by phlebotomy, and this treatment has been shown to effectively decrease serum ferritin levels, normalize elevated liver enzymes and arrest liver fibrosis [207], reduce risk of diabetes and glucose tolerance [228] and even reduce RBC-unit requirement in some MDS patients [216]. Furthermore, in low- and intermediate-1 risk IPSS MDS patients, overall survival was significantly and dramatically improved in those patients receiving ICT with deferoxamine as compared to those who did not, with median survival not reached at 160 months, compared to 40 months, respectively and 80% of MDS patients receiving deferoxamine survived to 4 years, compared to 40% without ICT [229]. 6.9.6.6.3 Deferasirox (Exjade) Deferasirox, an orally bioavailable iron chelator with a long half-life, allowing a once-daily dosage, has recently been approved by the FDA and the EMEA as second line therapy for patients with transfusional iron overload, who do not respond to, or cannot tolerate treatment with, deferoxamine. The recent development

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of an oral iron chelator deferasirox (Exjade) simplifies treatment of iron overload as compared to the conventional 12 h subcutaneous infusion regimens of the previously used iron chelator deferoxamin (Desferal). Large phase III randomized trials demonstrated that deferasirox was equally efficacious as deferoxamine in reducing iron burden in beta-thalassemic patients [230, 231], and also reduces iron burden in MDS patients [232, 233]. Patient-reported satisfaction and convenience were significantly higher for the once-daily, oral ICT deferasirox than for deferoxamine infusions, indicating a positive impact of deferasirox on patient quality of life [234]. Deferasirox has a dose-dependent effect on iron burden and stabilizes serum ferritin levels and liver iron concentration at a dosage of 20 mg/kg/day, while a dose of 30 mg/kg/day achieves a negative iron balance and reduces serum ferritin and liver iron concentrations [235]. Deferasirox also proved effective in mobilizing iron deposits from the liver and heart in MDS patients [233]. It is important to keep in mind that within the first 4 weeks of treatment, the values of ferritin may markedly increase by approximately 26%, before the continuous decline begins, as has recently been demonstrated in a phase-II setting of deferasirox in MDS patients [233]. This should not lead to an early dose increase, and dose adjustments by 5 mg/kg are not recommended before the third month of therapy. Starting dose is 20 mg/kg/day and the maximum dose of 30 mg/kg should not be exceeded. Side effects are usually mild, comprise transient nausea, vomiting, abdominal pain, diarrhea and skin rash as well as mild and non-progressive increases in liver values and creatinine levels, most of which do not require dose interruptions and all of which were reversible upon drug discontinuation [233]. Mild, often reversible elevations of creatinine levels occur quite often, and the drug should not be discontinued too early. A rise of creatinine levels of less than 1/3 of the initial value is acceptable, but creatinine levels must be monitored closely. Higher increases of creatinine levels above baseline, should lead to dose reduction or drug discontinuation, depending on the extent and the dynamics of creatinine elevation. Contraindications for deferasirox treatment include a creatinine clearance of less than 60 ml/min, as recommended by the manufacturer. Recently deferasirox has been reported to result in improvement of hemoglobin levels and a reduction in RBC transfusional requirements in up to 10% of patients after approximately 12 months of treatment [216, 236]. The biological mechanism of action for this effect was recently shown to be due to deferasirox mediated upregulation of JAK2.

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6.10 Low-Dose Palliative Chemotherapy in MDS Low-dose chemotherapy regimens have been used in MDS with different aims in mind. High-risk MDS patients and/or patients transforming into AML with multilineage dysplasia who are ineligible for allogeneic stem cell transplantation or conventional induction/consolidation-chemotherapy regimens, be it due to comorbidity, age or other reasons, have been treated with either best supportive care or low-dose chemotherapy in the preepigenetic era.

6.10.1 Low-Dose Melphalan Low-dose melphalan (2 mg/day) induced complete remissions in 33% of 21 consecutive elderly patients with RAEB or RAEB-t without causing pancytopenia or marrow suppression [237]. Median duration of CR was 14.5 months and responding patients included those with hypocellular marrow and normal karyotype. The therapeutic effect was attributed to maturation induction as well as to cytotoxic effects against the neoplastic clone [237]. Others have also observed beneficial activity of this drug [238 240] in patients with high-risk MDS or AML with multilineage dysplasia, with overall response rates of 35% [238].

6.10.2 Low-Dose Cytosine-arabinoside (Ara-C) Low-dose cytosine-arabinoside (Ara-C) (2 10 mg/m2/ day s.c. d1-14) was combined with GM-CSF (given either simultaneously or sequentially) in high-risk MDS patients suffering from RAEB or RAEB-t [241]. This EORTC trial reported a complete remission rate of 17% and an overall response rate of 46% [241]. Hemorrhages and infections occurred in 25% and 23%, respectively, irrespective of whether GM-CSF was given simultaneously or sequentially. The toxic death rate of 15% was surprisingly high and clearly within the range of high-dose Ara-C þ anthracycline regimens (see Sect. 6.11.5). The same approach was pursued in a three-armed randomized EORTC trial for patients with high-risk MDS (i.e., 10 30% marrow blasts and hematopoietic failure) [242]. Patients were treated with low-dose Ara-C either alone or combined with GM-CSF or IL-3 [242]. These cytokines were not only incapable of improving the CR rate (39%) but also increased infection rates [242]. Therefore, the combination of low-dose Ara-C with GM-CSF or IL-3 cannot be recommended [242], although previous uncontrolled small trials had reported substantial benefit from priming the blasts of elderly AML or high-

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risk MDS patients with GM-CSF prior to low- or intermediate doses of Ara-C [243]. Recently the results of a randomized phase III clinical trial were published, where in a significant improval in overall survival (24.5 months versus 15.3 months) for patients treated with azacytidine as compared to low-dose Ara-C was demonstrated [244] (see also Sect. 6.12.2), further diminishing the indication for low-dose Ara-C in patients with high-risk MDS. Insummary, low-dosechemotherapy regimensare not an attractive alternative for AML-like regimens orfordemethylating agents for most patients. Furthermore, concepts of priming the neoplastic cells with growth factors in order to simultaneously drive the MDS cells into S-phase, thus hypothetically rendering them more vulnerable to the ensuing cytotoxic attack, have not translated in a response benefit.

6.11 Treatment of MDS with Curative Intention 6.11.1 Myeloablative Chemotherapy and Allogeneic Stem Cell Transplantation (SCT) Usually, allotransplantation follows high dose myeloablative chemotherapy which is capable of reducing the tumor burden. In eligible patients with high-risk MDS, allogeneic stem cell transplantation (SCT) is considered the therapy of choice. Approximately 30 40% of patients are cured by this approach (e.g., [245 248]). The role of dose intensity in allogeneic stem-cell transplantation for MDS patients has been controversially discussed. Both myeloablative and reduced intensity conditioning (RIC) regimens are effective therapeutic modalities in MDS, although the relative merits of each may differ in different settings, based on disease activity and response to prior chemotherapy. There are no randomized trials comparing myeloablative and RIC transplants, as most groups use standard myeloablative chemotherapy for eligible patients, and reserve RIC for those patients not qualifying for standard dose chemotherapy due to age or comorbidities. Several non-randomized comparisons suggest that these procedures are more or less equivalent, with RIC demonstrating a lower non-relapse mortality, but higher relapse rate and equivalent survival to conventional conditioning regimens [249 251] (for details see below). Currently, high dose chemotherapy with allogeneic stem cell support is the only curative approach in patients with MDS. However, only a proportion of patients will have a suitable donor and despite improvements over time, the method still suffers a substantial degree of treatment related mortality in the range of 25 30% even

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Currently no recommendations can be made regarding donor selection (related versus unrelated allogeneic SCT), stem cell source (bone marrow versus peripheral blood) pre-SCT induction chemotherapy or conditioning regimens (reviewed in [256]).

6.11.2 When to Transplant in the Course of Disease?

Fig. 6.22 Treatment results with high dose chemotherapy (HDCT) and allogeneic transplantation in MDS. TRM Treatment related mortality, DFS (EFS) disease free survival (event free survival), OS overall survival. Results refer to three of the largest series reported by Runde et al. (n 131) [515], de Witte et al. (n 885) [247] and Sierra et al. (n 452) [246]. TRM is given in %. DFS and OS is given in % 5 year DFS/OS for Runde, % 3 year DFS/OS for de Witte and Sierra

in patients with the lowest risk (see Fig. 6.22) [252]. This is why allogenic SCT has traditionally been restricted to patients G60 years, although the maximum incidence of the disease occurs in the 7th decade. The introduction of various non-myeloablative chemotherapy regimens followed by allogeneic stem cell transplantation has increased the target population of MDS patients potentially qualifying for such an approach [252]. In addition, the long overall survival time of low risk patients and their often long-lasting lifetime periods without disease-related symptoms have to be weighed against the substantial toxicity associated with various myelosuppressive chemo-immunotherapy regimens. Considering the increasing number of therapeutic tools for conventional treatment such as HDAC inhibitors, methyltransferase inhibitors, demethylating agents and immunomodulators, complex treatment decision making processes in terms of optimal selection of patients, time and type of allotransplantation are necessary [253, 254]. Allthough, allogeneic SCT remains the only curative approach for MDS patients, overall mortality rates of up to 50% due to disease relapse and treatment-related deaths, are daunting (see Fig. 6.22). Further extension of the donor pool, optimal pre-transplant chemotherapeutic conditioning regimens, tailored post-transplant immunotherapy, and amplification of graft-versus-leukemia (GvL) effect whilst minimizing graft-versus-host disease (GvHD), are hurdles which still need to be overcome [255].

If one sets 60 years as an upper age limit for the qualification for myeloablative therapy with allogeneic SCT, at most 25% of MDS patients could be considered candidates for this intervention. This number is further reduced once patients with other relevant contraindications have been excluded [115]. In a Markow decision analysis model 868 patients undergoing allotransplantation at the Fred Hutchinson Cancer Centre or reported to IBMTR1 or IMRAW2, were compared with a control group of 184 non-transplant patients [253]. Despite the fact that low risk patients transplanted early did significantly better in a single study [245], the Markov model clearly favored the delay of transplantation in patients with low- and intermediate-1 risk profiles. This was also true for patients younger than 40 years of age. A watch and wait strategy seems safe in these patients, particularly allowing them to potentially benefit from future therapeutic progress while simultaneously avoiding early death rates of 25 30% in a group with a rather long life expectancy (i.e., sometimes H10a). The results of this analysis are important, since early transplantation of intermediate-1 risk MDS patients seems to be favored by hematologists in the US. However, early SCT may be considered in selected patients with low or intermediate-1 IPSS scores at diagnosis with poor prognostic features not included in the IPSS (such as older age, refractory cytopenias, etc.) [245, 253, 256]. In patients with intermediate-2 and high-risk IPSS however, immediate allotransplantation, provided the patient meets the eligibility criteria and has an appropriately matched donor, seems justified due to the rapid transformation into overt AML, which usually occurs within 1 year [253]. Immediate transplantation in this high-risk cohort seems to maximize survival. SCT should be carried out prior to the development of full blown AML as this evolution may not always be adequately controlled. The occurrence of a new cytogenetic anomaly, development of transfusion dependence or an increase in the IPSS score may be regarded as triggers for the change to an active attitude and initiation of allogeneic transplantation, provided a suitable donor has been found. Of course, the individual 1

IBMTR: International bone marrow transplant registration. IMRAW: International MDS risk analysis workshops.

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risk profiles of patients should be included in such a decision.

6.11.2.1 Factors Associated with Allogeneic SCT Outcome Several factors have been associated with disease outcome. Older age [257], longer duration of disease [257], more advanced stages of MDS [253, 257 259], intensive pre-treatment including induction chemotherapy prior to transplantation, as well as unfavorable cytogenetics [260], predict for a worse outcome of the procedure. Cytogenetics may provide the most important individual risk factor. In particular, patients with isolated chromosome anomalies do significantly better than those with a combination of karyotypic anomalies, mostly due to higher relapse rates in the latter group [261]. In fact, patients with poor-risk cytogenetics seem to fare better with best supportive care than with allogeneic SCT [248]. In a retrospective analysis, age, stage of disease, cytogenetics and ferritin were found to have a prognostic impact and were formed into a prognostic score predicting 5a OS rates of 56%, 22% and 5%, respectively [262]. In addition, the stem cell source may significantly impact on relapse rates as well as on the degree and severity of GvHD, with G-CSF mobilized peripheral blood stem cells as a source leading to better relapse free survival, but also to an increased frequency/severity of chronic, but not acute, GvHD [263, 264]. The cumulative acute and chronic GvHD rate of 80% and 82%, respectively, may be reduced to 40% when G-CSF-mobilized stem cell transplants are combined with pre-transplant ATG [252, 265]. This GvHD rate matches that reported for the use of bone marrow as a stem cell source. In addition, despite initial scepticism, cord blood stem cells may be prove an interesting future resource of stem cells [266, 267]. Results Table 6.17: Results with unrelated donor and cord blood transplants n

(age) Source of stem cells 118 24a Voluntary unrelated donors 198 Voluntary unrelated donors 510 38a Voluntary unrelated donors 13 40a Cord blood

TRM DFS (EFS)

Relapse rates

Ref.

58%

28% (2a)

35% (2a)

[498]

58%

25% (3a)

41% (3a)

[247]

54%

29% (2a)

14% (2a)

[499]

23%

with unrelated donors (Table 6.17) have improved significantly, at least at some centres, due to high-resolution HLA-typing with nearly identical overall results and a trend towards lower relapse rates as compared to those observed in recipients of sibling transplants [252].

6.11.3 Reduced Intensity Conditioning (RIC) Reduced intensity conditioning (RIC) with ensuring allogeneic transplantation is increasingly used for the treatment of MDS patients, particularly due to the lower acute treatment-related mortality than with conventional myeloablative conditioning. The high average age of MDS patients is another reason why RIC is increasingly used. The interpretation of results is significantly hampered by the fact that trials (i) usually include only small numbers of patients, (ii) apply highly different conditioning regimens and (iii) often include disease entities other than MDS and patients with highly different risk profiles. RIC regimens appear to be most effective in patients with a low tumor load and low blast counts at the time of transplantation (e.g., [268, 269]). In a retrospective analysis, 215 MDS patients receiving RIC prior to SCT from sibling donors, showed 3-year NRM (non-relapse mortality) rates of 22%, 3-year DFS (disease-free survival) of 45%, 3-year relapse or progression rates of 33%, and 3year OS (overall survival) rates of 41%, respectively [270]. Furthermore, advanced age does not seem to be a contraindication to RIC and SCT even with the use of unrelated donors [271]. In a recent study, the 3 year DFS rate of MDS Table 6.18: Univariate analysis of variables significantly affecting TRM, DFS and OS in MDS patients treated with Allo RIC (adapted from [271]) MDS subtypes and risk groups WHO stage RCMD RAEB I/II CMML IPSS Low/Int 1 Int 2/high Disease status CMR PR/progressive HCT-CI 0 2 3 or more

3-Year TRM (%)

3-Year DFS (%)

3-Year OS (%)

24 44 65

55 18 14

59 18 14

19 48

55 22

55 26

27 50

43 19

46 19

27 47

45 11

48 11

76.2% (2a) 23% ( 1a) [500]

DFS Disease free survival; EFS event free survival

HCT CI Hematopoietic stem cell comorbidity index; TRM treatment related mortality; DFS disease free survival; OS overall survival

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patients was 34% (31% for patients transplanted from a matched unrelated donor and 45% for patients receiving thetransplantofasiblingdonor)[272].Theresultsobtainedin some larger trials are shown in Table 6.18 and seem to be promising, but it is not clear whether this is due to a possible bias in patient selection. Relapse is still the major cause of treatment failure after RIC and allogeneic SCT. Donor lymphocyte infusions (DLI) may be used successfully for the reestablishment of full donor chimerism in most relapsing patients, allowing the restoration of cytogenetic remission in 50% of cases [273]. Although TRM (treatment-related mortality) is lower than with conventional myeloablative conditioning regimens and complete remissions may be induced in up to 88% of patients treated by sequential, RIC for allogeneic SCT, and prophylactic DLIs, the leukemic death rate remains high (25%) and 2-year overall and leukemia free survival are 40% [274]. RIC requires the induction of a graft-versus-leukemia effect (GvL), which may require several months to evolve. Patients who do not achieve CR with prior chemotherapy and still have significant remaining disease activity, are likely to experience early disease relapses after allo-RIC, outpacing the development of effective GvL [249]. Maintaining the balance between the soughls GvL effect and the feared GvHD remains a critical challenge of allogeneic SCT. The development of chronic GvHD not only reduces relapse, but is also one of the strongest predictors of disease free survival and 4-year overall survival [275]. Improvements in the rate of severe acute and chronic GvHD rates may be achieved using alemtuzumab-containing regimens, but CMV reactivation rates of up to 45% remain a significant problem [276 278]. Apparently, there is substantial room for further development of the technique and improvement of results. In conclusion, early non-relapse mortality has substantially been reduced with RIC, but this has been accomplished at the cost of significantly higher risk of relapses. Therefore, patients with no contraindications for standard myeloablative conditioning should not receive RIC outside of prospective randomized trials.

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men [254]. No upper age limit should be set, but careful consideration of the patients comorbidities and general health status are essential. RIC and specific GvHD prophylaxis may achieve TRM rates of 20% in patients transplanted from unrelated donors, a percentage very similar to that for younger patients receiving myeloablative chemotherapy with sibling transplantation [281]. Patients relapsing after HDCT (high dose chematherapy) with an AML like regimen without subsequent allotransplant, may again undergo induction chemotherapy and receive RIC in CR. RIC has also been successfully performed in patients with persistent neutropenia or even residual leukemia [254]. However, according to the data generated by Shimoni et al., RIC cannot be recommended for patients who do not achieve CR with prior chemotherapy [249]. RIC may also be considered as an option for patients relapsing after HDCT with either prior autologous or conventional allogeneic transplantation, since a second standard induction with high dose chemotherapy would exert excessive toxicity [282], and RIC might be the only chance for long-term survival. However, the chances of a myeloablative chemotherapy with allotransplant should not be easily exchanged for a lower acute toxicity rate accompanied by RIC, particularly in patients around 50 60 years. Relapse rates are high when e.g., very low dose induction regimens are used together with intensive GvHD prophylaxis. Furthermore, the frequent mixed chimerism occuring with very low dose RIC, is difficult to overcome with DLI [283, 284]. Survival rates thus may range between 53% for more intensive RIC regimens containing two alkylating agents with unrelated PBSC grafts [281] and only 10% for single low-dose melphalan monotherapy regimens [285, 286]. Currently there are insufficient data to make a recommendation for an optimal conditioning regimen intensity, and the optimal approach will likely depend on disease and patient characteristics such as age, comorbidities and response to prior chemotherapy [256].

6.11.3.1 Patient Selection for RIC

6.11.4 Induction of a T-cell Response Against the Malignant Clone

Age beyond 60 is usually considered a contraindication for myeloablative HCT with SCTX, since TRM may approach 50% or more [264, 279]. However, RIC with allotransplantation of stem cells of unrelated donors has been successfully performed in patients up to the age of 75 years [280, 281]. As a result, some suggest to recommend this procedure in all patients with an adequate indication and who are otherwise fit and able to withstand a conventional AML-like induction regi-

Immunosuppressive therapies try to counteract the humoral and T-cell mediated suppression of hematopoiesis (for details see Sect. 6.3.3 and Fig. 6.1.a, b, as well as Sect. 6.13). Immunosuppression mostly remains an option for low risk MDS subtypes according to the WHO- or FAB-classification, as well as the IPSS. Attempts to attack the malignant clone via cytotoxic T cells follow an inverse strategy. Allogeneic T-cells in the form of donor lymphocyte infusions (DLI) are used

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for fighting the neoplastic clone. Myelosuppresive chemotherapy or minimal total body irradiation (2 Gy) with subsequent allogeneic SCT and graft versus host prophylaxis with cyclosporin A and mycophenolic acid, allows the successful engraftment of peripheral blood-derived hematopoietic stem cells and may restore normal hematopoiesis with subsequent induction of mixed chimerism [287]. In fact, allogeneic T-cells are capable of exerting a profound antineoplastic graft-versus-leukemia (GvL) effect elegantly demonstrated by minimally myelosuppressive whole body irradiation (2 Gy) plus fludarabine, an immunosuppressive regimen which exerts nearly no direct antineoplastic activity but solely relies on the effect of donor lymphocytes [288]. Similarly, the infusion of donor lymphocytes in patients with a high risk of relapse after HDCT with subsequent allotransplant significantly reduced the risk of relapse, pointing to a valid GvL effect [289]. This principle was further developed to allogeneic stem cell transplantation with reduced intensity conditioning and donor lymphocyte infusion [252, 290], although a more recent analysis could demonstrate no advantage for cellular therapy with DLI compared to chemotherapy for MDS or AML patients who relapse or progress after RIC [291]. However, graft-versus-host disease (GvHD) remains the major complication of donor lymphocyte infusions [289, 292], and new strategies will have to focus on harnessing the GvL activity of donor T-cells, whilst minimizing the toxicity of GvHD. One such possibility is currently being explored in a phase I/II clinical trial of infusions of donor lymphocytes transduced with the herpes simplex virus thymidine kinase in MDS/AML and ALL patients [293]. Vaccination strategies represent another way to therapeutically raise an immunological response against the malignant clone. Examples include vaccination protocols with repeated application of WT-1 another PR-1 antigens, which was not only well tolerated in a phase I setting, but also led to an increase in WT-1/PR-1 specific CD 8+ CTLs with concomitant significant reduction of leukemic blasts [30 32].

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6.11.5 AML-like Chemotherapy in MDS In the absence of allogeneic donors, patients with MDS have been treated with AML-like regimens including high-dose cytosine-arabinoside and idarubicin or daunorubicin (see Table 6.19). While complete remission rates of 15 65% have been achieved, the duration of these responses has been short, namely 16 months in patients with normal cytogenetics and only 4 months for those with abnormal karyotypes [294]. Recent trials have reported CR rates between 47% and 68% by such approaches [258] (Table 6.19). Subgroup analysis from the MDACC point to an inferior outcome in terms of DFS in patients treated for RAEB as compared to those treated for RAEB-t or AML [295]. As in AML, the mortality of high-dose cytosin-arabinoside containing induction regimens is high in patients with high risk MDS, reaching 17% in a series of 510 patients treated at the MDACC [296]. Therefore, attempts have been made to lower toxicity, attributed to anthracyclines by introducing combinations of topotecan and AraC. In 77 patients treated with this combination, the induction death rate was only 6% as compared to 17% for Ara-C combined with idarubicin fludarabine, and the complete remission rate achieved was 56% [296]. Particularly in elderly patients beyond 65 years the mortality rate associated with the regimen was extremely low (i.e., 8%), whereas it increased to 31% in patients treated with Ara-C and fludarabine [296]. Topotecan is equivalent with Ara-C in terms of survival but its lower induction mortality rate make this regimen a valuable alternative for patients considered candidates for high dose therapy with or without subsequent stem cell transplantation.

6.11.6 High-Dose Chemotherapy (HDCT) with Autologous Stem Cell Rescue The possibility to mobilize primitive polyclonal [297] or cytogenetically normal CD34 þ hematopoietic progenitor cells [298] from the marrow of MDS patients has provided the basis for attempting autologous stem cell support after

Table 6.19: High dose AML like chemotherapy regimens for the treatment of MDS n pts MDS/AML Age Regimen 134 47 50 158 77

6569 54 46 60 63

CR (%)

Ara CþG CSF Fludarabine 71 vs. 65 Zorubicine þ Ara C 47 Idarubicin þ Ara C 54 Idarubicin þ Ara C or Fludarabine þ Ara C G CSF 65 Ara C þ Tepotecan 56

DFS a

23% vs. 16% 11 months 11 months 5 121 months

CR Complete response; DFS disease free survival; OS overall survival; Ara C cytosine arabinoside FLAG (þfludarabine) vs. AG ( fludarabine); b% 2 year OS; c% 5 year OS

a

OS a,b

39% vs. 24% 14 months 15 months 8%c

Ref. a,b

[496] [294] [258] [497] [296]

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high-dose chemotherapy. Bone marrow from MDS patients in clinical remission was xenotransplanted into NOD/SCD mice and led to the outgrowth of cytogenetically normal precursor cells [54]. These data provided the rational to intensify MDS treatment with high dose chemotherapy with autologous stem cell rescue for consolidation after remission induction. Peripheral blood stem cell collection seems to yield higher numbers of stem cells and hematological reconstitution tended to be faster than marrow collection, and may further improve the percentage of autografted MDS patients [299]. Low treatment related mortality rates of less than 10% and CR rates of 41 50% further demonstrate that sufficient numbers of hematopoietic stem cells with adequate repopulation capacity can be recovered in MDS patients [299, 300]. A retrospective, observational database study comparing the outcomes of 668 autotransplants with 476 unrelated donor transplants in patients with AML showed that autotransplantation yielded higher 3-year survival rates for AML patients transplanted either in first (57% vs. 44%) or second CR (46% vs. 33%) after induction chemotherapy, as compared to patients receiving unrelated donor grafts [301]. Similar results, demonstrating non-inferiority of autologue transplantation as compared to allogeneic SCT, were reported in a multicenter study of SAKK, GIMEMA, EORTC and EBMT. MDS patients with an HLA-identical sibling donor were scheduled for allogeneic transplantation, whereas patients lacking a donor were scheduled for autologous transplantation, after a common remission induction and consolidation course [302]. The event free survival rates were similar for recipients of auto- or allografts and a survival advantage could not be demonstrated for patients with a donor compared to patients without a donor [302]. When the results were compared with those obtained for 215 MDS or MDS/AML patients treated with high dose Ara-C for remission induction and lower doses of the identical induction regimens for further 6 12 months, the 4-year DFS was superior in the CR patients undergoing HDCT with SCT for consolidation in the European trial (29% vs. 17%, p ¼ 0.02) [303]. However the patients did not differ in terms of overall survival. Thus, the question concerning the optimal type and dose of consolidation chemotherapy remains open, and questions as to which may be the superior modality (auto- versus allo-transplantation) may be less important than the need to improve results for both [303]. Currently, AML-like induction regimens may be used for MDS patients undergoing transformation into AML, particularly since a debulking effect has been shown to favorably influence the outcome of subsequent allogeneic transplantation. In summary, intensive chemotherapy followed by autologous bone marrow or peripheral blood stem cell

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transplantation seems to be a valuable treatment option for patients with high-risk MDS/secondary AML lacking a suitable donor [256].

6.12 Epigenetic Therapies: DNA-Methyltransferase Inhibitors Epigenetic mechanisms such as DNA hypermethylation, which leads to selective gene silencing, have become increasingly attractive as targets in the treatment of MDS. Histone acetylation has been shown to be a critical factor in promoting gene transcription, in that histone hyperacetylation facilitates binding of transcriptional activators due to conformational relaxation of tightly coiled DNA, whereas histone deacetylation leads to gene repression.

6.12.1 Hypermethylation in MDS Hypermethylation of the p15INK4b gene can be detected early at diagnosis and is seen in 65% of high-risk MDS patients [304] and in 75% of patients who advance to AML [305]. Hypermethylation-mediated silencing of genes such as CDKN2B, which encodes for p15INK4b, is of particular importance in poor-risk MDS and predicts transformation into AML [306, 307]. Transcriptional silencing of p15INK4b is further thought to be closely associated with MDS pathogenesis and it is thought that hypermethylation of p15INK4b allows the neoplastic cells to escape inhibitory signals from the bone marrow microenvironment [307]. Selective silencing of p15INK4b has further been shown to be significantly correlated with bone marrow blast count and disease progression and may predict poor prognosis in early stage MDS patients, as well as in patients with deletions of 7q and therapy-related MDS [306 310]. DNA methyltransferase inhibitors are incorporated into DNA and inhibit DNA-methyltransferase, thereby reverting hypermethylation and restoring function of a number of genes involved in tumor suppression and differentiation. Demethylation of p15INK4b has been observed with arsenic trioxide or decitabine [304, 311, 312]. So far two hypomethylating agents have been developed clinically and have received FDA and EMEA approval for the treatment of MDS, namely 5-azacytidine (Vidaza), which can be used both i.v. and s.c., and 5-aza-20 -deoxycytidine (decitabine) which is applied exclusively via the i.v. route (for review see [313]). Recent data from ASH 2009 give early insights into possible future oral formulations of azacitidine and subcutaneous application of decitabine (ASH

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2009, #3797, #1772, #117, #119, #3804). In lower doses, these drugs act via DNA hypomethylation, restoring normal growth and differentiation, while in higher doses they exert cytotoxic effects resulting from incorporation into DNA and RNA [314 316]. Selective p15INK4b hypermethylation can be reversed with reestablishment of normal p15 protein expression by decitabine in MDS patients, and seems to be associated with treatment response [304, 311, 312]. A decrease of p15INK4b methylation was observed in 9/12 patients treated with at least one course of decitabine, and was associated with clinical response [304]. In patients treated with 5-azacytidine however, reduced methylation of CDKN2B did not correlate with response [317].

6.12.2 Hypomethylating Agents 6.12.2.1 5-Azacitidine (Vidaza) Azacitidine was developed as a cytarabine analogue more than 40 years ago, and has been the subject of renewed interest in the era of epigenetic therapy [318]. Vidaza was the first drug to receive FDA approval for the treatment of all types of myelodysplastic syndromes on May 19th 2004 (summarized in [319]). The basis for this approval was set by a randomized controlled trial comparing subcutaneously administered azacitidine with best supportive care [CALGB 9221 trial], as well as by two single-arm trials in which azacytidine was administered s.c. or i.v. [CALGB 8921 and 8421 trials] [320 322]. In the randomized CALGB 9221 trial [320], 5-azacitidine was compared with best supportive care alone, with the possibility of a cross-over to the 5azacytidine arm in patients with progressive disease with BSC alone. The response rate was impressive (60% vs. 5%) in patients receiving azacitidine and the response duration was 14 months [320]. Responses occurred early and time to death or transformation into AML was also significantly improved (i.e., 21 months vs. 12 months) as was the trend for overall survival (20 months vs. 14 months). Transfusion requirements were substantially reduced, and not surprisingly, quality of life parameters such as fatigue, dyspnea, and physical functioning ameliorated significantly [323]. Combined re-analysis of all three trials that led to FDA approval (n ¼ 309), using the WHO classification criteria for MDS and AML and the IWG criteria for response assessment, reveals OR rates of 40 47%, CR rates of 10 17%, rare partial remissions and hematologic improvement rates of 23 36%. The median numbers of cycles to first response was 3, 75% of patients achieved a response by cycle 4 and 90% of patients responded by

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cycle 6, but responses as late as cycle 17 were also observed [244, 322]. Importantly, azacitidine did not increase the rate of infections or bleeding events above the rate caused by the underlying disease [322], and coincided with a significant increase in the quality of life, reported in a CALGB companion study [323]. Very recently the full publication of the largest randomized trial to date was published by Fenaux et al. [244]. Patients with predominantly high-risk MDS were randomized to either 5-azacitidine 75 mg/m2 for 7 days or one of three different conventional care regimens, i.e., BSC (G-CSF and antibiotics for infections, transfusions for anemia), low-dose Ara-C or standard AML therapy with induction and consolidation. Survival increased significantly from 15 months for conventional care to 24.4 months for 5-azacitidine. The differences in survival were 12.9, 9.1 and 8.7 months for 5-azacitidine versus BSC, low-dose Ara-C and AML-like treatment, respectively, with the first two comparisons achieving statistical significance. Survival increased irrespective of good, intermediate and poor-risk IPSS status [244]. Although patients with karyotypic abnormalities fare worse than those with normal cytogenetics, patients with monosomy 8 gain the same survival benefit as those with normal karyotypes and patients with monosomy 7 may respond in up to 50% of cases, despite the usually bad prognosis of this karyotypic subset [313]. This high response rate of patients with chromosome 7 abnormalities has been confirmed in a high risk group of MDS patients in which 7/12 patients achieved a complete remission, 3/12 a partial remission and 1/12 a hematologic improvement [324]. Patients with monosomy 7 did better than those with complex chromosomal aberrations including monosomy 7. In high risk patients qualifying for a curative approach with allogeneic stem cell transplantation, primary treatment with 5-azacytidine may not only be beneficial in terms of a significant postponement of allotransplantation, but may also lead to a better outcome due to reduction in severe graft-versus-host disease [325]. Importantly, treatment with 5-azacitidine is advised until loss of response or occurrence of unacceptable side effects. Retreatment (after a period of 11 months after the last course of initial treatment) of previously azacitidine-responsive patients at the time of disease-recurrence, was merely found to result in a response rate of 55%, and the qualitiy and duration of second disease remissions were inferior ([326] and confirmed by our own experience with the drug). Various regimens have been tested in a randomized manner in low-risk RA/RARS patients requiring transfusions [327]. Alternative applications and dosages have been tested in clinical trails, including (i) the 5-2-2 regimen (i.e., 75 mg/m2 for 5 days followed by a two-

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day rest and 2 days 75 mg/m2) (ii) the 5-2-5 regimen (50 mg/m2 for 5 days followed by 2 days rest and 50 mg/ m2 for 5 days) or (iii) a 5-day regimen using 75 mg/m2 for 5 days. Transfusion independence was obtained in 55%, 60% and 67% of cases, respectively. Although the regimens caused identical rates of remission and hematologic improvement, grade 3/4 hematologic toxicities were lowest with the 5-day regimen (18% vs. 44% for the 5-2-2 regimen and 33% for the 5-2-5 regimen). For patients with CMML 5-day regimens with 100 mg/m2 have been tested in a phase-2 trial [328] (for details see Chapter 7). The currently approved dosage remains 75 mg/m2 s.c. or i.v., applied 7 days consecutively, to be repeated every 4 weeks. In clinical practice it is important to note that patients develop a worsening of cytopenia during the first 2 months prior to responding to 5-azaciticine, but this does not seem to be associated with an increased rate of infections or hemorrhages. Some patients experience painful bumps at the local site of injection when given subcutaneously, but these usually disappear within 2 3 days, and are usually perceived as negligible by most patients, and especially so by those who obtain transfusion independence. In early pilot trials, combination with other drugs such as ethanercept which targets increased TNF-a levels seem promising [329] (see Sect. 6.14.3). A pilot pharmacokinetic phase 1 study of oral azacitidine was recently conducted [330] and demonstrated sufficient oral bioavailability of the drug, and several clinical trials testing this oral formulation are currently recruiting patients. It will be of interest to see whether gastrointestinal side effects occur more frequently with oral azacitidine. In conclusion, azacitidine is an effective therapy for MDS, directly impacts the course of disease, rather than merely managing the symptoms, and leads to a significant overall survival benefit on top of improving quality of life for a substantial amount of MDS patients treated with this drug.

6.12.2.2 5-Aza-20 -Deoxycytidine (Decitabine) (Dacogen) Decitabine is a hypomethylating agent with significant activity in MDS and is one of the three disease modifying drugs (azacitidine, decitabine and lenalidomide) currently FDA-approved for treatment of MDS patients. On May 2nd 2006, the US Food and Drug Administration approved i.v. decitabine (Dacogen) for the treatment of patients with MDS, including previously treated and untreated, de novo and secondary MDS of all FAB subtypes with either intermediate-1, intermediate-2 or highrisk IPSS category. Decitabine has been granted orphan drug status in Europe by the EMEA.

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Decitabine induces differentiation in AML cell lines and in primary blasts in vitro. This translates into a reduction of peripheral blasts and a gradual increase in the absolute numbers of mature cells in both the bone marrow as well as peripheral blood in MDS patients [331 333]. Although there does not seem to be a difference in general DNA hypomethylation between responders and non-responders (e.g., [334]), specific decreases of p15INK4b methylation were associated with clinical response [304]. Safety and efficacy were demonstrated in an openlabel, multi-center, randomized phase II clinical trial, in which a total of 170 patients were randomized to receive decitabine plus best supportive care, versus best supportive care alone [335]. The rate of complete and partial responses according to the IWG-criteria was higher in the decitabine arm (17% vs. 0%), median duration of response was 288 days, and the median time to response was 93 days [335]. Overall response rates including hematological improvement of 30 73% have been observed with this drug [335 337]. As decitabine is an S-phase drug, the development of drug schedules was based on the assumption that prolonged exposure times would be associated with improved response. However, 72 h continuous infusion schedules are cumbersome, and the FDA approved schedule of 3-h infusions three times daily on three consecutive days every 6 weeks is not feasible on an outpatient basis. Several trials have suggested that decitabine is less active and more toxic when administered via continuous infusion, whereas increased dose intensity seems associated with higher response rates (reviewed in [338]). Therefore, optimal dosing regimens are still under heavy dispute and optimization is being sought [338 341]. The FDAapproved application regimen is 15 mg/m2 infused over a 4 h period every 8 h on days 1, 2 and 3 of week 1 every 6 weeks (1 cycle). Importantly, and similarly to treatment with azacytidine, prolonged decitabine administration until relapse or unacceptable toxicity seems necessary. In patients in whom decitabine was cessated after two consolidating courses after best response, retreatment with decitabine after relapse resulted in only 45% objective responses in previously decitabine responsive patients, and the quality and duration of second disease remissions were inferior [326]. Allogeneic stem cell transplantation after prior treatment with decitabine is not only feasible, but does not increase toxicity and may even improve outcome [325, 342]. It has even been suggested, that allografting after low-dose decitabine and subsequent reduced intensity conditioning may be a valid alternative to standard chemotherapy [343].

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Clinically significant responses to decitabine have been observed in patients previously treated with, and non-responsive to, azacitidine without significant toxicity [334]. Major toxicities include myelosuppression, nausea and other gastrointestinal side effects, hyperglycemia and alterations in serum electrolytes.

6.12.3 Histone Deacetylase Inhibitors (HDAC-I) and Combination Therapy with Other Epigenetic Drugs or Differentiation Inducer ATRA (Vesanoid) Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are involved in chromatin histone interactions and thereby regulate gene transcription. Histone deacetylation by HDACs results in a tight interaction of DNA with histones, which hinders access of transcription factors and thereby maintains the chromatin in a transcriptionally silent state. In a simplified model, HDAC inhibitors disrupt DNA histone interactions, resulting in a more open DNA conformation and enabling easy access of transcription factors. This leads to derepression of tumor suppressor genes and consecutive induction of either differentiation or cell cycle growth arrest and apoptosis (e.g., [344]). Abnormal activity of both HATs and HDACs have been observed in MDS/AML [345, 346], setting the stage for numerous in vitro and in vivo assays, as well as for a plethora of clinical trials testing HDAC-inhibitors alone, or in combination with chemotherapy or other epigenetic or differentiation inducing therapeutics [347 355]. The HDAC-inhibitor valproic acid (VPA) has been combined with differentiation inducer all-trans-retinoic acid (ATRA) by numerous groups [347 349, 352 354, 356]. Overall response rates of 16 27% with higher rates of diminished marrow blast counts and peripheral blast clearance as well as clinical benefit rates with striking 30% platelet transfusion independence lasting several months have been reported. It was concluded, that future trials should combine VPA with chemotherapy or demethylating agents [348, 349]. In vitro studies suggested that the combination of 5azacytidine and VPA with or without ATRA should have significant antileukemic activity in vivo [357]. This led to several clinical trials testing this combination in patients with MDS/AML [358, 359]. Overall response rates of 42 52% were observed, depending on whether patients had been pre-treated or were treatment-na€ıve. The median number of courses to response was 1 and median remission duration was 26 weeks [359]. Dose-limiting toxicity was reversible neurotoxicity [359].

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5-azacytidine has also been combined with HDAC-I sodium phenylbutyrate. Combination of the two substances in vitro led to an increased level of re-expressed p15INK4b [312]. In a pilot study, 10 MDS and AML patients were sequentially treated with 5-azacytidine (75 mg/m2/day for 7 days) and sodium phenylbutyrate (200 mg/kg/day i.v. for 5 days). Fifty percent of patients had either a partial remission or stable disease. The combination was well tolerated. Patients experienced typical side effects of 5-azacytidine and somnolence/ fatigue resulting from sodium phenylbutyrate [360].

6.13 Immunosuppressive Treatment in MDS As mentioned in the introductory section (see Sect. 6.3.2), the immune system is significantly disturbed in MDS. These immune defects not only play a role in MDS initiation and/or perpetuation, but are also associated with poorer overall survival due to leukemic progression and/or susceptibility to opportunistic infections [144]. The frequency of autoimmune phenomena in MDS [17, 371, 372] supports the view of a pathologically activated immune system with expansion of cytotoxic T-cell clones capable of suppressing hematopoietic progenitors [373, 374] and causing pancytopenia and bone marrow failure (see Sect. 6.3.2 and Fig. 6.1.a, b). Suppression of this pathologically activated T-cell response has therefore been developed as therapeutic strategy in MDS. This concept is further supported by the observation that marrow failure in aplastic anemia, which is associated with similar alterations of the immune system, such as skewing of the CD4/CD8 ratios and the T-cell receptor repertoire, responds well to ATG [375, 376]. ATG is an important tool in this regard, since this polyclonal serum not only reacts with T- and NK-cells, but also with components of the microenvironment such as integrins and chemokine receptors [377]. This strategy is of particular value since it aims to ameliorate the cumbersome sequelae of bone marrow failure often occurring in lowrisk MDS patients.

6.13.1 Treatment with Anti-thymocyte Globulin (ATG) Treatment with ATG has been shown to eliminate clonal T-cells and to restore a normal TCR repertoire [378]. As outlined in Table 6.20, hematological responses have been observed in 17 67% of MDS patients [33, 378 384]. Time to response may vary between 1 and more than 10 months and may be

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Table 6.20: Results with ATG in patients with MDS n

MDS subtype

96

RA 77%

19a of 34

All with trisomy 8

35

RA 69%

14c

RA 71%

32

RA or RARS 56% RAEB 32% RA 61% RARS 12% RCMD 29% RA 65% RARS 15% RAEB 20% not given

82 (69 treated with ATG, 13 with cyclosporin A) 30 (20 evaluable at 6 months) 32 (12 MDS, 13 AA, rest too early to evaluate)

Age in years (range) 54.7 (19 75) RARS 7% RAEB 16% 57.0 (14 76) 4 evolving from previous aplastic anemia 63 (41 75) RAEB and CMML 31% 67 (32 81) RAEB 29% 59 (28 79)

Hematological response 42%

Response duration (range) 31.5 months (6 92)

Ref. [381]

67%b

Not reported

[380]

34%

9 þ months (1 17þ)

[384]

46%

(up to H3 years)

[383]b

17%

[385]

59 (n.g)

29%

Durable remissions from 12 to 60þ months 31 months

[33]

54.5 (31 73)

50%

15.5 months (2 42þ)

[382]

63 (42 80)

33%

3.7 months (2.5 4.6)

[386]

a

34 patients, all with trisomy 8 were studied, 19 of them were treated with ATG; bIn patients with trisomy 8 as the sole karyotypic anomaly and de novo MDS; cPatients received ATG and subsequent etanercept

achieved with different preparations of ATG (e.g., from horse or rabbit [382]). Horse ATG may be given at 15 mg/kg/day for 5 days, whereas rabbit ATG is usually dosed at 3.75 mg/kg/day for 5 days [382]. ATG treatment seems to be feasible for a broad range of patients, when performed in specialized centres and by experienced physicians. Patients with MDS-RA and short duration of disease seem to respond best [382]. Care has to be given to avoid neutropenic and opportunistic infections and bleeding disorders, as patients can remain pancytopenic for weeks. A number of parameters have been associated with response to ATG. Presence of MDS subtypes RA [382] and RARS [33, 383], low IPSS scores, bone marrow hypoplasia and/or short duration of the disease [379, 382], are positive predictors of response to ATG. Patients with trisomy 8 as the sole karyotypic abnormality seem to respond especially well to immunosuppressive therapy with ATG, with durable reversal of cytopenias, loss of Vb-clonal T-cell expansion and restoration of transfusion independence in 67% [378]. In addition, T-cells in these patients seem to specifically suppress the neoplastic cells carrying the trisomy 8 anomaly, while sparing normal hematopoietic cells. It has been hypothesized that DR15/DR2 typing may identify a subset of MDS patients with immune mediated marrow failure and a more favorable prognosis. In fact, significant correla-

tions between overrepresentation of antigenic frequencies of HLA-DR2 and its serologic split HLA-DR15 with clinically relevant responses to immunosuppressive therapy have been reported for MDS and PNH [33]. However, in the same study 29% of non-responders were HLADR15 positive and 17% of responders were HLA-DR15 negative [33], and HLA-DR status was not found to be predictive of response to ATG by others [385]. In fact, this small trial had to be terminated early due to insufficient response and substantial toxicity (serum sickness in all patients, transient neutropenia and thrombocytopenia, diarrhea, vomiting, and syncope with a generalized seizure) [385]. Among these non-responders were also patients with hypoplastic marrows and the HLA-DR15 allele [385].

6.13.2 Immunosuppressive Treatment with Cyclosporin A (CyA) Immunosuppressive treatment with cyclosporin A (CyA) leads to major erythroid responses in up to 58 82% of MDS patients [386, 387], although small case studies report no efficacy in 6/6 patients [388]. One report of 17 patients noted a complete trilineage recovery in 27% of patients [387]. In a retrospective multivariate analysis of 72 patients treated with CyA, shorter disease duration and higher CyA doses (H5 mg/

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kg/day) were significantly associated with response [386]. Initially it was suspected, that the presence of oligoclonally expanded T-cells in aplastic anemia, hypoplastic MDS, AA/MDS/PNH-overlap syndromes, together with reported responses of aplastic anemia to immunosuppressive therapy (see Chapter 10), would predict for a relatively selective response of hypoplastic MDS patients to immunosuppressive treatment. Although some reports on the efficacy of CyA being active especially in MDS patients with hypoplastic marrow exist (e.g., [389 391]), these had low numbers of patients included (n ¼ 6, n ¼ 11 and n ¼ 2). Instead it seems, that the presence of expanded IFNg-expressing CD4þ -cells is more important than bone marrow cellularity per se [390]. This is in line with the hypothesis that the expanded oligoclonal T-lymphocytes observed in MDS mediate suppression of hematopoiesis and play an essential role in the pathogenesis and the development of cytopenias in MDS (see Sect. 6.3.2). Accordingly, the observed in vitro effects of CyA, which significantly decreased the number of IFNgproducing cells [390] and enhanced colony growth [389], presents one of the mechanisms of action of CyA in MDS. This further enforces the rational of immunosuppressive treatment of MDS patients with (oligo)-clonally expanded T-cells patients. The abovementioned larger retrospective analysis of 72 patients could not show any significant associations between response and bone marrow features such as erythroid hypoplasia or hypoplastic marrow. It was thus concluded that CyA may be an effective therapy for any type of low-risk MDS, irrespective of bone marrow cellularity [386]. It has been proposed, that future trials select patients according to presence of PNH-clones, inhibitory cytotoxic T-cells or autoimmune phenomena [387, 388]. Importantly, CyA does not influence immunity mediated by macrophages or neutrophilic granulocytes. Therefore, the risk of infectious complications is lower than with other immunosuppressive agents [387]. One of the putative caveats of immunosuppression in MDS, may be the possibility of reducing anti-tumor immunity, thereby facilitating leukemic transformation. However, enhanced disease progression during treatment with CyA has neither been reported, nor studied adequately.

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sponse to steroid therapy or immunosuppressive agents is generally good.

6.14 Targeting Bone Marrow Microenvironment in MDS 6.14.1 Thalidomide Thalidomide exerts pleiotropic effects on the bone marrow of patients with MDS. It is anti-angiogenic, immunomodulatory and capable of modifying apoptosis. In their pivotal publication, Raza et al. [361] used rising doses of thalidomide (100 400 mg/day). 19% of patients (31% of evaluable patients) showed hematologic improvement with 12% achieving transfusion independence, although no complete or partial responses were observed [361]. Lower pre-therapy blast counts, lower duration of platelet transfusion dependence and higher platelet counts predicted for better response. These results led to a widespread but uncontrolled use of thalidomide in clinical practice in the pre-epigenetics era. In another larger trial involving 42 low-risk IPSS and 30 high-risk patients, only 1/72 patients achieved a partial response and only 7/72 showed hematological improvement [362]. Due to the side effect profile, which typically includes fatigue, painful peripheral neuropathy and constipation, only 44% of patients completed treatment. In conclusion, the degree of efficacy of thalidomide is modest, while toxicity is clinically relevant. Serious and fatal complications may result form the combination of thalidomide with erythropoietin-stimulating agents, both of which are thrombogeneic [153, 154]. We recommend that this combination be strictly avoided. Interestingly, a recently published trial tested the combination of 5-azacytidine (75 mg/m2 day 1 5 every 4 weeks) and thalidomide (50 mg 100 mg/day) 42% hematologic improvement and 14% stable disease in 40 patients with MDS/AML were noted [363], but these percentages do not seem to be much higher than for treatment with 5-azacytidine alone, at least at first glance. Thalidomide is currently not approved for the treatment of MDS by the FDA or the EMEA.

6.14.2 Lenalidomide (Revlimid) 6.13.3 Treatment of MDS Associated Autoimmune Manifestations Most autoimmune manifestations associated with MDS are asymptomatic. In the rare symptomatic patient, re-

Lenalidomide is an orally administrable thalidomide analogue with immunomodulatory, antiangiogenic and antiproliferative properties, as well as a potential for direct clonal suppression or extinction of myelodysplastic clones, and especially those bearing the dele-

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tion 5q. For details on the 5q syndrome, see Sect. 6.19.1. Lenalidomide is dramatically more efficient in counteracting TNF (tumor necrosis factor a) than thalidomide and lacks the neurotoxic side effects such as somnolence, peripheral neuropathy, and obstipation. Responses with lenalidomide are clearly superior to those achieved with thalidomide in the pre-lenalidomide era [364]. Lenalidomide is FDA-approved since 12/2005 for the treatment of transfusion-dependent anemia in patients with low or intermediate-1-risk MDS with a chromosome 5q deletion with or without additional cytogenetic abnormalities, based on the excellent results reported by List et al. [365]. As such, lenalidomide is a shining example of a cytogenetics based treatment approach. The reasons for superior efficacy of lenalidomide in the del(5q) patient subset are currently unknown. Gene signatures are under investigation which predict the erythropoietic response to lenalidomide [366]. 75% of patients with isolated del(5q31) achieved a complete cytogenetic response, and 83% achieved an erythroid response with a more than 2 g/dl increase (median increase 5.4 g/dl) in hemoglobin, compared to 57% of patients with normal karyotype, or 12% of those with other chromosomal abnormalities [365]. However, whilst 57% responders in the normal karyotype subgroup may be significantly less than the 83% observed in the del(5q31) subgroup, this is still a substantial degree of remitting activity. In fact, a large phase II trial with 214 low risk and IPSS intermediate-1 risk MDS patients with karyotypes other than 5q , who required treatment as defined by transfusion dependence (2 Units within 8 weeks prior to treatment), were treated with two different regimens of lenalidomide [367]. After a median of 5 weeks, an overall hematologic improvement of 43% was noted with a median hemoglobin increase of 3.2 g/dl and 26% of patients achieved transfusion independence, with the median duration of transfusion independence being 41 weeks [367]. These results are highly promising, particularly since they were observed in a population of patients usually considered poor responders to erythropoietin stimulating agents (only 6 14% of patients with del(5q) respond to erythropoietin treatment [118, 119]). Erythroid responses to lenalidomide are highest in lowrisk to intermediate-1 IPSS patients, particularly in those with del(5q) (reviewed in [119]). However, high-risk patients may also experience significant responses to lenalidomide. The proportion of myeloblasts is reduced to less than 5% in 74% of patients with RAEB, as is the proportion of ringed sideroblasts in patients with RARS, after a median of 24 weeks [368]. Patients with complex karyotypes including del(5q31)

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are characterized by a dismal prognosis. The efficacy of lenalidomide in this patient population is not conclusive to date [369], but analysis thereof would be of special interest. It should be kept in mind that patients with del(5q31) do not respond well to erythropoietin substitution, with response rates ranging from 6 to 14% in this MDS population, as compared to 20 37% in the general MDS population [118, 161]. Similar results have been reported for the combination of G-CSF and erythropoietin [168]. Possibly this lack of response to ESA may be due to the usually observed high endogenous erythropoietin serum levels in del(5q) patients [112]. The response to treatment with lenalidomide is usually rapid. Therefore, treatment should be stopped after 4 months if no response can be documented. Lenalidomide also reverts the typical bone marrow morphological changes of megakaryocytes, which are characteristically hypolobulated, mononuclear, and often small in size (micro-megakaryocytes) in patients with del(5q31) [114]. Successful treatment results in normalization of size and (re)occurrence of multilobulated nuclei. Moderate to severe neutropenia (55%) and thrombocytopenia (44%) are the most common side effects occurring early in the course of treatment, resulting in treatment interruption or necessity of dose reduction. Due to the observed 2% mortality due to neutropenic fever, one should consider myeloid growth factors in the initial weeks of treatment. With adequate dose adjustment, lenalidomide may also be safe and efficient in patients with severe renal impairment [370].

6.14.3 Direct Targeting of TNF-a: Inﬂiximab and Ethanercept TNF-a (tumor necrosis factor a) is a central key player in the pathologic activation of the immune system in MDS patients. TNF-a causes increased sensitivity of hematopoietic precursor cells towards pro-apoptotic signals along the Fas- and TNF-pathway [43, 392]. While TNF-receptors TNFR-I and TNFR-II are upregulated in RA patients, only TNFR-II expression and signaling is increased in RAEB, RAEB and AML patients [393]. In good correlation, TNF-a is also overexpressed in early phases of MDS [394], where its expression levels correlated with the degree of anemia and microvessel density of bone marrow. Therefore, inactivation of TNF-a or its signaling pathways is assumed to correlate with amelioration of cytopenia in MDS patients. Targeting of TNF-a has been attempted in small phase II trials using inactivating antibodies such as infliximab [395, 399] or cA2 (anti-TNF-antibody) [397], or the

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soluble TNF receptor fusion protein ethanercept [398 400]. Decreases in apoptotic stem cells in the marrow [396, 397, 401], reduction of TNF-a levels in longterm bone marrow cultures, increases in the hematopoiesis-supportive capacity of long-term marrow culture adherent cells, and substantial increases in CFU-GM were reported [397, 398]. Clinical effects varied between these trials with the majority demonstrating no [400] or only very moderate responses [397 399] and only very few patients achieved short-term transfusion independency. In general, TNF-targeting is still an interesting option but remains experimental and probably a part of multi-agent strategies to be developed in the future.

6.14.4 Antiangiogenetic Therapies As mentioned above, the neoangiogenic cascade is significantly upregulated in MDS [402] and this process is under the control of VEGF (vascular endothelial growth factor) which additionally acts as an autocrine growth factor of abnormally localized myeloid precursors [47]. In good correlation, VEGF staining in the marrow of patients with MDS increases with increasing stage [48] and sVEGFR1 (soluble VEGF receptor 1) serves as a prognostic factor in MDS and AML patients [403]. However it should be mentioned, that the neoangiogenetic process seems to differ between MDS and AML. Microvessel density significantly decreases in MDS patients transforming into AML. These MDS/AML cases show lower microvessel density than de novo AML cases [46]. In addition, the pattern of angiogenic molecules and their receptors differs between MDS and AML [46, 404, 405]. Therefore, although there is a rational to target neoangiogensis in myeloid neoplasias, different strategies may be required for MDS versus AML. Antiangiogenic therapies have been used in phase I/II trials including MDS and AML patients. Small molecules inhibiting tyrosine kinases of various receptors have been applied. PTK787/ZK222584 inhibits kinase activity of VEGFR1, VEGFR2, VEGFR3, PDGFR and c-kit, but responses were only reported in AML patients simultaneously treated with chemotherapy [406]. AG013736 has a nearly identical profile of receptor tyrosine kinase inhibition, but exerted only minimal effects in elderly patients with high-risk AML and MDS [407]. Modest activity was observed for SU5416 (Sutent) in a phase II trial for patients with refractory AML and MDS [408, 409]. From the results of the currently evaluable trials it may be concluded that antiangiogenic treatment strategies may require combination treatment. Such trials are being conducted.

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6.15 Induction of Differentiation – Retinoic Acids Many attempts have been made to utilize in vitro effects of various agents on the induction of maturation of myelodysplastic stem cells into mature hematopoiesic cells for in vivo treatment of MDS. Substances with such a potential for the induction of differentiation have either been used alone or in combination with cytotoxic drugs, particularly since some of these drugs exert maturation effects by themselves (i.e., melphalan, cytosin-arabinoside, etc., see above). 13-cis Retinoic acid was among the first drugs utilized for maturation induction. When used alone and tested against a placebo-control, however, the drug failed to improve the outcome of relevant target parameters. However, side effects such as mild skin reactions were observed in up to 90% of patients [410]. Retinoic acid is still being tested however, particularly in its all-trans form (ATRA) and in combination with other drugs such as valproic acid, and/or cytosine-arabinoside (Ara-C).

6.16 Molecular Therapies Using Kinase-Inhibitors 6.16.1 Farensyltransferase Inhibitors (FTIs): Tipifarnib (Zarnestra) and Lonafarnib (Sarasar) Mutations within the Ras oncogene occur in up to 25% of patients with MDS or AML [411, 412], but the true frequency may be underestimated since not all of the three genes, i.e., H-Ras, N-Ras and K-Ras were included in the majority of analyses (see also Sect. 6.3.4.4). Furthermore, Ras and downstream signaling events may be activated even in the absence of Ras mutations [413]. To acquire transforming capacity, Ras molecules must be transferred to the cell membrane via farensylation. This provides the rationale for the development of farnesyltransferase inhibitors (FTIs) for targeting pathologic Ras activation. However, during treatment with Farnesyltransferase inhibitors some Ras molecules become geranylgeranylated, pointing to an alternative pathway of Ras activation, possibly circumventing the effect of FTIs [414]. However, a number of experimental data support the importance of the FTI inhibition of Ras for suppression of cancer growth [415]. In addition, clinical and translational research underline the independence of the clinical effect of FTIs on the presence of absence of Ras mutations [416]. Of interest, downregulation of TNFa may be associated with the clinical response to FTIs [416].

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In phase I/II trials, tipifarnib (Zarnestra) has shown efficacy in doses of 300 mg b.i.d. as induction therapy [416]. The response rates of 32% [417] and 30% [416] are noteworthy. Particularly in high-risk patients, the 15% CR rate, 17% hematologic improvement and 45% stabilization rate seem relevant. Although myelosuppression was a frequent side effect with 74% grade III/IV neutropenia and thrombocytopenia each, patients responding in one or two hematopoietic lineages never worsened in another lineage [417]. A quarter of patients became independent of platelet transfusions and 11% became independent of red blood cell transfusions. This beneficial result is supported by the fact that the median time to progression was 12.4 months in the CR patients. Taken together, these results compare favorably with the results obtained with 5-azacytidine and decitabine in high-risk patients and make further trials with FTIs such as tipifarnib (Zarnestra) and lonfarnib (Sarasar) [418] reasonable.

6.16.2 FLT3-Antagonist Tandutinib (MLN518/CT53518) Tandutinib is an orally applicable inhibitor of type III tyrosine kinases, namely FLT3, PDGFRb and KIT, with very limited activity outside this family of receptor-associated kinases. Activating mutations of FLT3 (internal tandem duplications or activating point mutations in the kinase activation loop of the receptor) are found in 20 30% of AML and generally portend poor prognosis in high-risk MDS and AML. A phase I trial with tandutinib in patients with these entities demonstrated evidence of antileukemic efficacy with decreases in both peripheral blood and bone morrow blasts. Dose limiting toxicity was reversible generalized muscular weakness and/or fatigue [419]. Other side effects included grade I vomiting and diarrhea, a possible prolongation of the QT interval, as well as peripheral and periorbital edema. The latter is interestingly also observed with imatinib and has been attributed to inhibition of PDGF. The optimal dosage determined was 525 mg b.i.d.

6.17 Targeting NF-kB NF-kB activates multiple downstream genes with antiapoptotic properties. High-risk MDS and MDS/AML are characterized by a low intramedulllary apoptotic tendency and strong, constitutive NF-kB activation [422], which is confined to cells carrying MDS associated genetic aberrations [421]. NF-kB activation correlates with blast

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counts and suppression of apoptosis, thus contributing to malignant transformation and disease progression [421]. Targeting NF-kB results in rapid induction of apoptosis in primary bone marrow and immature myeloid cells in patients with high-risk MDS [421, 422]. In contrast, enhanced apoptosis is typical of low-risk MDS in which NF-kB is not activated. Thus, NF-kB activation seems to be vital for MDS blasts and at least partly responsible for the suppression of apoptosis, therefore contributing to malignant transformation.

6.17.1 Bortezomib (Velcade) In accordance, NF-kB inhibition by bortezomib results in down-regulation of apoptosis inhibitory NF-kB target genes and subsequent cell death in bone marrow blasts from high-risk, but not low-risk, MDS patients [421]. Partial response rates of up to 35% have been observed in MDS-patients receiving bortezomib [425]. A phase I/II clinical trial revealed 24% response rate, including 3/37 patients with CR in intermediate-2 and high-risk MDS patients treated with bortezomib and low-dose cytarabine [426]. Furthermore, platelet budding from megakaryocytes is a process that depends in part on NF-kB, providing the therapeutic rational for bortezomib in MDS with thrombocytosis. Bortezomib leads to absolute decreases in platelet counts by 60%, by a mechanism which is though to transiently affect megakaryocyte function through alterations in cytokine levels, rather than a lethal effect on bone marrow megakaryocytes or decreased thrombopoietin production [423, 424]. In line with this, proteasome inhibition prevents activation of NF-kB, thus reducing the levels of potent thrombopoiesis factors IL-6 and TNF-a. Furthermore, major erythroid response and normalization of platelet counts was reported in a patient with 5q syndrome with thrombocytosis treated with bortezomib [424].

6.17.2 Arsenic Trioxide (Arsenox) Arsenic trioxide by induces apoptosis in the neoplastic clone and causes major alterations in the microenvironment. In fact, NF-kB is upregulated in the mononuclear and CD34 þ cells of patients with high stage MDS as compared to cells of RA/RARS and this stage-dependent activity of NF-kB seems to be under the control of exogenous TNF-a [427]. Arsenic trioxide downregulates the NF-kB activity even in the presence of exogenous TNF-a and subsequently

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downregulates NF-kB-dependent anti-apoptotic proteins like Bcl-2, BclXL, XIAP, FLICE and FLIP. It thus causes a mitochondrial membrane depolarization, release of cytochrome C, production and release of reactive oxygen species and induction of apoptosis [428, 429]. Furthermore, arsenic trixoxide directly targets the neoangiogenic vasculature in the microenvironment. The microvasculature is significantly expanded in the marrow of MDS patients. The density of microvessels increases with the stage of disease and transformation into acute leukemia, as does the production of VEGF. VEGF is in the center of an auto- and paracrine signaling pathway in which leukemic blasts produce VEGF which causes endothelial cells to secrete cytokines like IL-6, IL-7, IL-10 and GM-CSF. These cytokines deliver survival and proliferative signals to the neoplastic clone [430]. Arsenic trioxide is capable of interrupting this loop by inducing apoptosis in endothelial cells of new blood vessels [431]. Two major single agent Arsenic trioxide trials were performed in MDS. Hematologic responses were obtained in 19% of 115 European [432] and 20% of 70 US patients [433]. These responses were observed in all lineages, with some predominance of the erythroid lineage. In the US trial, hematologic improvement was nearly exclusively observed in low risk patients (34% vs. 6%), but this difference was not apparent in the European trial. Survival was longer in responding as compared to non-responding patients, both in the low-risk and in the high-risk MDS group [432]. Side effects were usually mild and QT prolongations varied significantly between the two trials. Due to the moderate effects in single agent trials, combinations of arsenic trioxide with thalidomide [434] plus/minus retinoic acid [435] were carried out. Twenty-five percent of patients responded in the doublet trail (arsenic trioxide thalidomide), whereas 48% responded in the triplet trails (arsenic trioxide þ thalidomide þ retineic acid). Indirect comparison pointed to some survival benefit in the RAEB-t group for the triple combination. Currently, all Arsenic trioxide applications remain experimental and require proof of benefit in phase III trials. Future trials will have to focus on the best combination partners and on refining the patient selection. In this regard it is noteworthy, that high expression levels of EVI-1, although regarded as an unfavorable prognostic marker (particularly in patients with inv3(q21q26.2)), predicted for the response to the combination of arsenic trioxide with thalidomide [434].

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6.18 Modulation of Pro-Apoptotic Cytokines with Pentoxiphylline, Dexamethasone and Ciproﬂoxacine The overproduction of proapoptotic cytokines, especially TNF-a, soluble Fas-ligand, MIP-1a, TGF-b, IL-6, IFNg and IL-1b, is measurable in the bone marrow microenvironment and serum of MDS patients. Inhibition of these cytokines in combination with cytoprotection is a relatively new therapeutic approach, which seems to provide substantial palliation for IPSS low-risk MDS patients. The ultimate motive in this treatment approach is that suppression of proapoptotic cytokines with subsequent reduction in apoptosis is presumed to lead to an improvement in peripheral cytopenias. However, this must be seen with great scepticism, as reduction in apoptotic capacity of dysplastic cells due to additionally acquired genetic events has been shown to coincide with, and potentially induce, leukemic transformation [12, 436]. In good accordance with this hypothesis, only patients with low-risk IPSS seem to respond to anti-apoptotic treatment. Data concerning transformation rates for patients receiving this sort of therapy was not made available. Furthermore, the suppression of merely one proapoptotic cytokine is not likely to be the most promising approach, due to the plethora of cytokines involved and the redundancy of apoptotic pathways in MDS. Modulation of proapoptotic cytokines with pentoxiphylline, dexamethasane and ciprefloxacine leads to responses in at least one lineage in 52 76% of patients, with median time to response being 4 weeks. Several small studies indicate that this combination is well tolerated, the most prominent side effects being nausea (57%), vomiting (10%) and hepatic toxicity with elevations of transaminases [437]. The rational for combining these three substances is given by: (i)

(ii) (iii)

(iv)

(v)

the reduction of activity of proapoptotic cytokines due to interference of pentoxiphylline with the used signaling pathway; the reduction in translation of TNF-a mRNA by dexamethasone (4 8 mg/day p.o); the reduction of hepatic degradation of pentoxiphylline (400 800 mg 3 times / day) due to adjuvant administration of ciprofloxacine (500 mg twice/day); the amifostine-mediated (200 400 mg/m2 i.v. 3 times / week) suppression of apoptosis, TNF-aand IL-6 production; the stimulation of hematopoiesis and cytoprotection.

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Summary Box 2: MDS treatment options Supportive/palliative treatment of cytopenias RBC transfusions (irradiated, leukocyte depleted) PLT transfusions (irradiated) * Erythropoietin * G-CSF, pegylated G-CSF, (GM-CSF) * Danazol * Vitamin D3 * AMG-531 * IL-3 * Recombinant pegylated MGF (1) * Amifostine þ pentoxyfylline þ ciprofloxacin Treatment of iron overload (ferritin levels H1,500 mg/l) * Deferasirox * Deferoxamine * Deferiprene First-line treatment of transfusion-dependent MDS * 5-Azacytidine * Decitabine First-line treatment of transfusion dependent MDS with 5q syndrome * Revlimid Second-line treatment of transfusion dependent MDS * Thalidomide * Tipifarnib * Arsen trioxide Treatment of MDS/AML Palliative * Low dose melphalan * Low dose cytarabine * Low dose etoposide * Oral idarubicine * CPT11 * Gemcitabine * Topotecan Curative treatment of MDS/AML * Cytarabine þ Ara-C * Cytarabine þ daunomycin * Cytarabine þ daunomycin þ etoposide * Cytarabine þ daunomycin þ thalidomide * Cytarabine þ daunomycin þ topotecan þ thalidomide * Daunomycin þ topotecan þ thalidomide * Cytarabine þ daunomycin þ gabapentin * Cytarabine þ idarubicin * Cytarabine þ idarubicin þ amifostine * Cytarabine þ idarubicin þ topotecan * Cytarabine þ idarubicin þ fludarabine þ G-CSF * Cytarabine low-dose þ etoposide * *

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Cytarabine low-dose þ ATRA þVitamin D3 Hemaotopietic stem cell transplantation * Matched related * Matched unrelated * Mismatched related * Nonmyeloablatice/reduced intensity conditioning (RIC) * Autologous Treatment of hypoplastic MDS * Antithymocyte globulin (ATG) * Cyclosporin A * Glucocorticoids * Mycophenolate mofetil * And combinations thereof Experimental targeted therapies MDS * Inhibitors of TNF-a (enbrel, infliximab, etc.) * Targeting of VEGF (bevacizumab, etc.) * Wilms Tumor peptide vaccination * Amifostine * Gemtuzumab ozogamicin * Hexamethylene bisacetamide * IFN-a * Imatinib mesylate * Phenylbutyrate * SU5416 *

Italics indicate substances that have not been approved by the EMEA or FDA in these indications (yet)

Patients who responded to treatment showed improvement of cytopenia(s) in parallel to an increase in Bcl-2 expression, a decrease in TNF-a-expression, and a decreased rate of apoptosis. However, as no complete responses were observed, such approaches are insufficient by themselves. It has been proposed that an initial improvement of cytopenias by an anticytokine/proapoptotic approach, may be consolidated by consecutive use of cytotoxic agents [45]. Summary Box 2 gives an overview of MDS treatment options.

6.19 MDS Subtypes Associated with Certain Cytogenetic Features 6.19.1 5q Syndrome Deletions of the long arm of chromosome 5 have been repeatedly reported to be the most frequent structural abnormality in de novo MDS (30%), occurring either as an isolated aberration (10%) or within a complex karyotype (20%). However, deletions of 5q are not restricted to de novo and therapy-related MDS, but are

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also found in AML and chronic myeloproliferative disorders (CMPDs). The WHO recognizes a distinct syndrome within a minor subgroup (10%) of MDS patients with isolated deletions of the long arm of chromosome 5q, which was first described in 1974 as a distinct clinical entity. The precise deletion end points have recently been mapped, revealing a cluster of 8 proximal break points in band q14.3 and a distal cluster comprising 11 breakpoints between bands q33.2 and q34 [438], with the common deleted region being located at 5q32 [439]. Chromosome 5 is rich in genes encoding cytokines and their receptors such as interleukines (IL-4, IL-5, IL-3, IL-9), IRF-1 (interferon regulator factor-1), GMCSF (gramolocyte-monocyte colony stimulating factor) and EGR-1 (early growth response-1). It has been hypothesized, that deletion of 5q leads to altered cytokine levels, thereby increasing proliferation of megakaryocytes. In accordance both TNF-a (tumor necrosis factor a) and IL-4 are negative regulators of megakaryopoiesis. Until very recently, no single underlying tumor suppressor had been identified among the known 41 genes within the critical region, although many putative genes had been suggested (e.g., [440 442]). At the ASH-meeting 2007, Ebert presented strong evidence that the 5q syndrome is caused by haploinsufficiency of the single gene RPS14, resulting in defective ribosomal protein function [443]. The 5q syndrome is a karyotypically, morphologically and clinically distinct entity, characterized by a striking female preponderance, an isolated 5q interstitial deletion, transfusion dependent severe hypoplastic macrocytic anemia, normal or increased platelet counts, bone marrow hyperplasia of hypolobulated micro-megakaryocytes, less than 5% bone marrow myeloblasts [439], low incidences of neutropenia and leukemic transformation (5 16%) [444, 445] and a relatively indolent clinical course. Interestingly, only 6 14% of del(5q) patients respond to erythropoietin therapy, in contrast to 10 37% of unselected MDS patients [118, 119]. This phenomenon is currently unexplained. Dramatic responses to thalidomide and especially its more potent analogue lenalidomide are common in MDS patients with del(5q) as the sole karyetypic anomaly. Furthermore, patients with del(5)(q13q31) have significantly longer survival than patients with other 5q deletions. MDS patients with 5q syndrome, del(5q) excluding 5q syndrome or (5) have median overall survival times of 6000 days, 510 days and 210 days, respectively. It is noteworthy, that none of the 5q syndrome patients died of leukemic progression [113]. In contrast, del(5q) is among the worst prognostic indicators in AML [446]. This may be due to additional

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cytogenetic abnormalities or involvement of a malfunctioning different tumor-suppressor gene for AML with del (5q). For further details and treatment of MDS-patients with deletions of 5q see Sect. 6.14.2.

6.19.2 MDS with Isolated del(20q) MDS with isolated deletion of the long arm of chromosome 20 was proposed to be a subtype of MDS with distinct hematological and prognostic features in 1993 by Wattel [447], although it has thus far not been defined as a specific entity by the WHO. These patients commonly present with thrombocytopenia. In fact, del(20q) was the most frequent cytogenetic abnormality found in MDS-patients with isolated thrombocytopenia [448]. Patients with isolated deletions of 20q seem to be characterized by minimal morphological dysplasia [449], and importantly, a tendency towards lower incidences of anemia, as well as lower levels of excess blasts and a lower incidence of transformation to AML [450]. These beneficial clinical features translate into longer overall survival for the del(20q) subgroup, which was only surpassed by patients with the prognostically favorable isolated del(5q) anomaly [447]. In fact, the morphological changes in this subgroup of patients are so subtle, that cytogenetic analysis of chromosome 20 is expected to be helpful in avoiding the misdiagnosis of ITP.

6.19.3 Monosomy 7 Syndrome The monosomy 7 syndrome has been described in young children and is characterized by refractory anemia, leucocytosis, thrombocytopenia and common evolution to AML [451, 452]. Recently, a marked increase in the percentage of immature platelet fraction of H10%, despite the absence of severe thrombocytopenia, has been found to be a marker of karyotypic abnormalities associated with poor prognosis, including chromosome 7 abnormalities and monosomy 7 [453].

6.19.4 MDS with Isolated Trisomy 8 Trisomy 8 (sub)clones occur in approximately 5% of patients with MDS, and appear to be a relatively benign chromosomal abnormality, which can often be detected long before MDS manifests. In patients in whom MDS has evolved from aplastic anemia, trisomy 8 is associated with a relatively good prognosis. In particular, the incidence of leukemic transformation seems to be especially low [454]. Furthermore, de novo MDS with isolated trisomy 8 is

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associated with oligoclonal CTL-expansions, suggestive of an antigen-driven pathophysiology [378] (see also Sect. 6.3.2). In line with these observations, trisomy 8 MDS responds well to immunosuppressive therapy. Fasexpression seems to be an intrinsic property of trisomy 8 cells, rendering them more sensitive to Fas-mediated apoptosis [455]. This correlates well with increased apoptotic markers in trisomy 8 disease. MDS patients with trisomy 8 often show HLA-DR15 positivity which has also been reported to go hand in hand with good responsiveness to immunosuppressive treatment (see also Sect. 6.13.1). Sloand et al. documented durable reversion of cytopenias and restoration of transfusion independence to ATG in 67% of patients with trisomy 8 as the sole karyotypic anomaly [378]. The same group demonstrated significant clonal CD8þ CTL expansions with selective use of one or more TCR-Vb subfamilies in all trisomy 8 MDS patients. This high response to immunosuppressive therapy, is achieved by reduction of trisomy-8-specific CTLs, thereby alleviating CTL-mediated suppression of hematopoiesis.

6.19.5 17p Syndrome The 17p syndrome has been rarely described and is characterized by dysgranulopoiesis, pseudo-Pelger-Huet hypolobulation and the presence of small granules in granulocytes. Clinically, MDS patients with deletions of the short arm of chromosome 17 seem to have a more aggressive disease which coincides with treatment resistance and short survival [456, 457].

6.19.6 3q21q26 Syndrome Inv(3)(q21q26) and t(3;3)(q21;q26) are specifically found in myeloid neoplasias characterized by disturbances of thrombopoiesis and megakaryocyte development. These aberrations involving 3q 21q 26 have been described in all FAB subtypes of AML (with the exception of M3), as well as in MDS, megakaryoblastic crisis in CML, PV and MMM [458 461]. These aberrations occur in 0.5 3.6% of AML patients (mostly M1), 50 55% of which have a brief phase of preceding MDS and/or exposure to mutagenic/carcinogenic agents such as topoisomeraseII-inhibitors. Trilineage dysplasia, an excess of micromegakaryocytes, normal or elevated platelet counts, as well as young age (G55 years), extremely poor response rates to first-, second- and third-line therapy with accordingly poor median OS rates of 5.5 months, are typically associated with the 3q21q26 syndrome. It has been hypothesized that transformation is a consequence of inappropriate expression of the EVI-1 gene (ectopic viral integration site-1), which maps to

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3q25 26. Most patients with chromosomal abnormalities involving 3q26 overexpress EVI-1 [462], but not all patients with overexpression of EVI-1 have structural abnormalities of 3q26, which has also been associated with del(7) [64, 461]. Furthermore, GATA-2, a gene in the breakpoint cluster region of 3q21 which plays a crucial role in hematopoietic differentiation, is overexpressed in the majority of patients with the 3q21q26 syndrome, suggesting that deregulation of GATA-2 also plays an important role in the pathophysiology of the disease in these patients [463]. Furthermore, mutations and translocations involving the RAF1 gene, located on 3p25, have been shown to abrogate apoptotic suppression by Bcl-2 [464].

6.20 MDS Variants 6.20.1 Therapy-Related MDS Therapy-related MDS is a severe life threatening complication of cytotoxic therapy. It is estimated that 10 20% of MDS and AML cases diagnosed at major centres are therapy-related [465]. Many different agents used in the treatment of neoplastic diseases have shown leukemogenic potential, with alkylating agents and topoisomerase-II inhibitors being among the most important ones (Table 6.21). Relative risks for MDS/AML in patients treated for breast cancer with alkylating agents have been shown to increase by tenfold and even by 17-fold when alkylating agents are combined with radiotherapy [466, 467]. In 7,110 patients treated with epirubicin containing regimens, the 8-year cumulative risk of developing MDS/AML was 0.55%. Clear-cut associations of MDS-risk with cumulative dosages of epirubicin (720 mg/m2), cyclophosphamide (6.300 mg/m2) but also with epirubicin doses (H100 mg/m2 per cycle or 33 mg/m2/ week) have been shown [466]. More recently, the use of G-CSF has been associated with an increased risk of treatment-associated MDS/AML [466, 468, 469]. Drugs and their association with treatment-related MDS and AML are shown in Table 6.21. In addition, a number of other factors predisposing to treatmentrelated MDS/AML have been demonstrated. Examples include lifestyle factors such as smoking, alcohol and use of hair-dyes, environmental factors such as viruses, chemicals, air pollution, benzene and irradiation, but also genetic factors such as Down-Syndrome, Blooms-Syndrome, enzymatic and DNA repair polymorphisms (for review see e.g., [470]). Overall, the life-time risk for secondary MDS/AML in patients

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Table 6.21: Drugs associated with the induction of treatment related MDS/AML Drug/regimen

Disease entity

Reference (comment)

Chlorambucil (C6)

Polycythemia rubra vera Rheumatoid arthritis

Mustargen (MOPP) vs. non MOPP containing Cylophosphamide

Morbus Hodgkin

[501, 502] (Associated with chromosome 5,7 anomalies and poor prognosis, occurring after 6.5 g Cb over 2 20 years) [503]

Breast cancer Rheumatoid arthritis Polycythemia vera

[502] (occurring after 118 g cyclophospha mide over 2 20 years) [504]

Breast cancer Testicular cancer Childhood acute lymphoblastic leukemia

[470, 505] [506] (at cumulative doses H650 mg) [507] (simultaneous application of G CSF and Asparaginase as risk factors) [470] [466, 467, 470, 472]

Pipobroman Busulphan Mitoxantrone Cisplatin Etoposide G CSF Alkylating agents (and others (HDCT þ SC transplantation) Radiophosphorus (P32) Zevalin Radiotherapy

Breast cancer adjuvant treatment Follicular lymphoma Multiple myeloma, Breast cancer Polycythemia vera Lymphoma Testicular cancer

[504] [506]

MOPP mechlovethamine, oncerin, pracarbatine, predmisene

with solid cancers, such as breast or germ cell cancers, is 1 5% with a sharp decrease after 10 years [470]. Adequate weighting of risk/benefit ratios in primary treatment of malignancies, optimized counselling concerning the control of additional risk factors and thorough observation during follow-up are therefore mandatory. Whereas most patients with de novo AML present with normal karyotypes, secondary MDS/AML cases display chromosomal abnormalities in 90% of cases, with only 10% of patients remaining with apparently normal karyotypes (Fig. 6.23). Grouping treatment-related 60 50 40 30 20 10 0

de novo MDS

t-MDS

de novo AML

t-AML

Fig. 6.23 The frequency of normal karyotypes in primary and therapy-induced MDS and acute leukemias (adapted from [467]). Numbers depicted on Y axis represent frequencies of normal kar yotypes in %, de novo diseases are depicted in beige. Treatment related MDS and AML are depicted in brown. The frequency of normal karyotypes is much lower in treatment related MDS/AML than in de novo MDS/AML

MDS according to the karyotypic pattern shows unbalanced cytogenetics (e.g., 5q /5, 7q /7 or þ 8) in 50 70% of secondary MDS (i.e., a roughly tripled frequency in comparison to de novo cases) and balanced cytogenetics (e.g., 11q23, 21q22, 17q21 and 16q22) in 40 50%. As show in Fig. 6.23, cases with normal karyotypes are much less frequent in treatment related MDS/ AML than in de novo cases of MDS and AML. It is assumed that the molecular pathogenesis of treatmentrelated MDS is similar to that of de novo cases, but that frequencies of well known molecular pathways are different. As mentioned in 6.3.4. at least three different events seem to be important in the multistep carcinogenesis, i.e., (i) mutations within the tyrosine kinase RAS/B-RAF pathway with subsequent increases in cell proliferation (class I mutations), (ii) loss-of-function mutations in hematopoietic transcription factors and the transcription regulatory protein NPM1 leading to altered maturation and differentiation (class II mutations) and (iii) inactivating mutations in p53. There is strong evidence for cooperation of class I and class II events in the leukemogenic process. Therapy-related MDS is known to more rapidly transform into AML and this event may be under the control of point mutations within the AML-1 and the Ras genes, respectively [465, 471]. According to the differences in causative agents, eight further distinct molecular pathways have been described (Table 6.22). Particular cytogenetic patterns are not only associated with the various agents involved in leukemogenesis

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Table 6.22: Molecular pathways currently recognized in treatment related MDS (modified according to [467, 508 511]) Type of signaling pathway

Characteristic karyotypic anomaly

I

* * *

II

* * *

III

*

IV

*

*

V

*

VI VII

*

VIII

*

*

7q / 7 but Normal chromosome 5 No balanced aberrations 5q / 5 No balanced aberrations Complex chromosome rearrangements Translocations 11q23

Balanced translocation 21q22 or inv (16) 17q21 Translocations 11p15 Normal karyotype

Chromosome 8 aberrations

Involved gene(s)

Causative agents

Comment

AML1 point mutations

Alkylating agents

t MDS or t AML

Mutated p53

Alkylating agents

t MDS or t AML

MLL gene involved in many translocations with different partner genes N Ras. K Ras or B Raf mutations are common Chimeric rearrangement of core binding factor genes AML1, CBFB c kit muations RARA Intern tandem duplications of FLT3 NUP98 rearrangements NPM1 mutation; intern tandem duplication of FLT3; point muta tions of CEBPA; Ras mutations; internal tandem duplications of MLL Rare involvement of Ras/B Raf mutations

Topoisomerase II inhibitors (par ticularly epidopophyllotoxins)

Predominantly FAB M4 or FAB M5

Anthracyklines

Mostly overt AML

Mitoxantrone (in breast cancer)

FAB M3

Topoisomerase II inhibitors Radiotherapy only or cytostatics

t MDS or tAML Clinically atypical cases

No specific previous therapy associated

t MDS

(see Table 6.21), but also occur with different lag-times after chemotherapy. For example, deletions in chromosomes 5q or 7q typically emerge after 4 7 years [472], while MDS involving 11q23 develops more rapidly (1 4 years) after treatment [473]. Cytogenetics are of major importance in predicting the course of the disease and prognosis in treatment-related

28

29

14 8

Fig. 6.24 Median survival times by karyotypic anomaly in treatment-related MDS (adapted from [476]). All numbers de picted represent survival times in months, depending on which cytogenetic aberration is present

MDS/AML. An analysis of 511 cases showed a range of survival times between 8 and 29 months, respectively (Fig. 6.24), depending on the karyotypic anomaly present [474]. The predictive and prognostic power of cytogenetics may also be demonstrated by the fact that the prognosis was worst for patients with 11q23 involvements, despite the younger age of these patients and their more frequent treatment with blood or marrow transplantation [474]. In fact, translocations involving 11q23 were found in 30% of treatment related MDS/acute leukemia. Clinically these patients developed ALL more often, with a shorter latency period, and shortest overall survival of all cytogenetic aberrations found in this study [474]. These patients more often received alkylating agents or topoisomerase-II inhibitors than patients with t-MDS/AML with other cytogenetic aberrations. Treatment of therapy-related MDS/AML closely follows the principles of the relevant de novo cases, although these patients typically do not respond well to any type of treatment and usually rapidly progress. Allogeneic bone marrow or peripheral blood stem cell transplantation seems of particular importance in patients carrying the worst prognosis, i.e., those with 11q23 anomalies. In these patients the 1 year survival rate has been doubled by allotransplantation [474].

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6.20.2 Hypocellular or Hypoplastic MDS The borderline condition of hypoplastic MDS shows features of both aplastic anemia and MDS. Hypocellular MDS represents 12 17% of MDS cases and is characterized by a bone marrow cellularity of less than 25 30% [475, 476]. Hypoplastic MDS appears to be a distinct clinicopathologic entity characterized by age-corrected bone marrow hypoplasia [477], macrocytosis, severe leukopenia and thrombocytopenia, low incidence of progression to acute leukemia, and unresponsiveness to conventional therapy [476, 478 480]. It is currently a matter of dispute as to whether patients with hypoplastic MDS have a better prognosis than patients with normocellular MDS. Tuzuner et al. demonstrated no difference in prognosis between hypo- and normo-/hypercellular MDS [478], whereas others have seemingly shown a better prognosis for this MDS subgroup [481]. Importantly, hypoplastic MDS must be differentiated from aplastic anemia, as not only treatment but also prognosis differs substantially between these two disease entities. Hypocellular MDS (see Chapter 10.3.1) can usually be differentiated from aplastic anemia due to the characteristic presence of morphologically and karyotypically abnormal dysplastic marrow cells, particularly dysmegakaryopoiesis. In a pancytopenic patient, the presence of distinct dysmegakaryopoiesis, increased circulating blasts, clusters of blasts in the bone marrow, or a clonal cytogenetic abnormality definitely point to the diagnosis of MDS [476, 482]. Normal or increased percentages of CD34þ cells [483] as well as lower levels of TNF-receptors I and II [484] have also been shown to adequately distinguish between hypoplastic MDS and aplastic anemia. Patients with aplastic anemia typically have reduced bone marrow CD34þ cell numbers [483] and greater TNFR-I and TNFR-II expression levels [484]. Additionally MRI is useful in the distinction of these disease entities (see respective chapter on aplastic anemia and overlap syndromes, Chapter 10). As mentioned above, distinction between is important, as the clinical course and the therapeutic management differs. Hypocellular MDS seems to respond especially well to immunosuppressive treatment ([391] and see chapter on immunosuppressive treatment in MDS Sect. 6.13). Some patients with hypoplastic MDS may have a remarkably indolent clinical course [476]. A recent publication comparing 37 hypoplastic MDS patients with a cohort of 152 non-hypoplastic MDS patients revealed that the IPSS is applicable for hypoplastic MDS, and that IPSS low and intermediate-1 hypoplastic MDS patients had a superior survival than

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non-hypoplastic MDS patients [481]. Furthermore, chromosomal abnormalities occurred more often in the non-hypoplastic MDS patients and different patterns of WHO-disease subtypes were reported, with MDS-RA being the predominant subtype in hypoplastic MDS (57%) and RAEB being the most common subtype in non-hypoplastic MDS (45%) [481]. However, there is currently no consensus as to whether patients with hypoplastic MDS should be treated differently than patients with normo/hyper-cellular MDS. It has been proposed however, that allogeneic bone marrow transplantation may not be the treatment of choice in the hypoplastic subgroup of MDS patients, given the assumed indolent course and immune-mediated pathogenesis [480].

6.20.3 Hyperﬁbrotic MDS MDS with increased marrow fibrosis is seen as a distinct clinico-pathological entity by some [487]. Patients are often pancytopenic and have bone marrow trilineage dysplasia with atypical megakaryocyte proliferation and severe bone marrow fibrosis. As in primary myelofibrosis (PMF, see Chapter 4), the intense fibroblastic proliferation is thought to be the result of abnormal liberation of cytokines, in particular TGF-b and PDGF [485]. Absence of splenomegaly and a generally rapid progressive clinical course set this entity apart from PMF [485]. The distinction from accelerated phase CML and AML (especially M7) can prove more difficult, however. In a retrospective analysis of 352 MDS patients, myelofibrosis was found in approximately 17% of cases [486]. Whilst focal myelofibrosis and reticulin fibrosis occurred frequently, collagen deposits were only rarely found [486]. Increasing myelofibrosis grades coincided with an elevated frequency of cytogenetic aberrations, higher incidences of CMML, lower peripheral hemoglobin and platelet counts, increased dysmegakaryopoiesis, as well as enhanced rates of leukemic transformation and a dramatically reduced life expectancy (9.6 months vs. 17.4 months in MDS patients with or without myelofibrosis, respectively) [486].

6.20.4 Familial MDS Rare familial occurrence of MDS, AML or both has been reported (reviewed in [487]). Variability in the age of disease onset has been interpreted as evidence of anticipation [488]. In familial MDS/AML one must distinguish between those groups without or with an underlying hereditary monogeneic disorder, such as Fanconi anemia,

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Low risk + transfusion-dependent

Favorable ESA-response Profile*

Del(5q) Non del(5q) karytotype abnormality

ESA ± G-CSF

ESA Failure

Unfavorable ESA-response profile

Lenalidomide

Failure

Normal karyotype 5-azacitdine or decitabine

Intermediate/high risk

Failure AML-like induction/consolidation

Allogeneic Transplant

Allo-Transplant RIC

< 65a (and no CI)

> 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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Chap. 6

Myelodysplastic Syndromes

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Myelodysplastic Syndromes

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Chap. 6

Myelodysplastic Syndromes

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Myelodysplastic Syndromes

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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)

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Chronic Myelomonocytic Leukemia (CMML)

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.

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Chronic Myelomonocytic Leukemia (CMML)

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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Chap. 7

Chronic Myelomonocytic Leukemia (CMML)

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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

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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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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

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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)

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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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associated with therapy intensification. Blood 111: 2556 2562 Pileri SA, Grogan TM, Harris NL et al. (2002) Tumours of histiocytes and accessory dendritic cells: an immunohis tochemical approach to classification from the International Lymphoma Study Group based on 61 cases. Histopathology 41: 1 29 Hornick JL, Jaffe ES, Fletcher CD (2004) Extranodal histiocytic sarcoma: clinicopathologic analysis of 14 cases of a rare epithelioid malignancy. Am J Surg Pathol 28: 1133 1144 Kairouz S, Hashash J, Kabbara W, McHayleh W, Tabbara IA (2007) Dendritic cell neoplasms: an overview. Am J Hematol 82: 924 928 Ferringer T, Banks PM, Metcalf JS (2006) Langerhans cell sarcoma. Am J Dermatopathol 28: 36 39 Misery L, Godard W, Hamzeh H et al. (2003) Malignant Langerhans cell tumor: a case with a favorable outcome associated with the absence of blood dendritic cell prolifera tion. J Am Acad Dermatol 49: 527 529 Efune G, Sumer BD, Sarode VR, Wang HY, Myers LL (2009) Interdigitating dendritic cell sarcoma of the parotid gland: case report and literature review. Am J Otolaryngol 30: 264 268 Gaertner EM, Tsokos M, Derringer GA, Neuhauser TS, Arciero C, Andriko JA (2001) Interdigitating dendritic cell sarcoma. A report of four cases and review of the literature. Am J Clin Pathol 115: 589 597 Chera BS, Orlando C, Villaret DB, Mendenhall WM (2008) Follicular dendritic cell sarcoma of the head and neck: case report and literature review. Laryngoscope 118: 1607 1612 Abidi MH, Tove I, Ibrahim RB, Maria D, Peres E (2007) Thalidomide for the treatment of histiocytic sarcoma after hematopoietic stem cell transplant. Am J Hematol 82: 932 933 Schwartz RH, Hayden GF, Rodriguez WJ, Sait T, Chhabra O, Golub J (1986) Leukocyte counts in children with acute otitis media. Pediatr Emerg Care 2: 10 14 Pouchot J, Sampalis JS, Beaudet F et al. (1991) Adult Stills disease: manifestations, disease course, and outcome in 62 patients. Medicine (Baltimore) 70: 118 136 McBride JA, Dacie JV, Shapley R (1968) The effect of splenectomy on the leucocyte count. Br J Haematol 14: 225 231 Christensen RD, Hill HR (1987) Exercise induced changes in the blood concentration of leukocyte populations in teenage athletes. Am J Pediatr Hematol Oncol 9: 140 142 Dale DC, Fauci AS, Guerry D, IV, Wolff SM (1975) Comparison of agents producing a neutrophilic leukocytosis in man. Hydrocortisone, prednisone, endotoxin, and etiocho lanolone. J Clin Invest 56: 808 813 Boggs DR, Joyce RA (1983) The hematopoietic effects of lithium. Semin Hematol 20: 129 138 dePalma L, Delgado P (1996) Clin Chem Acta 244: 83 90 Tanaka KR, Valentine WM (1960) NEJM 262: 912 918 Stinson RA, Mcphee J (1985) NEJM 312: 1642 1643

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-

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De novo Classic PNH

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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

CD55 CD59

Eculizumab

2 NO

C5 convertase

C3 convertase

C3

Free Hb

C3b

C5

C3a

C5a

Procoagulant activity of n.gr.↑

C5b

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Va

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Heme

L-Arginine NO scavenging

O2 TNFα

•MAC-mediated PLT - activation & granule secretion - PLT membrane blebbing - Procoagulant surfaces ඍ

TG↑

NOS ROS ↑

Citrulline ඏ

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NO Peroxinitrile

•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

Vasoconstriction

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

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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

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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

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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-

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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-

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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.

L. Pleyer and R. Greil

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

(10000) [Erys] FL1 Log - ADC

(10000) [Erys] FL1 Log - ADC

[Ungated] SS Log/FS Log - ADC 103

53

59 CD55

CD59 100.0%

100.0% 2

FS Log

10

101

CD55 neg 43.5%

Erys 96.1%

CD59 neg 43.1%

100

100

101

102

103

100

b

101

102

103

100

CD55 FITC

SS Log

54

1023

103

59

CD55 100.0%

CD59 100.0%

CD55 neg 69.5%

CD59 neg 69.4%

SS Lin

Grans 40.0%

102

(5000) [Grans] FL1 Log - ADC

(5000) [Grans] FL1 Log - ADC

[Ungated] FS Lin/SS Lin - ADC

101

CD59 FITC

0

0

1023

FS Lin

100

101

102

CD55-FITC

103

100

101

102

103

FL1 Log

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].

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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

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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-

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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

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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.

L. Pleyer and R. Greil

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.

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De novo Classic PNH

[122] Karim A, Ahmed S, Siddiqui R, Mattana J (2001) Methemo globinemia complicating topical lidocaine used during endo scopic procedures. Am J Med 111: 150 153 [123] Fincher ME, Campbell HT (1989) Methemoglobinemia and hemolytic anemia after phenazopyridine hydrochloride (Pyr idium) administration in end stage renal disease. South Med J 82: 372 374 [124] de Torres JP, Strom JA, Jaber BL, Hendra KP (2002) Hemo dialysis associated methemoglobinemia in acute renal fail ure. Am J Kidney Dis 39: 1307 1309 [125] Bjorge L, Ernst P, Haram KO (2003) Paroxysmal nocturnal hemoglobinuria in pregnancy. Acta Obstet Gynecol Scand 82: 1067 1071 [126] Frakes JT, Burmeister RE, Giliberti JJ (1976) Pregnancy in a patient with paroxysmal nocturnal hemoglobinuria. Obstet Gynecol 47: 22S 24S [127] Lange JG, Griever GE, Brand A, van Roosmalen J (1998) Paroxysmal nocturnal hemoglobinuria in pregnancy. Ned Tijdschr Geneeskd 142: 2308 2311 [128] Solal Celigny P, Tertian G, Fernandez H et al. (1988) Preg nancy and paroxysmal nocturnal hemoglobinuria. Arch In tern Med 148: 593 595 [129] Svigos JM, Norman J (1994) Paroxysmal nocturnal haemo globinuria and pregnancy. Aust N Z J Obstet Gynaecol 34: 104 106 [130] Takagi H, Imai A, Kawabata I, Sumi H, Shiraki S, Tamaya T (1989) A good outcome pregnancy in a patient with paroxysmal nocturnal hemoglobinuria. J Med 20: 163 170 [131] Heilmann L, Siekmann U, Ludwig H (1980) Paroxysmal nocturnal haemoglobinuria (PNH) and pregnancy (authors transl). Geburtshilfe Frauenheilkd 40: 682 687 [132] Buisson MP, Quereux C, Palot M, Pignon B, Wahl P (1991) Nocturnal paroxysmal hemoglobinuria disclosed during

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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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Fig. 10.1 Overview of overlap syndromes

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

Chap. 10 Clonal Bone Marrow Failure Overlap Syndromes

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

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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.