The Myelodysplastic Syndromes

The Myelodysplastic Syndromes

Judit Várkonyi Editor

The Myelodysplastic Syndromes

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Editor Prof. Judit Várkonyi 3rd Department of Internal Medicine Semmelweis University Kútvölgyi út 4, 1125 Budapest Hungary [email protected]

ISBN 978-94-007-0439-8â•…â•…â•… e-ISBN 978-94-007-0440-4 DOI 10.1007/978-94-007-0440-4 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2011921323 © Springer Science+Business Media B.V. 2011 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Cover design: deblik, Berlin Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

“In memory of my late father Zoltán Várkonyi and with love and gratitude to my dear husband Janos Quittner and son Zoltán Quittner”

Preface

Myelodysplastic syndromes (MDS) are the most common hematological malignancies involving mainly the elderly. They are defined as clonal stem cell disorders and characterized by ineffective hematopoiesis involving one to all bone marrow cell lineages [1]. The dominant morbidity of MDS relates to symptomatic cytopenias. According to various reports the annual incidence of MDS ranges widely from 2 to 12 per 100,000, increasing to 30–50 cases per 100,000 among persons aged 70 or older. It is believed that the true incidence of MDS has been underestimated and appears to be comparable to that of multiple myeloma and chronic lymphocytic leukemia [2, 3]. MDS may arise de novo, or as a result of previous environmental damage, or chemo-or radiotherapy with a peak incidence at 2–4 years following the initial exposure [4]. It might therefore be hypothesized that MDS arises due to cumulative environmental exposure in genetically predisposed individuals [5]. MDS may be regarded as a progression model in which the acquisition of genetic events occur by gain or loss of genetic material. MDS was previously named “preleukemia” or “smoldering leukemia” with a lack of terminal cells due to high apoptosis rate and the subsequent failure of differentiation. In about 25% of all cases when MDS progresses to AML—stem cell apoptosis stops and the cells fail to differentiate, a process that has been widely studied. In the past decade much progress had been achieved. We know more about disease pathophysiology resulting in increased emphasis on patient care and the evolution of targeted therapy. The chapters of this book offer updated knowledge on all clinically important aspects of the disease. Topics of great current interest are discussed by leading authors on MDS from different parts of the world. We would like to recommend this book to all those interested in this exciting and rapidly expanding field of hematology, including. medical students and postgraduates. Although MDS is a clonal disease it is not yet recognized as a malignant disease by the majority of health care systems. Consequently, access of MDS patients to novel, expensive and targeted therapeutic modalities is generally unsatisfactory. It is hoped that the present volume will increase awareness of the necessity of opti-

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mal treatment beyond simple supportive measures and facilitate the introduction of optimal treatment adequately sponsored by decision makers in health care systems.

References 1.╇Corey SJ, Minden MD, Barber DL, Kantarjian H, Wang JCY, Schimmer AD (2007) Myelodysplastic syndromes: the complexity of stem-cell diseases. Nat Rev Cancer 7:118–129 2.╇Rollison DE, Howlader N, Smith MT, Strom SS, Merritt WD, Ries LA, Edwards BK, List AF (2008) Epidemiology of myelodysplastic syndromes and chronic myeloproliferative disorders in the United States, 2001–2004, using data from the NAACCR and SEER programs. Blood 2008 112(1):45–52 3.╇Germing U, Aul C, Niemeyer CM, Haas R, Bennett JM (2008) Epidemiology, classification and prognosis of adults and children with myelodysplastic syndromes. Ann Hematol 87:691–699 4.╇Raposa T, Várkonyi J (1987) The relationship between sister chromatid exchange induction and leukemogenicity of different cytostatics. Cancer Detect Prev 10(1–2):141–151 5.╇Willman C (2001) MDS/AML: models of genetic progression and clues to etiology. Leukemia Res 25(1):1 (Abstr. Or3)

Budapest, Hungary

Prof. Judit Várkonyi

Contents

1  ╇ The History of the Myelodysplastic Syndromes ����������������������������������尓� ╅╇ 1 Ray M. Lowenthal 2  ╇Susceptibility to MDS: DNA Repair and Detoxification Genes ���������� ╅╇ 5 Claire Seedhouse and Nigel Russell 3  ╇Myelodysplastic Syndromes/Neoplasms: Morphological and Immunohistochemical Features and Standard Evaluation �����������������   25 H.-P. Horny and P. Valent 4  ╇Diagnostic Criteria and Classification of Myelodysplastic   Syndromes ����������������������������������尓������������������������������������尓�������������������������   43 Peter Valent, Friedrich Wimazal, Wolfgang R. Sperr and Hans-Peter Horny 5  ╇ Cytogenetics of MDS ����������������������������������尓������������������������������������尓��������   55 Detlef Haase, Christina Ganster, Christian Steidl, Katayoon Shirneshan, Friederike Braulke and Julie Schanz 6  ╇ Molecular Changes in Myelodysplastic Syndrome ����������������������������� â•… 87 Florian Nolte and Wolf-K. Hofmann 7  ╇ Prognostic Scoring in MDS ����������������������������������尓���������������������������������� ╇ 103 Michael Pfeilstöcker 8  ╇Flow Cytometry in Myelodysplastic Syndromes ����������������������������������尓 ╇ 121 C. Alhan, T. M. Westers, G. J. Ossenkoppele and Arjan A. van de Loosdrecht 9  ╇MDS as an Autoimmune Process ����������������������������������尓������������������������� ╇ 145 W. Ingram, Y. Kordasti and G. J. Mufti  he Myelodysplastic Overlap Syndromes ����������������������������������尓���������� ╇ 159 10  T W. Ingram and G. J. Mufti ix

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11  I ron and Copper Metabolism in the Myelodysplastic Syndromes ����� ╇ 175 Judit Várkonyi, Gabriella Bekő, Zoltán Prohászka and István Karádi 12  P  athogenesis and Management of Iron Overload in MDS ������������������ ╇ 187 Chaim Hershko 13  C  ytokines in MDS: Abnormalities and Treatment ������������������������������ ╇ 205 Howard S. Oster, Drorit Neumann and Moshe Mittelman 14  T  herapeutic Modalities and New Molecular Targets in MDS ������������ ╇ 219 Guillermo Garcia-Manero 15  H  aematopoietic Stem Cell Transplantation in MDS for Adults ��������� ╇ 239 Tamás Masszi 16  J MML and Myelodysplastic Syndrome in Children ��������������������������� ╇ 253 Henrik Hasle Appendix ����������������������������������尓������������������������������������尓������������������������������������尓 ╇ 279 Index ����������������������������������尓������������������������������������尓������������������������������������尓������� ╇ 285

Contributors

C. Alhan╇ Department of Hematology, VU Institute for Cancer and Immunology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Gabriella Bekő╇ 3rd Department of Internal Medicine, Semmelweis University, Kútvölgyi út 4, 1125 Budapest, Hungary Friederike Braulke╇ Abteilung Hämatologie/Onkologie, Georg-AugustUniversität Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany e-mail: [email protected] Christina Ganster╇ Abteilung Hämatologie/Onkologie, Georg-August-Universität Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany e-mail: [email protected] Prof. Chief Guillermo Garcia-Manero MD╇ Section of Myelodysplastic Syndromes, Department of Leukemia, MD Anderson Cancer Center, University of Texas, 1515 Holcombe Blvd, PO Box 428, Houston, TX 77025, USA e-mail: [email protected] Detlef Haase╇ Abteilung Hämatologie/Onkologie, Georg-August-Universität Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany e-mail: [email protected] Dr. Henrik Hasle╇ Department of Pediatrics, Skejby Hospital, Aarhus University, 8200 Aarhus N, Denmark e-mail: [email protected] Chaim Hershko MD╇ Department of Hematology, Shaare Zedek Medical Center, P. O. Box 3235, Jerusalem, Israel e-mail: [email protected] Wolf-K. Hofmann╇ Department of Hematology and Oncology, University Hospital Mannheim, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany

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H.-P. Horny╇ Institute of Pathology, Escherichstrasse 6, 91522 Ansbach, Germany e-mail: [email protected] Dr. W. Ingram╇ Department of Haematology, University Hospital of Wales, Heath Park, Cardiff, CF14 4XW, UK e-mail: [email protected] István Karádi╇ 3rd Department of Internal Medicine, Semmelweis University, Kútvölgyi út 4, 1125 Budapest, Hungary Y. Kordasti╇ Department of Haematological Medicine, King’s College London, London, UK Arjan A. van de Loosdrecht, MD, PhD╇ Department of Hematology, VU Institute for Cancer and Immunology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands e-mail: [email protected] Ray M. Lowenthal AO, MBBS, MD, FRCP, FRACP, FAChPM╇ Department of Clinical Haematology and Medical Oncology and Menzies Research Institute Tasmania, Royal Hobart Hospital, Hobart, TAS, Australia Department of Medicine, University of Tasmania, 7000 Hobart, Australia e-mail: [email protected] Tamás Masszi╇ Department of Haematology and Stem Cell Transplantation, St. István and St. László Hospital of Budapest, St. László Campus, Gyáli út 5-7, 1097 Budapest, Hungary e-mail: [email protected] Moshe Mittelman╇ Department of Medicine, Tel Aviv Sourasky Medical Center, 6 Weizmann St., 64239 Tel Aviv, Israel e-mail: [email protected] Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel G. J. Mufti╇ Department of Haematological Medicine, King’s College London, London, UK Drorit Neumann╇ Department of Cell and Developmental Biology, Tel Aviv University, Sackler School of Medicine building, Ramat Aviv, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Florian Nolte╇ Department of Hematology and Oncology, University Hospital Mannheim, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany e-mail: [email protected] G. J. Ossenkoppele╇ Department of Hematology, VU Institute for Cancer and Immunology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Howard S. Oster╇ Department of Medicine, Tel Aviv Sourasky Medical Center, 6 Weizmann St., 64239 Tel Aviv, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv

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Michael Pfeilstöcker╇ 3rd Medical Department and L. Boltzmann Institute for Leukemia Research & Hematology, Hanusch Hospital, H. Collinstr 30, 1140 Vienna, Austria e-mail: [email protected] Zoltán Prohászka╇ 3rd Department of Internal Medicine, Semmelweis University, Kútvölgyi út 4, 1125 Budapest, Hungary Prof. Nigel Russell╇ Department of Academic Haematology, University of Nottingham, Clinical Sciences Building, Nottingham University Hospitals—City Campus, NG5 1PB Nottingham, UK Julie Schanz╇ Abteilung Hämatologie/Onkologie, Georg-August-Universität Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany e-mail: [email protected] Dr. Claire Seedhouse╇ Department of Academic Haematology, University of Nottingham, Clinical Sciences Building, Nottingham University Hospitals—City Campus, NG5 1PB Nottingham, UK e-mail: [email protected] Katayoon Shirneshan╇ Abteilung Hämatologie/Onkologie, Georg-AugustUniversität Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany e-mail: [email protected] Wolfgang R. Sperr╇ Division of Hematology & Hemostaseology, Department of Medicine I, Medical University of Vienna and Ludwig Boltzmann Cluster Oncology, Waehringer Guertel 18-20, 1090 Vienna, Austria Christian Steidl╇ Department of Pathology, British Columbia Cancer Agency, 600 W 10th Avenue, V5Z 4E6 Vancouver BC, Canada e-mail: [email protected] P. Valent MD╇ Division of Hematology & Hemostaseology, Department of Medicine I, Medical University of Vienna and Ludwig Boltzmann Cluster Oncology, Waehringer Guertel 18-20, 1090 Vienna, Austria e-mail: [email protected] Judit Várkonyi╇ 3rd Department of Internal Medicine, Semmelweis University, Kútvölgyi út 4, 1125 Budapest, Hungary e-mail: [email protected] T. M. Westers╇ Department of Hematology, VU Institute for Cancer and Immunology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Friedrich Wimazal╇ Division of Hematology & Hemostaseology, Department of Medicine I, Medical University of Vienna Waehringer Guertel 18-20, 1090 Vienna, Austria Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria

Chapter 1

The History of the Myelodysplastic Syndromes Ray M. Lowenthal

The conditions that now come under the rubric of the myelodysplastic syndromes (MDS) have been recognised for over a century under a variety of names. Indeed, few haematological conditions seem to have excited as much interest in their history. Every review of MDS, it seems, is accompanied by a historical overview. So, how to provide information that is different? Where to begin? Perhaps we should look earlier than the first possible description of the disorder. The portrayal of MDS as an entity has relied on many preceding breakthroughs in the development of the speciality of haematology. One of the first was the recognition of the bone marrow as being the site of blood formation, a discovery attributed to Ernst Neumann (1834–1918) [1, 2]. The first comprehensive description of the various blood cells and their morphology was given by the German scientist/ physician Paul Ehrlich (1854–1915) in 1879 [3]. Ehrlich as a young man developed staining techniques which allowed the ‘colourless’ corpuscles (the leucocytes) to be visualised. This he achieved by utilising aniline dyes which contemporaneously were being developed by the German chemical industry for textile manufacturers [4]. Amongst other things he was the first to describe aplastic anaemia and distinguish it from other forms of anaemia. Ehrlich himself relied on developments in microscopy which took place over several centuries [5], and include particularly the name of Antonj van Leeuwenhoek (1632–1723). Another early discovery that has relevance to the MDS story was made in 1898. It was in that year that Marie and Pierre Curie described the element radium and its radioactivity, a discovery which in turn led to the development of radiotherapy. In the early part of the twentieth century radiotherapy was commonly used to treat the splenomegaly of some types of leukaemia and presumably also MDS; an irony, given that we now know that ionising radiation itself can cause leukaemia and MDS [6]. Indeed both Marie Curie and her elder daughter Irène, who carried on her R. M. Lowenthal () Department of Clinical Haematology and Medical Oncology and Menzies Research Institute Tasmania, Royal Hobart Hospital, Hobart, TAS, Australia Tel.: +61-3-6222-8157 Fax: +61-3-6226-4894 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_1, ©Â€Springer Science+Business Media B.V. 2011

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mother’s work on radioactivity after her mother’s death, are said to have died of ‘leukaemia’. Even more ironically Marie’s final illness, as reported in the biography written by her younger daughter Eve, could well be interpreted as MDS, particularly as she is said to have been anaemic for years beforehand. According to Eve, quoting the physician who cared for her, Marie died of ‘aplastic pernicious anaemia of rapid, feverish development. The bone marrow did not react’ [7]. Layton and Mufti (1986) provide a comprehensive bibliography of the important advances in the history of MDS up to that time [8]. They claim that the first account of MDS can be attributed to von Leube in 1900 who described a case of megaloblastic anaemia which preceded the onset of leukaemia [9]. Early appellations for the conditions which now come under the heading of MDS included pseudo-aplastic anaemia [10], a term that was based on the presence of peripheral blood cytopenias with a cellular rather than aplastic marrow; and refractory anaemia [11] on the grounds of the patient having a form of anaemia that did not respond (was ‘refractory’) to the known haematinics, iron, folic acid and vitamin B12 [12]. The first winner of the Harvey Cushing prize for the best essay on a topic in medical history was Jean Captain Sabine, who in 1938, wrote “A History of the Classification of Human Blood Corpuscles.” In it he summarised all the work up to that point [13]. In 1942 Chevallier and colleagues described a case they called odo-leukemia, odo allegedly being Greek for ‘threshold’ (indicating the condition being on the threshold of—or almost—leukaemia [14]. Interestingly they attributed their case to excessive exposure to benzene. In the late 1940s it was recognised that some cases of peripheral blood cytopenias ultimately transformed into acute leukaemia (usually of the myeloid type), thus leading to the terms preleukaemia or preleukaemic anaemia [15]. Then in the 1950s came the appreciation that cases of ‘refractory’ leucopenia or thrombocytopenia had a similar significance to refractory anaemia; this led to broadening of the terminology to include refractory cytopenia [16]. Further study of the natural history of the refractory cytopenias showed that they by no means all transformed to leukaemia, indeed only a minority did so, leading to the appreciation that the term ‘preleukaemia’ was prognostically misleading and that an alternative term was needed. Other early terms used (before 1976) included sideroblastic anaemia [17], smouldering leukaemia [18, 19] and dysmyelopoietic anaemia—this latter term continues to have currency [20]. The term subacute myeloid leukaemia probably refers to a subset of MDS [21]. The first FAB classification of the leukaemias, which included MDS, was proposed in 1976 [22] and a revision which expanded the system as it applied to MDS was introduced in 1982 [23]. The initial WHO classification was developed in 2000 [24] and revised in 2008 [25, 26]. Highlights in the scientific understanding of MDS include the first verification of the clonality of the condition, by G6PD isoenzyme studies [27] and the discovery of cytogenetic abnormalities including 5q- [28, 29]. In 1997 Greenberg et€al. introduced an international prognostic scoring system (IPSS) [30] that has become widely used; it was created in an attempt to synthesise information from several previously used scoring systems including those devised in Bournemouth, UK [31], Valencia, Spain [32], and Dusseldorf, Germany [33].

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The history of the treatment of MDS is unfortunately much shorter and less satisfactory than the history of its discovery and biology. Highlights include the use of bone marrow transplantation [34], the first report of use of growth factors, with GM-CSF [35]; the introduction of azacytidine, the first demethylating agent brought into clinical practice [36]; and the use of lenalidomide [37]. Hopefully when the history of the myelodysplastic syndromes is re-written in decades hence, much more will be said about the success of treatments.

References ╇ 1. Neumann E (1869) Über die Bedeutung des Knochenmarkes für die Blutbildung. ein Beitrag zue Entwicklungsgeschichte der Blutkörperchen. Arch Heilkd 10:68–102 ╇ 2. Tavassoli M (1980) Bone marrow: the seedbed of blood. In: Wintrobe MM (ed) Blood, pure and eloquent. McGraw-Hill, New York, pp€57–79 ╇ 3. Ehrlich P (1879) Methodologische Beiträge zur Physiologie und Patholgie der verschiedenen Formen der Leukocyten. Z Klin Med 1:553–560 ╇ 4. Ehrlich P (1877) Beitrag zur Kenntnis der Anilinfärbungen und ihrer Verwendung in der mikroskopischen. Technik Arch Mikr Anat 13:263–277 ╇ 5. Wintrobe MM (1980) Blood, pure and eloquent. McGraw-Hill, New York ╇ 6. Matsuo T, Tomonaga M, Bennett JM, Kuriyama K, Imanaka F, Kuramoto A, Kamada N, Ichimaru M, Finch SC, Pisciotta AV et€al (1988) Reclassification of leukemia among A-bomb survivors in Nagasaki using French-American-British (FAB) classification for acute leukemia. Jpn J Clin Oncol 18:91–96 ╇ 7. Curie E (1937) Madame Curie. Doubleday, Garden City ╇ 8. Layton DM, Mufti GJ (1986) Myelodysplastic syndromes: their history, evolution and relation to acute myeloid leukaemia. Blut 53:423–436 ╇ 9. von Leube W (1900) Rapid verlaufende schwere Anämie mit gleichzeitiger leukämischer Veränderung des Blutbildes. Klin Wochenschr 37:85–97 10. Luzzatto AM (1907) Sull anemia grave megiloblastica sensa reporto ematologica correspondente (anemia pseudoaplastica). Riv Veneta Med Venezia 47:193 11. Rhoades CP, Barker WH (1938) Refractory anemia: an analysis of one hundred cases. J Am Med Assoc 110:794–796 12. Bomford RR, Rhodes CP (1941) Refractory anemia. Q J Med 10:175–281 13. Sabine JC (1940) A history of the classification of human blood corpuscles. Bull Hist Med 8:696–720, 785–805 14. Chevallier P, LaMotte M, Umdenstock R (1942) Trois cas d’anémie-leucose benzolique. Le Sang 15:391–405 15. Hamilton-Paterson JL (1949) Preleukemia anemia. Acta Hematol 2:309–316 16. Block M, Jacobson LO, Bethard WF (1953) Preleukemic acute human leukaemia. J Am Med Assoc 152:1018–1028 17. Björkman SE (1956) Chronic refractory anemia with sideroblastic bone marrow; a study of four cases. Blood 11:250–259 18. Rheingold JJ (1974) Acute leukemia. Its smoldering phase, or leukemia never starts on thursday. J Am Med Assoc 230:985–986 19. Rheingold JJ, Kaufman R, Adelson E, Lear A (1963) Smoldering acute leukemia. N Engl J Med 268:812–815 20. Rosenthal DS, Moloney WC (1984) Refractory dysmyelopoietic anemia and acute leukemia. Blood 63:314–318

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21. Cohen JR, Creger WP, Greenberg PL, Schrier SL (1979) Subacute myeloid leukemia: a clinical review. Am J Med 66:959–966 22. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C (1976) Proposals for the classification of the acute leukaemias. French-American-British (FAB) cooperative group. Br J Haematol 33:451–458 23. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C (1982) Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51:189– 199 24. Bennett JM (2000) World Health Organization classification of the acute leukemias and myelodysplastic syndrome. Int J Hematol 72:131–133 25. Swerdlow S, Campo E, Harris NL, Jaffe ES, Pileri S, Stein H, Thiele J, Vardiman JW (eds) (2008) WHO classification of tumours of the haematopoietic and lymphoid tissues. IARC, Lyon 26. Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, Harris NL, Le Beau MM, Hellstrom-Lindberg E, Tefferi A, Bloomfield CD (2009) The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 114:937–951 27. Raskind WH, Tirumali N, Jacobson R, Singer J, Fialkow PJ (1984) Evidence for a multistep pathogenesis of a myelodysplastic syndrome. Blood 63:1318–1323 28. Van den Berghe H, Cassiman JJ, David G, Fryns JP, Michaux JL, Sokal G (1974) Distinct haematological disorder with deletion of long arm of no. 5 chromosome. Nature 251:437– 438 29. Haase D (2008) Cytogenetic features in myelodysplastic syndromes. Ann Hematol 87:515– 526 30. Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G, Sanz M, Vallespi T, Hamblin T, Oscier D, Ohyashiki K, Toyama K, Aul C, Mufti G, Bennett J (1997) International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 89:2079–2088 31. Mufti GJ, Stevens JR, Oscier DG, Hamblin TJ, Machin D (1985) Myelodysplastic syndromes: a scoring system with prognostic significance. Br J Haematol 59:425–433 32. Sanz GF, Sanz MA, Vallespi T, Canizo MC, Torrabadella M, Garcia S, Irriguible D, San Miguel JF (1989) Two regression models and a scoring system for predicting survival and planning treatment in myelodysplastic syndromes: a multivariate analysis of prognostic factors in 370 patients. Blood 74:395–408 33. Aul C, Gattermann N, Heyll A, Germing U, Derigs G, Schneider W (1992) Primary myelodysplastic syndromes: analysis of prognostic factors in 235 patients and proposals for an improved scoring system. Leukemia 6:52–59 34. Tallman MS, McGuffin RW, Higano CS, Starkebaum G, Collins SJ, Johnston H, Singer JW, Perry DJ, Kunath A (1987) Bone marrow transplantation in a patient with myelodysplasia associated with diffuse eosinophilic fasciitis. Am J Hematol 24:93–99 35. Vadhan-Raj S, Keating M, LeMaistre A, Hittelman WN, McCredie K, Trujillo JM, Broxmeyer HE, Henney C, Gutterman JU (1987) Effects of recombinant human granulocytemacrophage colony-stimulating factor in patients with myelodysplastic syndromes. N Engl J Med 317:1545–1552 36. Chitambar CR, Libnoch JA, Matthaeus WG, Ash RC, Ritch PS, Anderson T (1991) Evaluation of continuous infusion low-dose 5-azacytidine in the treatment of myelodysplastic syndromes. Am J Hematol 37:100–104 37. List A, Kurtin S, Roe DJ, Buresh A, Mahadevan D, Fuchs D, Rimsza L, Heaton R, Knight R, Zeldis JB (2005) Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J Med 352:549–557

Chapter 2

Susceptibility to MDS: DNA Repair   and Detoxification Genes Claire Seedhouse and Nigel Russell

Introduction The myelodysplastic syndromes (MDS) are a collection of heterogeneous disorders arising from a clonal myeloid stem cell. They are characterised by ineffective haematopoiesis and are frequently associated with genetic instability manifested as chromosomal abnormalities. MDS progresses through a pathway of one or more dysplasias and ends in myeloid leukaemia—the risk of developing acute myeloid leukaemia (AML) depends on the subtype of MDS. Similar to other malignant diseases, the stepwise disease progression is likely to be the consequence of the accumulation of mutations, probably due to an increased DNA damage burden and/or reduced ability to deal with the damage. MDS risk has previously been associated with benzene exposure or exposure to a number of other environmental toxins. In addition a subset of patients develop the disease following chemotherapy or radiotherapy treatment for a primary disease (therapy-related MDS (t-MDS)). Mammalian cells have a number of efficient systems designed to metabolise and inactivate harmful genotoxic agents, or if the agents manage to induce damage then complex DNA repair mechanisms effectively remove the damage. Importantly, if the damage is excessive, DNA damage response proteins will trigger the apoptotic pathway to get rid of the cell for the good of the whole organism. These systems are not only required for protection from exogenous damaging agents but also for the constant damage cells receive from endogenous cellular processes, predominantly oxidative stress. MDS incidence increases with increasing age suggesting that the genotoxic burden on DNA eventually reaches a critical level resulting in disease. What, however, predisposes some individuals to the development of MDS when they have encountered a similar level of damage as an individual who remains healthy? A large body C. Seedhouse () Department of Academic Haematology, University of Nottingham, Clinical Sciences Building, Nottingham University Hospitals—City Campus, NG5 1PB Nottingham, UK Tel.: +44-115-8231822 Fax: +44-115-8231820 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_2, ©Â€Springer Science+Business Media B.V. 2011

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of research suggests that the answer, at least in part, is genetic variation in key genes involved in (1) protection of cellular entities from damage and (2) repair of DNA damage. Mutations in these genes are rare but of high penetrance and lead to serious genetic diseases; one example of a disease arising from mutations in DNA repair genes is Fanconi Anaemia which carries a strong predisposition to MDS. Much more common are genetic polymorphisms that occur in many of the genes which function to protect our genomes. This chapter aims to summarise research published on polymorphisms in detoxification and DNA repair genes in MDS. Therapy-related MDS is rarely investigated alone but is commonly grouped with therapy-related AML and indeed t-AML and t-MDS are now classified together by the World Health Organisation. There is a great deal of research that has been done on polymorphisms in t-AML/MDS (reviewed in Seedhouse and Russell [67]), so we will concentrate on work performed on de novo MDS samples and only positive associations of polymorphisms with t-AML/MDS will be discussed.

Detoxification Pathways The first line of defence to genotoxic agents is detoxification—this should occur before the agents are able to damage cellular molecules. Metabolism of endogenous and exogenous agents occurs by the same pathways and is divided into two phases. Phase I involves activation of substrates into electrophilic intermediates; these reactions are predominantly catalysed by the cytochrome P450 (CYP) protein family, many of which harbour polymorphisms which affect their function. The products of the phase I reactions are highly reactive and liable to cause severe cellular damage and the phase II enzymes (conjugation) are required to inactivate the phase I products. Enzymes that participate in phase II include the glutathione S-transferases (GST) and NAD(P)H:quinine oxidoreductase-1 (NQO1). These enzymes not only detoxify reactive phase I products but also act on genotoxic agents that do not require phase I activation. The balance of phase I and II activity is critical and a consequence of high levels of phase I activity with low levels of phase II activity is the production of deleterious metabolites which will damage cell components, especially DNA. It follows that polymorphisms affecting the function of either phase I or II proteins, or indeed proteins from both phases, may upset the balance of detoxification activity and predispose individuals to high levels of damaging agents.

Phase I: Cytochrome P450 Enzymes The cytochrome P450 superfamily comprises a large and diverse group of membrane-associated haem-proteins divided into 18 families. They are responsible for the metabolism of both endogenous and exogenous substrates, largely via an oxidative reaction, creating highly reactive intermediates that cause damage to DNA

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unless detoxified by phase II proteins (reviewed in Nebert and Dalton [53]). The CYP proteins can be induced or inhibited resulting in highly variable expression. In addition polymorphisms exist in many of the family members which affect the function of the protein and contribute to high inter-individual variability. The allele frequencies of several of the polymorphic variants have been examined as potential disease risk-factors in MDS populations. CYP2E1 CYP2E1 harbours a C to T single nucleotide polymorphism (SNP) in its 5′ promoter (CYP2E1*5) which is associated with increased transcriptional activity [30]. CYP2E1 is known to play an important role in the metabolism of benzene, exposure to which is a known risk factor to MDS. The CYP2E1*5B polymorphism has been studied to establish whether the variant allele increases susceptibility to benzene poisoning and hence MDS but no association was found [64]. CYP3A4 An A to G SNP is present in the CYP3A4 5′ promoter (CYP3A4*1B). The variant was initially proposed to alter a regulatory element within the promoter with a resulting decrease in activity [63]. Conversely, further biological assays failed to demonstrate specific functional differences between the variant and wild-type alleles [76, 85]. Fabiani et€al. found no risk for MDS associated with CYP3A4*1B [23]. CYP3A5 Two SNPs exist within CYP3A5 (CYP3A5*3 and CYP3A5*6) both of which cause alternative splicing and truncated proteins resulting in the absence of CYP3A5 [40]. The frequencies of these polymorphisms have been studied in a Taiwanese MDS population. There was no difference in the distribution of the CYP3A5*3 allele in MDS when compared to a healthy control cohort and the CYP3A5*6 allele was not found in cases or controls [46]. Significant Findings of CYP Variants in t-AML/MDS A large number of studies have been published examining the distribution of several of the CYP variant alleles in t-AML/MDS (reviewed in Seedhouse and Russell [67]). However, the only significant findings have been concerning the CYP3A4 A to G 5′ promoter polymorphism. The polymorphic variant (CYP3A4*1B) was found to be under-represented in a group of 30 childhood t-MDS/leukaemia samples when compared to a control group of paediatric patients with de novo leukaemia (odds ratio (OR) 0.09; 95% confidence intervals (CI) 0.01–0.87) [24] suggesting that the

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variant allele may result in a decrease in DNA-damaging reactive intermediates and therefore protect against t-AML/MDS. These results were corroborated by Rund et€al. [65] when comparing healthy controls with adult t-AML/MDS patients.

Phase II: Glutathione S-transferases The glutathione S-transferase (GST) multigene family is a major class of phase II detoxification enzymes. Their substrates are manifold and include environmental mutagens, drugs and products of oxidative stress which may, or may not, be byproducts of a phase I reaction. The mode of action of the GSTs is conjugation of the reactive electrophilic substrates to glutathione. There are at least four cytosolic GST subfamilies: alpha (A), mu (M), pi (P) and theta (T) and functionally relevant polymorphisms exist in each subfamily. GSTM1 and GSTT1 GSTM1 and GSTT1 have attracted considerable interest because both genes are commonly deleted [61, 70]. The homozygous deletion GSTM1 polymorphism exists in approximately 50% of Caucasians and the GSTT1 deletion in 25%; these frequencies differ between races. Most studies examining the cellular consequences of the GSTM1 or GSTT1 deletions point towards reduced detoxification activity resulting in higher levels of DNA damage [15, 32, 73, 86, 87]. A large number of studies have examined the importance of the GSTM1 and GSTT1 deletions in MDS (summarised in Table€2.1) with some finding positive associations for either GSTM1 null, or GSTT1 null, or for the combined GSTM1 and GSTT1 null genotypes with MDS. Dahabreh et€al. have performed a meta-analysis of GSTM1 and GSTT1 deletion polymorphisms in MDS [20]. They identified 13 eligible studies for GSTT1 comprising a total of 1,471 cases and 1,907 controls. Using both fixed and random effects models the GSTT1 null genotype was shown to be significantly associated with an increased risk of MDS (Fixed effects OR 1.44, 95% CI 1.21–1.72, pâ•›<â•›0.0001; random effects OR 1.43, 95% CI 1.09–1.89, pâ•›=â•›0.01). This study provides strong evidence that the GSTT1 null genotype is associated with MDS susceptibility. For GSTM1, ten eligible studies were identified totalling 1,161 cases and 1,668 controls. Neither the fixed or random effects models showed an association between GSTM1 null and MDS. When MDS patients are divided into further sub-groups the majority of studies have shown a trend for the GSTT1 null genotype being more prevalent in the RA/ RARS FAB subtypes than the more aggressive RAEB/RAEB-T subtypes; however no study has reached statistical significance. Within the subgroup analyses one paper of particular interest is by Stavropoulou et€al. [77] who studied a large series of well characterised MDS patients (nâ•›=â•›323) enabling analysis of genotype

48 48 53 33.3 36.9 51 52 42.4 48.5 59.4 16 16 23 46 19 30 6.66 18.1 16 15 15.8 25.3 18.1 10.8 8.8 13.1 7 21 0 6.1

46 21 28 46 21 53 11.1 14.2 22 36 17.9 19.8 24.4 6.1 7.8 5.8 26 33 3 6.8

Control deletion frequency (%)

42 55 55 57.4 36.7 53 70 35.4 47.7 51.6

MDS deletion frequency (%)

Studies showing significant associations with MDS are shaded a Only Caucasian results shown, other racial groups had very low numbers (n<10) b Published gene frequencies of healthy human subjects sorted by race (n>15000)

GSTM1 deletion 92 MDS 201 cancer-free, similar age, sex and race 166 MDS 112 haematologically normal staff and patients 116 MDS 43 healthy volunteers 54 MDS 60 cancer-free, matched for age and sex 49 MDS 102 newborn 128 MDS 239 healthy, similar sex and race a b 73 MDS Garte et al. [26] 302 MDS 330 healthy, matched for age and sex 86 MDS 99 hospitalised controls, matched for age and sex 157 MDS 155 cancer-free, matched for age and sex GSTT1 deletion 92 MDS 190 cancer-free, similar age, sex and race 166 MDS 112 haematologically normal staff and patients 57 MDS 100 haematologically normal staff and patients 59 MDS 50 normal controls 158 MDS 100 blood donors, similar age and sex 116 MDS 43 healthy volunteers 54 MDS 60 cancer-free, matched for age and sex 49 MDS 102 newborn 128 MDS 239 healthy, similar sex and race a b 73 MDS Garte et al. [26] 323 MDS 330 healthy, matched for age and sex 86 MDS 99 hospitalised controls, matched for age and sex 156 MDS 155 cancer-free, matched for age and sex Combined GSTM1 and GSTT1 deletions 49 MDS 102 newborn 128 MDS 239 healthy, similar sex and race 86 MDS 99 hospitalised controls, matched for age and sex a b 73 MDS Garte et al. [26] 116 MDS 43 healthy volunteers 54 MDS 60 cancer-free, matched for age and sex 294 MDS 330 healthy, matched for age and sex

Control population

Table 2.1↜渀 Summary of de novo MDS studies on GSTM1 and GSTT1 polymorphisms

1.2 (0.2–4.7) Not given, non-significant Not given, non-significant 2.86, p < 0.001 1.84 (0.81–4.19) – P = 0.830

4.3 (2.5–7.4) 0.72 (0.4–1.34) Not given, non-significant Not given, non significant Not given, non-significant 2.65 (1.27–5.52) p < 0.01 1.75, not significant 0.7 (0.2–1.8) Not given, non-significant 1.81, p < 0.001 p = 0.518 Not given, non-significant 1.46 (0.82–2.62), p = 0.22

0.8 (0.5–1.3) 0.89 (0.5–1.43) 0.80 (0.62–2.43) 2.70, p < 0.01 0.9. (0.5–1.9) Not given, non-significant 2.32, p = 0.002 p = 0.086 Not given, non-significant 0.73 (0.45–1.17), p = 0.2

OR (95% CI)

[6] [27] [81] [78] [66] [80] [77]

[16] [7] [9] [59] [62] [66] [80] [6] [27] [78] [77] [81] [23]

[16] [7] [66] [80] [6] [27] [78] [77] [81] [23]

Reference

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frequencies within specific cytogenetic groups. They discovered that none of the five patient samples with an isolated 5q deletion were GSTT1-null and also noted that, in a previous publication, there were 0/10 del5q patients who had a GSTT1 null genotype [62]. By increasing their data set to 47 samples (22 of which had an isolated 5q-, 4 a 5q- with one additional abnormality and 21 a -5/del5q as part of a complex karyotype) Stavropoulou et€al. found only two samples with a GSTT1 null genotype. This was highly significant when comparing to MDS samples with other karyotypic abnormalities or to the normal controls. These striking findings were explained by the fact that exposure to organic solvents had previously been associated with chromosome 5q abnormalities and that GSTT1 enzyme activity is required for some organic solvents to form mutagenic metabolites. Hence Stavropoulou et€al. hypothesised that the presence of the GSTT1 gene is not always protective but may confer MDS risk following particular chemical exposure by promoting specific genetic damage. GSTP1 and GSTPA1 The third polymorphic GST which has been widely studied in disease-risk is GSTP1. This gene has a G to A SNP which results in an ile to val amino acid change at codon 105 of the protein [1]. Codon 105 resides in the electrophile-binding site of the GSTP1 protein and the altered properties of the val amino acid affects both the catalytic activity of the enzyme, in a substrate-dependent manner, and its thermal stability. Two studies have been published examining the contribution of the GSTP1-105 polymorphism to de novo MDS risk, the first found no positive association [66], whilst Fabiani et€al. in the latter study found an increased risk of MDS associated with the presence of at least one variant val allele (OR1.66, 95% CI 1.03–2.67; pâ•›=â•›0.04) [23]. Furthermore patients with low/intermediate risk MDS (IPSS risk group low/intermediate-1) had a higher probability of overall survival if they harboured a GSTP1-105€val allele (pâ•›=â•›0.008). Fabiani and colleagues also examined the GSTPA1-C69T promoter variant in their study. This polymorphism has been shown to be correlated with GSTA1 expression [19]. There was no association between the GSTP1A-69T variant and MDS risk. Significant Findings of GST Polymorphisms in t-AML/AML A comprehensive summary of GST polymorphisms in t-AML/MDS can be found in Seedhouse and Russell [67]. Two studies demonstrated significant associations between the GSTT1 null polymorphism and t-AML/MDS disease incidence [2, 66]. In addition Allan et€al. showed that individuals with at least one GSTP-105Val allele were significantly over-represented in t-AML/MDS when compared with either a control or a de novo AML group. The odds ratio was increased if only those patients

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who had received chemotherapy were considered and further increased in the subgroup of patients who had prior exposure to a known GSTP1 substrate. Subgroup analysis by Haase et€al. [27] also provided interesting results with a highly significant increase in the risk of developing t-MDS/AML following breast cancer treatment when the double GSTM1/GSTT1 null genotype was present. This was further pronounced when considering patients who had been treated with chemotherapy for their breast cancer. NAD(P)H: Quinone Oxidoreductase (NQO1) NQO1 uses NADH or NADPH to catalyse 2- or 4-electron reductions of its quinone substrates thereby producing less reactive hydroquinones. This activity plays a critical role in detoxification preventing the generation of ROS and free-radicals that would otherwise go on to damage DNA and other cellular components. The NQO1 gene has a well studied C to T SNP at position 609 resulting in a pro to ser amino acid substitution at codon 187 [79]. The variant ser-containing protein has negligible enzymatic activity and is no longer inducible in bone marrow cells following benzene metabolite exposure. An allele dosage effect occurs with the ser/ser homozygote having no activity and the pro/ser heterozygote showing intermediate activity between the homozygote variant and wild type proteins [51, 74]. Benzene poisoning is associated with a very strong risk of MDS development and the NQO1 serine allele increases the risk of benzene poisoning [64], there has therefore been interest in the distribution of the NQO1 polymorphism in MDS. Whilst no relationship has been found between the NQO1 SNP and de novo MDS risk [23, 43], these results are contrary to findings from several studies on the therapy-related disease (see below). Significant Findings of NQO1-Pro187Ser in t-AML/MDS Larson et€al. found a significant over-representation of the NQO1-187ser allele in a t-AML/MDS cohort: interestingly 7/8 of the ser/ser homozygotes had abnormalities of either chromosome 5 and/or 7 [43]. Naoe et€al. [52] confirmed these findings in 58 t-AML/MDS patients with the ser variant homozygous genotype conferring an odds ratio of 2.62 (95% CI 2.16–3.08) although chromosome 5 and/or 7 abnormalities were not over-represented in their variant homozygote samples.

DNA Repair Cells encounter constant attack from molecules which can damage DNA. Efficient detoxification mechanisms limit cellular damage but, even so, DNA cannot be entirely spared and therefore complex pathways are present to repair DNA damage

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and minimise its deleterious effects. The balance of DNA repair is critical—whilst too little repair can result in the acquisition and persistence of mutations and genetic instability, too much repair can be equally harmful by inhibiting the apoptotic pathway and enabling a cell with badly damaged DNA to attempt repair, possibly misrepair and survive. DNA damage is known to accumulate with age and eventually overloads the repair systems leading to haeamatopoietic stem cell (HSC) exhaustion. A number of studies using mouse models have effectively demonstrated the importance of DNA repair genes, involved in a number of different repair pathways, in maintaining HSC function (reviewed in Niedernhofer [55]). MDS is primarily a disease of the elderly and some cases can occur following large genotoxic insults such as chemotherapy drug treatment. If the damage levels sustained with increasing age or genotoxic insult are unrepaired and reach a certain threshold, protective cell senescence or apoptosis should be triggered. If these mechanisms fail then the genomic instability may prime stem cells for further mutation development and functional cellular abnormalities ultimately resulting in MDS and/or leukaemia. There is a significant body of research demonstrating high levels of DNA damage in MDS samples. Increased levels of the major oxidative damage product, 7,8-dihydro-8-oxoguanine (8-oxoG), have been found in MDS patient bone marrow samples when compared to normal bone marrow controls [33] and increased levels of oxidised pyrimidine nucleotides are seen in CD34+ enriched MDS bone marrow samples compared to either CD34− MDS samples or CD34+ bone marrow cells from normal subjects [60]. Novotna et€al. have also shown higher levels of oxidative damage and genetic instability in samples from MDS patients when compared to age matched controls [56, 57]. It is not clear whether the increased DNA damage is a cause, or a result, of the MDS disease. Whilst genetic instability is one of the main prerequisites for disease development, once the disease is established iron overload in transfusion-dependent patients and reactive oxygen species generation from inflammation also have to be considered among important factors in determining the burden of DNA damage. Whatever the case, the additional DNA damage burden means that aberrant activities of DNA repair genes are likely to be particularly important in MDS.

Double Strand Break Repair DNA double strand breaks (DSBs) are the most important class of DNA damage because, if unrepaired, they can result in a loss of genetic material, chromosome abnormalities and possibly cell death. The two major mechanisms that repair DSBs are the homologous recombination and non-homologous end-joining pathways. Non-homologous End Joining Non homologous end joining (NHEJ) is active in all phases of the cell cycle and is considered to be the most important DSB repair pathway in mammalian cells. In

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NHEJ the DSB is recognised by the Ku70/Ku80 heterodimer which then forms a complex with DNA-PKcs. Following recruitment of further proteins and DNA end processing, a XRCC4-ligase IV complex re-ligates the break. A number of patients have been reported who have a mutation in the ligase IV gene—Ligase IV syndrome—and bone marrow abnormalities, including MDS, are amongst the many clinical disorders (reviewed in Chistiakov et€al. [17]). Polymorphisms in NHEJ genes have been identified but have not been studied in either de novo MDS or t-AML/MDS. However a recent study found differences in the expression of several of the NHEJ proteins in MDS samples [22]. Expression of ligase 4 was significantly elevated in MDS bone marrow samples compared to control bone marrow with the high expression appearing to be associated with a good risk karyotype. Conversely, the Ku70 protein levels were significantly lower in patients with a good risk karyotype. Homologous Recombination Homologous recombination (HR) repair is a tightly regulated, high-fidelity process. It uses a second, intact copy of the chromosome as a template to copy the information lost at the DSB site. A number of polymorphic genes involved in the pathway have been studied in the context of MDS. RAD51 and XRCC3 RAD51 is a central protein in the HR repair pathway binding to DNA and promoting ATP-dependent homologous pairing and strand transfer reactions. XRCC3 also participates in the pathway interacting with, and stabilising, RAD51. Polymorphisms are present in the RAD51 and XRCC3 genes. RAD51 has a G to C polymorphism at position −135 of the 5′ promoter of the gene [83]. Characterisation of the RAD51 promoter demonstrated that the −135 variant C allele was associated with increased promoter activity [29]. However, a further study suggested that the effect of the polymorphism was due to alternative splicing within the RAD51 5′-untranslated region [4]. The substitution of C for G abolishes a splice site resulting in low transcript levels of the longer RAD51 isoform (isoform 2). The authors suggested that as isoform 2 lacks a translation-inhibitory GC-rich region then it would be expected to have increased translation efficiency. As the polymorphic variant results in less isoform 2, subsequently, a reduction in RAD51 protein may occur. The XRCC3 gene is also polymorphic and a thr to met substitution occurs at codon 241 [72]. Whilst the variant protein has been shown to complement the DNA repair defect in a XRCC3-deficient cell line [5], further work determined that the variant protein was unable to apoptotically eliminate aberrant cells with mitotic defects resulting in genetic instability [45]. Two studies have found no differences in the distribution of the RAD51-G135C and XRCC3-thr241met polymorphisms in MDS samples compared to controls

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[10, 23]. Baumann et€al. also found no difference if the two polymorphisms were analysed in combination. In t-AML/MDS we found an over-representation of the RAD51-135C allele when compared with a control group (OR 2.66, 95% CI 1.17– 6.02, pâ•›=â•›0.02) [69]. Whilst there was no difference in the distribution of the XRCC3 polymorphism, when we looked at the combination of variant RAD51 and XRCC3 alleles we found a prominent synergistic effect resulting in an odds ratio of more than 8 (OR 8.11, 95% CI 2.22–29.68, pâ•›=â•›0.002). BLM, TOP3A and RMI1 Bloom syndrome is a rare autosomal recessive disorder characterised by growth retardation, sensitivity to light and a predisposition to the development of many cancers including leukaemia and MDS. BLM is a RecQ helicase which prevents illegitimate recombination in mitotic cells. The BLM gene is mutated in Bloom syndrome resulting in an elevated frequency of exchange between homologous chromosomes and sister chromatids. BLM interacts physically and functionally with both topoisomerase IIIa (TOP3A) and RMI1/BLAP75 (reviewed in [75]. Polymorphisms in BLM, RMI1 and TOP3A have been studied in MDS. RMI1 harbours a G to A polymorphism which results in a ser to asn change at codon 455 of the protein. Ser455 is evolutionarily conserved however the functional consequences of the polymorphism are unknown [13]. The presence of the polymorphic asn residue was demonstrated to be a risk factor for MDS (OR 1.9, 95% CI 1.1–3.3) [13]. Interestingly the effect was stronger in those patients over the age of 65 years which the authors suggested may reflect the fact that mitotic recombination increases with age and aberrations of proteins involved in this process may therefore be expected to have an increased influence as mitotic recombination increases. A second study by the same group examined a further 26 polymorphisms in RMI1, BLM and TOP3A in a mixed group of MDS and AML samples (37% MDS; 12% AML secondary to MDS; 51% AML). Of particular attention when analysed singly were TOP3A GA rs12945597 where the homozygous variant was associated with a 4.6-fold increase risk of MDS/AML (95% CI 1.7–14) and BLM AC rs6496724 for which the variant homozygote was less frequent in MDS/AML (OR 0.34, 95% CI 0.12–0.95) [14]. Following analyses of multiple myeloma, bladder and breast cancer samples in addition to the MDS/AML cohort, combinations of polymorphisms were chosen. The TOP3A rs12945597 A allele in combination with the BLM rs2532105 variant T allele resulted in an odds ratio of 2.4 for MDS/AML risk (95% CI 1.1–5.4). None of the functional effects of the polymorphisms, if any, are known.

Base Excision Repair: hOGG1 The base excision repair (BER) pathway corrects individually damaged bases which can occur via a number of different mechanisms but predominantly via

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oxidation. The major product of oxidative damage to DNA is 8-oxoG. 8-oxoG is highly mutagenic as it has a tendency to mispair with adenine thereby generating GC to TA transversions. The human glycosylase responsible for repairing this base adduct is hOGG1 (reviewed in Klungland and Bjelland [36]). A SNP exists at position 1,245 of the hOGG1 gene resulting in a ser to cys amino acid change at codon 326 in exon 7 of the corresponding protein. The cys-encoding protein has been shown to have a reduction in its repair activity compared to the wild type ser protein [38]. Jankowska et€al. have provided a comprehensive analysis of the importance of 8-oxoG in MDS including the genotyping of 146 MDS patients for the hOGG1 ser326cys polymorphism [33]. When comparing the distribution of the hOGG1 SNP in MDS to that of a cohort of 350 controls, significantly increased risks for MDS were found for both heterozygote (OR 1.7, 95% CI 1.1–2.5, pâ•›=â•›0.02) and homozygote cases (OR 2.8, 95% CI 1–7.2, pâ•›=â•›0.05). The cys allele was associated with conferring a particularly strong risk for advanced MDS and additionally there was a significant positive relationship between the presence of the cys allele and the frequency of chromosomal abnormalities (pâ•›<â•›0.02). The authors also compared hOGG1 mRNA expression between the different hOGG1 genotype groups with the carriers of hOGG1-cys326 exhibiting significantly higher hOGG1 levels when compared to the wild type hOGG1 ser/ser cases (pâ•›=â•›0.008). They suggested that this could be due to a feedback mechanism attempting to compensate for the reduced activity of the cys allele. XRCC1 also participates in the BER pathway acting as a scaffold and recruiting other BER proteins to the repair site. A common variation in the gene substitutes arg for met at position 399 of the protein, a region which comprises the BRCT domain [72]. The variant Gln residue has been shown to result in significant conformational changes to the XRCC1 protein [50] and a large number of studies have shown that cells harbouring the variant gln allele have a decreased capacity to repair DNA damage resulting in increased DNA damage levels [8, 21, 41, 44, 47, 82, 84]. We have demonstrated that the presence of a variant XRCC1399 gln allele was actually protective for t-AML/MDS (OR 0.44, 95% CI 0.20– 0.93, pâ•›=â•›0.03) [68]. We consider this result indicates a strong gene-environmental interaction. The genotoxic therapy a patient receives for their primary condition is likely to cause very high DNA damage levels in some HSCs. Cells with reduced BER capacity, that is a XRCC1-399gln allele, are more likely to be driven towards apoptosis whilst the wild type cells may attempt repair but misrepair resulting in mutations and a clone which can initiate t-AML/MDS. This geneenvironment (XRCC1-399gln: high DNA damage) hypothesis is supported by a study of XRCC1-399gln in non-melanoma skin cancer which is also associated with high levels of DNA damage; similar to t-AML/MDS a protective effect of the gln allele was found [54]. Furthermore in a meta-analysis of XRCC1-399 in smoking-related cancers, the variant gln allele was found to confer an increased risk in light smokers, but was protective among heavy smokers who presumably have high levels of DNA damage [31].

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Nucleotide Excision Repair Whereas BER removes the products of minor structural base damages, nucleotide excision repair (NER) removes bulky damages which distort the DNA helix. The NER repair pathway consists of removing segments of ssDNA containing the bulky lesion followed by repair synthesis by a DNA polymerase and ligation. Kuramoto et€al. measured the mRNA expression levels of several genes involved in the NER pathway, namely ERCC1, ERCC3, ERCC5 and XPC, and found a reduction in expression in at least one gene in more than 20% of the MDS samples when compared to normal samples. Additionally, patients with high risk MDS were more likely to have a reduction in NER gene expression [42]. The authors suggested that such a decrease in DNA repair gene expression may predispose individuals to chromosomal instability and underlie the pathophysiology of the disease. The ERCC2 (XPD) gene harbours a lys to gln change at codon 751 [72]. Like many of the polymorphisms in DNA repair genes the functional consequences of the ERCC-751gln variant allele are still unclear and a review of the literature by Clarkson and Wood [18] questions whether a causal relationship between the ERCC2 gene and reduced DNA repair occurs; instead the SNP may be in linkage with another functional SNP. Whatever the consequences, the ERCC2-751gln homozygote variant has been associated with a significant increase of developing chemotherapy-induced t-AML/MDS (OR 2.22, 95% CI 1.04–4.74) [3].

Mismatch Repair Mismatch repair (MMR) corrects mismatched DNA bases that can result from misincorporation errors that have avoided polymerase proof-reading activity during DNA replication. In addition MMR can also process some types of DNA damage. MMR is reasonably easy to study because deficiencies in the pathway result in an elevated rate of mutations which can be measured in simple repetitive DNA sequences (microsatellites). There have been a number of studies which have searched for MMR deficiency (microsatellite instability; MSI) in both de novo MDS and t-MDS/AML. A summary of the de novo MDS studies is shown in Table€2.2. Noteworthy is the study by Kaneko et€al. (Table€2.2) because serial samples from the same patients were analysed demonstrating that MSI, when it occurs, is an early event and may then contribute to the pathogenesis of MDS. There are also a large number of studies looking at the incidence of MSI in t-AML/MDS (reviewed in Seedhouse and Russell [67]). The incidence in the tAML/MDS samples is much higher (approximately 50%) than for de novo MDS suggesting that a prerequisite of MSI in MDS is a significant burden of DNA damage. Along these lines is work done on the development of AML/MDS following azathioprine immunosuppressive treatment after organ transplant: Offman et€ al. proposed that AML/MDS development may reflect the selection and expansion of MMR-deficient clones within the bone marrow which are resistant to drug-induced damage. The lack of MMR is expected to lead to the accumulation of mutations

2╅ Susceptibility to MDS: DNA Repair and Detoxification Genes Table 2.2↜渀 Summary of microsatellite instability (MSI) studies in de novo MDS Number of samples Number of loci studied (up to) % of samples with MSIa 6 4 33 19 10 16 9 22 11 12 12 8b 7 14 0 23 18 4b 29 10 0b 23 9 0b

17

Reference [34] [35] [39] [71] [12] [49] [48] [28]

The proportion of loci showing MSI differs between studies. Results are shown for two or more loci displaying MSI and results indicate further samples have MSI at just one locus a

b

which may subsequently result in a clonal haematopoietic disorder [58]. Further support of the importance of MMR in t-AML/MDS is a study showing that the variant allele of the MSH2 IVS12-6 T to C polymorphism [25] is over-represented in t-AML/MDS patients who had previously received alkylating therapy (OR 4.02, 95% CI 1.4–11.37) [88]. Strikingly both of the t-MDS/AML variant homozygotes had MSI, although not all of the MSI-positive samples had the polymorphism. The functional consequence of the polymorphism is unknown, although it resides in the splice acceptor site of exon 13 and may therefore alter splice site recognition.

Discussion Protection from DNA damage, either by preventing (detoxification or apoptosis) or repairing it, is paramount in keeping our cells healthy and this is particularly so for haematopoietic stem and progenitor cells which often come into close proximity to damaging agents. An accumulation of unrepaired genomic damage in HSCs will results in an increased risk of a clonal stem cell disease. MDS is a disease characterised by DNA damage and genetic instability, a state that is expressed as gross karyotypic abnormalities in 50% of patients. Whilst many polymorphisms only have subtle functional effects, gene-environment interactions are likely to magnify these effects making polymorphisms worthwhile of study. We are now accumulating evidence demonstrating a contribution of polymorphisms in detoxification pathways and DNA repair pathways to MDS susceptibility, however there is still more work to be done.

Sample Size and Controls for Polymorphism Studies Much of the work on susceptibility to MDS has focused on polymorphisms in the DNA repair and detoxification genes. Many of the polymorphic genes have only been investigated in single small studies, whilst those polymorphisms which have

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been assessed by more than one group have produced contradictory results. The answer in improving this body of work is by increasing sample sizes; very large cohorts of well characterised MDS patients are needed. In this respect we are participating in a large European study in which we have already collected more than 1,000 MDS samples and determined the genotypes at a number of polymorphic sites. Many sources of control samples have been used in the studies described herein. Appropriately matched controls, whilst often not easy to obtain, are an absolute requirement for epidemiological studies. However, we also consider it important to compare subgroups within MDS and suggest that this will help improve our knowledge on understanding MDS genetic susceptibility. For example are some polymorphic variants more prevalent in high risk MDS? Or does a particular polymorphism confer risk to a specific cytogenetic abnormality such as the GSTT1 5qstory? There are many research avenues still to be followed.

Functional Consequences of Polymorphic Variants A further caveat in polymorphism studies is the lack of data regarding their functional effects. Whilst many of the detoxification gene variants have been categorically shown to have reduced activity, for most of the DNA repair polymorphisms it is not known whether the sequence variant is functionally significant and more research into this question is required. The possibility of linkage disequilibrium between polymorphisms should also be considered.

Combinations of Polymorphisms Another area of importance, only possible with very large studies, is the combination of variant genotypes. Detoxification and repair processes are complex and more than one protein/pathway can detoxify or repair a particular entity. This functional redundancy illustrates the critical importance of these processes and also poses the question of whether we are likely to see a noteworthy susceptibility to MDS if just one variant gene is studied. It is much more probable that MDS would result from the accumulation of minor phenotypic abnormalities caused by more than one variant protein. A few studies combining detoxification and repair genes have already been published in t-AML/MDS with the achievement of high odds ratios for the combinations [11, 69], although the number of samples was small. Once again, large sample sizes are required for adequate power for combination studies because the more polymorphisms combined the smaller the subgroups. High throughput genotyping techniques will aid this goal. We also suggest that such studies should be hypothesis-driven rather than a ‘fishing’ exercise where a lot of statistical tests upon a sample cohort are prone to a degree of false positivity. The hypothesis may take the form of aberrant detoxification genotypes with variant DNA repair genotypes of

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Bone marrow Stem cell

DNA damaging agents Efficient detoxification

Efficient DNA repair

Inefficient detoxification (Phase I and/or II) resulting in DNA damage

Aberrant DNA repair (HR, NHEJ, BER, NER, MMR)

Mutated cells

Clonal expansion

MDS

Fig. 2.1↜渀 Pathway to MDS. Possible mechanism of increased susceptibility to MDS. Deficient detoxification mechanisms increase the amount of DNA damage which aberrant DNA repair mechanisms fail to repair. The resulting genetic instability may result in clonal expansion of mutated stem cells resulting in MDS

proteins expected to repair the particular damage resulting from the aberrant detoxification, or instead variants of more than one gene that are able to detoxify the same substrate or repair the same damage type (Fig.€2.1).

Which Polymorphic Genes Confer the Greatest Susceptibility to MDS? We still do not know the answer to this question. Whilst GSTT1 appears to play a role and evidence is accumulating suggesting that members of the BLM complex may also be important, much of the data is contradictory and significant findings

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often only generate odds ratios of between one and two. This is a long way from the relative risk factor of ten suggested by Knudsen et€al. [37] as a safety factor to allow for individual susceptibility. We are of the opinion that we are unlikely to find high risks associated with single gene studies but that the study of more than one gene from the detoxification/DNA repair pathways would be more successful. Hopefully larger studies and combination assessments will soon allow us to conclusively identify those genes which play the most important role in conferring susceptibility to MDS. Acknowledgements╇ The authors are very grateful to the Nottinghamshire Leukaemia Appeal and the James Skillington Challenge for funding for their research.

References ╇ 1. Ali-Osman F et€al (1997) Molecular cloning, characterization, and expression in Escherichia coli of full-length cDNAs of three human glutathione S-transferase Pi gene variants. Evidence for differential catalytic activity of the encoded proteins. J Biol Chem 272:10004–10012 ╇ 2. Allan JM et€al (2001) Polymorphism in glutathione S-transferase P1 is associated with susceptibility to chemotherapy-induced leukemia. Proc Natl Acad Sci U S A 98:11592–11597 ╇ 3. Allan JM et€al (2004) Genetic variation in XPD predicts treatment outcome and risk of acute myeloid leukemia following chemotherapy. Blood 104:3872–3877 ╇ 4. Antoniou AC et€ al (2007) RAD51 135G→C modifies breast cancer risk among BRCA2 mutation carriers: results from a combined analysis of 19 studies. Am J Hum Genet 81:1186– 1200 ╇ 5. Araujo FD et€ al (2002) Variant XRCC3 implicated in cancer is functional in homologydirected repair of double-strand breaks. Oncogene 21:4176–4180 ╇ 6. Arruda VR et€al (2001) Increased risk for acute myeloid leukaemia in individuals with glutathione S-transferase mu 1 (GSTM1) and theta 1 (GSTT1) gene defects. Eur J Haematol 66:383–388 ╇ 7. Atoyebi W et€al (1997) Glutathione S-transferase gene deletions in myelodysplasia. Lancet 349:1450–1451 ╇ 8. Au WW et€al (2003) Functional characterization of polymorphisms in DNA repair genes using cytogenetic challenge assays. Environ Health Perspect 111:1843–1850 ╇ 9. Basu T et€al (1997) Glutathione S-transferase theta 1 (GSTT1) gene defect in myelodysplasia and acute myeloid leukaemia. Lancet 349:1450 10. Baumann Kreuziger LM, Steensma DP (2008) RAD51 and XRCC3 polymorphism frequency and risk of myelodysplastic syndromes. Am J Hematol 83:822–823 11. Bolufer P et€al (2007) Profile of polymorphisms of drug-metabolising enzymes and the risk of therapy-related leukaemia. Br J Haematol 136:590–596 12. Boyer JC et€al (1998) Stability of microsatellites in myeloid neoplasias. Cancer Genet Cytogenet 106:54–61 13. Broberg K et€al (2007) Genetic variant of the human homologous recombination-associated gene RMI1 (S455N) impacts the risk of AML/MDS and malignant melanoma. Cancer Lett 258:38–44 14. Broberg K et€al (2009) Association between polymorphisms in RMI1, TOP3A, and BLM and risk of cancer, a case-control study. BMC Cancer 9:140 15. Butkiewicz D et€al (1998) Modulation of DNA adduct levels in human mononuclear white blood cells and granulocytes by CYP1A1 CYP2D6 and GSTM1 genetic polymorphisms. Mutat Res 415:97–108

2â•… Susceptibility to MDS: DNA Repair and Detoxification Genes

21

16. Chen H et€al (1996) Increased risk for myelodysplastic syndromes in individuals with glutathione transferase theta 1 (GSTT1) gene defect. Lancet 347:295–297 17. Chistiakov DA et€al (2009) Ligase IV syndrome. Eur J Med Genet 52:373–378 18. Clarkson SG, Wood RD (2005) Polymorphisms in the human XPD (ERCC2) gene, DNA repair capacity and cancer susceptibility: an appraisal. DNA Repair (Amst) 4:1068–1074 19. Coles BF et€ al (2001) Effect of polymorphism in the human glutathione S-transferase A1 promoter on hepatic GSTA1 and GSTA2 expression. Pharmacogenetics 11:663–669 20. Dahabreh IJ et€al (2010) GSTT1 and GSTM1 polymorphisms and myelodysplastic syndrome risk: a systematic review and meta-analysis. Int J Cancer 126:1716–1723 21. Duell EJ et€al (2000) Polymorphisms in the DNA repair genes XRCC1 and ERCC2 and biomarkers of DNA damage in human blood mononuclear cells. Carcinogenesis 21:965–971 22. Economopoulou P et€al (2010) Expression analysis of proteins involved in the non homologous end joining DNA repair mechanism, in the bone marrow of adult de novo myelodysplastic syndromes. Ann Hematol 89:233–239 23. Fabiani E et€al (2009) Polymorphisms of detoxification and DNA repair enzymes in myelodyplastic syndromes. Leuk Res 33:1068–1071 24. Felix CA et€ al (1998) Association of CYP3A4 genotype with treatment-related leukemia. Proc Natl Acad Sci U S A 95:13176–13181 25. Fishel R et€al (1993) The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 75:1027–1038 26. Garte S et€al (2001) Metabolic gene polymorphism frequencies in control populations. Cancer Epidemiol Biomarkers Prev 10:1239–1248 27. Haase D et€al (2002) Increased risk for therapy-associated hematologic malignancies in patients with carcinoma of the breast and combined homozygous gene deletions of glutathione transferases M1 and T1. Leuk Res 26:249–254 28. Harada S et€al (1998) Microsatellite instability is rare in the clinical course of myelodysplastic syndrome studied with DNA from fresh and paraffin-embedded tissues. J Cancer Res Clin Oncol 124:231–235 29. Hasselbach L et€al (2005) Characterisation of the promoter region of the human DNA-repair gene Rad51. Eur J Gynaecol Oncol 26:589–598 30. Hayashi S et€al (1991) Genetic polymorphisms in the 5′-flanking region change transcriptional regulation of the human cytochrome P450IIE1 gene. J Biochem 110:559–565 31. Hung RJ et€al (2005) Genetic polymorphisms in the base excision repair pathway and cancer risk: a HuGE review. Am J Epidemiol 162:925–942 32. Izzotti A et€al (2001) Increased DNA alterations in atherosclerotic lesions of individuals lacking the GSTM1 genotype. FASEB J 15:752–757 33. Jankowska AM et€al (2008) Base excision repair dysfunction in a subgroup of patients with myelodysplastic syndrome. Leukemia 22:551–558 34. Kaneko H et€al (1995) Microsatellite instability is an early genetic event in myelodysplastic syndrome. Blood 86:1236–1237 35. Kaneko H et€al (1996) Microsatellite instability is an early genetic event in myelodysplastic syndrome but is infrequent and not associated with TGF-beta receptor type II gene mutation. Leukemia 10:1696–1699 36. Klungland A, Bjelland S (2007) Oxidative damage to purines in DNA: role of mammalian Ogg1. DNA Repair (Amst) 6:481–488 37. Knudsen LE et€al (2001) Risk assessment: the importance of genetic polymorphisms in man. Mutat Res 482:83–88 38. Kohno T et€al (1998) Genetic polymorphisms and alternative splicing of the hOGG1 gene, that is involved in the repair of 8-hydroxyguanine in damaged DNA. Oncogene 16:3219– 3225 39. Krskova-Honzatkova L et€al (2002) Microsatellite instability in hematological malignancies. Leuk Lymphoma 43:1979–1986 40. Kuehl P et€ al (2001) Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat Genet 27:383–391

22

C. Seedhouse and N. Russell

41. Kuptsova N et€al (2007) Polymorphisms in DNA repair genes and therapeutic outcomes of AML patients from SWOG clinical trials. Blood 109:3936–3944 42. Kuramoto K et€al (2002) Chromosomal instability and radiosensitivity in myelodysplastic syndrome cells. Leukemia 16:2253–2258 43. Larson RA et€al (1999) Prevalence of the inactivating 609C→T polymorphism in the NAD(P) H:quinone oxidoreductase (NQO1) gene in patients with primary and therapy-related myeloid leukemia. Blood 94:803–807 44. Li Y et€al (2009) Effect of the XRCC1 codon 399 polymorphism on the repair of vinyl chloride metabolite-induced DNA damage. J Carcinog 8:14 45. Lindh AR et€al (2006) Mitotic defects in XRCC3 variants T241€M and D213€N and their relation to cancer susceptibility. Hum Mol Genet 15:1217–1224 46. Liu TC et€al (2002) Polymorphism analysis of CYP3A5 in myeloid leukemia. Oncol Rep 9:327–329 47. Lunn RM et€al (1999) XRCC1 polymorphisms: effects on aflatoxin B1-DNA adducts and glycophorin A variant frequency. Cancer Res 59:2557–2561 48. Ma SK et€ al (2000) Absence of microsatellite instability in primary myelodysplastic syndrome. Int J Mol Med 5:159–163 49. Maeck L et€al (2000) Genetic instability in myelodysplastic syndrome: detection of microsatellite instability and loss of heterozygosity in bone marrow samples with karyotype alterations. Br J Haematol 109:842–846 50. Monaco R et€al (2007) Conformational effects of a common codon 399 polymorphism on the BRCT1 domain of the XRCC1 protein. Protein J 26:541–546 51. Moran JL et€al (1999) A potential mechanism underlying the increased susceptibility of individuals with a polymorphism in NAD(P)H:quinone oxidoreductase 1 (NQO1) to benzene toxicity. Proc Natl Acad Sci U S A 96:8150–8155 52. Naoe T et€ al (2000) Analysis of genetic polymorphism in NQO1, GST-M1, GST-T1 and CYP3A4 in 469 Japanese patients with therapy-related leukemia/myelodysplastic syndrome and de novo acute meyloid leukemia. Clin Can Res 6:4091–4095 53. Nebert DW, Dalton TP (2006) The role of cytochrome P450 enzymes in endogenous signalling pathways and environmental carcinogenesis. Nat Rev Cancer 6:947–960 54. Nelson HH et€al (2002) The XRCC1 Arg399Gln polymorphism, sunburn, and non-melanoma skin cancer: evidence of gene-environment interaction. Cancer Res 62:152–155 55. Niedernhofer LJ (2008) DNA repair is crucial for maintaining hematopoietic stem cell function. DNA Repair (Amst) 7:523–529 56. Novotna B et€al (2008) DNA instability in low-risk myelodysplastic syndromes: refractory anemia with or without ring sideroblasts. Hum Mol Genet 17:2144–2149 57. Novotna B et€al (2009) Oxidative DNA damage in bone marrow cells of patients with lowrisk myelodysplastic syndrome. Leuk Res 33:340–343 58. Offman J et€al (2004) Defective DNA mismatch repair in acute myeloid leukemia/myelodysplastic syndrome after organ transplantation. Blood 104:822–828 59. Okada M et€ al (1997) Glutathione S-transferase theta 1 gene (GSTT1) defect in Japanese patients with myelodysplastic syndromes. Int J Hematol 66:393–394 60. Peddie CM et€ al (1997) Oxidative DNA damage in CD34+ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumour necrosis factor-alpha concentration. Br J Haematol 99:625–631 61. Pemble S et€al (1994) Human glutathione S-transferase theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphism. Biochem J 300 (Pt 1):271–276 62. Preudhomme C et€al (1997) Glutathione S transferase theta 1 gene defects in myelodysplastic syndromes and their correlation with karyotype and exposure to potential carcinogens. Leukemia 11:1580–1582 63. Rebbeck TR et€al (1998) Modification of clinical presentation of prostate tumors by a novel genetic variant in CYP3A4. J Natl Cancer Inst 90:1225–1229

2â•… Susceptibility to MDS: DNA Repair and Detoxification Genes

23

64. Rothman N et€al (1997) Benzene poisoning, a risk factor for hematological malignancy, is associated with the NQO1 609C→T mutation and rapid fractional excretion of chlorzoxazone. Cancer Res 57:2839–2842 65. Rund D et€al (2005) Therapy-related leukemia: clinical characteristics and analysis of new molecular risk factors in 96 adult patients. Leukemia 19:1919–1928 66. Sasai Y et€al (1999) Genotype of glutathione S-transferase and other genetic configurations in myelodysplasia. Leuk Res 23:975–981 67. Seedhouse C, Russell N (2007) Advances in the understanding of susceptibility to treatmentrelated acute myeloid leukaemia. Br J Haematol 137:513–529 68. Seedhouse C et€ al (2002) The genotype distribution of the XRCC1 gene indicates a role for base excision repair in the development of therapy-related acute myeloblastic leukemia. Blood 100:3761–3766 69. Seedhouse C et€ al (2004) Polymorphisms in genes involved in homologous recombination repair interact to increase the risk of developing acute myeloid leukemia. Clin Cancer Res 10:2675–2680 70. Seidegard J et€al (1988) Hereditary differences in the expression of the human glutathione transferase active on trans-stilbene oxide are due to a gene deletion. Proc Natl Acad Sci U S A 85:7293–7297 71. Sheikhha MH et€al (2002) High level of microsatellite instability but not hypermethylation of mismatch repair genes in therapy-related and secondary acute myeloid leukaemia and myelodysplastic syndrome. Br J Haematol 117:359–365 72. Shen MR et€al (1998) Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans. Cancer Res 58:604–608 73. Shields PG et€al (1993) Polycyclic aromatic hydrocarbon-DNA adducts in human lung and cancer susceptibility genes. Cancer Res 53:3486–3492 74. Siegel D et€ al (1999) Genotype-phenotype relationships in studies of a polymorphism in NAD(P)H:quinone oxidoreductase 1. Pharmacogenetics 9:113–121 75. Singh DK et€ al (2009) Roles of RECQ helicases in recombination based DNA repair, genomic stability and aging. Biogerontology 10:235–252 76. Spurdle AB et€al (2002) The CYP3A4*1B polymorphism has no functional significance and is not associated with risk of breast or ovarian cancer. Pharmacogenetics 12:355–366 77. Stavropoulou C et€al (2008) Low frequency of the glutathione-S-transferase T1-null genotype in patients with primary myelodysplastic syndrome and 5q deletion. Leukemia 22:1643–1646 78. Sutton JF et€al (2004) Increased risk for aplastic anemia and myelodysplastic syndrome in individuals lacking glutathione S-transferase genes. Pediatr Blood Cancer 42:122–126 79. Traver RD et€al (1992) NAD(P)H:quinone oxidoreductase gene expression in human colon carcinoma cells: characterization of a mutation which modulates DT-diaphorase activity and mitomycin sensitivity. Cancer Res 52:797–802 80. Tsabouri SE et€al (2000) Increased prevalence of GSTM(1) null genotype in patients with myelodysplastic syndrome: a case-control study. Acta Haematol 104:169–173 81. Varkonyi J et€ al (2008) Glutathione S-transferase enzyme polymorphisms in a Hungarian myelodysplasia study population. Pathol Oncol Res 14:429–433 82. Vodicka P et€al (2004) Genetic polymorphisms in DNA repair genes and possible links with DNA repair rates, chromosomal aberrations and single-strand breaks in DNA. Carcinogenesis 25:757–763 83. Wang WW et€al (2001) A single nucleotide polymorphism in the 5′ untranslated region of RAD51 and risk of cancer among BRCA1/2 mutation carriers. Cancer Epidemiol Biomarkers Prev 10:955–960 84. Wang Y et€al (2003) From genotype to phenotype: correlating XRCC1 polymorphisms with mutagen sensitivity. DNA Repair (Amst) 2:901–908 85. Westlind A et€al (1999) Interindividual differences in hepatic expression of CYP3A4: relationship to genetic polymorphism in the 5′-upstream regulatory region. Biochem Biophys Res Commun 259:201–205

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C. Seedhouse and N. Russell

86. Wiencke JK et€al (1990) Human glutathione S-transferase deficiency as a marker of susceptibility to epoxide-induced cytogenetic damage. Cancer Res 50:1585–1590 87. Wiencke JK et€al (1995) Gene deletion of glutathione S-transferase theta: correlation with induced genetic damage and potential role in endogenous mutagenesis. Cancer Epidemiol Biomarkers Prev 4:253–259 88. Worrillow LJ et€ al (2003) An intron splice acceptor polymorphism in hMSH2 and risk of leukemia after treatment with chemotherapeutic alkylating agents. Clin Cancer Res 9:3012– 3020

Chapter 3

Myelodysplastic Syndromes/Neoplasms: Morphological and Immunohistochemical Features and Standard Evaluation H.-P. Horny and P. Valent

Introduction Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterised by dysplasia of blood cell precursors, signs of bone marrow insufficiency despite hypercellular marrows, and peripheral cytopenia. Refractory anemia is a key finding in most patients with MDS irrespective of the MDS variant. In advanced MDS, the major differential diagnosis is acute myeloid leukemia (AML) although a broad range of other neoplastic and also non-neoplastic states have been identified as potential mimickers of MDS. The combination of cytomorphology and histology including immunohistochemistry but also cytogenetics is considered as standard approach for diagnosis and subtyping of MDS. While dysplastic features of neutrophilic cells and erythroblasts can best be assessed by cytomorphological investigation of smear preparations, histological analysis of a bone marrow trephine biopsy specimen provides information on the degree of cellularity and fibrosis, but can also reveal minute infiltrates of blast cells, a finding that can not be assessed in bone marrow smears. Moreover, some rare subtypes of MDS that are not defined in the WHO classification system can only be diagnosed histologically: hypocellular MDS (“MDS-hypo”), fibrotic MDS (“MDS-f”) and MDS associated with systemic mastocytosis (SM or SM-AHNMD, respectively). It is strongly recommended that before a diagnosis of unclassifiable MDS (MDS-u) is established such cases should also be evaluated histologically using antibodies against CD34, CD117/KIT, and tryptase.

H.-P. Horny () Institute of Pathology, Escherichstrasse 6, 91522 Ansbach, Germany Tel.: +49-981-488830 Fax: +49-981-4888310 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_3, ©Â€Springer Science+Business Media B.V. 2011

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Materials and Methods The following patient-derived material should be examined in order to establish (or exclude) a diagnosis of MDS: 1. Air-dried smear preparations of blood and bone marrow. 2. Formalin-fixed bone marrow trephine biopsy specimen with a minimum length of 2€cm; for immunohistochemical investigations mild overnight decalcification with edetic acid is strongly recommended. The following stains are recommended for smear preparations: 1. Blood: Wright-Giemsa, naphthol AS-D chloroacetate esterase/CAE (Leder’s stain), and non-specific esterase/NE. 2. Bone marrow: Wright-Giemsa, Prussian blue, CAE, NE. The following stains are recommended for basic histological and immunohistochemical evaluations: 1. Giemsa 2. Prussian blue 3. Gömöri’s silver impregnation 4. CAE 5. Anti-CD34 6. Anti-CD42/61 7. Anti-tryptase 8. Anti-CD117/KIT There is a limited panel of antibodies that should be used in all cases of suspected MDS for immunohistochemical analysis of the bone marrow: 1. Anti-CD34: CD34 is a stem cell-related antigen expressed by normal hematopoietic progenitor cells, by blast cells in neoplastic states and also by endothelial cells. Thus, the staining of blood vessels and sinuses by anti-CD34 serves as perfect internal control. However, it is important to note that CD34 is usually expressed more strongly in endothelial cells than in progenitor/blast cells. Therefore, blast cells can only be visualized by anti-CD34 antibodies when a microwave oven retrieval step has been included. In case of a weak endothelial stain by anti-CD34, a negative blast stain should raise the suspicion of a falsenegative result. Although not all blast cells of all cases of MDS are detected by anti-CD34 it is generally accepted that immunostaining with anti-CD34 provides a perfect tool for the estimation of the numbers of loosely scattered progenitor/ blast cells and to detect even very small compact blast cell infiltrates that are otherwise not detectable in smear preparations. It should be noticed that expression of CD34 by immature megakaryocytes does not reflect overt immunophenotypical atypia but is found more often in neoplastic states of MDS or AML than in the reactive bone marrow. The number of CD34-positive progenitor cells in normal or reactive bone marrow almost never exceeds 1–2% of all nucle-

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ated cells, and groups or even compact infiltrates of CD34-expressing cells are never encountered in the normal bone marrow. In various myeloid neoplasms, including low risk MDS and myeloproliferative neoplasms, a slight increase in CD34-positive cells is often found. An excess of blast cells is defined as significant increase in CD34-positive cells exceeding 5% of all nucleated cells. A slight or moderate increase in CD34-positive cells is observed in MDS subtypes refractory anemia (RA) with excess of blasts (RAEB-1 and RAEB-2) but also in accelerated phases or incipient blast crisis of myeloproliferative neoplasms. Expression of CD34 by more than 20% of the nucleated bone marrow cells is usually indicative of acute leukemia and is observed in both AML and acute lymphoblastic leukemia/ALL. Altogether, anti-CD34 has proved to be one of the most important immunohistochemical markers in diagnostic hematopathology. In cases with blast cells that are obviously CD34-negative (despite repeated stains), anti-CD117/KIT antibodies are recommended as alternative blast celltargeting reagents. 2. Anti-CD61: CD61 (integrin beta-3) is a platelet-associated antigen expressed by megakaryocytes in all stages of maturation, even by megakaryoblasts. Although it is also expressed on endothelial cells this is not of relevance for immunohistochemical diagnosis. Anti-CD61 enables not only an exact and reproducible quantitative assessment of the numbers of megakaryocytes, but also detection of small groups of normal or atypical megakaryocytes. Cytological atypia can easily be evaluated in CD61-expressing megakaryocytes including immature and even blast forms that cannot be detected in conventional stainings. 3. Anti-tryptase: Tryptase is serine protease predominantly but not exclusively expressed in tissue mast cells. It could be demonstrated that tryptase is also expressed by immature basophilic granulocytes in neoplastic states, including chronic myeloid leukemia and MDS. Anti-tryptase is extremely useful in diagnostic hematopathology and clearly facilitates the estimation of numbers of loosely scattered mast cells. Anti-tryptase is also helpful to detect very small compact mast cell infiltrates, a finding which is essential to establish a diagnosis of SM. However, a tryptase-positive cell is not a mast cell unless coexpression of CD117 (KIT) has been demonstrated. Tryptase and CD117 are antigens found in normal/reactive mast cells in all stages of maturation and in neoplastic mast cells. A round cell expressing tryptase but neither CD117 nor CD34 is not a mast cell but has to be classified as an immature basophil, whereas a tryptase+ round cell that co-expresses CD117 and CD34 represents a myeloblast. However, a spindle-shaped tryptase+ cell can be regarded as mast cell, especially when forming clusters and aggregates which are found almost exclusively in SM. To summarize, a round tryptase-positive cell can be a mast cell, but can also be a basophilic granulocyte or even a tryptase+ myeloblast. A rare finding is the presence of compact infiltrates consisting exclusively of round tryptase-expressing cells. Such infiltrates may be focal or diffuse and have been termed TROCI-bm. TROCI-bm is only observed in myeloid neoplasms but includes a variety of usually very rare disorders like common-type SM (focal type), well-differentiated SM (focal type), myelomastocytic leukemia (diffuse type), basophilic leukemia

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(diffuse type) and mast cell leukemia (diffuse type). When compact mast cell infiltrates are identified in a patient with MDS, the diagnosis SM-AHNMD/ MDS has to be established. Mast cells in SM usually exhibit an aberrant immunophenotype with expression of CD25, an antigen not found on normal/reactive mast cells.

Classification System of MDS (WHO 2008) 1. Refractory anemia with unilineage dysplasia (a) Refractory anemia (RA) (b) Refractory neutropenia (RN) (c) Refractory thrombocytopenia (RT) 2. Refractory anemia with ring sideroblasts (RARS) 3. Refractory cytopenia with multilineage dysplasia (RCMD) 4. Refractory anemia with excess of blasts (RAEB) 5. MDS associated with isolated del(5q) 6. MDS, unclassifiable (MDS-u) 7. Childhood MDS Rare subvariants not included in the WHO classification system but with characteristic histomorphological features: 1. Hypocellular MDS (“MDS-hypo”) 2. MDS with fibrosis (“MDS-f”) 3. MDS associated with systemic mastocytosis (SM-AHNMD/MDS) Note╇ Histological evaluation of a bone marrow trephine biopsy specimen is recommended for all subtypes of MDS. However, the investigation is crucial for the following subvariants: MDS-u, MDS-hypo, MDS-f and SM-MDS. The typical histomorphological features of the main subtypes of MDS are listed in the Table€3.1. Table 3.1↜渀 Morphological features of MDS Diagnosis Cell NG EP MEG BC FIB Iron RA + − + − − − + − +â•›+ − − − +â•›+ RARS +â•›+ RAEB +â•›+â•›+ +â•›+ + −/+ +â•›+ + + RCMD +â•›+ + + + − − +â•›+ MDS del(5q) + − − +â•›+ − − + MDS-hypo −â•›− −â•›− −â•›− −â•›− + −/+ + MDS-f +/+â•›+ + + + + +â•›+ +â•›+ +â•›+â•›+ marked increase, +â•›+ moderate increase, + slight increase, − normal or absent, −â•›− decreased, CELL cellularity, NG neutrophilic granulocytpoiesis, EP erythrocytopoiesis, MEG megakaryocytes, FIB fibrosis

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General Considerations Although morphological aspects of MDS may vary even within a defined subvariant the common denominator is at least a unilineage dysplasia of a major myeloid cell line. Regarding neutrophilic cells the most common atypia is hypolobation of the nucleus in a significant proportion of circulating granulocytes. Such cells are termed “pseudo-Pelger” cells. In the rare event that all neutrophils exhibit hypolobation this is not MDS but is indicative of the inborn Pelger-Huet anomaly. The most important anomaly detected in the erythroid series in MDS are ring sideroblasts exhibiting at least a semicircular often a complete circular arrangement of siderin particles around the nucleus in the Prussian blue stain. In RA with ring sideroblasts (RARS), ring sideroblasts must represent at least 15% of all nucleated erythroid cells. A very characteristic abnormality of megakaryocytes is a marked hypolobation of their nuclei. When most or all megakaryocytes are hypolobated, MDS with isolated del(5q) is the most likely diagnosis. Histological evaluation of a bone marrow trephine biopsy specimen should be regarded as standard diagnostic approach in all patients with suspected MDS. The following findings are to be recorded in every patient: 1. Cellularity (Figs.€ 3.1, 3.2): Moderate to marked hypercellularity is found in most patients with MDS, but hypocellular variants have been described in up to 10% of cases. MDS-hypo is almost always a secondary disease occurring after radio- and/or chemotherapy. Estimating the degree of cellularity is crucial in all

Fig. 3.1↜渀 Myelodysplastic syndrome. An extremely hypercellular bone marrow contains an increased number of atypical small megakaryocytes and a prominent left-shifted erythrocytopoiesis. Neutrophilic granulocytpoiesis seems hypoplastic. Histopathological aspects allow a diagnosis of a myeloid neoplasm to be favored but definitive subtyping is not possible. Regarding the cytomorphological aspects on blood and bone marrow smears a diagnosis of refractory cytopenia with multilineage dysplasia was established. H&E

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Fig. 3.2↜渀 Myelodysplastic syndrome. A slightly hypercellullar bone marrow with left-shifted neutrophilic granulocytopoiesis and erythrocytopoiesis dominates the picture. Loosely scattered megakaryocytes are atypical but occur in normal numbers. Naphthol AS-D chloroacetate esterase (CAE) histochemistry nicely allows to confirm the aspect of a disturbed microarchitecture but rules also out the presence of compact (CAE-negative) blast cell infiltrates. Based on conventional bone marrow histology alone a myelodysplastic syndrome can be suspected but definitive diagnosis and subtyping are not possible. Immunostaining with anti-CD34 revealed a slight increase in loosely dispersed blast cells ranging between 5 and 10% of nucleated bone marrow cells (not depicted). Diagnosis of refractory anemia with excess of blasts (RAEB-1) was established. CAE

patients. The age of the patient must be taken into consideration as the physiologic cellularity of the marrow varies markedly in different age groups. 2. Microarchitecture: Alterations in the microarchitecture of the bone marrow may be subtle but are present in almost all patients with MDS. Common findings are prominent peritrabecular proliferation zones of the neutrophilic granulocytopoiesis, varying size of erythroid cell clusters, and grouping (not clustering) of megakaryocytes. Note that such findings alone are not sufficient to establish a diagnosis of MDS. 3. Reticulin fibers (Figs.€3.3a, b): In many cases of MDS there is a mild often focal reticulin fibrosis while marked fibrosis is rarely seen. The rare cases with reticulin fibrosis as leading feature may be termed MDS-f. 4. Iron stores (Fig.€3.4): In almost all patients, MDS is associated with a marked or even extensive hemosiderosis. Note that the Prussian blue stain in histological sections often yields lower amounts of siderophages than the Prussian blue stain in bone marrow smears in the same patient. Depletion of iron stores is almost incompatible with the primary diagnosis of MDS. 5. Neutrophilic granulocytopoiesis: Neutrophilic granulocytopoiesis is almost always increased and left-shifted with accumulations of immature cells in peritrabecular and perivascular sites. Perivascular proliferation zones have been misleadingly termed “atypically localized immature precursor” cells (ALIP) a finding which is usually very hard to detect or reproduce in modern times

3â•… Myelodysplastic Syndromes/Neoplasms: Morphological and Immunohistochemical Fig. 3.3a, b↜渀 Myelodysplastic syndrome. Silver staining allows to determine the degree of reticulin fibrosis. a Depicts a focal slight increase in reticulin fibers while b shows a marked diffuse reticulin fibrosis. Reticulin fibrosis is a very non-specific finding seen in variuos reactive and neoplastic states. While a shows a case of refractory anemia (RA), b represents one of the rare cases of true MDS with fibrosis (MDS-f) especially there was no increase in blast cells or clustering of megakaryocytes. Gömöri’s silver impregnation

Fig. 3.4↜渀 Myelodysplastic syndrome. In a moderately hypercellular bone marrow an increased number of siderophages can easily be detected. Moderate to marked siderosis is almost always seen in MDS but also in reactive states of anemia of chronic didease. On the basis of cytomorphological aspects on bone marrow smears this case was subtyped as refractory anemia with multilineage dysplasia (RCMD). Prussian blue

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Fig. 3.5↜渀 Myelodysplastic syndrome. Hypercellular bone marrow with markedly dilated sinuses is shown. Note a very pronounced increase in small immature to blast-like clustering megakaryocytes that are only detectable by an appropriate immunostaing with anti-CD61. Also note the prominent erythrocytopoiesis with signs of intravascular hematopoiesis. The neutrophilic granulocytopoiesis is hypoplastic. This case was originally diagnosed as MDS-f due to focal marked fibrosis but rather represents refractory anemia with excess of blasts (RAEB). Anti-CD61 (ABC method)

of immunhistochemistry. However, a complete maturation arrest is not found unless the disease has progressed to overt AML. 6. Erytrocytopoiesis: In most patients, erythropoiesis is increased (hyperplastic) and irregularly distributed with unusually varying size of the erythra. There is also marked left-shifting, but cellular atypia is difficult to recognize in histological sections. Ring sideroblasts cannot be identified in histological sections because mitochondrial iron deposits are destroyed during routine processing in water-containing fixatives (in contrast to the lysosomal hemosiderin). 7. Megakaryocytes (Fig.€3.5): There is almost always an irregular distribution of megakaryocytes with a tendency to group together, but true clusters are usually not seen in MDS. Immature megakaryocytes are often found in increased numbers in MDS. The hypolobated small megakaryocytes characteristic for MDS with isolated del(5q) are easily detectable in histological bone marrow sections. 8. Mast cells: Increased numbers of loosely scattered mast cells are a common feature in MDS while the association with systemic mastocytosis (SM-AHNMD) characterised by compact infiltrates is rarely detected. In a few cases, a slight increase of spindle-shaped loosely scattered mast cells exhibiting an aberrant immunophenotype and expression of CD25 is present. In such cases, SM-MDS should only be diagnosed when the activating point mutation KITD816V is also detected thus enabling the diagnosis of SM to be based on three minor diagnostic criteria. 9. Basophilic granulocytes: Increased numbers of basophilic granulocytes are found in many cases of MDS. Although basophils cannot be seen in routinely processed trephine biopsy specimens because their specific granules are water-soluble

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( in contrast to the likewise metachromatically reacting mast cell granules), it is possible to visualize these cells by their peculiar immunophenotypical characteristics. Small round tryptase-positive cells not coexpressing CD117 (KIT) are basophils (and not mast cells). An increase in basophils is almost exclusively seen in neoplastic states but not pathognomonic for MDS. Highest numbers of tryptase-expressing small round cells are seen in chronic myeloid leukemia. There are two antibodies recognizing specific basophil antigensand that can be applied in all cases of suspected basophilia: anti-basogranulin (BB1) and anti2D7. Both markers work well in paraffin-embedded bone marrow sections but are not commercially available. 10.╇Blast cells (Fig.€ 3.6a–d): The number of CD34-expressing progenitor cells/ blast cells varies in MDS patients. In case of CD34-negative blasts, CD117/ KIT can be recommended as an alternative blast cell marker.

Figs. 3.6a–d↜渀 Myelodysplastic syndrome. Figures 3.6a–d cover the aspects of immunostaining with anti-CD34. a Shows a case of MDS with a significant diffuse increase in loosely scattered CD34+ blast cells without forming groups or clusters. Quantitatively this case fulfills criteria of refractory anemia with excess of blasts (RAEB-1). b Shows a case with marked in increase in CD34+ blast cells forming dense aggregates. RAEB-2 transforming into overt AML was diagnosed here. c and d Exhibit a case of hypocellular MDS. c Reveals an overview with dilated sinuses, subtotal depletion of blood cell precursors and some CD34+ blast cells. d Depicts another marrow space of the same case with a significant increase in blast cells illustrating the difficulties to separate MDS-hypo with excess of blasts (RAEB-2) from overt AML. Anti-CD34 (ABC method)

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Altogether, typical cases of MDS are histologically characterised by a hypercellular bone marrow with left-shifted neutrophilic granulycytopoiesis and erythrocytopoiesis, atypical megakaryocytes and increased iron stores.

Differential Diagnoses MDS has to be separated from a great variety of reactive and neoplastic states. It should be emphasized that MDS is a disease of the elderly patient (median: about 70 years) and characterised by signs of bone marrow insufficiency with cytopenia in at least one major bone marrow lineage (usually refractory anemia). Regarding morphology alone the list of reactive states includes the following: 1. Myeloid hyperplasia 2. Granulomatosis 3. Aplastic syndromes 4. Vitamin B12 and/or folic acid deficiency 5. Congenital disorders: dyserythropoietic anemia 6. Viral infections: HIV and parvovirus B19 7. Cytoreductive therapy 8. Hemopoietic growth factors In order to avoid misinterpretation of morphological findings it is crucial to be aware of the case history and all available hematological parameters. Some of the above mentioned disorders are perfect mimickers of MDS exhibiting major cytological atypia of erythroblasts (giant proerythroblasts) in parvovirus B19 infections and the often marked cytological atypia of all blood cell precursors encountered in vitamin B12 deficiency. It is noteworthy to be aware of the fact that cytological atypia encountered in vitamin B12 deficiency may be more pronounced than ever seen in true MDS. In some cases of MDS small granulomas are detected without other indication of a systemic granulomatosis. This phenomenon is best termed as paraneoplastic granulomatosis. Aplastic syndromes may be extremely difficult to be separated from hypocellular (secondary) MDS without immunostainings (antiCD34!). In the following a list of neoplastic states is presented. All these diseases can be easily interchanged with MDS making it necessary to strictly observe defined morphological criteria and all available hematological/cytogenetic data before a diagnosis of MDS is established definitively: 1. AML 2. Acute panmyelosis with myelofibrosis 3. Systemic mastocytosis (SM-AHNMD) 4. Hairy cell leukemia 5. MDS/MPN: RARS-T, chronic myelomonocytic leukemia 6. Primary myelofibrosis 7. Megakaryoblastic leukemia

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Separation of MDS from AML is crucial and mainly depends on the number of blast cells: 1. MDS other than RAEB: <5% blasts 2. MDS RAEB-1: >5% <10% blasts 3. MDS RAEB-2: >10% <20% blasts 4. AML: >20% blasts Although these parameters appear to be simple to be assessed it may be extremely difficult to reliably determine blast cell numbers in daily routine practice. It is of major prognostic and therapeutic significance to be sure that blast cell numbers are below the threshold of 20% of all nucleated bone marrow cells. An advantage of the histology is that even small groups or infiltrates of CD34-expressing blast cells can be detected signalling an adverse prognostic event with rapid progression into AML in many patients. Even more challenging is the separation of MDS, especially MDS-f, from acute panmyelosis with fibrosis which represents a very rare subtype of AML on the one hand and from megakaryoblastic leukemia (FABM7) which also exhibits almost always marked fibrosis and therefore was termed acute myelosclerosis in previous times on the other. Acute panmyelosis shows blastic transformation of all three major myeloid cell lines and accordingly an increase in myeloblasts, megakaryoblasts (which may dominate the picture) and para-proerythroblasts while megakaryoblastic leukemia shows a marked increase only in megakaryoblasts. Since megakaryoblasts are not a prominent feature in MDS-RAEB the diagnosis should be possible after appropriate immunostaining using at least antibodies against CD34, CD61 and glykophorin A (as an erythroid antigen of diagnostic relevance for immunohistochemistry). Primary myelofibrosis (formerly: agnogenic myeloid metaplasia or chronic idiopathic myelofibrosis) may mimick MDS, especially MDS-f, at first glance but presence of groups and clusters of highly pleomorphic megakaryocytes and a significant splenomegaly definitively excludes MDS. Systemic mastocytosis may morphologically mimick MDS and granulomatosis but may also be associated with MDS in the setting of a SM-AHNMD. It should be noticed that true SM-MDS is much less frequent than other subtypes of SMAHNMD like SM-CMML or SM-AML. All major subvariants of MDS have been observed in SM-MDS. Presence of compact mast cell infiltrates and activating point mutation KITD816V are definitive proof of SM while reactive increase in mast cells (= mast cell hyperplasia) never is associated with compact mast cell infiltrates and/or KITD816V. Clinically, malignant lymphoma may also mimick MDS. This is especially true for hairy cell leukemia which almost always shows marked cytopenia. However, presence of splenomegaly and diffuse-compact infiltration of bone marrow by neoplastic medium-sized B cells often with wide pale cytoplasm enables the definitive diagnosis to be made. Disorders belonging to the group of MDS/MPN may be very difficult to be separated from MDS because by definition blood cell precursors exhibit signs of atypia. Formerly, the most frequent subtype of MDS/MPN was even included amongst

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MDS: chronic myelomonocytic leukemia (CMML). Using stains like UE or antiCD14/CD163 to visualize a significant increase in monocytes/promonocytes in blood and bone marrow is sufficient to assess or exclude CMML since a marked increase in monocytes is not a characteristic feature of MDS. It should be pointed out that diagnosis of MDS and its separation from all potential differential diagnoses is dependent on (1) a reasonable amount and quality of the bone marrow biopsy specimen, (2) good quality smears (including at least five unstained ones), (3) peripheral blood smears, (4) and sufficient information about all relevant clinical and laboratory findings.

 orphology of MDS Subvariants with Special Emphasis M on Histopathological Aspects (Subvariants 1.–6. Are Defined by FAB/WHO Criteria) 1. Refractory Cytopenia (RC): RC must not be diagnosed on the basis of histopathological bone marrow findings alone. Moreover, refractory neutropenia and refractory thrombocytopenia are recently defined very rare subentities of RC. Therefore there is very limited if any experience regarding their histomorphological features in routinely processed bone marrow biopsy specimens. Since cytological atypia of neutrophilic cells and erythroblasts cannot be recognized with certainty in histological sections RC of all types should be diagnosed in bone marrow smear preparations considering the presence of significant cytopenia (for at least 6 months), exclusively unilinear cytological atypia and cytogenetic abnormalities as major criteria. Cytological atypia without cytopenia or significant cytopenia without major dysplasia both do not allow such cases to be classified as MDS but should rather be put in categories like idiopathic dysplasia of unknown significance (IDUS) or idiopathic cytopenia of unknown significance (ICUS). Regarding histopathology in RC the bone marrow is almost always hypercellular with marked hemosiderosis. RC is mostly a diagnosis of exclusion, in particular an increase in CD34+ blast cells and significant fibrosis are not compatible with a diagnosis of RC. 2. RC with Multilineage Dysplasia (RCMD): RCMD belongs to the more common subtypes of MDS. Histopathological aspects include a marked hypercellularity of bone marrow in most cases. There is a left-shifted atypical neutrophilic granulocytopoiesis. In most patients, erythrocytopoiesis is prominent but not strongly increased. The number of megakaryocytes is usually slightly increased with a tendency to form small groups. Megakaryocytes may exhibit marked cytological atypia seen in histological sections of bone marrow. Megakaryocyte clusters and an increase in CD34+ blasts are not found in RCMD. The density of retculin fibers is often slightly increased but a significant fibrosis is not observed. RCMD must be diagnosed definitively only when significant cellular

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Fig. 3.7a, b↜渀 Myelodysplastic syndrome. Cytology in a Pappenheim stain reveals a hypercellular bone marrow with prominent erythrocytopoiesis which is normoblastic and markedly left-shifted. Note the abundance of basophilic erythroblasts and proerythroblasts. There is no excess of blast cells. Prussian blue stain shows a significant increase in ring sideroblasts enabling a diagnosis of “Refractory anemia with ring sideroblasts” to be established

atypia of more than one major myeloid cell line is observed in bone marrow and blood smears. 3. Refractory Anemia with Ring Sideroblasts (RARS; Figs.€ 3.7a, b): Since the pathognomonic ring sideroblasts can only identified in bone marrow smears but not in routinely processed trephine biopsy specimens RARS must not be diagnosed on the basis of a histological investigation of bone marrow alone. In contrast to the lysosomal hemosiderin mitochondrial iron deposits of ring sideroblasts are destroyed during processing. Cytological atypia, in particular of neutrophilic cells and megakaryocytes are not a prominent feature of the disease. Since ring sideroblasts are also found in patients with lead intoxication, the diagnosis of RARS in younger adults should only be made after having excluded lead-induced dysplasia. The histological picture of the bone marrow shows an increased left-shifted normo- or slightly macroblastic erythrocytopoiesis and a marked hemosiderosis. A differential diagnosis of hemolytic anemia

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or even of a hemophagocytic syndrome is not possible in most cases unless reticulocyte counts are taken into consideration. Reticulocytosis clearly favors a reactive state of hemolytic anemia. Number of CD34-positive blast cells is not increased and there is also no significant reticulin fibrosis. Usually there is a marked increase in loosely scattered reactive mast cells whereas SM associated with RARS is rarely seen. The number of megakaryocytes is usually normal or slightly increased and there is a left-shifted neutrophilic granulocytopoiesis. In the rare cases with excess of ring sideroblasts and significant thrombocytosis this is not true RARS but rather a special and recently defined entity belonging to the myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) termed RARS-T. RARS-T has found to be associated with the presence of the activating point mutation JAK2-V617F which is very rarely seen in RARS. 4. Refractory Anemia with Excess Blasts (RAEB): Pending on the number of blast cells two types of RAEB must be separated: (1) RAEB-1 with blast cell numbers >5% but <10%, and (2) RAEB-2 with blast cell numbers >10% but <20%. Blast cell numbers exceeding 20% of nucleated bone marrow cells allow a diagnosis of AML to be made. Since in almost all cases of RAEB blast cells express CD34 diagnosis and subtyping of the disease are not only possible but often crucial in histological bone marrow sections. It is not a very rare finding when small groups or infiltrates of blast cells are detected, especially in RAEB-2. Neutrophilic granulocytopoiesis is usually markedly atypical and left-shifted. Cytological atypia of erythroblasts and megakaryocytes are common, the number of megakaryocytes varies greatly. Using antibodies against platelet-associated antigens like CD61 enables detection of megakaryoblasts which are never seen in reactive states. Iron stores are increased and the degree of reticulin fibrosis is often low to moderate. 5. MDS with isolated del(5q) (Fig.€3.8): Although this seems a cytogenetic-based diagnosis the morphology of megakaryocytes is quite characteristic allowing a diagnosis of MDS with del(5q) to be established or at least strongly suspected

Fig. 3.8↜渀 Myelodysplastic syndrome. Bone marrow histology shows an abundance of small evenly distributed megakaryocytes expressing CD61. There are no groups or clusters of megakaryocytes. Note the uniform hypolobated nuclei of megakaryocytes which is seen only in MDS with del(5q)

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from histological investigation of the bone marrow. Almost all megakaryocytes which are loosely scattered throughout appear to be immature exhibiting hypoor even non-lobated round nuclei. An increase in blast cells and reticulin fibers are not found in most cases of MDS-del(5q). Moreover, cytological atypia of neutrophilic cells and erythroblasts is minimal or even missing while slight hypoplasia of erythrocytopoiesis is common. 6. Unclassifiable MDS (MDS-u): MDS-u is a waste-basket for a few rare hematopathological findings that do not allow to be put in one defined category of MDS or another myeloid neoplasm like MDS/MPN. Accordingly, a common histological picture of the bone marrow does not exist for MDS-u. It is therefore strongly recommended in every patient with suspected diagnosis of MDS-u to investigate a bone marrow trephine biopsy specimen in order not to miss any underlying reactive or neoplastic process undetectable in smear preparations, for example hypocellular MDS, mastocytosis, or even hairy cell leukemia. 7. Hypocellular MDS (MDS-hypo) (Fig.€ 3.6a–d): MDS-hypo is not listed as a separate entity in the WHO system of classification but represents a distinctive morphological subvariant of the disease with bad prognosis. Not more than 10% of MDS cases belong to the hypocellular variant which can only be recognised histologically while smear preparation of the marrow show a dry tap. The cytopenia is often very severe. MDS-hypo is almost always secondary event in contrast to the other categories of MDS and seen after radiochemotherapy with an interval up to 20 years. MDS-hypo is definitively recognizable when an increase in CD34-positive blast cells is present thus enabling a refined classification of MDS-hypo RAEB type. Since the paucicellularity of the bone marrow does not allow any statements about the presence of cytological atypia of the blood cell precursors immunostaining with anti-CD34 is of crucial importance in all hypocellular bone marrows. Usually the clear-cut distinction between MDS-hypo RAEB-2 and hypocellular AML is not possible because the constant presence of lymphocytes, plasma cells and macrophages in such hypoplastic marrows interferes with precise quantitative statements concerning the blast cell numbers amongst all nucleated blood cell precursors. 8. Fibrotic MDS (MDS-f): MDS-f is an ill-defined subvariant of MDS regarding both degree of fibrosis and separation from other MDS subtypes. For diagnosis of MDS-f at least a moderate diffuse increase in reticulin fibers should be present. Fibrosis has been described as an adverse prognostic feature in MDS patients. However, many cases of MDS-f show an excess of blast cells (quantitatively allowing them to be put into categories RAEB-1 or RAEB-2, respectively). The term myelofibrosis should not be used to describe MDS-f in order to avoid misleading interpretation as primary myelofibrosis which belongs to the myeloproliferative neoplasms. 9. Systemic mastocytosis associated with MDS (SM-AHNMD): Although a marked increase in mast cells is often seen in MDS, especially in RARS, an association of MDS and SM is rare. Almost all defined subtypes of MDS have been detected in the setting of SM-AHNMD, enabling diagnoses of SM-RCMD, SM-RAEB, or SM-RARS to be established. Diagnosis of SM basically implies the presence

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of at least one compact mast cell infiltrate as the one and only major criterion. In cases with prominent spindling of mast cells and immunophenotypical anomalies with expression of CD25 the diagnosis of SM can also be established when the activating point mutation KITD816V is detected. It is of major importance to apply an anti-tryptase antibody in all cases of myeloid tumors including MDS not only to be able to detect small mast cell infiltrates indicative of SM but also to find an increase in basophilic granulocytes which is almost never seen in reactive states. However, it has to be considered that basophilia also may occur in other myeloid neoplasms and is very prominent in chronic myeloid leukemia. In addition, in all patients with suspected SM-AHNMD/SM-MDS, KIT mutation analysis should be performed. The diagnosis of SM-MDS represents a challenging one since minor cellular atypia can also be seen in SM, in particular its smoldering subtype (SSM) which assumes an intermediate position between indolent and aggressive SM. This emphasizes the necessity to strictly apply defined diagnostic criteria for both SM and MDS before a diagnosis of SM-MDS must be established definitively.

References 1.

Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C (1982) Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51: 189–199 2. Orazi A, Bennett JM, Germing U, Brunning RD, Bain BJ, Thiele J (2008) Myelodysplastic syndromes. In: Swerdlow SH et€al (eds) World health organization classification of tumours. Pathology & genetics. Tumours of haematopoietic and lymphoid tissues. IARC Press, Lyon 3. Bennett JM (2005) A comparative review of classification systems in myelodysplastic syndromes (MDS). Semin Oncol 32:S3–S10 4. Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G et€al (1997) International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 89:2079–2088 5. Lambertenghi-Deliliers G, Annaloro C, Oriani A, Soligo D (1992) Myelodysplastic syndrome associated with bone marrow fibrosis. Leuk Lymphoma 8:51–55 6. Horny H-P, Wehrmann M, Schlicker HU, Eichstaedt A, Clemens MR, Kaiserling E (1995) QBEND10 for the diagnosis of myelodysplastic syndromes in routinely processed bone marrow biopsy specimens. J Clin Pathol 48:291–294 7. Baur AS, Meuge-Moraw C, Schmidt PM, Parlier V, Jotterand M, Delacretaz F (2000) CD34/ QBEND10 immunostaining in bone marrow biopsies: an additional parameter for the diagnosis and classification of myelodysplastic syndromes. Eur J Haematol 64:71–99 8. Horny H-P, Greschniok A, Jordan JH, Menke DM, Valent P (2003) Chymase expressing bone marrow mast cells in mastocytosis and myelodysplastic syndromes: an immunohistochemical and morphometric study. J Clin Pathol 56:103–106 ╇ 9. Horny H-P, Sotlar K, Sperr WR, Valent P (2004) Systemic mastocytosis with associated clonal haematological non-mast cell lineage diseases: a histopathological challenge. J Clin Pathol 57:604–608 10. Valent P, Horny H-P, Bennett JM, Fonatsch C, Germing U, Greenberg P, Haferlach T, Haase T, Kolb H-J, Krieger O, Loken M, van de Loosdrecht A, Ogata K, Orfao A, Pfeilstöcker M, Rüter B, Sperr WR, Stauder R, Wells DA (2007) Definitions and standards in the diagnosis and treatment of the myelodysplastic syndromes: consensus statements and report from a working conference. Leuk Res 31:727–736

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11. Tricot G, De Wolf-Peeters C, Vlietinck R, Verwilghen RL (1984) Bone marrow histology in myelodysplastic syndromes. II. Prognostic value of abnormal localization of immature precursors in MDS. Br J Haematol 58:217–225 12. Oriani A, Annaloro C, Soligo D, Pozzoli E, Cortelezzi A, Lambertenghi Deliliers G (1996) Bone marrow histology and CD34 immunostaining in the prognostic evaluation of primary myelodysplastic syndromes. Br J Haematol 92:360–364 13. Horny H-P, Sotlar K, Stellmacher F, Krokowski M, Agis H, Schwartz LB, Valent P (2006) The tryptase-positive compact round cell infiltrate of the bone marrow (TROCI-bm): a novel histopathological finding requiring the application of lineage-specific markers. J Clin Pathol 59:298–302 14. Thiele J, Quitmann H, Wagner S, Fischer R (1991) Dysmegakaryopoiesis in myelodysplastic syndromes (MDS): an immunomorphometric study of bone marrow trephine biopsy specimens. J Clin Pathol 44:300–305 15. Agis H, Krauth MT, Böhm A, Mosberger I, Müllauer L, Simonitsch-Klupp I, Walls AF, Horny H-P, Valent P (2006) Identification of basogranulin (BB1) as a novel immunohistochemical marker of basophils in normal bone marrow and patients with myeloprolferative disorders. Am J Clin Pathol 125:1–9 16. Agis H, Krauth MT, Mosberger I, Müllauer L, Simonitsch-Klupp I, Schwartz LB, Printz D, Böhm A, Fritsch G, Horny H-P, Valent P (2006) Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific antibody 2D7. J Clin Pathol 59:396–402 17. Orazi A, O’Malley DP, Jiang J, Vance GH, Thomas J, Czader M, Fang W, An C, Banks PM (2005) Acute panmyelosis with myelofibrosis: an entity distinct from acute megakaryoblastic leukemia. Mod Pathol 18:603–614 18. Imbert M, Nguyen D, Sultan C (1992) Myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) with myelofibrosis. Leuk Res 16:51–54 19. Horny H-P, Sotlar K, Valent P (2007) Diagnostic value of histology and immunohistochemistry in myelodysplastic syndromes. Leuk Res 31:1609–1616 20. Brunning RD, Bennett JM, Flandrin G, Matutes E, Head D, Vardiman JW et€al (2001) Myelodysplastic syndromes. In: Jaffe ES, Harris NL, Stein H, Vardiman JW (eds) World health organization classification of tumours. Pathology & genetics. Tumours of haematopoietic and lymphoid tissues, vol€1. IARC Press, Lyon, pp€61–73

Chapter 4

Diagnostic Criteria and Classification   of Myelodysplastic Syndromes Peter Valent, Friedrich Wimazal, Wolfgang R. Sperr and Hans-Peter Horny

Introduction Myelodysplastic syndromes (MDS) are hematopoietic neoplasms characterized by a maturation-defect in myelopoietic progenitor cells, peripheral cytopenia, and clonal instability with an enhanced risk to transform into secondary acute myeloid leukemia (sAML) [2, 4–6, 9, 17, 18]. Until 2001, MDS have primarily been classified according to criteria provided by the French-American-British (FAB) working group [1]. In 2001, the World Health Organization (WHO) has extended and reestablished the MDS classification, and in 2008 an update of this classification was presented [4, 5]. The WHO classification provides robust criteria for the discrimination of MDS variants from each other. In addition, minimal diagnostic criteria for MDS have been proposed in a Working Conference in Vienna in 2006 [26, 27]. These criteria are important and helpful for the discrimination between MDS and all other reactive and neoplastic disorders that can produce cytopenia or/and dysplasia [26, 27]. In most patients with MDS, the bone marrow smear reveals marked dysplasia in one or more major hematopoietic cell lineage/s (erythroid, neutrophilic, megakaryocytic) [1, 2, 4, 5, 16]. Monocytosis or/and an increase in blasts may also be detected [1, 4, 5, 16]. In addition, typical blood count abnormalities such as macrocytic anemia or abnormal neutrophils (Pseudo-Pelger-Huet cells) may be found in these patients. Together, in most patients, the diagnosis MDS can be established quite easily by a thorough examination of blood and bone marrow smears [1, 16]. In other patients, no prominent dysplasia is found but an abnormal karyotype is detected, leading to the conclusion the patient is suffering from MDS [4, 5, 27]. However, there are also patients in whom it is quite difficult to define whether (mild) P. Valent () Division of Hematology & Hemostaseology, Department of Medicine I, Medical University of Vienna and Ludwig Boltzmann Cluster Oncology, Waehringer Guertel 18-20, 1090 Vienna, Austria Tel.: +43-1-40400-5488 Fax: +43-1-40400-4030 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_4, ©Â€Springer Science+Business Media B.V. 2011

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Table 4.1↜渀 Step-wise approach in the diagnosis and prognostication in MDS 1. Minimal diagnostic criteria Establish the diagnosis MDS 2. FAB and WHO classification Establish the disease variant 3. IPSS, WPSS and other scores Establish the risk of AML transformation 4. Patient-related risk factors Estimate survival 5. Therapy-related scores Establish the treatment plan MDS myelodysplastic syndromes, FAB French-American-British working group, WHO World Health Organization, IPSS international prognostic scoring system, WPSS WHO-adapted prognostic scoring system

cytopenia or dysplasia would indeed result from an underlying MDS, a prephase of MDS, or from another hematologic or even non-hematologic disease, especially when the karyotype is normal or is not available [27]. In a group of patients, it may be difficult to discriminate between advanced MDS and AML, or MDS and an overlap (MDS/myeloproliferative) disorder. From a practical point of view, the diagnosis MDS should be established in a step-wise fashion. In a first step, minimal diagnostic criteria have to be documented. Then, the variant, preferably WHO and FAB (but at least FAB) should be defined [25]. In a third step, the patient is examined for individual risk factors and scores, in order to establish the overall risk profile, preferably be IPSS and WPSS. In a last step, treatment-scores are applied to define the optimal therapeutic approach (Table€4.1).

Minimal Diagnostic Criteria Minimal diagnostic criteria for MDS were discussed and proposed in a Working conference in 2006 [27]. By consensus, minimal diagnostic criteria include (1) marked and constant (>6 months) peripheral cytopenia in at least one major hematopoietic lineage (erythroid, neutrophil, platelet), (2) MDS-related bone marrow features, i.e. one or more of the following: dysplasia ≥10% of all cells in one (or more) major hematopoietic lineage(s), ring sideroblasts ≥15%, myeloblasts ≥5%, or an MDS-related karyotype, and (3) exclusion of all other hematopoietic and non-hematopoietic disorders as primary reason for dysplasia and/or cytopenia [26, 27]. Based on this proposal, all patients with suspected MDS must undergo a bone marrow investigation [27]. The diagnosis may be difficult to substantiate in those patients in whom MDS co-exists with another bone marrow neoplasm that can also cause cytopenia or even dysplasia (example: coexisting mastocytosis). In these patients, it is important to look at bone marrow sections to define whether cytopenia could be caused by MDS or (also) by the co-existing disease, and to document additional criteria for MDS [26, 27]. In a group of cytopenic patients with suspected MDS, disease-related criteria are not fulfilled although clinical features are highly suggestive of MDS (e.g. macrocytic transfusion-dependent anemia) [26, 27]. In these patients, it is important to

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exclude all differential diagnoses and to ask for MDS-co-criteria, including aberrant phenotypes, abnormal stem cell function, and clonality of bone marrow cells [26, 27]. Stem cell function is almost invariably reduced in MDS and is best examined by colony-formation of progenitor cells (CFC) [3, 8, 26, 27]. Notably, a normal CFC count almost excludes MDS, whereas markedly reduced CFC are confirmatory for this diagnosis [8, 22]. In patients with a normal karyotype, monoclonality can be documented by the presence of (somatic) mutations in critical target genes, such as RUNX, RAS, or JAK2 [6, 17, 18, 27, 28]. Another approach is to screen for “monoclonal” patterns in mRNA expression profiles or proteomics [12, 18, 20, 28]. However, these techniques are not standardized and are not specific for MDS. Another approach is to determine cell surface antigen patterns in blast cells and maturing myeloid cells by flow cytometry [14, 27, 29]. Again, results are not specific for MDS but may help in reaching the conclusion the patient is suffering from a myeloid neoplasm resembling MDS [27, 29]. If one or more co-criteria are documented in a cytopenic patient with typical blood findings (e.g. severe macrocytic anemia) and all other causes for cytopenia have been excluded, the provisional diagnosis of MDS can be established even if no dysplasia and no abnormal karyotype are found [27].

ICUS and IDUS: The Diagnostic Interface A diagnostic challenge are patients who do not fulfil minimal diagnostic criteria for MDS but are suffering from constant (>6 months) cytopenia or from unexplained dysplasia without marked cytopenia [25–27, 30]. In these patients repeated investigations of the bone marrow and an extensive search for an underlying disease may be required [25, 27, 30]. Repeated tests may reveal an underlying hematologic or non-hematologic disease or an imminent MDS. If this is not the case, a provisional diagnosis should be established: in those with marked and constant cytopenia (hemoglobin <10€g/dl and/or neutrophils <1,000/µl and/or platelets <100,000/µl) but no evident dysplasia (dysplasia in less than 10% of cells in all three major lines) the diagnosis Idiopathic Cytopenia of Undetermined (Uncertain) Significance (ICUS) should be considered [26, 30]. In those patients who have marked dysplasia (>10% in a major cell lineage) with or without an MDS-related karyotype but no or only mild cytopenia, the term Idiopathic Dysplasia of Undetermined (Uncertain) Significance (IDUS) should be applied [26] (Table€4.2). By definition the presence of both ICUS and IDUS is exclusive since coexistence of these conditions is diagnostic and meets criteria for MDS [26] (Fig.€4.1). Nevertheless, for patients with mild cytopenia (hemoglobin ≥10€g/dl; neutrophils ≥1,000/µl; platelets ≥100,000/µl) but clearly demonstrable dysplasia (>10%), the diagnosis IDUS is appropriate [26]. Some of these patients progress to frank MDS over time, whereas others may progress to a myeloproliferative neoplasm (MPN) or an overlap disease (MPN/MDS) (unpublished observation). All patients with ICUS and IDUS should have a hematologic

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Table 4.2↜渀 Features defining idiopathic cytopenia (ICUS) and idiopathic dysplasia (IDUS) Term Definition Other features Older patients low EPO level Constant marked cytopeniaa Idiopathic cytopenia of FISH may reveal a small unknown significance No MDS found by criteriab clone in the BM (ICUS) No dysplasia, no karyotypec d No Co-criteria No other disease as reason for cytopenia found No constant marked cytopeniaa Younger patients usually Idiopathic dysplasia of unknown significance detected in a routine No MDS found by criteriab (IDUS) blood test (e.g. Pelger or Dysplasia and/or karyotypec macrocytosis) No other disease as reason for Dysplasia/karyotype detected

MDS myelodysplastic syndromes, EPO erythropoietin, FISH fluorescence in situ hybridization, BM bone marrow a Constant marked: for at least 6 months: hemoglobin <10€g/dl, neutrophils <1,000/µl blood, platelets <100,000/µl b Criteria refer to reference [26, 27] c Diagnostic dysplasia: ≥10% of cells; karyotypes typically found in MDS d If one or more co-criteria are found, the condition should be termed “highly suspective for MDS or a related myeloid neoplasm”

follow up in order to document or exclude evolution to MDS [26, 27, 30]. One important parameter in ICUS-patients is the serum erythropoietin (EPO) level. In most patients with ICUS, serum erythropoietin levels are (inadequately) low even if the kidney function is (otherwise) normal [7, 23, 26]. In these patients, treatment with recombinant EPO can correct the anemia similar to patients with low risk MDS who have a low endogenous EPO level [23]. All in all, it is standard to manage patients with ICUS and IDUS in the same way as patients with low risk MDS [27, 30]. An important diagnostic approach in patients with ICUS is fluorescence in situ hybridization (FISH) of bone marrow interphases [30]. In several of these patients, FISH may reveal the presence of a small population of clonal cells carrying an MDS-related cytogenetic defect [30]. Sometimes, when recorded over time, the size of the clone (number of “FISH-positive” metaphases) increases, bone marrow function (number of CFC) decreases, and MDS can be diagnosed [30]. Similarly, in patients with IDUS, FISH may reveal the presence of an MDS-related cytogenetic abnormality [24]. Although IDUS may not be a rare condition, the numbers of well documented cases are very low [26, 24]. In fact, these patients have only mild or no cytopenia. Several of them are referred because of unexplained macrocytosis or unexplained Pseudo-Pelger-Huet cells in a blood film [24, 26]. Similar to patients with ICUS, patients with IDUS should have a hematologic follow up in order to document or exclude evolution to MDS. This is important as patients with IDUS usually are younger than those with ICUS [24, 26]. Bone marrow investigations should be repeated as soon as cytopenia develops or other signs for an imminent MDS are found in these patients. However, not all patients with IDUS develop an MDS even when recorded over many years [24]. In contrast to patients with

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Normal Hematopoietic Stem Cell

Clonal expansion of a premalignant progenitor cell population leading to ineffective (impaired) erythropoiesis / hematopoiesis Disease manifestation may depend on EPO production (kidney function↓? androgens↓?)

+ EPOresponsive progenitors

IDUS EPO-response lost +/- blasts ↑ → MDS

EPO production adequate and sufficient to prevent anemia

IDUS no MDS detected as no anemia develops – these patients have dysplasia without cytopenia = IDUS

Inadequate ´EPO-response´ to ineffective erythropoiesis → anemia

Normal hematopoiesis in the healthy elderly

10-30% Low EPO in elderly → Anemia / AOE

+ ICUS LOW RISK MDS often responsive to EPO therapy (15-25% of patients)

ICUS

progression/clonal expansion HIGH RISK MDS (cytopenia found invariably) Further oncogenic hits that lead to maturation arrest and proliferation SECONDARY ACUTE MYELOID LEUKEMIA

Fig. 4.1↜渀 Role of erythropoietin (EPO) production in the manifestation of MDS. Myelodysplastic syndromes are considered to arise from hematopoietic (neoplastic) stem cells and to develop in a step-wise process. Apart from clone-specific factors, also patient-related factors and the specific microenvironment play a role in disease manifestation and disease evolution. In a pre-phase of MDS, the neoplastic clone may first replace normal hematopoiesis but still may not produce anemia, because clonal (sometimes also residual non-clonal) erythroid progenitor cells are responsive to EPO. As long as EPO production is normal in these patients (adequate and able to counteract severe anemia) they will not develop anemia/cytopenia so that criteria for MDS are not fulfilled. These patients are called idiopathic dysplasia of uncertain significance (IDUS). Later, when EPO production declines or clonal progenitor cells no longer respond to EPO, these patients develop frank MDS. EPO production may decrease in the elderly normal population, and may then lead to anemia: this form of anemia has also been termed anemia of the elderly (AOE). If in these patients, a dysplastic clone develops, the patient may rapidly develop overt MDS, and when clonal BFU-E are responsive, these patients will respond to therapy with exogenous EPO. The equation in the hypothesis thus is: IDUS╛+╛ICUS╛=╛MDS

ICUS, the erythropoietin level in patients with IDUS is usually adequate and can increase in response to anemia. Moreover, in patients with IDUS, BFU-E retain responsiveness against EPO (unpublished observation). All these observations point to a pathogenetic model, in which younger individuals develop an MDS-related clone and thus IDUS in a relatively early phase of their life. Most of these sub-

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jects are not diagnosed because no cytopenia is found despite clonal hematopoiesis. Later, when these patients are older and therefore develop ICUS (or renal anemia), frank anemia occurs and thus an overt MDS is diagnosed, following the equation: IDUS╛+╛ICUS╛=╛MDS (Fig.€4.1) [26]. It seems clear that exactly these patients are those with low risk MDS who respond to exogenous EPO for a certain time period.

Important Differential Diagnoses in Patients   with Suspected MDS The diagnosis MDS is based on both MDS-related (positive defining) criteria and exclusion of other disorders as primary reason for cytopenia or dysplasia [26, 27]. The “exclusion criterion” is of importance as also other hematopoietic and non-hematopoietic disorders can present with cytopenia and/or myelodysplasia. In patients with increased blast counts, differential diagnoses include acute myeloid leukemia and MDS/MPN. In patients with low blast counts, important differential diagnoses include aplastic anemia, toxic bone marrow damage, copper deficiency, vitamin B12 or folate deficiency, chronic autoimmune disorders, and chronic infectious diseases including HIV infection. In each case it is mandatory to exclude all these potential differential diagnoses by appropriate investigations [27] (Table€4.3). A bone marrow biopsy with conventional histology and immunohistochemistry is an essential step in the diagnostic work-up in patients with suspected MDS [13, 25, 27]. Likewise, the provisional diagnosis REAB-2 established from a bone marrow smear will change to AML when the pathologist has convincingly documented that the bone marrow contains multifocal disseminated blast cell aggregates or is fully packed with blast cells in a CD34-stained bone marrow section. Bone marrow histology is also crucial for the detection of certain (rare) variants of MDS, such as hypocellular (often secondary) MDS, MDS with fibrosis (MDS-F), and MDS associated with systemic mastocytosis (SM-MDS) [13]. A limited panel of antibodies has been shown to be sufficient for immunohistochemical studies of bone marrow cells in these patients [13, 27]. These include antibodies against progenitor (blast) cells (CD34 and CD117/KIT), antibodies against megakaryocytes (CD42 or CD61), and an antibody against tryptase with which mast cells and immature basophils can be labelled and counted [13]. In those patients in whom no MDS is found and clinical or laboratory feature point to another differential diagnosis such as a lymphoma or hairy cell leukemia, additional antibodies have to be applied.

Classification of MDS A first robust classification of MDS was proposed by the FAB working group [1]. This proposal is still in use since (a) it is independent of cytogenetic findings and (b) the diagnosis can be established rapidly. In addition, the FAB classification is

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Table 4.3↜渀 Laboratory studies required to exclude differential diagnoses in patients with suspected MDS Peripheral blood – Virus tests (CMV, HIV, HCV, EBV, others) – Auto-antibodies (ANA and others) – T cell receptor rearrangement (neutropenia suspected T/NK neoplasm) – Immunoglobulin rearrangement (suspected lymphoma/NHL) – Lymphoid surface markers by flow cytometry (DD lymphoma or NK neoplasm) – Copper level (in case of gastrointestinal disorder/symptoms) – Serum Tryptase – CFU-GM and BFU-E – TSH, T3, T4 (suspected thyroid disease) – Serum EPO levels and renal function parameters (anemia) – Hepatic function parameters, GOT, GPT, GGT, CHE – Vitamin B12 and folate concentration – LDH, bilirubin, haptoglobin, and PI-linked surface antigens (hemolysis) – Ferritin (in select patients) – Genetic testing for congential cytopenias and related syndromes (examples: GATA1, LPIN2, WAS, RSP19, FANC, BRCA2, DCK1, TERC, TERT, ELA-2, G-CSFR, others) – Somatic mutations: RAS, JAK2, others – Screen for various toxins depending on clinical symptoms and test results Bone marrow – Histology, cytochemistry, and immunohistochemistry – Standard stains on bone marrow smears including MGG and iron stain – Metachromatic stain (toluidine blue or other) – Cytogenetics and if required: FISH – Flow cytometry to detect aberrant immunophenotypes – Mutation analysis: RAS, KIT, JAK2, AML-related fusion genes, others Other parameters and tests – Ultrasound of liver and spleen – Analysis of family members (suspected hereditary disease) – Screen for malformations (suspected congenital cytopenia/syndrome) – Urinary analyses (creatinine clearance) MDS myelodysplastic syndrome(s), CMV cytomegaly virus, HIV human immunodeficiency syndrome virus, HCV hepatitis C virus, EBV Epstein barr virus, ANA anti-nuclear antibodies, NK natural killer cell, NHL Non Hodgkin’s Lymphoma, DD differential diagnosis, EPO erythropoietin, CHE cholinesterase, LDH lactate dehydrogenase, AML acute myeloid leukemia

of prognostic significance. However, the FAB classification has several limitations and does not include the karyotype or other key prognostic parameters. In 2001 the WHO classification has been established [4] and an update was presented in 2008 [5]. In this classification, a cytogenetically distinct variant, the 5q- syndrome, has been included [4, 5]. Today, it is standard to classify all MDS patients according to the WHO proposal. Sometimes, the FAB diagnosis is used as a provisional diagnosis, and as soon as all WHO-parameters have been recorded, the WHO classification is applied. This is of importance, as cytogenetic and molecular markers may reveal AML in suspected high risk MDS (e.g. when an AML-releated translocation

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is found). Therefore, from an academic point of view it must be regarded standard that in all patients with MDS (at least those with excess of blasts), all AML-related cytogenetic and molecular parameters proposed by the WHO, are applied.

Prognostic Scoring Systems and Additional Risk Factors In previous years, a number of different scoring systems have been proposed by various working groups [2, 23]. These scoring systems include various prognostic factors, some of which are not included in standard scores, such as the lactate dehydrogenase (LDH) level or age. In 1997, the International Prognostic Scoring System (IPSS) was published [10]. This score includes blast counts, cytogenetic abnormalities, and the number of cytopenias, and can still be regarded as gold standard for risk assessment in MDS. It should be pointed out that the IPSS is a superior score for predicting the probability of AML-free survival, whereas several survival-related prognostic parameters have not been included in this score [10]. Another disadvantage of the IPSS is that patients without available karyotype cannot be included. Moreover, the IPSS was established in the pre-WHO era. More recently, a WHO-based scoring system, the WPSS has been established [15]. This score includes transfusion dependence as an important prognostic parameter and may also be applicable in the follow up. Neither the IPSS nor the WPSS included patient-related risk denominators such as age or comorbidity [10, 15]. However, exactly these variables may be key factors predicting survival in elderly patients with MDS [19, 21]. More importantly, such patient-related factors may lead to the conclusion the patient is a candidate for intensive or targeted drug therapy or—on the other hand—may not be eligible. Therefore, we believe that new scores including major patient-related variables should be established, with the aim to better predict overall survival in these patients. Another aim is to improve AML-prediction and related scores by introducing refined cytogenetic analysis [11] and additional disease-related parameters such as the LDH.

 redictive Value of Therapy-Related Scores and Their Use P in Treatment Algorithms Today, specific and symptomatic therapy is available for most patients with MDS. In fact, there are several different (more or less) specific drugs and treatment approaches that can be considered in these patients, such as EPO, lenalidomide, demethylating agents (decitabine and azacytidine), polychemotherapy, ATG+CSA, and stem cell transplantation [2, 27]. For most therapeutic agents, factors predicting responses in patients with MDS have been defined (Table€4.4), which is of great importance as MDS is a heterogenous disease and only (sub)groups of patients are responding to certain drugs [2, 6, 27]. For most of these drugs, one or more prognos-

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Table 4.4↜渀 Factors predicting responses to certain therapies in patient with MDS Drug Factors predicting response Factors making a response less likely EPOâ•›>>â•›100€U/l EPO EPOâ•›<â•›100€U/l Low transfusion frequency Frequent transfusions Low risk MDS High risk MDS, high blast count ATGâ•›+â•›CSA Young patient Old or/and unfit patient HLA DR15 Complex karyotype PNH clone Low risk MDS Lenalidomide 5q- anomaly Complex karyotype (+/− 5q-) 5q- syndrome Increase in blasts Low risk MDS High risk MDS Demethylating agents Complex karyotype, -7 Rapid progression to AML Low risk MDS High risk MDS High-Dose-CT and/ AML-like disease High risk MDS or SCT Young fit patient Higher age, comorbidity Low/normal ferritin level Higher ferritin level CR after induction No CR after induction MDS myelodysplastic syndrome(s), EPO erythropoietin, ATG anti-thymocyte globulin, CSA cyclosporin-A, PNH paroxysmal nocturnal hemoglobinuria, AML acute myeloid leukemia, CT chemotherapy, SCT stem cell transplantation, CR complete remission

tic parameters predicting responses have been identified. Examples are the isolated 5q- anomaly that is highly predictive for responses against lenalidomide, provided that no other cytogenetic abnormalities and no increase in blast cells are found [27]. For other drugs, multi-factor score systems have been developed. These scoring systems are helpful for patient selection and for selection of most effective drugs. Examples are the Nordic-score (EPO-score) that is helpful in patient selection for EPO-therapy, or the transplantation scores that can also be applied to patients with AML. Table€4.4 provides a summary of factors and scores that are helpful for patient selection and drug selection and are thus regarded standard and an essential component in treatment algorithms.

Concluding Remarks and Future Perspectives Although parameters, assays, and score systems have improved markedly over the past two decades, appropriate diagnosis and optimal prognostication of MDS remains a challenge in practice. A recommended approach is to proceed in a step wise fashion: In a first step, the diagnosis MDS is confirmed by minimal diagnostic criteria. In a second step, the FAB and WHO classification is applied to define the disease variant. Then, the IPSS or WPSS are applied for prognostication. If this is not possible, alternative prognostic markers and scores should be employed. Finally, therapy-specific scores including the EPO-score or transplant-score are applied in

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order to learn what treatment options can be recommended in the individual patient. For patients with ICUS and IDUS, the general recommendation is to manage the patient in the same way as patients who have low risk MDS. These recommendations should facilitate the management and may improve the clinical outcomes in MDS. Acknowlegments╇ Supported by Research Grant “Myelodysplastic Syndromes” of the Medical University of Vienna.

References ╇ 1. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et€al (1982) Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51:189–199 ╇ 2. Bennett JM, Komrokji RS (2005) The myelodysplastic syndromes: diagnosis, molecular biology and risk assessment. Hematology 10:258–269 ╇ 3. Berer A, Jäger E, Sagaster V, Streubel B, Wimazal F, Sperr WR et€ al (2003) Circulating myeloid colony-forming cells predict survival in myelodysplastic syndromes. Ann Hematol 82:271–277 ╇ 4. Brunning RD, Bennett JM, Flandrin G, Matutes E, Head D, Vardiman JW et€al (2001) Myelodysplastic syndromes. In: Jaffe ES, Harris NL, Stein H, Vardiman JW (eds) World Health Organization classification of tumours. Pathology & genetics. Tumours of haematopoietic and lymphoid tissues, vol€1. IARC Press, Lyon, pp€61–73 ╇ 5. Brunning RD, Orazi A, Germing U, Le Beau MM, Porwit A, Baumann I et€al (2008) Myelodysplastic syndromes/neoplasms. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (eds) World Health Organization classification of tumours. Pathology & genetics. Tumours of haematopoietic and lymphoid tissues. IARC Press, Lyon, pp€88–107 ╇ 6. Fenaux P (2001) Chromosome and molecular abnormalities in myelodysplastic syndromes. Int J Hematol 73:429–437 ╇ 7. Ferrucci L, Guralnik JM, Bandinelli S, Semba RD, Lauretani F, Corsi A et€al (2007) Unexplained anaemia in older persons is characterised by low erythropoietin and low levels of pro-inflammatory markers. Br J Haematol 136:849–855 ╇ 8. Geissler K, Hinterberger W, Jäger U, Bettelheim P, Neumann E, Haas O et€al (1988) Deficiency of pluripotent hemopoietic progenitor cells in myelodysplastic syndromes. Blut 57:45–49 ╇ 9. Germing U, Aul C, Niemeyer CM, Haas R, Bennett JM (2008) Epidemiology, classification and prognosis of adults and children with myelodysplastic syndromes. Ann Hematol 87:691–699 10. Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G et€al (1997) International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 89:2079–2088 11. Haase D, Germing U, Schanz J, Pfeilstöcker M, Nösslinger T, Hildebrandt B et€ al (2007) New insights into the prognostic impact of the karyotype in MDS and correlation with subtypes: evidence from a core dataset of 2124 patients. Blood 110:4385–4395 12. Hofmann WK, de Vos S, Komor M, Hoelzer D, Wachsman W, Koeffler HP (2002) Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow. Blood 100:3553–3560 13. Horny HP, Sotlar K, Valent P (2007) Diagnostic value of histology and immunohistochemistry in myelodysplastic syndromes. Leuk Res 31:1609–1616 14. Loken MR, van de Loosdrecht A, Ogata K, Orfao A, Wells DA (2008) Flow cytometry in myelodysplastic syndromes: report from a working conference. Leuk Res 32:5–17

4â•… Diagnostic Criteria and Classification of Myelodysplastic Syndromes

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15. Malcovati L, Germing U, Kuendgen A, Della Porta MG, Pascutto C, Invernizzi R et€al (2007) Time-dependent prognostic scoring system for predicting survival and leukemic evolution in myelodysplastic syndromes. J Clin Oncol 25:3503–3510 16. Mufti GJ, Bennett JM, Goasguen J, Bain BJ, Baumann I, Brunning R et€al (2008) International working group on morphology of myelodysplastic syndrome. Diagnosis and classification of myelodysplastic syndrome: International Working Group on Morphology of myelodysplastic syndrome (IWGM-MDS) consensus proposals for the definition and enumeration of myeloblasts and ring sideroblasts. Haematologica 93:1712–1717 17. Nimer SD (2008) Myelodysplastic syndromes. Blood 111:4841–4851 18. Nolte F, Hofmann WK (2008) Myelodysplastic syndromes: molecular pathogenesis and genomic changes. Ann Hematol 87:777–795 19. Nösslinger T, Tüchler H, Germing U, Sperr WR, Krieger O, Haase D et€al (2010) Prognostic impact of age and gender in 897 untreated patients with primary myelodysplastic syndromes. Ann Oncol 21:120–125 20. Pellagatti A, Esoof N, Watkins F, Langford CF, Vetrie D, Campbell LJ et€ al (2004) Gene expression profiling in the myelodysplastic syndromes using cDNA microarray technology. Br J Haematol 125:576–583 21. Sperr WR, Wimazal F, Kundi M, Baumgartner C, Nösslinger T, Makrai A et€al (2010) Comorbidity as prognostic variable in MDS: comparative evaluation of the HCT-CI and CCI in a core dataset of 419 patients of the Austrian MDS Study Group. Ann Oncol 21:114–119 22. Tennant GB, Jacobs A, Bailey-Wood R (1986) Peripheral blood granulocyte-macrophage progenitors in patients with the myelodysplastic syndromes. Exp Hematol 14:1063–1068 23. Valent P (2008) Low erythropoietin production as non-oncogenic co-factor contributing to disease-manifestation in low-risk MDS: a hypothesis supported by unique case reports. Leuk Res 32:1333–1337 24. Valent P, Fonatsch C, Stindl R, Schwarzinger I, Haas OA, Sperr WR et€al (2004) Normal bone marrow function over 6 years in a patient with dysplastic hematopoiesis and a complex karyotype. Leuk Res 28:651–655 25. Valent P, Hofmann WK, Büsche G, Sotlar K, Horny HP, Haase D, Haferlach T, Kern W, Bettelheim P, Baumgartner C, Sperr WR, Nösslinger T, Wimazal F, Giagounidis AA, Lübbert M, Krieger O, Kolb HJ, Stauder R, Pfeilstöcker M, Gattermann N, Fonatsch C, Aul C, Germing U (2009) Meeting report: Vienna 2008 workshop of the German-Austrian working group for studying prognostic factors in myelodysplastic syndromes. Ann Hematol 88:607–611 26. Valent P, Horny HP (2009) Minimal diagnostic criteria for myelodysplastic syndromes and separation from ICUS and IDUS: update and open questions. Eur J Clin Invest 39:548–553 27. Valent P, Horny H-P, Bennett JM, Fonatsch C, Germing U, Greenberg P et€al (2007) Definitions and standards in the diagnosis and treatment of the myelodysplastic syndromes: consensus statements and report from a working conference. Leuk Res 31:727–736 28. Valent P, Wieser R (2009) Update on genetic and molecular markers associated with myelodysplastic syndromes. Leuk Lymphoma 4:1–8 29. van de Loosdrecht AA, Alhan C, Béné MC, Della Porta MG, Dräger AM, Feuillard J et€al (2009) Standardization of flow cytometry in myelodysplastic syndromes: report from the first ELNet working conference on flow cytometry in MDS (Amsterdam 2008). Haematologica 94:1124–1134 30. Wimazal F, Fonatsch C, Thalhammer R, Schwarzinger I, Müllauer L, Sperr WR et€al (2007) Idiopathic cytopenia of undetermined significance (ICUS) versus low risk MDS: the diagnostic interface. Leuk Res 31:1461–1468

Chapter 5

Cytogenetics of MDS Detlef Haase, Christina Ganster, Christian Steidl, Katayoon Shirneshan, Friederike Braulke and Julie Schanz

Introduction Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem [1, 2] and stroma cell disorders [3]. Chromosomal anomalies can be identified in 40–60% of de novo cases and up to 90% of secondary MDS. The detection sensitivity of cytogenetic changes in MDS is critically dependent on optimized culture conditions [4, 5]. The karyotype has important diagnostic and prognostic impact and gains more and more influence on therapeutic decisions. The examination of bone marrow cells by cytomorphology and cytogenetics still defines the gold standard for initial diagnosis and documentation of treatment response in MDS patients. The cytogenetic heterogeneity in MDS is profound. In a recent study, we identified clonal karyotype changes in 1,080 (52%) out of 2,072 patients with MDS with a total number of 2,370 clonal chromosomal abnormalities. Grouping and subdividing similar chromosomal abnormalities identified 684 different cytogenetic categories [6]. In future studies, however, it still has to be determined whether distinct cytogenetic abnormalities lead to similar phenotypes and biological features and to which extent additional abnormalities influence prognosis. Therefore it is critical to increase reliability and precision of future prognostic systems by defining the prognostic impact of every given karyotype.

Cytogenetic Features of MDS MDS show a characteristic genetic profile with predominance of unbalanced abnormalities. Most frequently a loss of genetic material with deletions and monosomies can be observed. The most frequent abnormalities leading to a loss of chromosomal D. Haase () Abteilung Hämatologie/Onkologie, Georg-August-Universität Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_5, ©Â€Springer Science+Business Media B.V. 2011

55

56 Table 5.1↜渀 Frequencies of unbalanced chromosome abnormalities in MDS Isolated, n (%)b With one additional Abnormality % Total abnormality, n (%)b incidencea Monosomies/deletions del(5q) 15.1 146 (46.8) 52 (16.7) -7/7q11.1 86 (37.4) 31 (13.5) -18/18q3.8 3 (3.8) 2 (2.6) del(20q) 3.6 36 (48.7) 10 (13.5) -5 3.3 1 (1.4) 4 (5.8) -Y 2.8 41 (70.7) 5 (8.6) -17/17p2.0 1 (2.4) 1 (2.4) -13/13q1.9 4 (12.5) 6 (15) -21 1.6 3 (9.1) 4 (12.1) -12 1.3 0 2 (7.7) del(12p) 1.2 7 (28) 6 (24) 11q1.1 8 (34.8) 4 (17.4) del(9q) 1.1 8 (34.8) 3 (13) -20 1.1 0 0 Partial/complete trisomies +8 8.4 81 (46.8) 37 (21.4) +21 2.2 5 (11.1) 18 (40) +1/1q 1.8 4 (10.8) 6 (16.2) +11 1.4 6 (21.4) 4 (14.3) +19 0.9 4 (22.2) 3 (16.7) +13 0.8 4 (25) 4 (25) +14 0.8 3(18.8) 4 (25) +10 0.6 1 (8.3) 0 +9 0.5 2 (20) 1 (10) a b

D. Haase et al.

As part of complex abnormalities, n (%)b 114 (36.5) 113 (49.1) 73 (93.6) 28 (37.8) 64 (92.8) 12 (20.7) 40 (95.2) 29 (72.5) 26 (78.8) 24 (92.3) 12 (48) 11 (47.8) 12 (52.2) 22 (100) 55 (31.8) 22 (48.9) 28 (75.7) 18 (64.3) 11 (61.1) 8 (50) 9 (56.2) 11 (91.7) 7 (70)

Related to all cases with MDS (n╛=╛2,072), according to Haase et€al. [6] Of cases with the respective abnormality

material are -5/5q-, -7/7q-, -18/18q-, 20q-, and -Y (Table€5.1) [6]. Gain of genetic material by total or partial trisomies is less frequent, but typical karyotypic changes involve chromosomes 1, 8, 11 and 21 (Table€5.1). Loss or gain of genetic material can also be the result of unbalanced translocations which are most frequent in MDS with multiple abnormalities. The higher frequency of loss of genetic material in comparison to gains gives rise to the hypothesis that a major molecular mechanism in MDS is the loss or inactivation of tumor suppressor genes while an oncogeneactivation seems to be less relevant in myelodysplasia. Balanced structural abnormalities like translocations and inversions are rare in MDS. Due to the pronounced genetic heterogeneity research has focused only on the most frequent abnormalities. The molecular background as well as the prognostic relevance of rare abnormalities still remains obscure in most cases and can only be clarified by large multicentric studies [6–9]. Another problem to delineate the prognostic impact of chromosomal changes is complicated by the fact that chromosome abnormalities can occur in three different settings: (1) as isolated abnormalities, (2) together with one addi-

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tional change and (3) as part of complex abnormalities with at least two additional cytogenetic alterations. Table€5.1 shows the incidence of the most frequent abnormalities and the frequency of accompanying alterations occuring in our GermanAustrian patient cohort. Further details on cytogenetic prognosis are given below characterizing the most common cytogenetic subtypes.

Distinct Cytogenetic Subgroups 5q-Deletions Chromosome 5q-deletions are the most frequent cytogenetic changes in MDS. They occur in up to 30% of abnormal cases (Table€5.1) [6–9]. This abnormality can be observed in low risk MDS as well as in high risk MDS. The deletions can have variable sizes with breakpoints between 5q12 and 5q34. The common deleted region (CDR) contains the chromosome band 5q31 [10], but two different non-overlapping deleted regions in 5q31 to 5q32 have been assumed. The first more centromeric one is possibly associated with bad prognosis, complex abnormalities, and high-risk-, as well as therapy-related MDS and rapid transformation to AML. The latter area is supposed to be related to the good risk 5q-syndrome [11]. Knowledge about the molecular background of this abnormality is increasing: the CDR could be narrowed to 1.5€Mb containing 40 genes [11]. One of them, a ribosomal subunit protein (RPS14) seems to be a candidate gene [12]. However, not haploinsufficiency of one single gene but the combined haploinsufficiency of several genes in 5q31 might be the decisive pathogenetic mechanism [13]. A true 5q-syndrome is cytogenetically characterized by an isolated deletion of chromosome 5q but can be observed in only 10% of patients with MDS. The clinical course is mild and long-lasting with a very low risk for leukemic transformation. Partial or total monosomy 5 can occur with equal incidences in de novo as well as secondary MDS. However, in a recent meta-analysis monosomy 5 was more frequent in t-MDS while isolated del(5q) was significantly more fequent in de novo MDS [14] (Table€ 5.2). Monosomy 5 nearly exclusively occurs as an element of chomplex aberrations. Chromosome 5 material involed in very complex unbalanced structural rearrangements might be misinterpreted as monosomy 5 [15]. Monosomy 7 Monosomy 7 is the second most frequent distinct chromosome abnormality in MDS occurring in 25% of abnormal cases (Table€5.1) [6, 7, 9]. It is more frequent in advanced MDS and manifests itself as total or partial monosomy. In the latter variable deletions of parts of the long arm were described. In the German-Austrian dataset 36% of cases with monosomy 7 were isolated, 14% displayed one additional abnormality and 50% occurred as part of complex abnormalities [6]. Several distinct regions of common deletions have been identified: 7q22, 7q31-32, and 7q36. A re-

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Table 5.2↜渀 Incidences (%) of chromosome abnormalities in primary vs. therapy-associated (t)-MDS related to abnormal cases. (According to Mauritzson et€al. [14]) Abnormality MDS t-MDS p-value RT alone Aâ•›±â•›RT A+Tâ•›±â•›RT p-value de novo (nâ•›=â•›252) (nâ•›=â•›44) (nâ•›=â•›115) (nâ•›=â•›73) (nâ•›=â•›1,377) -5 7 16 19 8 n.s. <0.001 18 5q25 25 n.s. 54 22 14 <0.001 Sole 5q13 6 0.001 18 4 1 0.02 -7 12 35 47 27 0.003 <0.001 20 Sole -7 6 12 20 8 0.008 <0.001 0 7q5 10 0.005 14 8 12 n.s. Der(1;7) 2 2 n.s. 0 4 3 n.s. Loss of 5 and/or 7 +8 21 10 9 7 n.s. <0.001 14 Sole +8 13 2 2 0 n.s. <0.001 5 Der(12p) 6 6 n.s. 7 7 4 n.s. -17 4 6 n.s 5 5 6 n.s. Der(17p) 4 7 0.02 5 9 6 n.s. 20q6 5 n.s. 5 5 4 n.s. Sole 20q4 1 0.01 0 2 0 n.s. Complex 24 39 36 37 n.s. <0.001 48 Balanced 14 17 n.s. 23 16 18 n.s. Unbalanced 95 96 n.s. 98 99 92 n.s. n.s.â•›=â•›not significant, RTâ•›=â•›radio therapy, Aâ•›=â•›alkylators, Tâ•›=â•›DNA topoisomerase II inhibitors

gional genetic instability of the long arm of chromosome 7 has been described [16, 17]. It can be assumed that this region harbours a cluster of genes with tumor suppressive features and that a deletion of any part of it has very similar biological consequences. In contrast to some hypotheses in the 5q-deletions as yet no prognostic differences have been elaborated between different regions of deletions [18]. Investigations of the molecular background of monosomy 7-MDS are currently ongoing. Overexpression of HOXA9, PRAME, BMI1, PLAB and the DNA-repair gene BRCA2 and parallel down-regulation of the tumor suppressor genes p21, GATA2, and MAP were observed [19]. Recently, EZH2 was suggested to be a canditate tumor suppressor gene involved in monosmy 7/7q-MDS [20]. In pediatric MDS monosomy 7 is the most frequent cytogenetic change. The prognosis in these cases is poor [21–23]. In a substantial portion of patients with -7/del(7q) a mutagen exposition was documented [14, 24–26]. There is increasing evidence that also secondary MDS and AML following immunosuppressive therapy are associated with monosomy 7 [14, 27, 28]. Trisomy 8 Trisomy 8 can be observed in 12–21% of abnormal cases in MDS (Table€5.1) [6, 7, 14]. It seems to be more frequent (17%) in MDS with transformation to AML

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[6]. In 47% of cases trisomy 8 occurred as a sole change, it was combined with one abnormality in 21% and was part of complex anomalies in 32%. The most frequent accompanying abnormality is a deletion of 5q. It was hypothesized that trisomy 8 is not the primary event because in AML, comparison of different karyotypes with trisomy 8 revealed no common characteristic profile of overexpressed genes [29]. In MDS, the size of cell clones with trisomy 8 tends to fluctuate during the course of the disease but is not always correlating with the blast counts or leukemic transformation [30]. The hypothesis of a secondary nature of trisomy 8 was supported by further studies [1, 2]. In cases with a constitutional trisomy 8 mosaicism the trisomy might be regarded as the first mutation in a multistep leukemogenesis process [30]. It was recently demonstrated that trisomy 8 clones show over-expression of the potential autoantigenes Cyclin D1 and Wilms tumor protein (↜WT-1) [19, 31]. CD34+ cells from patients with trisomy 8 furthermore display a pronounced expression of apoptotic markers like caspase-3 and FAS [32]. This phenomenon can be explained by an up-regulation of the antiapoptotic proteins survivin, CMYC and CD1 [33]. Chromosome 17 Abnormalities Abnormalities of chromosome 17 mostly occur as loss of material of the short arm and are observed in <5% of cases with clonal changes in MDS (Table€5.1) [6, 9, 34]. They are associated with advanced disease and an elevated blast count and are more frequent in therapy-associated MDS as compared to de novo MDS (Table€ 5.2) [14]. The great majority of these changes are unbalanced, leading to the deletion of varying parts of the short arm of chromosome 17 with a common loss of the band 17p13 harbouring the tumor suppressor gene TP53. In our German-Austrian database 77% of chromosome 17 changes were part of complex abnormalities, only 12% (n╛=╛12) occurred as isolated anomalies of which isochromosome i(17)(q10) was the most frequent one [6]. Since isolated anomalies of chromosome 17 are rare little is known about their prognostic relevance [9, 35, 36]. TP53 point mutations were associated with poor prognosis and bad response to chemotherapy in AML and MDS [34, 37, 38]. They display, as well as chromosome 17-anomalies in general, a strong association with complex abnormalities [6, 34, 39, 40]. An association with microsatellite instability and thus a replication error phenotype was described [39, 41]. Hence it is likely that TP53 mutations by themselves are causally related to genetic instability and accumulation of cytogenetic and molecular defects in MDS. 20q-Deletions Deletions of 20q including monosomy 20 occurred in 7% of abnormal MDS cases of our dataset (Table€5.1). They were equally distributed in all FAB-subtypes but were very rare in CMML (<1%). Deletions of 20q occurred as sole changes in 49%, with

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one additional anomaly in 13% and as part of complex alterations in 38%. Monosomy 20 was exclusively (22 of 22 cases) observed in complex abnormal karyotypes. Thus it can be assumed that as in monosomy 5 most if not all of these cases share similar features displaying complex, unbalanced cryptic abnormalities [6]. Recently, a CDR with the size of 10.4€ Mb and two common retained regions (CRR) were identified. The deletion may result in the loss of one or several tumor suppressor genes [42]. However, the target genes still remain unknown although several groups have reported candidate genes in the deleted region [43–45]. Recent activities focus on L3MBTL3 that has putative tumor suppressive functions and is probably involved in chromatin modeling and mitotic progression [46]. An isochromosome of the long arm of chromosome 20 with loss of interstitial material [ider(20q)] is a variant of the deletion of chromosome 20q and is a rare abnormality in MDS. Amplification of the genes HCK, TNFRSF6B and DIDO1 included in the CRR associated with loss of tumor suppressor genes in deleted regions could explain tumor progression and possibly the less favourable prognosis of ider(20q) compared with del(20q) [47].

Sex Chromosomes Abnomalities In MDS loss of the Y chromosome can be observed in 5% of abnormal cases (Table€5.1) [6, 7] and is seen with higher frequency in low risk MDS. It is still subject of debate to which extent the loss of the Y chromosome in MDS is an age-related or disease-associated, clonal phenomenon. On the one hand there are data suggesting that in elderly men -Y is not indicative of malignancy [48]. On the other hand several groups found hints that at least under certain conditions loss of the Y chromosome could also be a clonal marker in MDS [49, 50]. It was suggested that even in older males, loss of the Y chromosome in >75% of metaphase cells probably represents a disease-associated clonal population [51]. Furthermore, three different types of karyotype evolution observed in our laboratory as well as by others, clearly support the clonal nature of loss of the Y chromosome: (1) Emergence of cells with loss of the Y chromosome during disease progression in cases with an initially normal karyotype, (2) Emergence of -Y as secondary abnormality and (3) Emergence of secondary abnormalities in cells with loss of the Y chromosome in cases with an initial mosaic of normal and -Y cells (↜own unpublished observation). It thus can be concluded that at least under certain conditions loss of the Y chromosome is a clonal abnormality with relatively high frequency in MDS but also occurring in other hematologic malignancies. Candidate genes are not known as yet. Non-constitutional loss of one X chromosome as the sole cytogenetic change is a rare but recurrent abnormality observed in MDS. In the German-Austrian dataset isolated monosomy X occurred in only 4 of 1,080 patients with abnormalities and four further patients had monosomy X in combination with one additional abnormality [6]. The molecular background of this abnormality is not known.

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Balanced Abnormalities Balanced abnormalities are rare in MDS. The most frequent balanced abnormalities belong to a group of changes affecting the chromosome bands 3q21 (↜RPN1) and 3q26 (↜EVI1) such as inv(3)(q21q26), translocations t(1;3)(p36;q21) (fusion of MEL1/RPN1), t(3;3)(q21;q26), and t(3;21)(q26;q22) (↜EVI1/RUNX1). Their estimated incidence in MDS is <5% up to 10% of abnormal cases [18, 52, 53]. Heterogeneous translocations involving the MLL-gene localized on chromosome band 11q23 can be observed in <2% of abnormal cases in MDS, often in therapyassociated cases. A translocation t(5;12)(q33;p12), leading to the fusion of ETV6 and PDGFRB, can be observed in <1% of patients with CMML [54]. Another rare balanced translocation involves chromosome bands 3q25.1 (↜MLF1) and 5q34 (↜NPM1), resulting in a fusion protein, NPM-MLF1 [55, 56]. Translocations t(8;21) (q22;q212) with a fusion of RUNX1T1 on 8q22 and RUNX1 on 21q22, one of the most frequent clonal chromosome abnormalities in AML, have been described very rarely in MDS and could be misdiagnosed AML [57–59]. Pericentric inversion of chromosome 16 associated with AML M4Eo has been observed as well in rare cases of mostly advanced MDS [57, 60]. Chromosomal Manifestations of Gene Amplifications Gene amplifications in hematologic diseases can occur as extrachromosomal DNA (so-called “single or double minutes”) as well as so-called “homogeneously staining regions” attached to or inserted into chromosomes. In AML these rare findings have an incidence of <1% while there is no report about the exact frequency in unselected MDS cohorts. Chromosomal gene amplifications may occur as the sole karyotypic change or in association with chromosome abnormalities like 5q-deletions, trisomy 4 or trisomy 8 [61–63] but most frequently they are associated with complex changes. FISH analysis allows identification of genes within amplified regions. The genes most frequently amplified in MDS are MLL in 11q23, RUNX1 in 21q22 and MYC in 8q24 [64–66]. Such amplifications show an association with bad prognosis and therapy-related MDS/AML (Figs.€5.1, 5.2, 5.3) Independent Clones Independent (unrelated) clones showing completely different cytogenetic abnormalities occurring in one and the same patient are rare in hematologic malignancies, with a frequency of 0.9–7.2% of cases [67–73]. The frequency of independent clones was significantly higher in t-MDS (6.2%) as compared to de novo MDS (3.0%; pâ•›<â•›0.01) [69]. The incidence of distinct abnormalities observed in independent clones was comparable to the common MDS cohort, except of an overrepresentation of trisomy 8 [6, 7, 9]. The most common pattern of abnormalities in independent clones was the combination of del(5q) and trisomy 8 [69]. Complex

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Fig. 5.1↜渀 MLL-amplification (four fusion signals) in interphase cells in a case of t-MDS after breast cancer with complex abnormalities

Fig. 5.2↜渀 Multicolour FISH metaphase of the same case (Fig.€5.1) with complex abnormalities and a large marker chromosome containing three copies of MLL

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Fig. 5.3↜渀 Giemsa-banded karyogram of the same metaphase

abnormalities in at least one of the clones are infrequent, occurring in 4.5% of independent clones (↜own data, not published). The majority of patients (88%) display only two different clones. Interestingly, 89% of cases show unbalanced abnormalities exclusively [69]. Unfortunately, the prognostic impact of independent clones in MDS remains unclear as yet. Complex Chromosome Abnormalities According to ISCN-criteria complex chromomosome abnormalities are defined by the parallel existance of at least three independent abnormalities within one cell clone [74]. Complex abnormalities can be observed in 15% of all MDS patients (30% of all abnormal cases) [6, 7, 9]. They may represent the final step of a multistep process with sequential accumulation of abnormalities, called karyotype evolution. In most of these cases the aberration numbers exceed the threshold of three by far and cell-to-cell variations occur. It is conceivable that in many cases the process leading to complex abnormalities and genetic instability must proceed rapidly and likely involves defective DNA-repair and cell cycle control comparable to the situation in DNA-repair deficiency syndromes. Recent results of gene expression analyses in patients with MDS and complex chromosome abnormalities support these assumptions [40]. In a substantial portion of patients with complex abnormalities

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exposition to therapeutically applied mutagens can be derived from the medical history [25, 75]. There is a strong association of complex alterations with unbalanced structural abnormalities affecting most frequently chromosomes 5q and 7q. Genomic gains were observed for chromosome 8/8q, 11q and 21q [76]. Mutations of TP53 have been observed in up to 90% of patients with AML and complex abnormalities [40]. Recently, array comparative genome hybridization (aCGH)-analyses facilitated to unravel complex karyotype changes [77]. Karyotype Evolution In the vast majority of cytogenetic MDS studies investigations are focused on one distinct observation time point only, most often that of initial diagnosis. As MDS is a very dynamic disease at the morphologic, clinical and genetic level, it can be reasonably hypothesized that karyotype evolution (KE) could be indicative of disease progression, hence influencing therapy decision. The model of multistep pathogenesis in MDS [78–80] is based on the findings that MDS is a clonal stem cell disease [2, 6, 78, 79] and that karyotype abnormalities are only the second step following acquisition of clonality [81]. It was suggested that there are primary abnormalities leading to specific gene rearrangements and secondary changes that result in largescale genomic imbalances and are important in tumor progression [82]. As yet it is uncertain whether a distinct sequence or the amount of genetic changes accounts for disease progression [82]. On the cytogenetic level a stepwise progression manifests itself as an evolution of the karyotype, characterized by (a) the acquisition of a clonal abnormality in cases with an initially normal karyotype, (b) the expansion of a cell clone with a given abnormality or (c) the occurrence of secondary abnormalities in cases with a distinct primary karyotypic change. Johansson and Mitelman analysed the Catalog of Chromosome Aberrations in Cancer [71] and identified the most frequent secondary abnormalities related to seven primary abnormalities (Table€5.3) [82–84]. Karyotype evolution is also studied by repeated analyses of the karyotype during the course of the disease (Table€5.4). The earliest systematic study of KE in MDS patients was conducted by Tricot in 1985, defining four distinct reproducible patterns of disease evolution (Fig.€5.4) [85]. Considering the results of seven studies the incidence of KE in MDS ranges between 12% and 47% of patients examined sequentially (Table€5.4) [84–91]. ObTable 5.3↜渀 Primary and secondary abnormalities in MDS. (According to Johansson et al., Mallo et al., and Steidl et€al. [82–84])

Primary abnormality der(1;7)(q10;p10) der(3q21/q26) del(5q) -7 +8 del(20q)

Secondary abnormalities +8 -7 -7,+8, del(12p), der(17p), +21, -18/18q-, -Y del(20q), +8, +21 del(5q), -7, +8, -17/der(17p), +22, -Y -7/del(7q), +8, +21

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Table 5.4↜渀 Abnormalities gained during transformation to AML Number of patients Gained abnormalities % Of patients with karyotype with karyotype evolution/entire cohort evolution 5q-, -7/7q-, +8, complex abnormalities 21% 121/577 Abnormalities of chromosomes 2, 4, 7, 8, 7 10, 11, 17, and 21 inv(3), +8, +9, +13, +21, del(11p) 39% 7/18 12% 15/126 +1, der(1;7), t(1;18), +2, del(2q), del(5q), -5, +6, -7, +8, del(9q), +der(11) t(11;21), del(12p), +13, +14, +15, del(17q), r(18), -20, del(20q), +21, del(21q), -21, -Y, + mars 27% 17/63 dup(1q), del(1q), del(1p), del(3q), -5, -6, -7, add(7q), add(7p), +6, +8, -9, del(9q), add(10q), +10, -11, +11,╛+╛12, +13, +14, der(17), inv(16), -16, -18, +19, del(20p), +21, + mars t(1;5), t(1;7;8),+5, +8, t(8;20), t(11q23), 33% 10/30 +14, +15, 17p+, -16, +21, +22, -X, -Y t(1;3), del(5q), -7, +8, 9q+, -17, -18, 20% 9/46 18q-, +21, -21, +22

References [84] [89] [90] [91]

[86]

[88] [85]

viously the incidence of KE is depending on the composition of the patient cohorts as well as on the duration of observation time. In all analyses the occurrence of KE was associated with a dismal prognosis and leukemic transformation. In a recent multicentric analysis of our own German-Austrian group KE was observed in 155/577 patients with MDS (27%). Additional anomalies occurred in bone marrow blasts

AML -7/7q-, complex

D

Sole 5qtrisomy 8 C

5q-, + complex B

sole 5q-

normal karyotype A t

Fig. 5.4↜渀 Patterns of evolution in MDS modified according to the proposal by Tricot et€al. [85]

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121 patients (21%) and in 34 patients (6%) the abnormal clone expanded. In the latter patient subgroup the most frequent abnormalities were del(5q) (nâ•›=â•›9) and trisomy 8 (nâ•›=â•›7). The most frequent abnormality types later acquiring additional changes were complex (nâ•›=â•›22) or double anomalies (nâ•›=â•›11). KE was observed in 70 patients (12%) with an initially normal karyotype. The most frequent secondary abnormalities were -7/7q- (nâ•›=â•›23), del(5q) (nâ•›=â•›11) and trisomy 8 (nâ•›=â•›11). Patients with initially normal karyotype and KE had a significantly shorter survival in comparison to those with a stable normal karyotype [84]. KE could also be observed in vitro in long term bone marrow cultures from MDS patients and it was suggested that the expansion of small latent subclones could contribute significantly to leukemic transformation [92–94]. Other genetic abnormalities occurring during MDS evolution and associated with worsening of prognosis and leukemic transformation are double minutes [62, 95], telomere shortening [96], N-RAS gene mutations [87, 90], FLT3 internal tandem duplications [97], KIT mutations [98] and loss of heterozygosity on chromosome arms 6q, 7p, 10p, 11q, 14q, and 20q [99]. Microsatellite instability, a marker of defective mismatch-repair, was rarely observed in MDS appearing mostly early during the course of the disease and was not associated with disease progression in most cases examined [99–102]. New therapeutic options influencing the natural course of the disease will complicate studying the spontaneous evolution of MDS in the future. However, novel therapies warrant a narrow-meshed genetic monitoring of disease progression to better define the best time point for therapeutic decision and intervention. Since the potential sequence of traumatizing bone marrow biopsies is limited due to ethical reasons alternative methods allowing for a complete surveillance of the development of MDS are urgently required. Our group recently demonstrated the value of comprehensive FISH-analyses of circulating CD34 cells for such purposes [103]. A multicentric prospective diagnostic trial is currently under way to examine whether this method is appropriate for an initial diagnostic screening, to trace KE and to quantitate therapy response in two to three month intervals. On strength of these preliminary data generated by this technique, we remain hopeful to significantly increase our knowledge about genetic evolution in MDS. Cytogenetics in Therapy-Associated MDS Differnces of chromosomal abnormalities in primary vs. therapy-associated (t)MDS are shown in Table€5.2. With 37–50%, the aberration rate is much lower in de novo MDS [7–9, 14, 104, 105] compared to 82–94% in t-MDS [14, 25, 106]. Complex abnormalities and unbalanced chromosome 5- and/or 7-alterations were found to be significantly increased in t-MDS, while sole 5q-, trisomy 8 and sole 20q- were underrepresented [14, 25, 106]. Furthermore an increased frequency of dicentric chromosomes was observed in therapy-related MDS and AML compared to de novo cases [107]. Amplifications of MLL and RUNX1 were identified as rare but recurrent cytogenetic findings in t-MDS [108, 109].

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FISH-Analyses in MDS Cytogenetics in MDS is increasingly not only based on chromosome banding analysis alone but also on the use of different fluorescence in situ hybridization (FISH)techniques. Several studies have shown the usefulness of molecular cytogenetic techniques, such as FISH with specific probes for centromeric regions or distinct chromosomal loci (Fig.€5.1) and methods for the delineation of the whole chromosomal complement like spectral karyotyping (SKY) or multicolor FISH (M-FISH) (Fig.€5.2) to detect and more accurately define abnormal karyotypes. These studies demonstrate that FISH analysis can provide additional useful information in a substantial portion of cases.

Value of FISH as a Supplement to Banding Analyses There are several diagnostic situations where FISH proved to be a worthwhile supplement to banding analyses. These are (1) detection of cryptic abnormalities in cases with a seemingly normal karyotype by banding, (2) search for additional cryptic abnormalities in cases with a known chromosome abnormality, (3) the detection of chromosomal abnormalities in cases with no metaphases or metaphases of only minor quality and (4) more detailed characterization of complex abnormalities.

FISH in cases with a normal banding karyotype Occasionally occurring cryptic chromosomal mutations may be undetectable by banding analyses because of a poor bone marrow sample or preparation, bias of metaphase analysis, “true” cryptic/submicroscopic mutations, or lack of dividing neoplastic cells. Approximately 50% of MDS patients have normal results by conventional cytogenetic analysis, thus it was examined by several groups whether FISH panel tests can be utilized to identify overlooked or cryptic cytogenetic abnormalities. FISH panel tests on MDS patients with normal karyotype detected clonal abnormalities in 16–18% of patients [110, 111]. These patients showed a worse prognosis in comparison to patients without cryptic chromosomal changes [110]. In some cases it was observed that distinct cell clones were traced in interphase cells by FISH but could not be detected by banding analyses relying on dividing cells [112, 113]. This might be explained by differences in the proliferative capacity of normal versus abnormal cells. Comparison of Metaphase Cytogenetics and Interphase FISH By comparing banding analyses and examination by FISH, different detection frequencies were reported: Using FISH analysis, a Chinese group confirmed 3/46 cases

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of total or partial monosomy 7 that were previously detected by banding analyses, but found 6/46 additional cases with -7 or del(7q) [114]. A Swedish group confirmed monosomy 7 in 10/123 cases previously karyotyped and found only 4/123 further cases with a cryptic monosomy 7 [115]. In another study in 3/30 cases (10%) FISH analysis detected trisomy 8 which was not found by banding analysis [116]. By banding analyses, an Italian group detected clonal changes in 13/40 (32.5%) of patients while FISH with a panel of probes traced abnormalities in 14/40 (35%). The combination of both techniques increased the detection rate up to 40% [117]. In an ECOG study, a cytogenetic abnormality was detected by metaphase analysis in 37.5% of 48 patients while the FISH panel detected clonal changes in 35.4%. The authors concluded that interphase FISH is nearly as sensitive as banding analyses of metaphases [118]. Different clone sizes in cells with a 5q-deletion in interphase versus metaphase could be related to a different rate of mitoses in cells carrying or not the 5q deletion pointing to a low proliferation and a high apoptotic rate of the abnormal cell clone. As some cryptic abnormalities are detected by FISH only, and other abnormalities are detected by chromosome banding only, both methods should be combined. The International Cytogenetic Working Group of the MDS Foundation recently has launched an international multicentric prognostic trial to evaluate interphase FISH in comparison with conventional cytogenetic studies to detect chromosome abnormalities in peripheral blood and bone marrow of patients with MDS [119]. Characterization of Complex Abnormalities The wide variety of complex chromosomal aberrations (CCA) in patients with MDS is often difficult to define by conventional banding cytogenetics alone. Molecular cytogenetic techniques such as multiplex FISH (M-FISH) or spectral karyotyping (SKY) allow the comprehensive evaluation of CCA. They allow analyzing the origin of marker chromosomes, revealing cryptic rearrangements, and determining recurrent breakpoints and the structure of derivative chromosomes (Fig.€5.2) [15, 76, 120–124].

FISH-Analysis of Circulating CD34+ cells from Peripheral Blood Usually, chromosomal anomalies are detected in bone marrow cells by cytogenetic analyses of metaphases using conventional banding techniques. Most of these chromosome abnormalities can be detected by FISH-techniques too. To study karyotype evolution and molecular cytogenetic response to therapy with unprecedented accuracy and frequency our group has launched a seminal study to test a novel method to perform long-term highly sequential cytogenetic analyses in MDS patients without exposing the patient to the often unacceptable strain of frequently repeated bone marrow biopsies. The investigations focus on the analysis of CD34+ cells which

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are known to circulate in increased numbers in the peripheral blood of patients with MDS [125]. In preliminary data we could demonstrate, that this method is sensitive, reliable, and representative for the clonal cell population found in the bone marrow [103].

FISH in MDS—Conclusions Conventional cytogenetics, FISH panel testing and M-FISH all have their own specific analytical limitations. Conventional cytogenetics often cannot unravel the specific anatomy of marker chromosomes involving multiple rearrangements, and cryptic mutations can be missed or misidentified. Furthermore, it is much dependent on the quality and yield of short-term bone marrow cultures. Locus specific FISH analyses are limited only to the specific chromosomal regions to which the respective probes bind. Thus, mutations involving any other chromosome or other regions on the same chromosome will be overlooked. A great advantage of FISH techniques is the much larger number of cells that can be examined with reasonable expenditure as compared to banding analysis. For a quantification of a clone size with a known abnormality FISH is superior to conventional cytogenetics. The strength of M-FISH is in identifying cryptic translocations and additions. However, the resolution of M-FISH is limited, and subtle translocation events as well as smaller deletions, insertions or inversions can be missed. FISH and banding analyses are complementary and a combined application of conventional cytogenetics and FISH techniques adapted to the needs in specific analytical situations and cases significantly improves the diagnostic accuracy, reliability and sensitivity.

Prognostic Meaning of the Karyotype Since one of the earliest prognostic graduations was introduced in 1985 [126], cytogenetics was proven as an independent prognostic factor in large-scaled, multivariate analyses (Table€5.5) [6–9, 91, 104, 105, 120, 126–129], finally resulting in the IPSS score, which actually defines the gold standard in risk assessment in MDS [7]. The IPSS includes bone marrow blast count, peripheral cytopenias and cytogenetics as major prognostic variables to define the risk for overall and AML-free survival. Due to the profound heterogeneity of cytogenetic findings in MDS however, there are still difficulties to classify distinct cytogenetic abnormalities. About 50% of all patients show a normal karyotype, which is associated with a good prognosis [6, 7, 9, 104, 105, 121, 129]. In about 20–25% of patients, one of the most frequent abnormalities (5q-, -7/7q-, +8, 20q-, -Y), associated with an established prognostic significance, occurs [6, 9]. At least 8–18% [6–9] show complex abnormalities at the time of first diagnosis. According to the IPSS cytogenetic classification 14% of patients present with rare abnormalities with an unknown prognostic denotation.

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Table 5.5↜渀 Incidence and prognostic subgrouping of karyotype abnormalities in MDS References N= Abnormal Favourable Intermediate Unfavourable (%) [126] 174 66 (38) Normal (NN) Complex [128, 135] 144 63 (44) 5q-, 20q+8 -7/7q[127] 169 74 (44) NN, 5q+8 -7/7q[129] 247 106 (43) NN Complex [104] 408 151 (37) NN, 5q-, -Y, -7/7q-, 20q- +8 Complex [105] 401 200 (50) +8 -7/7qComplex [91] 198 75 (38) NN, 5q12p-, +21 Complex, +8, 20q[7] 816 327 (40) NN, 5q-, 20q-, -Y +8, all others Complex, abnormal #7 [9] 968 500 (51) NN, 5q-, 20q-, -Y, 11q-, 3q, +8, +9, t11q, Complex, -7/q-, 12p17pi17q [6] 2,072 1,080 (52) NN, 5q-, 20q-, -Y, +1/1q, Rea 3q, -7/7q-, Complex (>3), +8, 11q-, t1q, t7q, 12p-, +21, t(5q) 11q23, +19, -21 and others =3 anomalies [8] 1,029 485 (45) NN, 5q-, 20q-, -Y +8, other rare -7, 7q-, complex abnormalities

Thus, rare abnormalities are frequent findings in MDS and one of the main future goals in MDS cytogenetics is to define their prognostic relevance.

Distinct Frequent Abnormalities More than half of the MDS patients with clonal chromosomal abnormalities show isolated abnormalities without any additional changes [6, 9]. The prognostic relevance of the most frequent of these abnormalities is known. Deletions within the long arm of chromosome 5 are the most frequent cytogenetic changes in MDS, occurring in 10–30% of all abnormal cases [6–9]. With a median survival of 80 months isolated deletions of 5q are clearly associated with a good prognosis [6–9]. But the prognosis of 5q-deletions in MDS is significantly modified by the number of additional cytogenetic changes [6, 130]. Abnormalities of chromosome 7, appearing in 9–21% [6, 9] of all patients with clonal abnormalities, are associated with a poor prognosis. No significant differences in survival were observed between total and partial monosomy 7. In about 50% of all patients showing a -7/7q-, the abnormality is part of a complex karyotype at the time of first diagnosis with a poor prognosis and a median survival of <6 months [6, 7, 9]. Additionally, more than one third (37%) of all complex abnormal cases (↜own unpublished data) contain this type of abnormality. If monosomy 7 is found isolated or with only one additional abnormality, median survival is 14–16 months [6, 9], indicating an intermediate to poor prognosis.

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Trisomy 8 represents the third most frequent abnormality, occurring in 8–16% of all abnormal cases [6, 7, 9]. Median survival in patients with isolated +8 is 19–24 months [6, 8, 9]. Interestingly, patients with trisomy 8 and one additional abnormality show a slightly better prognosis with a median survival of 44 months [6], which can most likely be attributed to the fact that a deletion of 5q is the most frequent accompanying abnormality in these patients (↜own unpublished data). One third of patients with trisomy 8 show complex abnormalities, critically downgrading the prognosis to a median survival <6 months [6, 7, 9]. A deletion of 20q can be observed in 3–4% of abnormal cases [6, 9]. As an isolated abnormality, it is associated with a good prognosis [6–9, 104]. This aberration is very rarely (<1% of cases showing this abnormality) seen in a combination of two abnormalities [6], further data is needed to delineate the prognosis in this subgroup. Prognosis of cases with a deletion of 20q within complex abnormal karyotypes is dismal [6]. Loss of the Y chromosome as an isolated abnormality is associated with a good prognosis and median survival of 36–60 months. In combination with one other abnormality the median survival is 39.0 months [6, 9]. Within a complex abnormal karyotype, -Y is associated with a poor prognosis [6]. A study of the German-Austrian MDS study group (↜own unpublished data) showed no differences in survival or clone size between younger and older patients developing an isolated -Y. Rare abnormalities are detectable in about 15% of MDS patients [6, 7, 9]. They form a heterogeneous group, hence Table€5.6 shows an overview concerning details of their frequency and prognostic impact.

Table 5.6↜渀 Frequency and overall survival in rare abnormalities Abnormality Frequency (%)a MS (months)a t(1q) 0.6/34.7/der(3)(q21q26) 0.5/0.6 19.9/31.6 t(5q) 0.4/4.4/t(7q) 0.6/34.7/del(9q) 0.4/NR/del(11q) 0.9/0.7 15.9/45.6 t(11q) 0.5/0.6 20.0/26.6 del(12p) 0.8/1.3 108.0/NR del(17p) -/0.5 -/NR iso17q -/1.0 -/11.0 +19 -/-21 0.5/32.0/+21 1.1/0.8 108.0/13.9 -X 0.5/56.4/a b

German-Austrian/and Spanish-MDS database [6, 9] International rare abnormalities database [133]

MS (months)b NR NR 34.7 63.1 28.0 NR 21.7 35.0 15.7

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Double Abnormalities Double abnormalities are less frequent as single or complex abnormalities and occur in 9% of all patients (17% of abnormal cases) [6]. We observed a profound heterogeneity of combinations without recurrent patterns of abnormalities [131]. Due to this heterogeneity, it is challenging to develop a prognostic classification system for double abnormalities (Fig.€5.5). By revising a large database containing 2,664 primary, untreated MDS patients [6, 7, 9, 132], the most reliable system was to integrate double abnormalities into three prognostic subgroups: Double abnormalities including deletions of 5q (favourable), double abnormalities including -7/7q- (unfavourable) and any other combinations of two abnormalities (intermediate). The calculated median survival was significantly different within these groups with 44 months for combinations classified as good, 34 months for intermediate and 14 months for unfavourable. In a multivariate analysis of the data by using age, gender and bone marrow blast count as covariables, the Hazard ratio (normal karyotype as baseline with a HR of

-7/7qn=4

+8 n=6

del(5q) n=49

del(20q) n=3

Fig. 5.5↜渀 Composition of “double abnormalities” using the example of del(5q) in 49 cases. (↜Own unpublished data)

any other n=36

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1.0) was 1.14 for double abnormalities including 5q- (p not significant), 1.47 for any double (pâ•›<â•›0.05) and 2.84 for double including -7/7q- (pâ•›<â•›0.01) [133].

Complex Abnormalities Complex abnormalities, defined as three or more independent abnormalities within one cell clone [74], occur in about 15% of all cases [6, 7, 9]. It is well known since the first publications about the prognostic impact of cytogenetics in MDS (Table€5.5) [6–9, 104, 105, 126–129, 134, 135] that complex abnormalities are invariably associated with a poor prognosis. Anyhow, they comprise different prognostic subgroups: patient with exactly three abnormalities show a slightly better prognosis (median survival 16 months) as compared to those with more than three abnormalities (6 months) [6, 131]. In a multivariate approach with age, sex and bone marrow blast count as co-variables, the Hazard ratio was found to be 3.0 in complexâ•›=â•›3 as compared to 5.0 in complex >3 (baseline: normal karyotype), reflecting the impact of the number of abnormalities [133]. Furthermore, complex abnormalities including chromosomes 5 or 7 are associated with a worse prognosis [136] and higher risk for AML-transformation [9]. They show a higher degree of complexity allocating these cases to the group of cases with more than three abnormalities. In summary in cases with complex changes there is a direct correlation between the number of abnormalities and the worsening of prognosis (Fig.€5.6) [6, 133].

Cytogenetics Within the IPSS The IPSS includes the number of peripheral cytopenias, bone marrow blast count and cytogenetics as major prognostic variables of risk stratification in MDS [7]. Abn.per case 0 1 2 3 >3

1,0

Cum. survival

0,8

0,6

0,4

0,2

Fig. 5.6↜渀 Kaplan-Meyer analyses of cytogenetic subgroups defined by the number of abnormalities per case (own data, adapted from [6])

0,0 0,0

100,0

200,0 months

300,0

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Since its implementation, the weighting of cytogenetics as compared to the other variables, was discussed [137–140]. Most recently, it was suggested that the prognostic relevance of cytogenetics as compared to the bone marrow blast count is underestimated within the IPSS [9, 136, 140]. In order to stratify the cytogenetic risk, the IPSS established six cytogenetic categories and classified them to three prognostic subgroups: favourable (normal karyotype, isolated del(5q), del(20q), -Y), unfavourable (complex abnormalities, any chromosome 7-abnormalities), and intermediate (any other abnormalities) [4]. Abnormalities with an unknown prognostic relevance are summarized within the intermediate subgroup (14% of all patients). Efforts on the improvement of the cytogenetic part of the IPSS need to focus on the characterization of these rare abnormalities. A recent work of the German-Austrian-MDS-study group was able to integrate 92.5% of all patients into 22 cytogenetic subgroups by consistent analysis of rare abnormalities [133]. The main future goal is to create a powerful prognostic tool allowing an optimized risk-stratification for patients suffering from MDS.

Therapeutic Consequences of Cytogenetics Besides allogeneic stem cell transplantation, supportive care used to be the therapeutic standard in patients with MDS. Retinoic acid and cytarabine turned out to be insufficient [141]. However, recently a remarkably high response rate of MDS patients with 5q-deletions to the immunomodulating agent lenalidomide was reported [142]. These encouraging results could be confirmed on the basis of a large multicentric trial [143]. For patients with monosomy 7 conventional intensive chemotherapy bears a high risk of early death and non-response. Even complete remissions frequently are of only short duration with a high risk of early relapse [144]. Thus patients should be treated with allogeneic stem cell transplantation whenever possible although monosomy 7 displays a dismal prognosis in relation to event free survival and relapse rates also for allogenic stem cell transplantation [145]. In recent therapy trials demethylating agents like 5-Azacytidine have turned out to be an attractive therapeutic option for MDS patients with monosomy 7 [146–148]. MDS with trisomy 8 were found to be preferentially responsive to immunosuppressive therapy. The therapeutic effect seems to be related to an indirect effect of immune suppression on the trisomy 8 clone [32, 33, 149]. Furthermore, MDS cells harbouring trisomy 8 show an overexpression of c-myc that leads to an upregulation of survivin and Cycin D1. Knockdown of survivin and Cyclin D1 can eliminate the survival advantage of these cells [33]. Cyclin D1 can also be chemically inhibited by the styryl sulfone On 01910.Na that may prove to be a targeted therapy for patients with trisomy 8 [31]. Phase I/II trials are currently ongoing. For patients with complex abnormalities “AML-like” intensive chemotherapy is not beneficial [144]. Allogeneic stem cell transplantation, the only curative option for these patients, is only feasible for a minority of individuals due to age-related

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multimorbidity and organ dysfunctions. However, cytogenetic response has been reported to demethylating [153], deacetylating [134, 150] and immunomodulating agents [143, 151]. Further multicentric clinical trials are needed to confirm these observations.

Conclusion In MDS cytogenetics is indespensible for diagnosis, classification, prognostication and therapeutic decisions and has proven its value during the last three decades. While cytogenetic analyses at initial diagnosis of MDS are frequently performed and are now the basis of genetic research and clinical management in MDS, sequential analyses with the opportunity of studying the phenomenon of karyotype evolution are not performed systematically. New techniques like FISH of circulating CD34+ cells might help to overcome these limiations. Conventional chromosome banding has come to its ages and new methods based on fluorescence in situ hybridization have been developed and are used with increasing frequency. However, it can be concluded from comparative studies that both methods have their own advantages and disadvantages. They cannot replace each other but provide additive supplementary information. Today only their combined use enables an up to date cytogenetic analysis in MDS. The great variability of cytogenetic findings still hampers the prediction of the clinical course in individual patients. However, due to recent international cooperations substantial progress has been made towards a comprehensive cytogenetic prognostic scoring system. Very recently, novel molecular whole genome scanning technologies, comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) arrays have been introduced for a more detailed genetic characterization of MDS and other disorders. In first studies it was demonstrated that these methods can complement conventional cytogenetics as well as FISH by the detection of copy number changes and copy number-neutral loss of heterozygosity [152]. Even at this early timepoint it appears that comparable to FISH-analyses CGH- and SNP-arrays will not make conventional cytogenetics unnecessary but rather will complement the established genetic methods and thus lead to a comprehensive genetic characterization of MDS.

References ╇ 1. Haase D, Feuring-Buske M, Schäfer C, Schoch C, Troff C, Gahn B, Hiddemann W, Wörmann B (1997) Cytogenetic analysis of CD34+ subpopulations in AML and MDS characterized by the expression of CD38 and CD117. Leukemia 11:674–679 ╇ 2. Nilsson L, Astrand-Grundström I, Anderson K, Arvidsson I, Hokland P, Bryder D, Kjeldsen L, Johansson B, Hellström-Lindberg E, Hast R, Jacobsen SEW (2002) Involvement and functional impairment of the CD34+CD38-Thy-1+ hematopoietic stem cell pool in myelodysplastic syndromes with trisomy 8. Blood 100:259–267

76

D. Haase et al.

╇ 3. Blau O, Hofmann WK, Baldus CD, Thiel G, Serbent V, Schümann E, Thiel E, Blau IW (2007) Chromosomal aberrations in bone marrow mesenchymal stroma cells from patients with myelodysplastic syndrome and acute myeloblastic leukemia. Exp Hematol 35:221–229 ╇ 4. Haferlach C, Rieder H, Lillington DM, Dastugue N, Hagemeijer A, Harbott J, Stilgenbauer S, Knuutila S, Johansson B, Fonatsch C (2007) Proposals for standardized protocols for cytogenetic analyses of acute leukemias, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, and myelodysplastic syndromes. Genes Chromosomes Cancer 46:494–499 ╇ 5. Steidl C, Steffens R, Gassmann W, Hildebrandt B, Hilgers R, Germing U, Trümper L, Haase D (2005) Adequate cytogenetic examination in myelodysplastic syndromes: analysis of 529 patients. Leukemia Res 29:987–993 ╇ 6. Haase D, Germing U, Schanz J, Pfeilstöcker M, Nösslinger T, Hildebrandt B, Kündgen A, Lübbert M, Kunzmann R, Giagounidis AAN, Aul C, Trümper L, Krieger O, Stauder R, Müller TH, Wimazal F, Valent P, Fonatsch C, Steidl C (2007) New insights into the prognostic impact of the karyotype in MDS and correlation with subtypes: evidence from a core dataset of 2124 patients. Blood 110:4385–4395 ╇ 7. Greenberg P, Cox C, Le Beau MM, Fenaux P, Morel P, Sanz G, Sanz M, Vallespi T, Hamblin T, Oscier D, Ohyashiki K, Toyama K, Aul C, Mufti G, Bennett J (1997) International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 89:2079–2088 ╇ 8. Pozdnyakova O, Miron PM, Tang GL, Walter O, Raza A, Woda B, Wang SA (2008) Cytogenetic abnormalities in a series of 1029 patients with primary myelodysplastic syndromes: a report from the US with a focus on some undefined single chromosomal abnormalities. Cancer 113:3331–3340 ╇ 9. Solé F, Luño E, Sanzo C, Espinet B, Sanz GF, Cervera J, Calasanz MJ, Cigudosa JC, Millà F, Ribera JM, Bureo E, Martin ML, Arranz E, Florensa L (2005) Identification of novel cytogenetic markers with prognostic significance in a series of 968 patients with primary myelodysplastic syndromes. Haematologica 90:1168–1178 10. Van den Berghe H, Vermaelen K, Mecucci C, Barbieri D, Tricot G (1985) The 5q- anomaly. Cancer Genet Cytogenet 17:189–255 11. Boultwood J, Fidler C, Strickson AJ, Watkins F, Gama S, Kearney L, Tosi S, Kasprzyk A, Cheng JF, Jaju RJ, Wainscoat JS (2002) Narrowing and genomic annotation of the commonly deleted region of the 5q-syndrome. Blood 99:4638–4641 12. Ebert BL, Pretz J, Bosco J, Chang CY, Tamayo P, Galili N, Raza A, Root DE, Attar E, Ellis SR, Golub TR (2008) Identification of RPS14 as a 5q-syndrome gene by RNA interference screen. Nature 451:335–339 13. Graubert TA, Payton MA, Shao J, Walgren RA, Monahan RS, Frater JL, Walshauser MA, Martin MG, Kasai Y, Walter MJ (2009) Integrated genomic analysis implicates haploinsufficiency of multiple chromosome 5q31.2 genes in de novo myelodysplastic syndromes pathogenesis. PLoS One 4:e4583 14. Mauritzson N, Albin M, Rylander L, Billström R, Ahlgren T, Mikoczy Z, Bjork J, Strömberg U, Nilsson PG, Mitelman F, Hagmar L, Johansson B (2002) Pooled analysis of clinical and cytogenetic features in treatment-related and de novo adult acute myeloid leukemia and myelodysplastic syndromes based on a consecutive series of 761 patients analyzed 1976–1993 and on 5098 unselected cases reported in the literature 1974–2001. Leukemia 16:2366–2378 15. Galvan AB, Mallo M, Arenillas L, Salido M, Espinet B, Pedro C, Florensa L, Serrano S, Solé F (2010) Does monosomy 5 really exist in myelodysplastic syndromes and acute myeloid leukemia? Leukemia Res 34:1242–1245 16. Liang H, Fairman J, Claxton DF, Nowell PC, Green ED, Nagarajan L (1998) Molecular anatomy of chromosome 7q deletions in myeloid neoplasms: evidence for multiple critical loci. Proc Natl Acad Sci U S A 95:3781–3785 17. Tosi S, Scherer SW, Giudici G, Czepulkowski B, Biondi A, Kearney L (1999) Delineation of multiple deleted regions in 7q in myeloid disorders. Genes Chromosomes Cancer 25:384– 392

5â•… Cytogenetics of MDS

77

18. Olney HJ, Le Beau MM (2002) The cytogenetics and molecular biology of myelodysplastic syndromes. In: Bennett JM (ed) The myelodysplastic syndromes: pathobiology and clinical management. Marcel Dekker, New York, pp€89–119 19. Chen GB, Zeng WH, Miyazato A, Billings E, Maciejewski JP, Kajigaya S, Sloand EM, Young NS (2004) Distinctive gene expression profiles of CD34 cells from patients with myelodysplastic syndrome characterized by specific chromosomal abnormalities. Blood 104:4210–4218 20. Nikoloski G, Langemeijer SM, Kuiper RP, Knops R, Massop M, Tonnissen ER, van der Heijden A, Scheele TN, Vandenberghe P, de Witte T, van der Reijden BA, Jansen JH (2010) Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nature Genet 42:665–667 21. Emanuel PD (1999) Myelodysplasia and myeloproliferative disorders in childhood: an update. Br J Haematol 105:852–863 22. Luna-Fineman S, Shannon KM, Lange BJ (1995) Childhood monosomy 7: epidemiology, biology, and mechanistic implications. Blood 85:1985–1999 23. Passmore SJ, Hann IM, Stiller CA, Ramani P, Swansbury GJ, Gibbons B, Reeves BR, Chessells JM (1995) Pediatric myelodysplasia: a study of 68 children and a new prognostic scoring system. Blood 85:1742–1750 24. Levine EG, Bloomfield CD (1992) Leukemias and myelodysplastic syndromes secondary to drug, radiation, and environmental exposure. Semin Oncol 19:47–84 25. Pedersen-Bjergaard J, Pedersen M, Roulston D, Philip P (1995) Different genetic pathways in leukemogenesis for patients presenting with therapy-related myelodysplasia and therapyrelated acute myeloid leukemia. Blood 86:3542–3552 26. West RR, Stafford DA, White AD, Bowen DT, Padua RA (2000) Cytogenetic abnormalities in the myelodysplastic syndromes and occupational or environmental exposure. Blood 95:2093–2097 27. Huebner G, Karthaus M, Pethig K, Freund M, Ganser A (2000) Myelodysplastic syndrome and acute myelogenous leukemia secondary to heart transplantation. Transplantation 70:688– 690 28. Knipp S, Hildebrandt B, Richter J, Haas R, Germing U, Gattermann C (2005) Secondary myelodysplastic syndromes following treatment with azathioprine are associated with aberrations of chromosome 7. Haematologica 90:691–693 29. Schoch C, Kohlmann A, Dugas M, Kern W, Schnittger S, Haferlach T (2006) Impact of trisomy 8 on expression of genes located on chromosome 8 in different AML subgroups. Genes Chromosomes Cancer 45:1164–1168 30. Iwabuchi A, Ohyashiki K, Ohyashiki JH, Sasao I, Murakami T, Kodama A, Toyama K (1992) Trisomy of chromosome 8 in myelodysplastic syndrome: significance of the fluctuating trisomy 8 population. Cancer Genet Cytogenet 62:70–74 31. Sloand EM, Pfannes L, Reddy R, Reddy P, Groopman JS, Young NS (2007) Suppression of cyclin D1 by on 01910.Na is associated with decreased survival of trisomy 8 myelodyplastic bone marrow progenitors: a potential targetted therapy. Blood (ASH Annual Meeting Abstracts) 110:822 32. Sloand EM, Kim S, Fuhrer M, Risitano AM, Nakamura R, Maciejewski JP, Barrett AJ, Young NS (2002) Fas-mediated apoptosis is important in regulating cell replication and death in trisomy 8 hematopoietic cells but not in cells with other cytogenetic abnormalities. Blood 100:4427–4432 33. Sloand EM, Pfannes L, Chen GB, Shah S, Solomou EE, Barrett J, Young NS (2007) CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins. Blood 109:2399–2405 34. Laï JL, Preudhomme C, Zandecki M, Flactif M, Vanrumbeke M, Lepelley P, Wattel E, Fenaux P (1995) Myelodysplastic syndromes and acute myeloid leukemia with 17p deletion. An entity characterized by specific dysgranulopoiesis and a high incidence of p53 mutations. Leukemia 9:370–381

78

D. Haase et al.

35. Becher R, Carbonell F, Bartram CR (1990) Isochromosome 17q in Ph1-negative leukemia: a clinical, cytogenetic, and molecular study. Blood 75:1679–1683 36. Mc Clure RF, Dewald GW, Hoyer JD, Hanson CA (1999) Isolated isochromosome 17q: a distinct type of mixed myeloproliferative disorder/myelodysplastic syndrome with an aggressive clinical course. Br J Haematol 106:445–454 37. Kaneko H, Misawa S, Horiike S, Nakai H, Kashima K (1995) TP53 mutations emerge at early phase of myelodysplastic syndrome and are associated with complex chromosomal abnormalities. Blood 85:2189–2193 38. Wattel E, Preudhomme C, Hecquet B, Vanrumbeke M, Quesnel B, Dervite I, Morel P, Fenaux P (1994) p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies. Blood 84:3148–3157 39. Horiike S, Misawa S, Kaneko H, Sasai Y, Kobayashi M, Fujii H, Tanaka S, Yagita M, Abe T, Kashima K, Taniwaki M (1999) Distinct genetic involvement of the TP53 gene in therapyrelated leukemia and myelodysplasia with chromosomal losses of Nos 5 and/or 7 and its possible relationship to replication error phenotype. Leukemia 13:1235–1242 40. Schoch C, Kern W, Kohlmann A, Hiddemann W, Schnittger S, Haferlach T (2005) Acute myeloid leukemia with a complex aberrant karyotype is a distinct biological entity characterized by genomic imbalances and a specific gene expression profile. Genes Chromosomes Cancer 43:227–238 41. Ben-Yehuda D, Krichevsky S, Caspi O, Rund D, Polliack A, Abeliovich D, Zelig O, Yahalom V, Paltiel O, Or R, Peretz T, Ben-Neriah S, Yehuda O, Rachmilewitz EA (1996) Microsatellite instability and p53 mutations in therapy-related leukemia suggest mutator phenotype. Blood 88:4296–4303 42. Douet-Guilbert N, Basinko A, Morel F, Bris MJ, Ugo V, Morice P, Berthou C, Braekeleer M (2008) Chromosome 20 deletions in myelodysplastic syndromes and Philadelphia-chromosome-negative myeloproliferative disorders: characterization by molecular cytogenetics of commonly deleted and retained regions. Ann Hematol 87:537–544 43. Bench AJ, Nacheva EP, Hood TL, Holden JL, French L, Swanton S, Champion KM, Li J, Whittaker P, Stavrides G, Hunt AR, Huntly BJP, Campbell LJ, Bentley DR, Deloukas P, Green AR (2000) Chromosome 20 deletions in myeloid malignancies: reduction of the common deleted region, generation of a PAC/BAC contig and identification of candidate genes. UK Cancer Cytogenetics Group (UKCCG). Oncogene 19:3902–3913 44. Roulston D, Espinosa R, Stoffel M, Bell GI, Le Beau MM (1993) Molecular genetics of myeloid leukemia: identification of the commonly deleted segment of chromosome 20. Blood 82:3424–3429 45. Wang PW, Iannantuoni K, Davis EM, Espinosa R, Stoffel M, Le Beau MM (1998) Refinement of the commonly deleted segment in myeloid leukemias with a del(20q). Genes Chromosomes Cancer 21:75–81 46. MacGrogan D, Kalakonda N, Alvarez S, Scandura JM, Boccuni P, Johansson B, Nimer SD (2004) Structural integrity and expression of the L3MBTL gene in normal and malignant hematopoietic cells. Genes Chromosomes Cancer 41:203–213 47. Douet-Guilbert N, Laï JL, Basinko A, Gueganic N, Andrieux J, Pollet B, Plantier I, Delattre C, Crépin O, Corm S, Le Bris MJ, Morel F, De Braekeleer M (2008) Fluorescence in situ hybridization characterization of ider(20q) in myelodysplastic syndrome. Br J Haematol 143:716–720 48. United Kingdom Cancer Cytogenetics Group (UKCCG) (1992) Loss of the Y chromosome from normal and neoplastic bone marrows. Genes Chromosomes Cancer 5:83–88 (Erratum in: Genes Chromosom Cancer 85, 411) 49. Wong AK, Fang B, Zhang L, Guo XQ, Lee S, Schreck R (2008) Loss of the Y chromosome— an age-related or clonal phenomenon in acute myelogenous leukemia/myelodysplastic syndrome? Arch Pathol Lab Med 132:1329–1332 50. Zhang LJ, Shin ES, Yu ZX, Li SB (2007) Molecular genetic evidence of Y chromosome loss in male patients with hematological disorders. Chin Med J 120:2002–2005

5â•… Cytogenetics of MDS

79

51. Wiktor A, Rybicki BA, Piao ZS, Shurafa M, Barthel B, Maeda K, Van Dyke DL (2000) Clinical significance of Y chromosome loss in hematologic disease. Genes Chromosomes Cancer 27:11–16 52. Bernasconi P (2008) Molecular pathways in myelodysplastic syndromes and acute myeloid leukemia: relationships and distinctions—a review. Br J Haematol 142:695–708 53. Xiao Z, Zhang M, Liu X, Zhang Y, Yang L, Hao Y (2006) MEL1S, not MEL1, is overexpressed in myelodysplastic syndromes patients with t(1;3)(p36;q21). Leukemia Res 30:332– 334 54. Golub TR, Barker GF, Lovett M, Gilliland DG (1994) Fusion of PDGF receptor beta to a novel ets-like gene, TEL, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell 77:307–316 55. Raimondi SC, Dubé ID, Valentine MB, Mirro J, Watt HJ, Larson RA, Bitter MA, Le Beau MM, Rowley JD (1989) Clinicopathologic manifestations and breakpoints of the t(3;5) in patients with acute nonlymphocytic leukemia. Leukemia 3:42–47 56. Yoneda-Kato N, Look AT, Kirstein MN, Valentine MB, Raimondi SC, Cohen KJ, Carroll AJ, Morris SW (1996) The t(3;5)(q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1. Oncogene 12:265–275 57. Estey E, Trujillo JM, Cork A, O’Brien S, Beran M, Kantarjian H, Keating M, Freireich EJ, Stass S (1992) AML-associated cytogenetic abnormalities (inv (16), del (16), t(8;21)) in patients with myelodysplastic syndromes. Hematol Pathol 6:43–48 58. Kojima K, Omoto E, Hara M, Sasaki K, Katayama Y, Nawa Y, Kimura Y, Azuma T, Takimoto H, Harada M (1998) Myelodysplastic syndrome with translocation (8;21): a distinct myelodysplastic syndrome entity or M2-acute myeloid leukemia with extensive myeloid maturation? Ann Hematol 76:279–282 59. Taj AS, Ross FM, Vickers M, Choudhury DN, Harvey JF, Barber JCK, Barton C, Smith AG (1995) t(8;21) myelodysplasia, an early presentation of M2 AML. Br J Haematol 89:890–892 60. Narayanan MN, Geary CG, Harrison CJ, Cinkotai KI, Lewis MJ (1993) Three cases of the myelodysplastic syndrome with pericentric inversion of chromosome 16. Br J Haematol 85:217–219 61. Ohyashiki K, Murakami T, Ohyashiki JH, Kawakubo K, Fujimura T, Iwabuchi A, Toyama K (1995) Double-minute chromosomes appearing in a patient with myelodysplastic syndrome with disease evolution. Cancer Genet Cytogenet 79:169–172 62. Reddy KS, Sulcova V (1997) c-myc amplification in a preleukemia patient with trisomy 4 and double minutes: review of the unique coexistence of these two chromosome abnormalities in acute myelogenous leukemia. Cancer Genet Cytogenet 95:206–209 63. Sambani C, Trafalis DTP, Vessalas G, Politis G, Peristeris P, Nakopoulou L, Giannopoulou J, Michaelidis C, Ayoutantis M, Fantelias GE (1998) Trisomy 6 and double minute chromosomes in a case of chronic myelomonocytic leukemia. Cancer Genet Cytogenet 106:180–181 64. Mohamed AN, Macoska JA, Kallioniemi A, Kallioniemi OP, Waldman F, Ratanatharathorn V, Wolman SR (1993) Extrachromosomal gene amplification in acute myeloid leukemia; characterization by metaphase analysis, comparative genomic hybridization, and semi-quantitative PCR. Genes Chromosomes Cancer 8:185–189 65. Sait SNJ, Qadir MU, Conroy JM, Matsui SI, Nowak NJ, Baer MR (2002) Double minute chromosomes in acute myeloid leukemia and myelodysplastic syndrome: identification of new amplification regions by fluorescence in situ hybridization and spectral karyotyping. Genes Chromosomes Cancer 34:42–47 66. Streubel B, Valent P, Jäger U, Edelhäuser M, Wandt H, Wagner T, Büchner T, Lechner K, Fonatsch C (2000) Amplification of the MLL gene on double minutes, a homogeneously staining region, and ring chromosomes in five patients with acute myeloid leukemia or myelodysplastic syndrome. Genes Chromosomes Cancer 27:380–386 67. Han JY, Theil KS, Hoeltge G (2006) Frequencies and characterization of cytogenetically unrelated clones in various hematologic malignancies: seven years of experiences in a single institution. Cancer Genet Cytogenet 164:128–132

80

D. Haase et al.

68. Heim S, Mitelman F (1989) Cytogenetically unrelated clones in hematological neoplasms. Leukemia 3:6–8 69. Johansson B, Billstroem R, Broberg K, Fioretos T, Nilsson PG, Ahlgren T, Malm C, Samuelsson BO, Mitelman F (1999) Cytogenetic polyclonality in hematologic malignancies. Genes Chromosomes Cancer 24:222–229 70. Kobayashi H, Kaneko Y, Maseki N, Sakurai M (1990) Karyotypically unrelated clones in acute leukemias and myelodysplastic syndromes. Cancer Genet Cytogenet 47:171–178 71. Mitelman F (1998) Catalog of chromosome aberrations in cancer, 6th edn. Wiley-Liss, New York 72. Musilová R, Michalová K, Zemanová Z, Brezinová J (1996) Multiple unrelated clones in myelodysplastic syndrome and in acute myeloid leukemia. Cancer Genet Cytogenet 88:141–143 73. Pedersen-Bjergaard J, Timshel S, Andersen MK, Andersen AS, Philip P (1998) Cytogenetically unrelated clones in therapy-related myelodysplasia and acute myeloid leukemia: experience from the Copenhagen series updated to 180 consecutive cases. Genes Chromosomes Cancer 23:337–349 74. Mitelman F (1991) ISCN Guidelines for cancer cytogenetics, supplement to an international system for human cytogenetic nomenclature. Karger, Basel 75. Pedersen-Bjergaard J, Andersen MK, Christiansen DH, Nerlov C (2002) Genetic pathways in therapy-related myelodysplasia and acute myeloid leukemia. Blood 99:1909–1912 76. Trost D, Hildebrandt B, Beier M, Müller N, Germing U, Royer-Pokora B (2006) Molecular cytogenetic profiling of complex karyotypes in primary myelodysplastic syndromes and acute myeloid leukemia. Cancer Genet Cytogenet 165:51–63 77. Rücker FG, Bullinger L, Schwaenen C, Lipka DB, Wessendorf S, Frohling S, Bentz M, Miller S, Scholl C, Schlenk RF, Radlwimmer B, Kestler HA, Pollack JR, Lichter P, Döhner K, Döhner H (2006) Disclosure of candidate genes in acute myeloid leukemia with complex karyotypes using microarray-based molecular characterization. J Clin Oncol 24:3887–3894 78. Boultwood J, Wainscoat JS (2001) Clonality in the myelodysplastic syndromes. Int J Hematol 73:411–415 79. Cermák J, Belicková M, Krejcová H, Michalová K, Zilovcová S, Zemanová Z, Brezinová J, Sieglová Z (2005) The presence of clonal cell subpopulations in peripheral blood and bone marrow of patients with refractory cytopenia with multilineage dysplasia but not in patients with refractory anemia may reflect a multistep pathogenesis of myelodysplasia. Leukemia Res 29:371–379 80. Galili N, Cerny J, Raza A (2007) Current treatment options: impact of cytogenetics on the course of myelodysplasia. Curr Treat Options Oncol 8:117–128 81. Raskind WH, Tirumali N, Jacobson R, Singer J, Fialkow PJ (1984) Evidence for a multistep pathogenesis of a myelodysplastic syndrome. Blood 63:1318–1323 82. Johansson B, Mertens F, Mitelman F (1996) Primary vs, secondary neoplasia-associated chromosomal abnormalities—balanced rearrangements vs, genomic imbalances? Genes Chromosomes Cancer 16:155–163 83. Mallo M, Cervera J, Schanz J, Such E, García-Manero G, Luño E, Steidl C, Espinet B, Vallespí T, Germing U, Blum S, Ohyashiki K, Grau J, Pfeilstöcker M, Hernández JM, Noesslinger T, Giagounidis A, Aul C, Calasanz MJ, Martín ML, Valent P, Collado R, Haferlach C, Fonatsch C, Lübbert M, Stauder R, Hildebrandt B, Krieger O, Pedro C, Arenillas L, Sanz MA, Valencia A, Florensa L, Sanz GF, Haase D, Solé F (2010) Impact of adjunct cytogenetic abnormalities for prognostic stratification in patients with myelodysplastic syndrome and deletion 5q. Leukemia (in press) 84. Steidl C, Schabla R, Germing U, Hildebrandt B, Noesslinger T, Pfeilstoecker M, Giagounidis A, Kunzmann R, Haas P, Luebbert M, Truemper L, Haase D (2005) Sequential cytogenetic analyses of 577 patients with myelodysplastic syndromes: correlations between initial karyotype, cytogenetic dynamics, and clinical course. Blood (ASH Annual Meeting Abstracts) 106:2531

5â•… Cytogenetics of MDS

81

╇ 85. Tricot G, Boogaerts MA, De Wolf-Peeters C, Van den Berghe H, Verwilghen RL (1985) The myelodysplastic syndromes: different evolution patterns based on sequential morphological and cytogenetic investigations. Br J Haematol 59:659–670 ╇ 86. Ghaddar HM, Stass SA, Pierce S, Estey EH (1994) Cytogenetic evolution following the transformation of myelodysplastic syndrome to acute myelogenous leukemia: implications on the overlap between the two diseases. Leukemia 8:1649–1653 ╇ 87. Horiike S, Misawa S, Nakai H, Kaneko H, Yokota S, Taniwaki M, Yamane Y, Inazawa J, Abe T, Kashima K (1994) N-ras mutation and karyotypic evolution are closely associated with leukemic transformation in myelodysplastic syndromes. Leukemia 8:1331–1336 ╇ 88. Horiike S, Taniwaki M, Misawa S, Abe T (1988) Chromosome abnormalities and karyotypic evolution in 83 patients with myelodysplastic syndrome and predictive value for prognosis. Cancer 62:1129–1138 ╇ 89. Lai YY, Qiu JY, Zhang Y, Shi Y, He Q, Dang H, Lu DP (2004) Clinical significance of continuous karyotyping in myelodysplastic syndromes. Zhonghua Xue Ye Xue Za Zhi 25:645– 648 ╇ 90. Tien HF, Wang CH, Chuang SM, Lee FY, Liu MC, Chen YC, Shen MC, Lin KH, Lin DT (1995) Acute leukemic transformation of myelodysplastic syndrome—immunophenotypic, genotypic, and cytogenetic studies. Leukemia Res 19:595–603 ╇ 91. White AD, Hoy TG, Jacobs A (1994) Extended cytogenetic follow-up and clinical progress in patients with myelodysplastic syndromes (MDS). Leukemia Lymphoma 12:401–412 ╇ 92. Daniel CP, Ponting IL, Dexter TM (1989) Growth and development of haemopoietic cells: a deterministic process? Haematol Blood Transfus 32:172–177 ╇ 93. Kanamaru A, Tamura S (1993) Application of long-term marrow cultures for studying the leukemic transformation of myelodysplastic syndromes. Leukemia Lymphoma 11:345–352 ╇ 94. Tamura S, Kanamaru A, Takemoto Y, Kakishita E, Nagai K (1993) Clonal evolutions during long-term cultures of bone marrow from de novo acute myeloid leukemia with trilineage myelodysplasia and with myelodysplastic remission marrow. Br J Haematol 84:219–226 ╇ 95. Michalová K, Cermák J, Brezinová J, Zemanová Z (1999) Double minute chromosomes in a patient with myelodysplastic syndrome transforming into acute myeloid leukemia. Cancer Genet Cytogenet 109:76–78 ╇ 96. Ohyashiki JH, Ohyashiki K, Fujimura T, Kawakubo K, Shimamoto T, Iwabuchi A, Toyama K (1994) Telomere shortening associated with disease evolution patterns in myelodysplastic syndromes. Cancer Res 54:3557–3560 ╇ 97. Pinheiro RF, de Sá Moreira ED, Silva MRR, Alberto FL, Chauffaille M (2008) FLT3 internal tandem duplication during myelodysplastic syndrome follow-up: a marker of transformation to acute myeloid leukemia. Cancer Genet Cytogenet 183:89–93 ╇ 98. Lorenzo F, Nishii K, Mortma F, Kuwagata S, Usui E, Shiku H (2006) Mutational analysis of the KIT gene in myelodysplastic syndrome (MDS) and MDS-derived leukemia. Leukemia Res 30:1235–1239 ╇ 99. Mori N, Morosetti R, Hoflehner E, Lübbert M, Mizoguchi H, Koeffler HP (2000) Allelic loss in the progression of myelodysplastic syndrome. Cancer Res 60:3039–3042 100. Harada S, Komatsu H, Seto M, Ni HP, Xu JH, Hayami Y, Tsuboi K, Wakita A, Nitta M, Kato T, Ueda R (1998) Microsatellite instability is rare in the clinical course of myelodysplastic syndrome studied with DNA from fresh and paraffin embedded tissues. J Cancer Res Clin Oncol 124:231–235 101. Maeck L, Haase D, Schoch C, Hiddemann W, Alves F (2000) Genetic instability in myelodysplastic syndrome: detection of microsatellite instability and loss of heterozygosity in bone marrow samples with karyotype alterations. Br J Haematol 109:842–846 102. Tasaka T, Lee S, Spira S, Takeuchi S, Hatta Y, Nagai M, Takahara J, Koeffler HP (1996) Infrequent microsatellite instability during the evolution of the myelodysplastic syndrome to acute myelocytic leukemia. Leukemia Res 20:113–117 103. Braulke F, Schanz J, Jung K, Shirneshan K, Schulte K, Schuetze C, Steffens R, Truemper L, Haase D (2010) FISH analysis of circulating CD34+ cells as a new tool for genetic monitor-

82

104.

105.

106.

107.

108.

109. 110.

111.

112.

113. 114. 115. 116.

D. Haase et al. ing in MDS: verification of the method and application to 27 MDS patients. Leukemia Res 34:1296–1301 Morel P, Hebbar M, Laï JL, Duhamel A, Preudhomme C, Wattel E, Bauters F, Fenaux P (1993) Cytogenetic analysis has strong independent prognostic value in de novo myelodysplastic syndromes and can be incorporated in a new scoring system: a report on 408 cases. Leukemia 7:1315–1323 Toyama K, Ohyashiki K, Yoshida Y, Abe T, Asano S, Hirai H, Hirashima K, Hotta T, Kuramoto A, Kuriya S, Miyazaki T, Kakishita E, Mizoguchi H, Okada M, Shirakawa S, Takaku F, Tomonaga M, Uchino H, Yasunaga K, Nomura T (1993) Clinical implications of chromosomal abnormalities in 401 patients with myelodysplastic syndromes: a multicentric study in Japan. Leukemia 7:499–508 Le Beau MM, Albain KS, Larson RA, Vardiman JW, Davis EM, Blough RR, Golomb HM, Rowley JD (1986) Clinical and cytogenetic correlations in 63 patients with therapy-related myelodysplastic syndromes and acute nonlymphocytic leukemia: further evidence for characteristic abnormalities of chromosome 5 and chromosome 7. J Clin Oncol 4:325–345 Andersen MK, Pedersen-Bjergaard J (2000) Increased frequency of dicentric chromosomes in therapy-related MDS and AML compared to de novo disease is significantly related to previous treatment with alkylating agents and suggests a specific susceptibility to chromosome breakage at the centromere. Leukemia 14:105–111 Andersen MK, Christiansen DH, Kirchhoff M, Pedersen-Bjergaard J (2001) Duplication or amplification of chromosome band 11q23, including the unrearranged MLL gene, is a recurrent abnormality in therapy-related MDS and AML, and is closely related to mutation of the TP53 gene and to previous therapy with alkylating agents. Genes Chromosomes Cancer 31:33–41 Andersen MK, Christiansen DH, Pedersen-Bjergaard J (2005) Amplification or duplication of chromosome band 21q22 with multiple copies of the AML1 gene and mutation of the TP53 gene in therapy-related MDS and AML. Leukemia 19:197–200 Bernasconi P, Cavigliano PM, Boni M, Calatroni S, Klersy C, Giardini I, Rocca B, Crosetto N, Caresana M, Lazzarino M, Bernasconi C (2003) Is FISH a relevant prognostic tool in myelodysplastic syndromes with a normal chromosome pattern on conventional cytogenetics? A study on 57 patients. Leukemia 17:2107–2112 Rigolin GM, Bigoni R, Milani R, Cavazzini F, Roberti MG, Bardi A, Agostini P, Della Porta M, Tieghi A, Piva N, Cuneo A, Castoldi G (2001) Clinical importance of interphase cytogenetics detecting occult chromosome lesions in myelodysplastic syndromes with normal karyotype. Leukemia 15:1841–1847 Mallo M, Arenillas L, Espinet B, Salido M, Hernández JM, Lumbreras E, del Rey M, Arranz E, Ramiro S, Font P, González O, Renedo M, Cervera J, Such E, Sanz GF, Luño E, Sanzo C, González M, Calasanz MJ, Mayans J, García-Ballesteros C, Amigo V, Collado R, Oliver I, Carbonell F, Bureo E, Insunza A, Yañez L, Muruzabal MJ, Gómez-Beltrán E, Andreu R, León P, Gómez V, Sanz A, Casasola N, Moreno E, Alegre A, Martín ML, Pedro C, Serrano S, Florensa L, Solé F (2008) Fluorescence in situ hybridization improves the detection of 5q31 deletion in myelodysplastic syndromes without cytogenetic evidence of 5q. Haematologica 93:1001–1008 Trost D, Hildebrandt B, Müller N, Germing U, Royer-Pokora B (2004) Hidden chromosomal aberrations are rare in primary myelodysplastic syndromes with evolution to acute myeloid leukaemia and normal cytogenetics. Leukemia Res 28:171–177 Shen Y, Xue Y, Li J, Guo Y, Pan J, Wu Y (2001) Detection of monosomy 7 or 7q- in cases of myelodysplastic syndrome. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 18:255–258 Jakovleva K, Ogard I, Arvidsson I, Jacobsson B, Swolin B, Hast R (2001) Masked monosomy 7 in myelodysplastic syndromes is uncommon and of undetermined clinical significance. Leukemia Res 25:197–203 Panani AD, Pappa V (2005) Hidden chromosome 8 abnormalities detected by FISH in adult primary myelodysplastic syndromes. In Vivo 19:979–981

5â•… Cytogenetics of MDS

83

117. Romeo M, Chauffaille M, Silva MR, Bahia DM, Kerbauy J (2002) Comparison of cytogenetics with FISH in 40 myelodysplastic syndrome patients. Leukemia Res 26:993–996 118. Cherry AM, Brockman SR, Paternoster SF, Hicks GA, Neuberg D, Higgins RR, Bennett JM, Greenberg PL, Miller K, Tallman MS, Rowe J, Dewald GW (2003) Comparison of interphase FISH and metaphase cytogenetics to study myelodysplastic syndrome: an Eastern Cooperative Oncology Group (ECOG) study. Leukemia Res 27:1085–1090 119. Slovak ML, Dewald GW (2008) International Working Group on MDS cytogenetics: October 2007 meeting report. Leukemia Res 32:1329–1332 120. Barouk-Simonet E, Soenen-Cornu V, Roumier C, Cosson A, Laï JL, Fenaux P, Preudhomme C (2005) Role of multiplex FISH in identifying chromosome involvement in myelodysplastic syndromes and acute myeloid leukemias with complex karyotypes: a report on 28 cases. Cancer Genet Cytogenet 157:118–126 121. Crescenzi B, La Starza R, Romoli S, Beacci D, Matteucci C, Barba G, Aventin A, Marynen P, Ciolli S, Nozzoli C, Martelli MF, Mecucci C (2004) Submicroscopic deletions in 5qassociated malignancies. Haematologica 89:281–285 122. Li JY, Xiao B, Chen LJ, Pan JL, Xu W, Qiu HR, Li L, Xue YQ (2008) An analysis of complex chromosomal aberrations in seven cases of myelodysplastic syndromes by M-FISH and whole chromosome painting. Int J Hematol 88:369–373 123. Mohr B, Bornhäuser M, Thiede C, Schäkel U, Schaich M, Illmer T, Pascheberg U, Ehninger G (2000) Comparison of spectral karyotyping and conventional cytogenetics in 39 patients with acute myeloid leukemia and myelodysplastic syndrome. Leukemia 14:1031–1038 124. Van Limbergen H, Poppe B, Michaux L, Herens C, Brown J, Noens L, Berneman Z, De Bock R, De Paepe A, Speleman F (2002) Identification of cytogenetic subclasses and recurring chromosomal aberrations in AML and MDS with complex karyotypes using M-FISH. Genes Chromosomes Cancer 33:60–72 125. Vehmeyer K, Haase D, Alves F (2001) Increased peripheral stem cell pool in MDS: an indication of disease progression? Leukemia Res 25:955–959 126. Knapp RH, Dewald GW, Pierre RV (1985) Cytogenetic studies in 174 consecutive patients with preleukemic or myelodysplastic syndromes. Mayo Clinic Proc 60:507–516 127. Billström R, Thiede T, Hansen S, Heim S, Kristoffersson U, Mandahl N, Mitelman F (1988) Bone marrow karyotype and prognosis in primary myelodysplastic syndromes. Euro J Haematol 41:341–346 128. Nowell PC, Besa EC (1989) Prognostic significance of single chromosome abnormalities in preleukemic states. Cancer Genet Cytogenet 42:1–7 129. Pierre RV, Catovsky D, Mufti GJ, Swansbury GJ, Mecucci C, Dewald GW, Ruutu T, Vandenberghe H, Rowley JD, Mitelman F, Reeves BR, Alimena G, Garson OM, Lawler SD, de la Chapelle A (1989) Clinical-cytogenetic correlations in myelodysplasia (preleukemia). Cancer Genet Cytogenet 40:149–161 130. Giagounidis AAN, Germing U, Aul C (2006) Biological and prognostic significance of chromosome 5q deletions in myeloid malignancies. Clin Cancer Res 12:5–10 131. Steidl C, Schanz J, Le Beau MM, Bennett JM, Germing U, Greenberg PL, Haase D (2007) Coalescence of the German-Austrian and IMRAW cytogenetic MDS databases: modification of patient risk groups. Blood (ASH Annual Meeting Abstracts) 110:2468 132. Schanz J, Slovak ML, Ohyashiki K, Solé F, Mallo MD, Steidl C, Pfeilstoecker M, Noesslinger T, Hildebrandt B, Luebbert M, Andersen MK, Giagounidis A, Valent P, Fonatsch C, Germing U, Haase D (2008) New prognostic data on rare cytogenetic abnormalities in MDS: a collaborative study of the international working group on MDS cytogenetics. Blood (ASH Annual Meeting Abstracts) 112:2688 133. Schanz J, Tuechler H, Solé F, Mallo M, Hildebrandt B, Slovak ML, Ohyashiki K, Steidl C, Fonatsch C, Pfeilstoecker M, Noesslinger T, Valent P, Giagounidis A, Luebbert M, Stauder R, Krieger O, Le Beau MM, Bennett JM, Greenberg PL, Germing U, Haase D (2009) Cytogenetic risk features in MDS-update and present state. Blood (ASH Annual Meeting Abstracts) 114:1084

84

D. Haase et al.

134. Braulke F, Schanz J, Steidl C, Truemper LH, Haase D (2007) FISH-Analyses of cirulating CD34+cells in MDS-Patients—a suitable method to measure and predict response to 5-azacytidinie. Blood (ASH Annual Meeting Abstracts) 110:2466 135. Nowell PC, Besa EC, Stelmach T, Finan JB (1986) Chromosome studies in preleukemic states. V. Prognostic significance of single versus multiple abnormalities. Cancer 58:2571– 2575 136. Schanz J, Estey EH, Steidl C, Germing U, Hildebrandt B, Garcia-Manero G, Kantarjian HM, Haase D (2007) Multivariate analysis suggests that the prognostic impact of poor cytogenetics is potentially underestimated in the IPSS. Blood (ASH Annual Meeting Abstracts) 110:248 137. Bennett JM (2005) A comparative review of classification systems in myelodysplastic syndromes (MDS). Semin Oncol 32:S3–S10 138. Lee JH, Shin YR, Lee JS, Kim WK, Chi HS, Park CJ, Seo EJ, Lee KH (2003) Application of different prognostic scoring systems and comparison of the FAB and WHO classifications in Korean patients with myelodysplastic syndrome. Leukemia 17:305–313 139. Müller-Berndorff H, Haas PS, Kunzmann R, Schulte-Mönting J, Lübbert M (2006) Comparison of five prognostic scoring systems, the French-American-British (FAB) and World Health Organization (WHO) classifications in patients with myelodysplastic syndromes: results of a single-center analysis. Ann Hematol 85:502–513 140. Steidl C, Schanz J, Pfeilstoecker M, Noesslinger T, Hildebrandt B, Kuendgen A, Luebbert M, Kunzmann R, Giagounidis A, Aul C, Truemper L, Krieger O, Stauder R, Mueller T, Wimazal F, Valent P, Fonatsch C, Germing U, Haase D (2007) Growing Evidence for an Underestimation of Poor-Risk Cytogenetics in the International Prognostic Scoring System in Myelodysplastic Syndromes. Clin Leukemia 1:353–356 141. Giagounidis AAN, Haase S, Germing U, Schlegelberger B, Wilkens L, Büsche G, Kreipe HH, Wysk J, Grips KH, Grabenhorst U, Rothmann F, Lübbert M, Ganser A, Aivado M, Heinsch M, Aul C (2005) Treatment of myelodysplastic syndrome with isolated del(5q) including bands q31–q33 with a combination of all-trans-retinoic acid and tocopherol-alpha: a phase II study. Ann Hematol 84:389–394 142. List A, Kurtin S, Roe DJ, Buresh A, Mahadevan D, Fuchs D, Rimsza L, Heaton R, Knight R, Zeldis JB (2005) Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J Med 352:549–557 143. List A, Dewald G, Bennett J, Giagounidis A, Raza A, Feldman E, Powell B, Greenberg P, Thomas D, Stone R, Reeder C, Wride K, Patin J, Schmidt M, Zeldis J, Knight R, Myelodysplastic S (2006) Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion. N Engl J Med 355:1456–1465 144. Knipp S, Hildebrand B, Kundgen A, Giagounidis A, Kobbe G, Haas R, Aul C, Gattermann N, Germing U (2007) Intensive chemotherapy is not recommended for patients aged >60 years who have myelodysplastic syndromes or acute myeloid leukemia with high-risk karyotypes. Cancer 110:345–352 145. Nevill TJ, Fung HC, Shepherd JD, Horsman DE, Nantel SH, Klingemann HG, Forrest DL, Toze CL, Sutherland HJ, Hogge DE, Naiman SC, Le A, Brockington DA, Barnett MJ (1998) Cytogenetic abnormalities in primary myelodysplastic syndrome are highly predictive of outcome after allogeneic bone marrow transplantation. Blood 92:1910–1917 146. Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V, Finelli C, Giagounidis A, Schoch R, Gattermann N, Sanz G, List A, Gore SD, Seymour JF, Bennett JM, Byrd J, Backstrom J, Zimmerman L, McKenzie D, Beach CL, Silverman LR, International Vidaza High-Risk MDS Survival Study Group (2009) Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 10:223–232 147. Raj K, John A, Ho A, Chronis C, Khan S, Samuel J, Pomplun S, Thomas NSB, Mufti GJ (2007) CDKN2B methylation status and isolated chromosome 7 abnormalities predict responses to treatment with 5-azacytidine. Leukemia 21:1937–1944

5â•… Cytogenetics of MDS

85

148. Rüter B, Wijermans P, Claus R, Kunzmann R, Lübbert M (2007) Preferential cytogenetic response to continuous intravenous low-dose decitabine (DAC) administration in myelodysplastic syndrome with monosomy 7. Blood 110:1080–1082 149. Sloand EM, Yong ASM, Ramkissoon S, Solomou E, Bruno TC, Kim S, Fuhrer M, Kajigaya S, Barrett AJ, Young NS (2006) Granulocyte colony-stimulating factor preferentially stimulates proliferation of monosomy 7 cells bearing the isoform IV receptor. Proc Natl Acad Sci U S A 103:14483–14488 150. Cheson BD, Greenberg PL, Bennett JM, Lowenberg B, Wijermans PW, Nimer SD, Pinto A, Beran M, de Witte TM, Stone RM, Mittelman M, Sanz GF, Gore SD, Schiffer CA, Kantarjian H (2006) Clinical application and proposal for modification of the International Working Group (IWG) response criteria in myelodysplasia. Blood 108:419–425 151. Giagounidis AAN, Germing U, Strupp C, Hildebrandt B, Heinsch M, Aul C (2005) Prognosis of patients with del(5q) MDS and complex karyotype and the possible role of lenalidomide in this patient subgroup. Ann Hematol 84:569–571 152. Maciejewski JP, Mufti GJ (2008) Whole genome scanning as a cytogenetic tool in hematologic malignancies. Blood 112:965–974 153. Lübbert M, Wijermans P, Kunzmann R, Verhoef G, Bosly A, Ravoet C, Andre M, Ferrant A (2001) Cytogenetic responses in high-risk myelodysplastic syndrome following lowdose treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine. Br J Haematol 114:349–357

Chapter 6

Molecular Changes in Myelodysplastic Syndrome Florian Nolte and Wolf-K. Hofmann

Introduction Myelodysplastic syndromes (MDS) are heterogenous diseases which are characterized by ineffective hematopoiesis with an increased risk of evolution to acute myeloid leukemia (AML). Diagnosis of MDS is mainly based on morphological findings and cytogenetical analyses. However, little is known about the underlying pathomechanisms causing MDS. MDS in early stages with relatively slow progression to AML might be a prototype of the multistep concept in leukemogenesis with accumulation of cellular and molecular defects during initiation and disease progression. The model of Knudson postulates ‘two hits’ that are required for the development of cancer, e.g. MDS and consecutive AML. This concept bases on the observation in hereditary cancers, that loss or inactivation of one allele rarely is sufficient to result in the development of tumors or expansion of a malignant clone but loss of the corresponding allele or additional alterations are necessary for the penetrance of clonal cells [36]. Since conventional cytogenetics show structural or numerical aberrations in about 50% of MDS patients only, introduction of array based techniques in research was logical and necessary to identify more subtle genomic changes such as cryptic deletions, regions of loss of heterozygosity and uniparental disomy. Moreover, since gene silencing by DNA methylation has been recognized as an important feature in the initiation and progression of human cancer, the development of high density methylation arrays provides the possibility to analyze the methylation status of a vast amount of CpG islands of promoters of (tumor suppressor) genes and genes with a so far unknown role in leukemogenesis.

F. Nolte () Department of Hematology and Oncology, University Hospital Mannheim, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany Tel.: +49-621-383-6955 Fax: +49-621-383-6969 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_6, ©Â€Springer Science+Business Media B.V. 2011

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In the following article we will summarize and describe what is known about molecular changes in MDS and will in particular focus on the ‘new’ high throughput techniques.

 onventional Cytogenetics and Fluorescence   C In Situ Hybridization Chromosomal aberrations can be detected by conventional metaphase cytogenetics in about 50% of MDS cases. Detection of cytogenetic abnormalities is of prognostic and therapeutic relevance since it has been demonstrated that different abnormalities are associated with varying risks of evolution to AML and have a strong impact on the overall survival in these patients. Moreover, response to certain drugs has been linked to the presence of specific chromosomal aberrations in MDS such as deletion on the long arm of chromosome 5 or monosomy 7. Noteworthy, application of fluorescence in situ hybridization has been shown to provide additional information in MDS patients when conventional cytogenetics was unsuccessful but is of little value when applied in case of a cytogenetically normal karyotype [9]. The different chromosomal aberrations and their consequences are extensively discussed in this book by G. Mufti and D. Haase.

 lteration of Signal Transduction and Transcription A Factors Alteration of the RAS Pathway The RAS subfamily consists of the H-, N- and K-RAS genes (along with the RAL and RAP genes). Activating mutations, predominantly of N-RAS (in codons 12, 13 and 61) and less often of K-RAS (in codons 12 and 61), have been found in 10–15% of MDS but up to 40% in more advanced cases, and 15–25% of AML cases. Activation of RAS proteins leads to stimulation of its downstream targets with consecutive activation of transcription factors such as NF-IL6, ELK-1, c-Jun and c-Myc. MDS with mutant N-RAS may be associated with a worse prognosis. Moreover, RAS mutations have been linked to the progression to AML [3, 51, 55–57].

JAK2 Mutations Among the Jak kinase family JAK2 has recently gained a lot of interest since it seems to play a crucial role in the pathogenesis of myeloproliferative syndromes (MPS). Several groups [4, 32, 35] identified a valine to phenylalanin substitution at amino acid position 617 of JAK2 (JAK2V617F). This amino acid substitution is

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due to a G-to-T alteration in exon 12 of JAK2 which is located on the short arm of chromosome 9 [4]. Cells carrying JAK2V617F show a hypersensitivity to cytokines due to a constitutive phosphorylation activity of the kinase that promotes cytokine hypersensitivity [32]. JAK2V617F mutations are infrequent in typical MDS and are found in about 5% of cases [63, 68] (as compared to polycythaemia vera, essential thrombocythaemia and idiopathic myelofibrosis in which JAK2 is mutated in 97, 57 and 50% of cases, respectively). However, JAK2V617F is frequently found in the so called refractory anaemia with ringed sideroblasts and thrombocytosis (RARS-T) and is present in >60% of cases [6, 31, 62, 63, 68, 73, 74]. This entity contributes to the subgroup of overlapping syndromes MDS/MPS of the WHO classification presenting with both hallmarks of myelodysplastic and myeloproliferative syndromes. However, it still remains controversial whether RARS-T corresponds to two different simultaneous entities or to a unique disease.

AML1 Gene The human AML1 gene (also known as CBFA2 or RUNX1) encodes for one of two subunits of the human core binding factor (CBF). It is located on chromosome 21q22. AML1 is normally expressed in all hematopoietic lineages and acts to regulate the expression of various genes playing a pivotal role in myeloid differentiation [30]. Mutations of AML1 occur in about 25% of AML cases (mainly in AML-M0) and are frequently observed in MDS with excess of blasts [24]. Since sole loss of function of AML1 due to AML1 mutations has been shown not to be sufficient to induce MDS/ AML alone second hit events such as epigenetic alterations (methylation, deacetylation) could promote the development of AML1 mutation positive MDS/AML.

EVI1 Gene Ecotropic viral integration site 1 (EVI1) gene encodes for a large nuclear protein which has been shown to act as an aggressive oncoprotein. It is located on chromosome 3q26.2. There exist two forms of EVI1: the 145€kDa EVI1 gene and the larger MDS1/EVI1 gene which is a result of an in-frame splicing. EVI1 and MDS1/EVI1, respectively, are expressed in hematopoietic cells [54]. An inappropriate expression has been implicated in the development and progress of myeloid disorders, either AML or MDS [53]. Overexpression of EVI1 can result from 3q26 rearrangements such as translocations or inversions. However, several groups reported the frequent over expression of EVI1 in MDS without structural alteration of 3q26 [64, 75]. In MDS as in AML patients, EVI1 expression is a very poor prognostic marker and is associated with severe erythropoietin (EPO)-unresponsive anemia [61]. However, the mechanisms by which EVI1 alters gene expression and influences the biology of the disease are not yet fully understood.

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p53 Gene Alterations of the p53 gene occur in about 10% of MDS [41]. Changes are usually missense mutations of one allele with the second allele being lost. These mutations usually prevent the protein from being able to bind to DNA thus losing its ability to transactivate target genes such as the cyclin dependent kinase inhibitor (CDKI) p21WAF2. Mutations of p53 are associated with progression of the disease and poor prognosis.

TET2 Gene Several groups reported a high frequency of mutations of the tet oncogene family member 2 gene (TET2) in MDS and other myeloid malignancies. TET2 is mapped to chromosome 4q24 and frequently lies within a region of uniparental disomy as detected by SNP arrays. In MDS, TET2 mutations have been detected in 6–30% of MDS cases. Of importance, TET2 mutations have been identified as an independent favorable prognostic marker in MDS. Kosmider and co-workers [37] recently found that TET2 mutations in MDS were associated with a better overall survival after 5€years (77% with TET2 mutations [95% CI: 49–91] vs. 18% without TET2 mutations [95% CI: 4–41]) and a better 3-year leukemia-free survival in TET2 mutated patients (89% [95% CI: 63–97] vs. 63% [95% CI: 48–75]). In addition, the same group provided evidence, that TET2 mutations are in particular frequently found in patients with chronic myelomonocytic leukemia (CMML) [38]. However, the function of TET2 in normal hematopoiesis and leukemogenesis remains unclear. Since TET1 displays demethyl transferase activity TET2 might contribute to the pathogenesis of MDS by altering epigenetic patterns.

Epigenetic Alterations DNA Methylation and Histone Acetylation Methylation of CpG islands within gene promoters is a major epigenetic transcriptional control mechanism that is frequently dysregulated in human cancer and plays a critical role in the transcriptional silencing of tumor suppressor genes in cancer [26]. Several fundamental biological processes can be affected by this epigenetic event in human cancer such as DNA repair, cell cycle control, apoptosis and detoxification. Hypermethylation of CpG islands of genes involved in cell cycle control and apoptosis is a common feature particularly in high risk MDS. The cyclin dependent kinase inhibitors p15INK4b and p16INK4a are rarely mutated or deleted [50], but

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transcription of the p15INK4b gene is often silenced due to abnormal methylation of its promoter region and several studies indicate that roughly 50% of MDS patients show this alteration [59, 71]. Hopfer and co-workers [29] performed lineage specific promoter methylation analysis of key regulator genes involved in cell cycle control, DNA repair, apoptosis and differentiation. They could demonstrate an association of the methylation status of p16, survivin, CHK2 and WT1 with IPSS risk types. Moreover, for specific hematopoietic lineages and differentiation stages a methylation associated mRNA downregulation was shown, which provides evidence that lineage specific methylation followed by gene silencing contributes to MDS specific phenotypes and possibly to the different courses of MDS entities. DNA methylation seems to play a critical role in the progression of MDS into ouvert AML. Jiang and co-workers [34] used DNA methylation microarrays and compared the methylation status of early MDS samples to advanced MDS and AML samples and found an increase of methylated CpG loci in the MDS/AML group. Interestingly, chromosomal aberrations detected by SNP analysis showed no dramatic increase during progression (79% of early stage and 90% of advanced stage MDS). This led to the suggestion, that DNA methylation might be the dominant mechanism for MDS evolution to AML. In contrast to genetic deletions silencing of genes by DNA methylation is a reversible process and the introduction of demethylating agents (e.g. 5-azacytidine and 5-aza-2′-deoxycytidine) in the treatment of high risk MDS yielded encouraging results [10, 13, 66] by improving hematopoiesis and prolonging the survival time. Acetylation of amino acids of the histones catalyzed by histone acetyltransferases (HAT) leads to a destabilization of the histone-DNA interaction which in turn leads to an opening of the nucleosome structure and consecutively to a permission of transcription. These acetyl groups can be removed from histones by histone deacetylases (HDAC) which leads to a stabilization of the local chromatin structure and renders affected promoter sequences inaccessible resulting in silencing of genes in this region [14]. HDAC inhibitors such as valproic acid (VPA) were able to induce differentiation and apoptosis of leukemic cell lines in vitro. Kuendgen et€al. [39, 40] showed that VPA has some effect in MDS. Response rates were high (50%) in patients with a normal blast count in the bone marrow and a low IPSS score. Moreover, since it has been shown that DNA methylation and histone acetylation are closely connected, there is evidence that combination of VPA with demethylating agents has additional effects in advanced stages of MDS and AML [5, 19].

Array Comparative Genomic Hybridization (aCGH) in MDS Array comparative genomic hybridization (aCGH) is based on the comparison of fluorescence intensities between a test sample (e.g. MDS) and a reference sample (e.g. healthy) allowing for identification of copy number changes due to structural variations such as microdeletions.

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In MDS aCGH can provide additional information by identifying cryptic lesions in MDS patients. In 56% (17/30) of MDS patients Slovak and co-workers found new cytogenetic aberrations [67]. Moreover, in three patients cryptic RUNX1 deletions were found at the time of progression to AML, which underscores the importance of these high density approaches in the further characterization of MDS and identification of prognostic markers and therapeutic targets.

Single Nucleotide Polymorphism (SNP) Analysis in MDS As mentioned above, conventional cytogenetics yield genetic aberrations in only 50% of MDS patients. This may be due to the low resolution of routine metaphase cytogenetics. Moreover, metaphase karyotyping is restricted to dividing cells. Undisputable, the introduction of interphase fluorescent in situ hybridization (FISH) improved cytogenetic analysis. However, FISH utilizes a limited number of probes directed to already known lesions and therefore new chromosomal defects can not be detected. The introduction of high density single nucleotide polymorphism (SNP) microarrays offer the opportunity to perform a global analysis of genomic DNA and provide the possibility to detect so far cryptic lesions since they cover the whole genome and the resolution is much higher irrespective to cell division. Moreover, whole genome analysis by SNP microarrays allows for the detection of loss of heterozygosity (LOH) which arises either via hemizygous deletion, where a DNA segment is lost from one homolog while the other remains at one copy per cell, or uniparental disomy (UPD), wherein the retained homolog is duplicated so as to preserve two total copies per cell at the locus. Both types of somatic LOH have been observed in studies of various cancer types and may explain some of the mechanisms by which tumor suppressor genes are inactivated or activating mutations duplicated. Thus, analysis of recurrent LOH may point toward the presence of important recurrent mutations. Uniparental disomy (UPD) was initially described in developmental disorders. UPD occurs when an individual inherits both copies of chromosome pair from one parent and no copy from the other parent. If the two chromosome copies originate from the father it is called paternal UPD, if it originates from the mother it is called maternal UPD. UPD can occur either as a heterodisomy, in which sequences from both homologues from the transmitting parent are present or as isodisomy which refers to the situation in which two identical segments from the same homologue are present (see Fig.€6.1). In case of germline isodisomy two copies of a recessive mutation can be transmitted, which might result in pathological states. For several diseases homozygosity for a recessive mutation has been described such as cystic fibrosis, hemophila A and congenital adrenal hyperplasia. Heterodisomy as a result of germline alterations can cause abnormalities when the genes within the involved region are imprinted genes. Imprinting describes dif-

6╅ Molecular Changes in Myelodysplastic Syndrome Fig. 6.1↜渀 Uniparental disomy (UPD). UPD can occur due to somatic aberrations either by incomplete mitotic segregation or mitotic recombination. The most frequent mechanism leading to UPD is mitotic recombination, which can lead to UPD of a whole arm of a chromosome, of a part of an arm spanning to the telmere

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Both parental alleles present

Mitotic recombination

UPD of a whole arm or part to telomere

ferential gene expression which is dependent and specific for the transmitting parent. Disruption of normal imprinting due to UPD has been shown to play a role in several syndromes, such as the Prader-Willi syndrome and the Beckwith-Wiedemann syndrome. Beside the above mentioned germline alterations, UPD can occur due to somatic aberrations either by incomplete mitotic segregation or mitotic recombination. In case of UPD, the karyotype of affected patients appears normal when examined by conventional cytogenetics. This might be one reason why UPD played a minor role in concepts and models of tumorigenesis in the pre-microarray era. The introduction of microarrays allowed to perform high density whole genome SNP analysis and identification of regions displaying copy number changes and regions of copy number neutral changes such as UPD. UPD has been described in various myeloid and lymphoid malignancies and other neoplasms [15, 17, 23, 49, 60, 70].

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By using 250€K Sty chips Heinrichs et€al. [25] found regions of UPD in 6 out of 33 patients with MDS. Four patients had a normal bone marrow karyotype (in conventional cytogenetics), one patient had a complex karytype and one showed a t(11; 19) translocation. Since all regions of UPD extended entirely to the telomere Heinrichs et€al. suggested, that these regions of UPD resulted from a single recombination event. Remarkably, two patients with a normal karyotype showed regions of UPD on chromosome 7q. Moreover, although these patients had a low risk disease according to IPSS, they showed a deteriorating clinical course, underscoring that aberrations on chromosome 7 lead to a worse outcome. In addition, the presence of cytogenetically indetectable aberrations in MDS even in low risk patients could at least in part explain the wide intra-subtype variations concerning the prognosis despite patients belonging to the same IPSS risk group as impacted by conventional cytogenetics. Moreover, in an additional patient with a normal karyotype a UPD on 3q (the EVI1 gene is mapped to this region) was found and one patient with a complex karytype haboured a UPD on chromosome 17p both of which known to be associated with an adverse prognosis when cytogenetically abnormal. However, no follow up data about these two patients were reported. Compared to other groups, Heinrichs and co-workers [25] found less genomic changes in MDS with a normal ‘conventional’ karyotype and attributed this discrepancy to the lack of paired normal DNA samples in most previous analyses resulting in the detection of inherited copy number changes or regions of apparent homozygosity as somatically acquired genomic alterations within the MDS clone. Table€6.1 gives a brief overview of some regions of UPD in MDS reported so far. Langemeijer and co-workers [42] performed an extensive survey on 102 patients with MDS by using 250€K Nsp chips and sequencing techniques. In five patients they found large aberrations on the long arm of chromosome 4, four of them showed a UPD in this region and one a monoallelic deletion. These alterations could not be detected in a cohort of 1,015 healthy controls. Since the TET2 gene maps to the long arm of chromosome 4 all MDS samples were sequenced for TET2 mutations, which revealed TET2 mutations in 27 of 102 MDS patients (26%). The TET2 gene is ubiquitously and highly expressed in hematopoietic cells with the highest expression in granulocytes. Therefore, TET2 is suggested to function as a tumor suppressor gene. The exact function, however, remains elusive. The highest frequency of UPD in MDS has been reported by Mohamedali et€al. [47]. In 49% of 199 MDS patients they could detect regions of UPD of at least 2€Mb. UPD were most frequent in low risk MDS patients compared to high risk MDS. Furthermore, the same group has recently published data about the high prevalence of TET2 mutations in patients carrying a UPD4q [48]. Of nine patients four displayed a UPD4q24 (all of them had RCMD-RS) and one patient with a refractory anemia (RA) showed a UPD4q28. All of the RCMD-RS patients carried a major TET2 clone, whereas the patient with RA only yielded a very small TET2 mutated clone (13%). Since in CD3+ lymphocytes of one patient a small TET2 background clone was also detectable, it was suggested that TET2 mutations might be rather a first hit according to the Knodson model. Furthermore, Mohamedali et€al. [48]

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Table 6.1↜渀 Regions of uniparental disomy as detected by SNP array analysis in MDS Langemeijer Gondek et€al. Nowak et€al. Mohamedali Heinrichs [21] [52] et€al. [47] et€al. [42] et€al. [25] System used 250€K (Sty) 250€K (Nsp) 250€K (Nsp) 500€K 250€K (Sty) (Nsp+Sty) 500€K (Nsp+Sty) No. of patient 51 (according 102 (according 94 (according 13 (according 119 (according analyzed to WHO) to FAB) to WHO) to FAB) to WHO) 33 (65%) 62 (60%) 38 (40%) 9 (69%) 77 (65%) No. of pts. with normal karytype (NC) (%) UPD total 6 (12%) 9 (9%) 18 (19%) 13 (100%) 55 (46%) (%) 4 (12%) 5 (8%). 8 (21%) 9 (100%) 80 (67%) UPD in NC (%) UPD 7q in NC 2 (6%) 2 (3%) 1 (3%) – – (%) Total UPD4q – 4 (4%) – – 7 (9%) (%) UPD4q in NC 1 (3%) 2 (3%) – – – (%) UPD3q in NC 1 (3%) – 1 (3%) – – (%) – means either not done or no exact data available

hypothesized that TET2 mutations might be restricted to RCMD-RS patients based on the observation that RCMD-RS patients were exclusively affected in their cohort. However, other groups found a high frequency in other myeloid malignancies suggesting that TET2 mutations might be a common initiating defect in myeloid neoplasms. Jankowska and co-workers [33] found TET2 frequently mutated not only in MDS but especially in patients with overlap syndromes (MDS/myeloproliferative neoplasia [MPN]) and secondary AML, a finding which is in accordance to data published by other groups [1, 21]. In conclusion, although only few data on SNP analysis by high throughput techniques have been published, the data reported so far are encouraging, since the use of SNP arrays led to the identification of new genomic alterations and in case of the TET2 gene discovered a new tumor suppressor gene possibly involved in myeloid malignancies.

Gene Expression Analysis by Microarrays in MDS As mentioned previously, diagnosis of MDS relies on morphological and cytogenetical findings. However, morphological diagnosis is often difficult in MDS and depends on the experience of the hematologist and pathologist and in about 50% of

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MDS cases conventional cytogenetics show a normal karyotype. Gene expression profiling (as SNP analysis or methylation analysis) holds the promise to base the diagnosis on objective criteria such as gene expression patterns and become autonomous from observer-dependent criteria such as blast count and degree of dysplasia. Recently, Mills and co-workers [45] assessed the clinical utility of a microarraybased gene expression profiling (GEP) assay in the diagnosis and subclassification of 16 clinically recognized subtypes of acute and chronic leukemia and found an accuracy for subtyping leukemia of approximately 95%. However, the accuracy concerning MDS subtyping was only 50% whereas discordant samples were either classified as leukemia or “non-of-the-targets” (neither leukemia nor MDS). Interestingly, patients that were categorized as MDS by conventional diagnostics (morphology, cytogenetics) but classified as AML by GEP showed a deteriorating course of their disease with a rapid development of AML. Beside diagnosis, microarray techniques provide the possibility to identify genomic changes not detectable by conventional molecular approaches. Moreover, it has become clear, that altered gene expression of oncogenes or tumor suppressor genes, respectively, is not necessarily due to loss or gain of genetic material (i.e. deletions or amplifications) but a result of promoter methylation or histone deacetylation. Gene expression analysis by high density microarrays became powerfull tools to further elucidated the pathobiology not only of MDS but other hematologic malignancies and cancers. By performing gene expression analysis of isolated CD34+ cells from 122 MDS patients Pellegatti et€al. [58] identified the lymphoid enhancer-binding factor 1 (LEF1) as the most significant differentially expressed gene between early and advanced MDS. Decreasing LEF1 expression levels were associated with disease evolution and Pellagatti et€al. [58] suggested LEF1 as a molecular marker of disease progression. Several attempts have been and will be undertaken to identify gene expression patterns that could predict whether patients will respond to a certain drug or benefit from a chosen treatment. We and others have shown that microarray analysis can provide sufficient data to detect genes or gene patterns which are associated with alterations of specific cellular pathways or signal cascades in tumor cells including MDS [28, 43, 46]. The technique of gene expression profiling can also be used for subclassification of leukemias [65] and lymphomas [2]. However, the rapid progress in microarray techniques might further improve this direction of molecular diagnosis.

Next Generation Sequencing (NGS) The classical automated Sanger sequencing technique has led to major accomplishments in molecular research including the completion of the human genome sequence. Automated Sanger sequencing is referred to as the “first generation”

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technology. Development of newer technologies (referred to as “next generation”) provide the possibility to cheaply sequence the whole genome of many different individuals for example MDS patients in an attempt to enhance our understanding of how (subtle) genetic differences affect health and disease. Different NGS platforms do exist that differ with regard to template preparation procedures (emulsion PCR, solid-phase amplification etc.) and sequencing and imaging techniques (cyclic reversible termination, pyrosequencing etc.). Interested readers are directed to the excellent review by Metzker [44]. The possible impact these new techniques might have on the further research and classification of MDS was recently demonstrated by Grossmann and co-workers who screened 81 patients with chronic myelomonocytic leukemia for oncogene aberrations including the CBL, RUNX1, JAK2, MPL, KRAS, NRAS and TET2 gene [22]. In 12 of these patients known mutations detected by conventional methods (conventional sequencing or melting curve analyses) could be confirmed by NGS. Moreover, in patients without any known mutation either point mutations or large deletions were identified in all candidate genes. In only 20 of 81 CMML patients no mutation could be detected by NGS. These results give an impression what will be possible in the future in further molecular characterization of the MDS and other malignant diseases.

Summary MDS are heterogenous diseases. Several molecular aberrations have been described but none of them are specific for MDS only but occur in other myeloid neoplasms as well (see Table€6.2). The introduction of high-throughput technologies will enable researchers to go into further molecular detail aiming at the identification of subtle molecular defects which will hopefully lead to elucidation of underlying alterations and discovery of potential therapeutic targets. Table 6.2↜渀 Frequent gene mutations in MDS and other myeloid malignancies Gene Myeloid entity MDS AML MPN CMML TET2 JAK2 RAS AML1 ASXL1

10–25% [33, 37] 3% [63, 68] 10–15% [57] 15% [24] 11–17% [7]

12% [11] <5% [72] 15–25% [51] 13–25% [69, 24] 18% [7]

10–15% [11] 74% [35] 0% [18, 27] – 8% [8]

20–40% [11, 37] 6% [35] 57% [27] 19% [12] 43% [20]

RARS-T 26% [16] 79% [63, 68] – – 8% [68]

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References ╇ 1. Abdel-Wahab O et€al (2009) Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies. Blood 114:144–147 (July) ╇ 2. Alizadeh AA et€al (2000) Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403:503–511 (Feb) ╇ 3. Bacher U et€al (2007) A comparative study of molecular mutations in 381 patients with myelodysplastic syndrome and in 4130 patients with acute myeloid leukemia. Haematologica 92:744–752 (June) ╇ 4. Baxter EJ et€al (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365:1054–1061 (March) ╇ 5. Blum W et€al (2007) Phase I study of decitabine alone or in combination with valproic acid in acute myeloid leukemia. J Clin Oncol 25:3884–3891 (Sept) ╇ 6. Boissinot M et€ al (2006) The JAK2-V617F mutation and essential thrombocythemia features in a subset of patients with refractory anemia with ring sideroblasts (RARS). Blood 108:1781–1782 (Sept) ╇ 7. Boultwood J et€al (2009) Frequent mutation of the polycomb-associated gene ASXL1 in the myelodysplastic syndromes and in acute myeloid leukaemia. ASH Annu Meet Abstr 114:419 (Nov) ╇ 8. Carbuccia N et€al (2009) Mutations of ASXL1 gene in myeloproliferative neoplasms. Leukemia 23:2183–2186 (Nov) ╇ 9. Costa D et€al (2010) Do we need to do fluorescence in situ hybridization analysis in myelodysplastic syndromes as often as we do? Leuk Res 34(11):1437–1441 10. Daskalakis M et€al (2002) Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2′-deoxycytidine (decitabine) treatment. Blood 100:2957–2964 (Oct) 11. Delhommeau F et€al (2009) Mutation in TET2 in myeloid cancers. N Engl J Med 360:2289– 2301 (May) 12. Ernst T et€ al (2010) Transcription factor mutations in myelodysplastic/myeloproliferative neoplasms. Haematologica 95(9):1473–1480 (April) 13. Fenaux P et€al (2009) Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 10:223–232 (March) 14. Fenrick R, Hiebert SW (1998) Role of histone deacetylases in acute leukemia. J Cell Biochem Suppl 30–31:194–202 15. Fitzgibbon J et€al (2005) Association between acquired uniparental disomy and homozygous gene mutation in acute myeloid leukemias. Cancer Res 65:9152–9154 (Oct) 16. Flach J et€al (2010) Mutations of JAK2 and TET2, but not CBL are detectable in a high portion of patients with refractory anemia with ring sideroblasts and thrombocytosis. Haematologica 95:518–519 (March) 17. Forconi F et€al (2008) Genome-wide DNA analysis identifies recurrent imbalances predicting outcome in chronic lymphocytic leukaemia with 17p deletion. Br J Haematol 143:532–536 (Nov) 18. Gaidano G et€ al (1993) Mutations in the P53 and RAS family genes are associated with tumor progression of BCR/ABL negative chronic myeloproliferative disorders. Leukemia 7:946–953 (July) 19. Garcia-Manero G et€al (2006) Phase 1/2 study of the combination of 5-aza-2′-deoxycytidine with valproic acid in patients with leukemia. Blood 108:3271–3279 (Nov) 20. Gelsi-Boyer V et€al (2009) Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. Br J Haematol 145:788–800 (June) 21. Gondek LP et€al (2008) Chromosomal lesions and uniparental disomy detected by SNP arrays in MDS, MDS/MPD, and MDS-derived AML. Blood 111:1534–1542 (Feb)

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22. Grossmann V et€al (2009) Next-generation sequencing (NGS) in CMML, MDS and AML detects molecular mutations in oncogenes and allows the identification of balanced chromosomal abnormalities with extraordinary sensitivity and specificity. ASH Annu Meet Abstr 114:144 (Nov) 23. Gupta M et€al (2008) Novel regions of acquired uniparental disomy discovered in acute myeloid leukemia. Genes Chromosomes Cancer 47:729–739 (Sept) 24. Harada H, Harada Y, Niimi H, Kyo T, Kimura A, and Inaba T (2004) High incidence of somatic mutations in the AML1/RUNX1 gene in myelodysplastic syndrome and low blast percentage myeloid leukemia with myelodysplasia. Blood 103:2316–2324 25. Heinrichs S et€al (2009) Accurate detection of uniparental disomy and microdeletions by SNP array analysis in myelodysplastic syndromes with normal cytogenetics. Leukemia 23:1605– 1613 (Sept) 26. Herman JG, and Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349:2042–2054 27. Hirsch-Ginsberg C et€al (1990) RAS mutations are rare events in Philadelphia chromosomenegative/bcr gene rearrangement-negative chronic myelogenous leukemia, but are prevalent in chronic myelomonocytic leukemia. Blood 76:1214–1219 (Sept) 28. Hofmann WK et€al (2002) Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow. Blood 100:3553–3560 (Nov) 29. Hopfer O et€al (2009) Aberrant promotor methylation in MDS hematopoietic cells during in vitro lineage specific differentiation is differently associated with DNMT isoforms. Leuk Res 33:434–442 (March) 30. Ichikawa M et€al (2004) Runx1/AML-1 ranks as a master regulator of adult hematopoiesis. Cell Cycle 3:722–724 (June) 31. Ingram W et€al (2006) The JAK2 V617F mutation identifies a subgroup of MDS patients with isolated deletion 5q and a proliferative bone marrow. Leukemia 20:1319–1321 (July) 32. James C et€al (2005) A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434:1144–1148 (April) 33. Jankowska AM et€al (2009) Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms. Blood 113:6403–6410 (June) 34. Jiang Y et€al (2009) Aberrant DNA methylation is a dominant mechanism in MDS progression to AML. Blood 113:1315–1325 (Feb) 35. Jones AV et€al (2005) Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 106:2162–2168 (Sept) 36. Knudson AG (1996) Hereditary cancer: two hits revisited. J Cancer Res Clin Oncol 122:135– 140 37. Kosmider O et€ al (2009) TET2 mutation is an independent favorable prognostic factor in myelodysplastic syndromes (MDS). Blood 114:3285–3291 (Aug) 38. Kosmider O et€ al (2009) TET2 gene mutation is a frequent and adverse event in chronic myelomonocytic leukemia. Haematologica 94(12):1676–1681 (Oct) 39. Kuendgen A et€al (2004) Treatment of myelodysplastic syndromes with valproic acid alone or in combination with all-trans retinoic acid. Blood 104:1266–1269 (Sept) 40. Kuendgen A et€al (2005) Results of a phase 2 study of valproic acid alone or in combination with all-trans retinoic acid in 75 patients with myelodysplastic syndrome and relapsed or refractory acute myeloid leukemia. Ann Hematol 84(Suppl 1):61–66 (Dec) 41. Lai JL et€al (1995) Myelodysplastic syndromes and acute myeloid leukemia with 17p deletion. An entity characterized by specific dysgranulopoiesis and a high incidence of P53 mutations. Leukemia 9:370–381 (March) 42. Langemeijer SM et€al (2009) Acquired mutations in TET2 are common in myelodysplastic syndromes. Nat Genet 41:838–842 (July) 43. Lee YT et€al (2001) Transcription patterning of uncoupled proliferation and differentiation in myelodysplastic bone marrow with erythroid-focused arrays. Blood 98:1914–1921 (Sept) 44. Metzker ML (2010) Sequencing technologies—the next generation. Nat Rev Genet 11:31–46 (Jan)

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45. Mills KI et€al (2009) Microarray-based classifiers and prognosis models identify subgroups with distinct clinical outcomes and high risk of AML transformation of myelodysplastic syndrome. Blood 114:1063–1072 (July) 46. Miyazato A et€al (2001) Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction. Blood 98:422–427 (July) 47. Mohamedali A et€al (2007) Prevalence and prognostic significance of allelic imbalance by single-nucleotide polymorphism analysis in low-risk myelodysplastic syndromes. Blood 110:3365–3373 (Nov) 48. Mohamedali AM et€al (2009) Novel TET2 mutations associated with UPD4q24 in myelodysplastic syndrome. J Clin Oncol 27:4002–4006 (Aug) 49. Mullighan CG et€al (2007) Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature 446:758–764 (April) 50. Nakamaki T et€al (1997) Molecular analysis of the cyclin-dependent kinase inhibitor genes, p15, p16, p18 and p19 in the myelodysplastic syndromes. Leuk Res 21:235–240 (March) 51. Neubauer A et€al (1994) Prognostic importance of mutations in the ras proto-oncogenes in de novo acute myeloid leukemia. Blood 83:1603–1611 (March) 52. Nowak D et€al (2009) Genome-wide DNA-mapping of CD34+ cells from patients with myelodysplastic syndrome using 500€K SNP arrays identifies significant regions of deletion and uniparental disomy. Exp Hematol 37:215–224 (Feb) 53. Nucifora G, Laricchia-Robbio L, Senyuk V (2006) EVI1 and hematopoietic disorders: history and perspectives. Gene 368:1–11 (March) 54. Ohashi H et€al (2001) Relationship between methylation of the p15 gene and ectopic expression of the EVI-1 gene in myelodysplastic syndromes (MDS). Leukemia 15:990–991 (June) 55. Padua RA, West RR (2000) Oncogene mutation and prognosis in the myelodysplastic syndromes. Br J Haematol 111:873–874 (Dec) 56. Padua RA et€al (1998) RAS, FMS and p53 mutations and poor clinical outcome in myelodysplasias: a 10-year follow-up. Leukemia 12:887–892 (June) 57. Paquette RL et€al (1993) N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome. Blood 82:590–599 (July) 58. Pellagatti A et€al (2009) Marked downregulation of the granulopoiesis regulator LEF1 is associated with disease progression in the myelodysplastic syndromes. Br J Haematol 146:86– 90 (June) 59. Quesnel B et€al (1998) Methylation of the p15(INK4b) gene in myelodysplastic syndromes is frequent and acquired during disease progression. Blood 91:2985–2990 (April) 60. Raghavan M et€al (2005) Genome-wide single nucleotide polymorphism analysis reveals frequent partial uniparental disomy due to somatic recombination in acute myeloid leukemias. Cancer Res 65:375–378 (Jan) 61. Raza A et€ al (2004) Arsenic trioxide and thalidomide combination produces multi-lineage hematological responses in myelodysplastic syndromes patients, particularly in those with high pre-therapy EVI1 expression. Leuk Res 28:791–803 (Aug) 62. Remacha AF et€al (2006) Occurrence of the JAK2 V617F mutation in the WHO provisional entity: myelodysplastic/myeloproliferative disease, unclassifiable-refractory anemia with ringed sideroblasts associated with marked thrombocytosis. Haematologica 91:719–720 (May) 63. Renneville A et€al (2006) High occurrence of JAK2 V617 mutation in refractory anemia with ringed sideroblasts associated with marked thrombocytosis. Leukemia 20:2067–2070 (Nov) 64. Russell M et€al (1994) Expression of EVI1 in myelodysplastic syndromes and other hematologic malignancies without 3q26 translocations. Blood 84:1243–1248 (Aug) 65. Schoch C et€al (2002) Acute myeloid leukemias with reciprocal rearrangements can be distinguished by specific gene expression profiles. Proc Natl Acad Sci U S A 99:10008–10013 (July) 66. Silverman LR et€al (2002) Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B. J Clin Oncol 20:2429– 2440 (May)

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67. Slovak M et€ al (2009) C008 array-based comparative genomic hybridization as a clinical assay for genomic profiling in the myelodysplastic syndromes: validation by comparison with conventional cytogenetics and fluorescence in situ hybridization. Leuk Res 33:S35–S36 (May) 68. Szpurka H et€al (2006) Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), another myeloproliferative condition characterized by JAK2 V617F mutation. Blood 108:2173–2181 (Oct) 69. Tang JL et€ al (2009) AML1/RUNX1 mutations in 470 adult patients with de novo acute myeloid leukemia: prognostic implication and interaction with other gene alterations. Blood 114:5352–5361 (Dec) 70. Tyybakinoja A et€al (2008) Single nucleotide polymorphism microarray analysis of karyotypically normal acute myeloid leukemia reveals frequent copy number neutral loss of heterozygosity. Haematologica 93:631–632 (April) 71. Uchida T et€ al (1997) Hypermethylation of the p15INK4B gene in myelodysplastic syndromes. Blood 90:1403–1409 (Aug) 72. Vainchenker W, Dusa A, Constantinescu SN (2008) JAKs in pathology: role of Janus kinases in hematopoietic malignancies and immunodeficiencies. Semin Cell Dev Biol 19:385–393 (Aug) 73. Wang SA et€al (2006) Refractory anemia with ringed sideroblasts associated with marked thrombocytosis harbors JAK2 mutation and shows overlapping myeloproliferative and myelodysplastic features. Leukemia 20:1641–1644 (Sept) 74. Zipperer E et€ al (2008) MPL 515 and JAK2 mutation analysis in MDS presenting with a platelet count of more than 500â•›×â•›10(9)/l. Ann Hematol 87:413–415 (May) 75. Zoccola D et€al (2003) A discriminating screening is necessary to ascertain EVI1 expression by RT-PCR in malignant cells from the myeloid lineage without 3q26 rearrangement. Leukemia 17:643–645 (March)

Chapter 7

Prognostic Scoring in MDS Michael Pfeilstöcker

Introduction The management of patients with Myelodysplastic Syndromes is challenging. This is especially true in present times when a multitude of new promising therapeutic approaches becomes available and change from supportive care only to effective targeted treatment for most of the patients is on the horizon [86]. Difficulties and importance of patient assessment stems at least in part from the high degree of heterogeneity of the many different conditions grouped together under the name MDS based on common characteristics such as morphological findings or genetic changes [3, 39, 84, 89]. Lessons learned from the elucidation of pathophysiological processes as well as insights into the mechanisms of action of new therapies have to be incorporated for optimal patient management. Different pathophysiology in different patients warrants adapted treatment approaches, but choice depends not only from a possible benefit of treatment options, also possible toxicities of interventions have to be considered; the risk-benefit ratio has to be assessed using comparison with the projected natural course of the disease. Pathogenetic processes identified include genetic, epigenetic and immune-mediated mechanisms [86]. Despite these findings, diagnosis of MDS relies still mostly on morphological and clinical features. In absence of methods characterising individual pathophysiological pathways in individual patients and in the light that classifications of MDS do provide prognostic information but still leave space for improvement further instruments have been developed for estimation of patient’s prognosis or prediction of response to specific therapies. New parameters were generally described in a univariate pattern first, later incorporation in so called prognostic scores was performed. Classifications and prognostic instruments for MDS developed in the last decades reflect our ever M. Pfeilstöcker () 3rd Medical Department and L. Boltzmann Institute for Leukemia Research & Hematology, Hanusch Hospital, H. Collinstr 30, 1140 Vienna, Austria Tel.: +43-1-91021-85430 Fax: +43-1-91021-85439 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_7, ©Â€Springer Science+Business Media B.V. 2011

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evolving knowledge of MDS biology and have contributed to the successful implementation of new therapies. They provide the tools for adequate risk evaluation and treatment selection.

Aims and Criteria for Prognostic Factors The aims of prognosis may be defined by several aspects: the criterion (endpoint), the conditions of prognosis (e.g. type of patients analyzed, time point of testing etc.) and the intended level of use (description of a population series or prognostication in individual patients). The most common criteria used are overall survival and leukaemia free survival [2, 7, 24, 29, 44, 54, 61, 63, 69, 71, 73] whereas most prognostic systems are not specifically adapted to one of them. Further criteria could include disease related mortality and quality of life adjusted survival time, the latter may be especially relevant to patients. Prognostic systems for MDS are almost exclusively based on data of primary and untreated patients. They describe what could be seen as the “natural course of disease”, leaving aside the considerable influence of supportive care. Implicitly this estimates a risk inherent to the disease without considering possible effects of treatment. A comprehensive prognostic system instead would allow for prognosis under different conditions, including not only disease and patient features, but therapeutic options as well [96]. Prognostic systems may be used on the level of individual prognosis or as stratification variables in clinical trials and meta analyses. For individual prognosis current systems only permit estimation within large boundaries [71]. Candidate prognostic factors should meet several criteria. The feature, its measurement and its interpretation should be precisely defined and it should be reliable to measure, common availability and easy assessment are certainly advantages. A rationale linking to clinical outcome is not imperative but desirable. As long as the resulting model is understood as prognostic, without being causally explanatory, the factor needs not to be causal. If the measurement is categorised, categories should be clinically meaningful [97]. Ideally features should be independent of therapeutic options, changing clinical rules or subjective impression of the physician (e.g. when using transfusion needs). Variability in time has to be valuated depending on the aim of the prognostic model. Long term prognosis is preferably based on relatively stable features (e.g. cytogenetics), while short term prognosis may include more condition dependent, and therefore unstable characteristics [87]. Clinical use of a prognostic system combining more than one prognostic feature requires first of all a comprehensible measure of its prognostic power [34], also the target population has to be stated [42], as otherwise physicians cannot responsibly rely on it. Further the model has to be transparent regarding its components and score assignments to prevent “double counting” or neglect of features in a clinical decision based on prognostic system and further characteristics as observed by the physician [60]. Prognostic systems

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should be validated after its construction and publication, preferably by different researchers at different centers and thus on independent data [73].

Prognostic Factors in MDS A specific diagnostic parameter unequivocally establishing the diagnosis MDS is still lacking [86], such a parameter if meeting the requirements for prognostic factors as described above would represent the ideal prognostic marker. In absence of such a marker surrogate parameters are available, Table€7.1 provides an overview of prognostic markers used or proposed for MDS. Whereas some features such as bone marrow morphology parameters are clearly disease related, others such as age, gender and co-morbidity are clearly patient related. Parameters such as the performance status may be influenced by both consequences of the disease or the presence of co-morbidities. Most parameters may be subject to changes over time (evolution of the MDS disease, acquisition of further co-morbidities, increasing age), whereas others such as gender remain invariable. Parameters can be tested easily and reliably e.g. LDH, whereas others depend on the quality of the sample taken (e.g. bone marrow cellularity) or the subjective judgement of the persons assessing morphological features. Table 7.1↜渀 Prognostic factors in MDS Clinical features Performance status Age Gender Previous therapy (secondary MDS, treatment related MDS) Transfusion dependency Co-morbidity Morphological features Dysplasia, degree of dysplasia, unilineage versus multilineage Blast cell counts (bone marrow, peripheral blood) Cellularity Fibrosis ALIPs Eosinophils, basophils Classifications FAB WHO 2001 WHO 2008 Cytopenias Anemia, neutropenia, thrombocytopenia Chemistry LDH, ferritin Cytogenetics Aberrations Clonal evolution Molecular features Mutations Gene-expression profiles Epigenetics (methylation pattern, etc…) FACS parameters

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Morphology Morphologic features are used for separation of MDS from other entities and have become hallmarks of MDS classifications [3, 39, 84]. Classifications require bone marrow dysplasia and blast percentages besides other parameters such as cytogenetics and cytopenias. These features not only establish a diagnosis of MDS but also have prognostic significance. When MDS as a distinguishable clinical entity was established, different prognosis of patients according to blast counts was already evident and presence of ringed sideroblasts established an entity with better prognosis compared to refractory anemia [3, 13]. The value of consideration of multilineage dysplasia for prognostic purposes was shown in several case series demonstrating both shorter survival and increased risk of AML evolution [25, 37, 55]. Accordingly RA and RARS were stratified based on the presence of multilineage dysplasia in the WHO 2001 classification [39]. For RARS this stratification was abandoned in the WHO 2008 classification reuniting RARS and RCMD-RS [84]. Bone marrow biopsy can provide further diagnostic and prognostic information. Among others a specific feature—abnormal localization of immature precursors (ALIPs) was proposed as prognostic factor [56]. Another feature is presence of fibrosis in dysplastic marrow. Marrow fibrosis may predict early fatal marrow failure in MDS [5, 49] and increased staining for reticulin has been shown to carry independent prognostic information especially in a distinct subgroup with multilineage dysplasia and high transfusion requirement [16]. Furthermore, the presence of CD34+ cell clusters was confirmed as an independent risk factor for progression to acute leukemia [16].

Cell Counts Cell counts in peripheral blood especially cytopenias such as anemia, neutropenia and thrombocytopenia but also elevated cell counts such as monocytosis have been considered to be of prognostic value since the conception of MDS as an entity [3]. Despite this long experience there is still a debate on the inclusion of cytopenias of specific cell lineage in prognostic systems or on threshold values to be used [2, 7, 24, 29, 44, 54, 61, 63, 69, 71, 73]. Recently the additional prognostic role of eosinophil and basophil counts was observed, eosinophilia and basophilia predicted a significantly reduced overall survival without a significant impact on leukemia-free survival [93].

Other Laboratory Parameters As there is no marker unequivocally specific for MDS, laboratory parameters derived from blood chemistry underlie possible influence from other (patho)physi-

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ological processes. Examples are measurements of lactate-dehydrogenase (LDH) and ferritin. LDH values may reflect increased cellular turnover in the bone marrow and therefore disease activity of MDS, but may also be influenced by concomitant heart or liver diseases. Ferritin as a possible marker of iron overload due to changes in iron metabolism and chronic transfusion in MDS may also be increased in inflammation and infection. Despite these limitations the prognostic value of both parameters has been demonstrated: LDH is a prominent feature in the Düsseldorf scoring system [2]. Its prognostic power has been examined as a static and dynamic variable [94, 95] and has been used to add prognostic significance to the International prognostic scoring system (IPSS) [26]. The Spanish group showed in a large retrospective study that iron overload reflected by increased ferritin values is of independent prognostic importance [74] although there is still ongoing debate on this topic [10, 81].

Cytogenetics Chromosomal aberrations are a hallmark of MDS, identified in approximately half of all de novo MDS patients [62]. Due to the heterogeneity of the disease the MDS literature harbours an ever increasing number of recurrent abnormalities [32, 77]. The worse prognosis of complex cytogenetics has been established early and introduced into prognostic scores [61, 69]. In addition specific favourable single aberrations such as del(5)(q) [91], molecularly characterised in 2008 [18], or del(20)(q) [92] were described. Patients with isolated del(5)(q) and specific characteristics (<5% blasts in the bone marrow without Auer rods, thrombocytosis, typical dysmegakaryopoiesis, macrocytic anemia), named 5q- syndrome in the WHO classification [39, 84] typically have a low frequency of progression to AML and favourable survival compared with other MDS subgroups. Specific aberrations may carry different prognostic significance according to the complete karyotype. The good prognosis of isolated del(5)(q) is reversed if this aberration is part of a complex karyotype [28]. In the IPSS all chromosome 7 aberrations were set in the poor risk group [29]. This seems confirmed for monosmy 7, but not for translocations involving the long arm of this chromosome [32]. Finally recurrent cytogenetic aberrations were grouped according to their prognostic significance. The number of patients included in the case series used for establishing cytogenetic prognostic groups aberrations enables the refinement of cytogenetics as prognostic factor. Whereas the significance of complex karyotype was derived from series of less than 400 patients [61, 69], about the same number of patients that was used to construct the piscore [71], a purely cytogenetic score; more than 800 patients were included for establishing the more comprehensive IPSS [29], that identifies three cytogenetic subgroups. Further multicenter collaboration enabled identification of 684 different cytogenetic categories in 2,124 patients [32] that may be grouped in four or five prognostic subgroups [33]. Cytogenetics is a key variable of currently used MDS prognostic scoring systems, both the pres-

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ence and type of chromosomal alteration are of importance. In the IPSS a normal karyotype or evidence of -Y, del(5q), or del(20q) is classified as good risk, while the presence of chromosome 7 or complex abnormalities represents poor risk. All other abnormalities, constituting a very heterogeneous group, are considered intermediate risk [29]. The same criteria are also used in the WHO based prognostic scoring system (WPSS) [54]. Significance of the WPSS validates the IPSS risk groups, but more advanced cytogenetic risk data suggest, that an improved prognostication for MDS may be achieved by inclusion of more detailed cytogenetic data [32, 33, 77]. Cytogenetic evolution may be associated with a more progressive clinical course. A study on 153 patients showed that cytogenetic evolution significantly affected overall and progression free survival independently of other prognostic variables, interestingly del(7)(q) was the only secondary chromosomal defect which significantly affected progression free survival whereas trisomy 8 had only a moderate influence [4]. Besides prognosis, in recent years the specific predictive significance (i.e. predicting response to therapy) of certain aberrations has become evident. Positive predictive value has been described for del(5)(q) in transfusion dependent low or intermediate risk patients as a predictor for response to the immunomodulatory drug lenalidomide. In these cases not only hematologic responses, but also frequent cytogenetic remissions can be achieved [20, 51, 52]. Additional chromosomal aberrations as described above have also influence on the predictive information. In complex karyotypes involving del(5)(q) responses to lenalidomide treatment were more modest [52]. Evidence of a complex karyotype also predicts poor response to chemotherapy [46]. Interestingly epigenetic therapy with 5-Azacitidine retains a significant benefit also in cytogenetic subgroups previously considered high risk [21]. Concerning methodology, using cytogenetics as prognostic factor in MDS warrants standard metaphase cytogenetic analysis with certain quality standards [83]. Fluorescence in situ hybridization (FISH) may be useful to assess chromosomal changes in MDS not detected by standard cytogenetic analysis, particularly in cases with insufficient numbers of metaphases. However only alterations at defined loci can be detected by FISH, and specific FISH findings cannot rule out further aberrations. Therefore at present this technique can only be used as adjunct of conventional cytogenetics also in the setting of prognostication. Current MDS prognosis systems do not yet incorporate FISH or other molecular analyses.

Further Genetic Prognostic Factors The knowledge on MDS pathophysiology has been improved further during the last years. This is in part due to the refinement of methods elucidating these mechanisms. Classic Metaphase cytogenetics has been complemented by FISH as described above. A higher resolution allowing detection of minimal deletions is ob-

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tained by the SNP array technology. In addition gene expression profiling (GEP) has also been used in MDS. All new methods have been used to find new relevant pathways of MDS pathobiology as well as tools for separating MDS from other diseases or establishing meaningful MDS subtypes. A possible prognostic or predictive value has also been investigated. So identification of prognostic relevant deletions by SNP arrays has been demonstrated: Uniparental disomy on 4q was identified in a proportion of low risk MDS with normal cytogenetics with prognostic relevance in the univariate setting [59]. GEP studies have seperated MDS samples from normal ones or AML and identified new MDS relevant genes. Some studies were able to correlate profiles with conventional prognostic factors or scores [11, 35, 58, 70, 88]. In the MILE Study correlation of GEP defined MDS with cases according to classic morphologic classification was only observed in 50% of MDS (versus 90% in AML), others having an GEP of AML or “none-of-the-targets samples” type. However, in those identified as MDS a significant correlation of these samples with prognosis was observed, allowing the establishment of a classification model with prognostic relevance [57]. GEPs may also carry a predictive value, as shown for the erythroid response to lenalidomide in del(5)(q) patients [17], data still to be confirmed. A number of single genetic alterations have been assessed also for their predictive and prognostic significance. An example is the TET2 gene that has been analysed in different hematopoietic neoplasias [14, 85]. Its function—conversion of methylcytosine residues to the respective hydroxymethyl forms—has recently been elucidated [47], therefore a role in epigenetic instability has been proposed [23]. In MDS prognosis is better in patients carrying a mutated TET2 [48], of interest is a possible additional predictive effect for response to epigenetic therapy [38, 40]. Space restrictions do not allow a further comprehensive review of all genes found to be relevant in at least some MDS cases, for further data see chap. 7 (molecular changes in myelodysplastic syndromes) and [86]. The field of epigenetics has attained wide interest especially due to effective therapeutic interventions using epigenetic therapies [19]. In MDS development dynamic changes of DNA-methylation have been demonstrated [36]. DNA methylation as one of the epigenetic mechanisms can be assessed by different methods: screening methylation patterns of individual genes or assessment of whole methylation profiles of bone marrow cells. Results have been tested for a possible prognostic or predictive use: first data showed, that median overall survival in patients with MDS correlates with the methylation status of p15INK4b [72], a prognostic role of methylation was confirmed in many other genes such as FZD9 [41]. As a possible predictive factor DNA hypermethylation has been shown to be associated with poor response to intensive chemotherapy in older patients with high-risk MDS [30]. Concerning prediction of response to demethylating agents reduced methylation of phosphoinositide-phospholipase C beta1 was associated with clinical response in high risk MDS [22], however data presented at ASH 2009 could not achieve prediction of response using global DNA methylation measurements [6]. So a clear correlation of methylation with response to methylating agents cannot be considered to be definitely established to date.

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FACS Flow cytometry can help in the diagnostic process of MDS and standardization of flow methods in MDS is underway: several cytometric scoring systems have been proposed [12, 53, 76], the recently published report of an ELN working conference describes a consensus on sampling, handling and processing as well as definitions of minimal combinations of antibodies to define dysplasia [90]. Flow results correlate well with morphology, subclassification and prognostic factors, but further prospective validation and standardization in multicenter studies is still required. Particularly in patients otherwise considered low risk flow cytometry could be beneficial [76], but sensitivity and specificity are still inconsistent in different series [67]. FACS analysis has not yet been included in the WHO classification system, but will certainly add prognostic information in the future.

Transfusion Dependency As stated above cytopenias are of prognostic importance, and anemia at time of diagnosis was included in scoring systems such as the IPSS [29]. The Pavia group showed that transfusion dependency resulting from anemia significantly decreases probability of survival in MDS [8]. This increased mortality risk may be secondary to reduced bone marrow efficiency due to MDS or to sequelae of transfusion therapy such as iron overload when inadequate chelation therapy is present. Including development of transfusion dependency during the course of the disease as prognostic factor was not only significantly associated with shorter survival but also with increased risk of leukemic evolution, reflecting the likelihood of an adverse biology of the disease as contributing factor [55]. On the other hand chronic transfusions increase the risk of iron overload, which may affect survival due to cardiac or hepatic complications as well as the toxicity of labile plasma iron. So iron overload is being discussed as an independent prognostic factor [10, 74, 75]. The data on the prognostic value of transfusion dependence in MDS has been used for generation of a new prognostic scoring system (WPSS) [54]. In addition also a predictive impact of transfusion history on the outcome of allogeneic transplantation for MDS was suggested [1].

Patient Related Factors: Age, Gender, Performance Status, Comorbidity Age and gender per se affect overall survival of humans independently of disease associated factors. Early studies evaluating age and/or gender as prognostic factors

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in MDS had yielded significant results [2, 61, 75]. In addition in the original IPSS publication, age and gender were analyzed and discussed [29]. Their additional predictive importance was stated, but not quantified in the scoring system as disease related factors were considered more important than patient related factors. Morbidity and mortality rates in MDS patients are higher than those of age-matched normal subjects because of complications related to cytopenia [8], still age and gender modulate MDS prognosis, addition of these factors may improve prognostication [66]. Performance status is correlated with the outcome of chemotherapy or stem cell transplantation in MDS or AML [31, 43]. Lower performance status which may prevent eligibility for aggressive therapies is more prevalent in elderly patients and may be the consequence of pre-existing comorbidities. Disregarding specific therapies these co-morbidities may influence overall survival in MDS patients independently of disease specific prognostic factors. Pre-existing comorbidity scores developed for other clinical situations such as the hematopoietic cell transplant comorbidity index (HCTCI) [78] or the Charlson co-morbidity Index (CCI) [9] have been applied for MDS [79, 98] and MDS specific co-morbidity scores have been developed [15]. Taken together, geriatric assessment should be included in treatment decisions for MDS: age alone should not be considered a surrogate marker for functional decline or co-morbidities; there is still a need to improve co-morbidity scoring systems, individualized risk-assessment, and treatment algorithms for elderly patients with MDS [80].

Combinations of Prognostic Factors: Prognostic Scores Role of Classifications in Prognostication The aim of the development of classifications was to create meaningful sub-entities for clinical decision making. This includes also prognostic significance, but other features such as grouping patients by morphologic similarities or pathological findings were also of importance. Factors that per se also carry prognostic information in the univariate setting as described above in “Prognostic factors in MDS” were used in classifications: in the original FAB classification [3] bone marrow or peripheral blasts, ring sideroblasts and monocytes were used, in the first WHO classification multilineage dysplasia, del(5)(q) were added and blasts considered in a more differentiated way [39]. Applying very stringent criteria led to a high number of unclassified cases [65], therefore in the revision of WHO in 2008 besides further refinements cases with unilineage dysplasia were specified [84]. The prognostic value of classifications has been validated in large patient cohorts [25, 27, 37, 45, 50, 55, 64, 65] and there is still ongoing refinement [89]. While classification systems including prognostic factors are widely used, they do not always provide sufficient prognostic information and do not eliminate the need to use prognostic scores for prognostication and clinical decision making.

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Prognostic Scoring Systems In parallel to the development of classifications prognostic factors have been combined in so called prognostic scoring systems. Early scores like the Bournemouth score included only cell counts and percentage of blasts in bone marrow [63], the Spanish score included age [75], later LDH was included in the Düsseldorf score [2], as well as histopathologic features [7, 56]. When the prognostic significance of cytogenetics became apparent, these data were incorporated in the Lille score and Lausanne-Bournemouth score [61, 69]. International Prognostic Scoring System (IPSS) In 1997 the international prognostic scoring system (IPSS) was developed, based on a regression model including data from 816 patients with primary MDS [29]. It represents still the most widely used prognostic score for MDS at least in the context of clinical trials. It is based on bone marrow blasts percentage, number of cytopenias and three then newly defined cytogenetic risk groups: a normal karyotype or evidence of -Y, del(5)(q), or del(20)(q) is classified as good risk, while the presence of chromosome 7 or complex abnormalities represents poor risk. All other abnormalities are arbitrarily considered intermediate risk. The IPSS discriminates four risk categories with distinct survival and risk of AML evolution: Table€ 7.2, Fig.€7.1. The prognostic significance of the IPSS has been validated on independent study populations [e.g. 26, 71]. In a comparison with historic scores, the IPSS had the highest prognostic power of all scores tested [71]. Despite this improvement the limitations of the IPSS have to be taken into account when using this score. Generally it is only applicable using data from the time of diagnosis and it has not been validated for patients with secondary MDS. In addition the IPSS is a prognostic score and has no predictive relevance regarding specific therapies. Some limitations that offer opportunities for future improvements are founded in the limitations of the initial data. Analysis of larger databases has Table 7.2↜渀 IPSS. (Adapted after Greenberg et€al. [29]) 5–10 – Bone marrow <5 blasts (%) Poor risk Intermediate Cytogenetics Good risk Complex or chromorisk Normal some 7 aberrations -y, 5q-, 20q- All others 0/1 2/3 Number of cytopenias Hbâ•›<â•›10, ANCâ•›<â•›1,500, PLTâ•›<â•›100,000 Scoring points 0 0.5 1.0 Risk group Sum of points

Low 0

Intermediate-1 Intermediate-2 0.5–1.0 1.5–2.0

11–20

21–30

1.5

2.0

High 2.5–3.5

– –

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Fig. 7.1↜渀 Survival (a) and freedom from AML evolution (b) of MDS patients related to their classification by the IPSS for MDS: Low, INT-1, INT-2, and High (KaplanMeier curves). (Reproduced with permission from Greenberg [29])

International MDS Risk Classification 100 90

Survival

80 70

percent

60

Low

267 pts

Int-1

314 pts

Int-2

179 pts

High

56 pts

50 40 30 20 10 0

a 100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 years AML Evolution

90 80 70

percent

60 50

Low

235 pts

Int-1

295 pts

Int-2

171 pts

High

58 pts

40 30 20 10 0

b

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 years

increased our knowledge on the prognostic significance of cytogenetic abnormalities: whereas noncomplex rare cytogenetic abnormalities were assigned intermediate risk in the IPSS—a very heterogenous category, data presented from the German Austrian MDS study group analysing 2,124 MDS patients enabled recognition of infrequent aberrations and their assignment to prognostic subgroups [33]. Currently

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an improved cytogenetic score is being developed. Observation during longer follow up periods than the ones used in the original IPSS cohort, may provide the explanation why there should be a redistribution of the weight of bone marrow blasts versus cytogenetic risk groups in terms of score points which should be considered when deliberating refinements of the IPSS [82, 87]. Besides these adjustments using modified parameters already included in the IPSS, addition of further parameters, that have shown their possible utility in univariate analyses may increase its prognostic power: elevated lactate dehydrogenase (LDH) identified as an additional prognostic variable was used to refine the IPSS [26]. The IPSS+LDH was capable of separating a new group of “verylow-risk” patients. In addition, for patients in the Int-2 and high-risk groups, adding LDH as prognostic factor led to significant stratifications of these prognostic groups. Similarly addition of age and gender may be used to refine the IPSS. Risk factors for the four IPSS prognostic subgroups can be modified according to age and gender thus contributing to an improved and more individualized prognostication [66]. WHO Based Prognostic Scoring System WPSS The IPSS was developed on patients classified according to the FAB classification [29]. While the IPSS remains significant within WHO subgroups, there is redundancy: similar blast intervals are used in IPSS and WHO, peripheral cytopenias in IPSS are reflected in the number of dysplasias in WHO, only cytogenetics besides del(5)(q) provide additional prognostic information in the IPSS. In addition the WHO classification reclassifying RAEBt as acute leukemia leads to a substantial loss in the population the IPSS was initially based upon, especially in the higher risk groups. Therefore the development of an WHO based prognostic scoring system (WPSS) was performed (Tab.€7.3) [54]. Factors already included in the WHO subgroup or in the IPSS such as cytopenias, cytogenetic risk factors (the same subgroups as in the IPSS) and bone marrow blast counts were used. According to the data on the importance of transfusion dependency this feature replaced anemia. The WPSS discriminates five prognostic groups ranging from very low to very high risk with significant differences for overall and leukemia free survival. In patients older than 70 years survival for very low-risk MDS was comparable to that of the general population. Addressing the problem of identifying higher risk patients in “low risk” groups, this is accomplished by means of inclusion of multilineage dysplasia and transfusion need. The WPSS also allows for repeated assessment, providing a time-dependent score, enabling reassessment of the prognosis of a patient during the course of the disease and at time of progression. The WPSS publication has a learning sample/validation sample design offering internal validation of the score and has been independently validated as well [68]. However, in the light of further developments the final role of the WPSS remains open. Especially the fact that it still uses the limited cytogenetic subgroups of the IPSS warrants further improvements.

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Table 7.3↜渀 WPSS. (Adapted after Malcovati et€al. [54]) WHO subtype RA, RARS RCMD, RCMD-RS Intermediate risk Cytogenetics Good risk All others Normal -y, 5q-, 20qTransfusion No Yes requirement Scoring points 0 1 Risk group Sum of points

Very low 0

Low 1

RAEB-1 RAEB-2 Poor risk Complex or chromosome 7 aberrations 2

Intermediate High 2 3–4

3 Very high 5–6

Ongoing Improvement of Scoring Systems The problem of limited applicability of scores in the low risk subgroups as well as the need for dynamic scores allowing for reevaluation in patients with a prolonged life span has been also addressed by a further scoring systems by the MD Anderson group [24, 44]. Patients were assessed at time of referral not at time of diagnosis, therefore validation with patients at time of diagnosis is needed. In addition the upcoming cytogenetic data will have to be integrated. On the basis of the IPSS requiring refinement, the proposal of the WPSS and upcoming additional prognostic features an international working group has been convened to assess the utility of prognostic information and to finally construct a new improved scoring system. Probably at the International MDS Symposium 2011 first results may become available.

Conclusion: Use of Prognostication for Clinical Decision Making This review reflects the steady progress in prognostication for MDS. Different prognostic tools utilized over the years in clinical trials implicates that it can be a challenge to compare their results. Recently a more homogeneous use of classifications and prognostic criteria has been applied in clinical trials but knowledge of inclusion criteria used is still of importance for interpretation of study data. Prognostic scores have been improved over years and data are available showing the additional utility of additional approaches for further advances in this field. Collaboration of many centers is warranted to include as many patients as possible in validation studies in order to achieve meaningful results and to establish a comprehensive approach to prognostication also facilitating planning and performing clinical trials. Prognostic scores are only part of a meticulous patient assessment necessary before making therapeutic decisions. This assessment should include secure confirmation of the diagnosis MDS, a correct classification on the basis of expert morphology, the evaluation of prognostic factors, the consideration of predictive factors and assessment of comorbidity. As seen in this review the boundaries between these fields overlap, and we can expect changes and further developments as our

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knowledge on MDS will improve. At the time being the development in the field of prognostication so far has greatly benefited our MDS patients.

References ╇ 1. Alessandrino EP, Della Porta MG, Bacigalupo A et€al (2010) Prognostic impact of pre-transplantation transfusion history and secondary iron overload in patients with myelodysplastic syndrome undergoing allogeneic stem cell transplantation: a GITMO study. Haematologica 95(3):476–484 ╇ 2. Aul C, Gattermann N, Heyll A et€al (1992) Primary myelodysplastic syndromes: analysis of prognostic factors in 235 patients and proposals for an improved scoring system. Leukemia 1:52–59 ╇ 3. Bennett JM, Catovsky D, Daniel MT et€al (1982) Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51:189–199 ╇ 4. Bernasconi P, Klersy C, Boni M et€al (2010) Does cytogenetic evolution have any prognostic relevance in myelodysplastic syndromes? A study on 153 patients from a single institution. Ann Hematol 89:545–551 ╇ 5. Buesche G, Teoman H, Wilczak W et€al (2008) Marrow fibrosis predicts early fatal marrow failure in patients with myelodysplastic syndromes. Leukemia 22:313–322 ╇ 6. Byun HM, Triche T, Kim HJ et€al (2009) Genome-wide DNA methylation analysis of patients with myelodysplastic syndrome after azacitidine treatment. Blood (ASH Annual Meeting Abstracts) 114:600 ╇ 7. Cassano E, Giordano M, Riccardi A et€al (1990) Myelodysplastic syndromes: a multiparametric study of prognostic factors and a proposed scoring system. Haematologica 75:141– 145 ╇ 8. Cazzola M, Malcovati L (2005) Myelodysplastic syndromes—coping with ineffective hematopoiesis. N Engl J Med 352:536–538 ╇ 9. Charlson ME, Pompei P, Ales KL et€al (1987) A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis 40:373–383 10. Chee CE, Steensma DP, Wu W et€al (2008) Neither serum ferritin nor the number of red blood cell transfusions affect overall survival in refractory anemia with ringed sideroblasts. Am J Hematol 83:611–613 11. Chen G, Zeng W, Miyazato A et€al (2004) Distinctive gene expression profiles of CD34 cells from patients with myelodysplastic syndrome characterized by specific chromosomal abnormalities. Blood 104:4210–4218 12. Cherian S, Moore J, Bantly A et€al (2005) Peripheral blood MDS score: a new flow cytometric tool for the diagnosis of myelodysplastic syndromes. Cytometry B Clin Cytom 64:9–17 13. Coiffier B, Adeleine P, Viala JJ et€al (1983) Dysmyelopoietic syndromes. A search for prognostic factors in 193 patients. Cancer 52:83–90 14. Delhommeau F, Dupont S, Della Valle V et€al (2009) Mutation in TET2 in myeloid cancers. N Engl J Med 360:2289–2301 15. Della Porta MG, Kuendgen A, Malcovati L et€al (2008) Myelodysplastic syndrome (MDS)specific comorbidity index for predicting the impact of extra-hematological comorbidities on survival of patients with MDS. Blood (ASH Annual Meeting Abstracts) 112:2677 16. Della Porta MG, Malcovati L, Boveri E et€al (2009) Clinical relevance of bone marrow fibrosis and CD34-positive cell clusters in primary myelodysplastic syndromes. J Clin Oncol 27:754–762 17. Ebert BL, Galili N, Tamayo P, Bosco J et€ al (2008) An erythroid differentiation signature predicts response to lenalidomide in myelodysplastic syndrome. PLoS Med 5:e35

7â•… Prognostic Scoring in MDS

117

18. Ebert BL, Pretz J, Bosco J et€al (2008) Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature 451(7176):335–339 19. Esteller M (2008) Epigenetics in cancer. N Engl J Med 358:1148–1159 20. Fenaux P, Giagounidis A, Selleslag D et€al (2009) RBC transfusion independence and safety profile of lenalidomide 5 or 10 mg in Pts with low- or Int-1-risk MDS with Del5q: results from a randomized phase III trial (MDS-004). Blood (ASH Annual Meeting Abstracts) 114:944 21. Fenaux P, Mufti GJ, Hellstrom-Lindberg E et€ al (2009) Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 10:223–232 22. Follo MY, Finelli C, Mongiorgi S et€al (2009) Reduction of phosphoinositide-phospholipase C beta1 methylation predicts the responsiveness to azacitidine in high-risk MDS. Proc Natl Acad Sci U S A 106(39):16811–16816 23. Ganetzky RD, Jankowska AM, Prince C et€al (2009) TET2 mutation status is associated with distinct hypermethylation patterns; a marker of epigenetic instability? Blood (ASH Annual Meeting Abstracts) 114:215 24. Garcia-Manero G, Shan J, Faderl S et€al (2008) A prognostic score for patients with lower risk myelodysplastic syndrome. Leukemia 22:538–543 25. Germing U, Gattermann N, Strupp C et€ al (2000) Validation of the WHO proposal for a new classification of primary myelodysplastic syndromes: a retrospective analysis of 1600 patients. Leuk Res 24:983–992 26. Germing U, Hildebrandt B, Pfeilstöcker M et€ al (2005) Refinement of the international prognostic scoring system (IPSS) by including LDH as an additional prognostic variable to improve risk assessment in patients with primary myelodysplastic syndromes (MDS). Leukemia 19:2223–2231 27. Germing U, Strupp C, Kuendgen A et€al (2006) Prospective validation of the WHO proposals for the classification of myelodysplastic syndromes. Haematologica 91:1596–1604 28. Giagounidis AA, Germing U, Haase S et€al (2004) Clinical, morphological, cytogenetic, and prognostic features of patients with myelodysplastic syndromesand del(5q) including band q31. Leukemia 18:113–119 29. Greenberg P, Cox C, LeBeau MM et€al (1997) International system for evaluating prognosis in myelodysplasticsyndromes. Blood 89:2079–2088 (Erratum in Blood 91:1100, 1998) 30. Grövdal M, Khan R, Aggerholm A et€al (2007) Negative effect of DNA hypermethylation on the outcome of intensive chemotherapy in older patients with high-risk myelodysplastic syndromes and acute myeloid leukemia following myelodysplastic syndrome. Clin Cancer Res 13:7107–7112 31. Gupta V, Chun K, Yi QL et€al (2005) Disease biology rather than age is the most important determinant of survival of patients > or = 60 years with acute myeloid leukemia treated with uniform intensive therapy. Cancer 103:2082–2090 32. Haase D, Germing U, Schanz J et€al (2007) New insights into the prognostic impact of the karyotype in MDS and correlation with subtypes: evidence from a core dataset of 2124 patients. Blood 110:4385–4395 33. Haase D, Schanz J, Tuechler H et€al (2009) Updated cytogenetic risk features in MDS—present state. Leuk Res 33(Suppl 1):S9–S10 (10th Int Symp MDS abstracts) 34. Hemingway H, Riley RD, Altman DG (2009) Ten steps towards improving prognosis research. BMJ 339:b4184. doi:10.1136/bmj.b4184 35. Hofmann WK, de Vos S, Komor M et€al (2002) Characterization of gene expression ofCD34+ cells from normal and myelodysplastic bone marrow. Blood 100:3553–3560 36. Hopfer O, Komor M, Koehler IS et€al (2007) DNA methylation profiling of myelodysplastic syndrome hematopoietic progenitor cells during in vitro lineage-specific differentiation. Exp Hematol 35:712–723 37. Howe B, Porwit-MacDonald A, Wanat R et€al (2004) The WHO classification of MDS does make a difference. Blood 103:3265–3270

118

M. Pfeilstöcker

38. Itzykson R, Kosmider O, Cluzeau T et€al (2010) Presence of TET2 mutation predicts a higher response rate to azacitidine in MDS and AML post MDS. Blood (ASH Annual Meeting Abstracts) 2010 116: Abstract 439 39. Jaffe ES, Harris NL Stein H, Vardiman JW (eds) (2001) WHO classification of tumours: pathology and genetics of haematopoietic and lymphoid tissues. IARC Press, Lyon 40. Jankowska AM, Shaik M, Cazzolli H et€ al (2009) Impact of TET2 mutations on responsiveness to demethylating agents in MDS. Blood (ASH Annual Meeting Abstracts) 114:1606 41. Jiang Y, Dunbar A, Gondek LP, Mohan S et€al (2009) Aberrant DNA methylation is a dominant mechanism in MDS progression to AML. Blood 113:1315–1325 42. Justice AC, Covinsky KE, Berlin JA (1999) Assessing the generalizability of prognostic information. J Ann Intern Med 130:515–524 43. Kantarjian H, O’Brien S, Cortes J et€ al (2006) Results of intensive chemotherapy in 998 patients age 65 years or older with acute myeloid leukemia or high-risk myelodysplastic syndrome: predictive prognostic models for outcome. Cancer 106:1090–1098 44. Kantarjian H, O’Brien S, Ravandi F et€al (2008) Proposal for a new risk model in myelodysplastic syndrome that accounts for events not considered in the original International Prognostic Scoring System. Cancer 113:1351–1361 45. Kerkhofs H, Hermans J, Haak HL et€al (1987) Utility of the FAB classification for myelodysplastic syndromes: investigation of prognostic factors in 237 cases. Br J Haematol 65:73–81 46. Knipp S, Hildebrand B, Kündgen A et€al (2007) Intensive chemotherapy is not recommended for patients aged >60 years who have myelodysplastic syndromes or acute myeloid leukemia with high-risk karyotypes. Cancer 110:345–352 47. Ko M, Huang Y, Jankowska AM, Pape UJ et€al (2010) Impaired hydroxylation of 5– methylcytosine in myeloid cancers with mutant TET2. Nature (in press) 48. Kosmider O, Gelsi-Boyer V, Cheok M et€al (2009) TET2 mutation is an independent favorable prognostic factor in myelodysplastic syndromes (MDS). Blood 114:3285–3291 49. Lambertenghi-Deliliers G, Orazi A, Luksch R et€al (1991) Myelodysplastic syndrome with increased marrow fibrosis: a distinct clinico-pathological entity. Br J Haematol 78:161– 166 50. Lee J-H, Lee J-H, Shin Y-R et€al (2003) Application of different prognostic scoring systems and comparison of the FAB and WHO classification in Korean patients with myelodysplastic syndromes. Leukemia 17:305–313 51. List A, Dewald G, Bennett J et€al (2006) Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion. N Engl J Med 355:1456–1465 52. List A, Kurtin S, Roe DJ et€al (2005) Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J Med 352:549–557 53. Malcovati L, Della Porta MG, Lunghi M et€al (2005) Flow cytometry evaluation of erythroid and myeloid dysplasia in patients with myelodysplastic syndrome. Leukemia 19:776–783 54. Malcovati L, Germing U, Kuendgen A et€al (2007) Time-dependent prognostic scoring system for predicting survival and leukemic evolution in myelodysplastic syndromes. J Clin Oncol 25:3503–3510 55. Malcovati L, Porta MG, Pascutto C et€al (2005) Prognostic factors and life expectancy in myelodysplastic syndromes classified according to WHO criteria: a basis for clinical decision making. J Clin Oncol 23:7594–7603 56. Maschek H, Gutzmer R, Choritz H et€al (1994) Life expectancy in primary myelodysplastic syndromes: a prognostic score based upon histopathology from bone marrow biopsies of 569 patients. Eur J Haematol 53:280–287 57. Mills KI, Kohlmann A, Williams PM et€al (2009) Microarray-based classifiers and prognosis models identify subgroups with distinct clinical outcomes and high risk of AML transformation of myelodysplastic syndrome. Blood 114:1063–1072 58. Miyazato A, Ueno S, Ohmine K et€al (2001) Identification of myelodysplastic syndromespecific genes by DNA microarray analysis with purified hematopoietic stem cell fraction. Blood 98:422–427

7â•… Prognostic Scoring in MDS

119

59. Mohamedali A, Gäken J, Twine NA et€al (2007) Prevalence and prognostic significance of allelic imbalance by single-nucleotide polymorphism analysis in low-risk myelodysplastic syndromes. Blood 110:3365–3373 60. Moons KG, Altman DG, Vergouwe Y et€al (2009) Prognosis and prognostic research: application and impact of prognostic models in clinical practice. BMJ 338:b606. doi:10.1136/bmj.b606 61. Morel P, Hebbar M, Lai JL et€al (1993) Cytogenetic analysis has strong independent prognostic value in de novo myelodysplastic syndromes and can be incorporated in a new scoring system: a report on 408 cases. Leukemia 7:1315–1323 62. Mufti GJ (1992) Chromosomal deletions in the myelodysplastic syndrome. Leuk Res 16:35– 41 63. Mufti G, Stevens JR, Oscier DG et€al (1985) Myelodysplastic syndromes: a scoring system with prognostic significance. Br J Haematol 59:425–433 64. Müller-Berndorff H, Haas PS, Kunzmann R et€al (2006) Comparison of five prognostic scoring systems, the French-American-British (FAB) and World Health Organization (WHO) classifications in patients with myelodysplastic syndromes: results of a single-center analysis. Ann Hematol 85:502–513 65. Nösslinger T, Reisner R, Koller E et€ al (2001) Myelodysplastic syndromes, from FrenchAmerican-British to World Health Organization: comparison of classifications on 431 unselected patients from a single institution. Blood 98:2935–2941 66. Nösslinger T, Tüchler H, Germing U et€al (2010) Prognostic impact of age and gender in 897 untreated patients with primary myelodysplastic syndromes. Ann Oncol 21:120–125 67. Ogata K, Della Porta MG, Malcovati L et€ al (2009) Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study. Haematologica 94:1066–1074 68. Park MJ, Kim HJ, Kim SH et€al (2008) Is International Prognostic Scoring System (IPSS) still standard in predicting prognosis in patients with myelodysplastic syndrome? External validation of the WHO Classification-Based Prognostic Scoring System (WPSS) and comparison with IPSS. Eur J Haematol 81:364–373 69. Parlier V, van Melle G, Beris P et€al (1995) Prediction of 18-month survival inpatients with primary myelodysplastic syndrome: a regression model and a scoring system based on the combination of chromosome findings and the Bournemouth score. Cancer Genet Cytogen 81:158–165 70. Pellagatti A, Esoof N, Watkins F et€al (2004) Gene expression profiling in the myelodysplastic syndromes using cDNA microarray technology. Br J Haematol 125:576–583 71. Pfeilstöcker M, Reisner R, Nösslinger T et€al (1999) Cross-validation of prognostic scores in myelodysplastic syndromes on 386 patients from a single institution confirms importance of cytogenetics. Br J Haematol 106:455–463 72. Quesnel B, Guillerm G, Vereecque R et€al (1998) Methylation of the p15(INK4b) gene in myelodysplastic syndromes is frequent and acquired during disease progression. Blood 91:2985–2990 73. Reynolds T (2001) Prognostic models abound, but how useful are they? Ann Intern Med 135:473–476 74. Sanz G, Nomdedeu B, Such E et€al (2008) Independent impact of iron overload and transfusion dependency on survival and leukemic evolution in patients with myelodysplastic syndrome. Blood (ASH Annual Meeting Abstracts) 112:640 75. Sanz GF, Sanz MA, Vallespí T et€ al (1989) Two regression models and a scoring system for predicting survival and planning treatment in myelodysplastic syndromes: a multivariate analysis of prognostic factors in 370 patients. Blood 74:395–408 76. Scott BL, Wells DA, Loken MR et€al (2008) Validation of a flow cytometric scoring system as a prognostic indicator for post transplantation outcome in patients with myelodysplastic syndrome. Blood 112:2681–2686 77. Solé F, Luño E, Sanzo C et€al (2005) Identification of novel cytogenetic markers with prognostic significance in a series of 968 patients with primary myelodysplastic syndromes. Haematologica 90:1168–1178

120

M. Pfeilstöcker

78. Sorror ML, Maris MB, Storb R et€ al (2005) Hematopoietic cell transplantation(HCT)specific comorbidity index: a new tool for risk assessment before allogeneic HCT. Blood 106:2912–2919 79. Sperr WR, Wimazal F, Kundi M et€al (2010) Comorbidity as prognostic variable in MDS: comparative evaluation of the HCT-CI and CCI in a core dataset of 419 patients of the Austrian MDS study group. Ann Oncol 21:114–119 80. Stauder R, Nösslinger T, Pfeilstöcker M et€al (2008) Impact of age and comorbidity in myelodysplastic syndromes. J Natl Compr Cancer Netw 6:927–934 81. Steensma DP (2009) Myelodysplasia paranoia: iron as the new radon. Leuk Res 33:1158– 1163 82. Steidl C, Schanz J, Pfeilstöcker M et€al (2007) Growing evidence for an underestimation of poor-risk cytogenetics in the International Prognostic Scoring System in myelodysplastic syndromes. Clin Leuk 1:353–356 83. Steidl C, Steffens R, Gassmann W et€al (2005) Adequate cytogenetic examination in myelodysplastic syndromes: analysis of 529 patients. Leuk Res 29:987–993 84. Swerdlow SH, Campo E, Harris NL et€ al (eds) (2008) WHO classification of tumours of haematopoietic and lymphoid tissues. IARC Press, Lyon 85. Tefferi A, Lim KH, Abdel-Wahab O et€al (2009) Detection of mutant TET2 in myeloid malignancies other than myeloproliferative neoplasms: CMML, MDS, MDS/MPN and AML. Leukemia 23:1343–1345 86. Tefferi A, Vardiman W (2009) Myelodysplastic syndromes. N Engl J Med 361:1872–1885 87. Tüchler H, Makrai A, Nösslinger T et€al (2008) At which interval should prognosic scores for preleukemic duration and survival in myelodysplastic syndromes be reevaluated? Onkologie (DGHO abstracts) 31(Suppl 4):170 88. Ueda M, Ota J, Yamashita Y et€ al (2003) DNA microarray analysis of stage progression mechanism in myelodysplastic syndrome. Br J Haematol 123:288–296 89. Valent P, Horny HP, Bennet JM et€al (2007) Definitions and standards in the diagnosis and treatments of the myelodysplastic syndromes: consensus statements and report from a working conference. Leuk Res 31:727–736 90. van de Loosdrecht AA, Alhan C, Béné MC et€al (2009) Standardization of flow cytometry in myelodysplastic syndromes: report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromes. Haematologica 94:1124–1134 91. Van den Berghe H, Cassiman JJ, David G et€al (1974) Distinct haematological disorder with deletion of long arm of no. 5 chromosome. Nature 251:437–438 92. Wattel E, Laï JL, Hebbar M et€al (1993) De novo myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 20: a subtype of MDS with distinct hematological and prognostic features? Leuk Res 17:921–926 93. Wimazal F, Germing U, Kundi M et€al (2010) Evaluation of the prognostic significance of eosinophilia and basophilia in a larger cohort of patients with myelodysplastic syndromes. Cancer 116:2372–2381 94. Wimazal F, Sperr WR, Kundi M et€al (2001) Prognostic value of lactate dehydrogenase activity in myelodysplastic syndromes. Leuk Res 25:287–294 95. Wimazal F, Sperr WR, Kundi M et€ al (2008) Prognostic significance of serial determinations of lactate dehydrogenase (LDH) in the follow-up of patients with myelodysplastic syndromes. Ann Oncol 19:970–976 96. Windeler J (2000) Prognosis—what does the clinician associate with this notion? Stat Med 19:425–430 97. Wyatt JC, Altman DG (1995) Commentary: prognostic models: clinically useful or quickly forgotten? BMJ 311:1539–1541 (Dec) 98. Zipperer E, Pelz D, Nachtkamp K et€al (2009) The hematopoietic stem cell transplantation comorbidity index is of prognostic relevance for patients with myelodysplastic syndrome. Haematologica 94:729–732

Chapter 8

Flow Cytometry in Myelodysplastic Syndromes C. Alhan, T. M. Westers, G. J. Ossenkoppele and Arjan A. van de Loosdrecht

Introduction Maturation and differentiation of hematopoietic cells is a tightly controlled process, leading to highly conserved levels of antigen expression at different stages of development. In myelodysplastic syndromes (MDS), progenitor cell formation is affected resulting in deviation from the normal level of antigen expression in the (im) mature myelo-monocytic, erythroid and megakaryocytic cell lineages [29, 38, 42]. The gold standard for the diagnosis of MDS is the detection of dysplastic features in the erythroid, megakaryocytic and/or myeloid cell lineages in the bone marrow (BM) by morphology. Dysplasia can be very mild and in combination with a normal karyotype, the diagnosis of MDS can be challenging. In a consensus report from a working conference on MDS, flow cytometric analysis of cytopenic patients with inconclusive BM morphology and cytogenetics is included as a tool to establish a diagnosis of MDS [48]. Flow cytometry is based on intrinsic physical qualities of the cells such as size, corresponding with forward light scatter (FSC) and cytoplasmic granularity, corresponding with sideward light scatter (SSC). Furthermore, by labeling cells with fluorochrome-conjugated monoclonal antibodies it is possible to detect surface, cytoplasmic or nuclear antigens. Flow cytometry can detect minimal aberrancies in the differentiation of myelomonocytic cell populations by changes in antigen expression in BM of MDS patients that are otherwise not detected by morphology [42]. Since new therapeutic strategies are emerging for MDS, a more refined diagnostic and prognostic procedure is of importance. In this chapter the principles of flow cytometry in MDS and the value of flow cytometry for the diagnosis and prognosis A. A. van de Loosdrecht () Department of Hematology, VU Institute for Cancer and Immunology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Tel.: +31-20-444-2604 Fax: +31-20-444-2601 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_8, ©Â€Springer Science+Business Media B.V. 2011

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of MDS will be discussed with emphasis on technical issues and aberrancies that can be detected.

Flow Cytometric Evaluation of Dyspoiesis in MDS Application of flow cytometry for the diagnosis of MDS requires knowledge of changes in antigen expression in normal hematopoietic cell differentiation. Hematopoiesis is a tightly regulated process in which antigen expression patterns are highly conserved [51]. In MDS disturbances in hematopoiesis can be associated with disruption of antigen expression patterns [46, 53]. In a consensus document from the European LeukemiaNet (ELN) working group for standardization of flow cytometry in MDS, a minimal combination of antibodies is proposed to analyze dyspoiesis in the (im)mature myelo-monocytic cell compartments, Table€8.1 [50]. The aberrancies in hematopoiesis that can be observed in MDS are the expression of lymphoid antigens on myeloid cells, over, under and/or loss of antigen expression, the expression of immature antigens on mature cells and vice versa and abnormal differentiation patterns between antigens. However, non-clonal disorders such as vitamin deficiency related anemia can cause (transient) immunophenotypic changes in the BM that can also be found in MDS. Although some of the aberrancies that can be detected in MDS are not specific for MDS, the accumulation of aberrancies might discern MDS from non-clonal cytopenias [27, 30]. Antigen expression levels are defined as aberrant in MDS if a 0.5log decrease or increase is found as compared to antigen expression levels from hematopoietic cells of healthy individuals. Table 8.1↜渀 Combination of markers as recommended by the ELN working group for the standardization of flow cytometry in MDS. (van de Loosdrecht et€al. [50]) Erythroid CD71/CD235a/CD117 Optional: CD105, CD34/CD117, CD36 Myeloid progenitors CD34 in combination with CD117/CD11b/HLA-DR/CD15 Lineage infidelity markers: CD5, CD7/CD13, CD19, CD56 Optional: CD123, TdT Lymphoid progenitors CD34/CD19 Optional: CD10/CD19/CD38, TdT, CD79a Maturing myeloids CD11b/CD13/CD16, CD11b/CD117/HLA-DR/CD10, CD34 in combination with CD5, CD7, CD15, CD19, CD56, CD33/CD14 Optional: CD65, CD123 Monocytes CD11b/HLA-DR, CD34 in combination with CD5, CD7, CD19, CD56, CD64/CD14, CD33/CD14, CD33/CD36 Optional: CD64/CD36 A minimum panel for the diagnosis and/or prognosis of MDS. The common leukocyte antigen CD45 should be combined with the antigens that are listed in the table. The subpopulations in the BM should be gated based on CD45 and SSC

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Most studies analyze dyspoiesis in BM samples of MDS patients, although there is some data on flow cytometric analysis of peripheral blood (PB) in MDS. Analysis of aberrancies in antigen expression levels of mature cell populations in PB might be of value to diagnose MDS patients [7]. Increased expression of CD13 on mature neutrophils has been associated with an adverse clinical outcome [26]. In a study that compared PB with BM, antigen expression levels of CD36, CD56 and CD117 on neutrophils and monocytes differed between PB and BM [21]. The threshold for abnormal expression of CD56 and CD117 was higher in BM than PB. However, aberrancies in antigen expression levels of neutrophils and monocytes of MDS patients were more frequent in BM than in PB. The analysis of circulating myeloid progenitors in PB of MDS patients might hold prognostic significance [6]. Furthermore, it should be kept in mind that the nomenclature for cells that is used in morphology might not be applicable in flow cytometry. For example, an immature cell might be denominated as a blast by morphology and as a myeloid progenitor cell by flow cytometry. These and other studies demonstrate that disregulation of hematopoiesis in MDS can not only be detected by morphology but also by flow cytometry as will be discussed in detail below.

Myeloid Progenitors Flow cytometry can be used to identify distinct subpopulations of myeloid progenitor cells based on antigen expression levels. CD45 is a leukocyte common antigen and is expressed on all leukocytes. In combination with SSC, CD45 is a powerful means to delineate lymphocytes, monocytes, maturing myeloid cells and myeloid progenitors in normal BM. Normal myeloid progenitors are identified by CD45dim expression, low SSC, heterogeneous expression of CD34 and CD117, CD13, CD33, HLA-DR and absence of CD11b. CD34 is expressed on the most immature myeloid, lymphoid and erythroid cells and is rapidly lost at an early stage of differentiation. Immature myeloid and erythroid progenitors express CD117 and lose expression during myeloid and erythroid differentiation. CD13 is expressed at the myeloid progenitor stage, decreases during myeloid differentiation and is increased on the most mature myeloid cells to the level of expression on myeloid progenitors. All myeloid cells express CD33. Maturing myeloid cells have highest CD33 intensity compared with immature and mature myeloid cells. HLA-DR is lost at an early stage during myeloid differentiation. It is recommended to use a combination of antibodies, for the following reasons. B cell progenitors express CD34 and these should be excluded from myeloid progenitor analysis. Furthermore, B cell progenitors express CD19 and can be distinguished from myeloid progenitors by lower SSC properties and lower CD45 expression as compared to myeloid progenitors. Basophils are also low in SSC and CD45dim but can be identified by absence of HLA-DR and bright expres-

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sion of CD123. Therefore, the combination of markers that is recommended by the ELN working group for the standardization of flow cytometry in MDS to identify myeloid progenitors is by using scatter properties and CD45 in combination with CD34/CD117/HLA-DR and CD34/CD123/HLA-DR and CD11b/HLA-DR, Table€8.1 [50]. A combination of antibodies is also necessary to identify myeloid progenitors because CD45 and/or CD34 expression on myeloid progenitors can be aberrantly decreased or absent in MDS [31, 36, 37, 40, 49, 54]. Aberrancies that can be detected in the immature myeloid compartment are listed in Table€8.2 [23]. A common finding in MDS is the aberrant expression of antigens on immature myeloid cells that are normally expressed on mature myeloid cells, such as CD11b and/or

Table 8.2↜渀 Common immunophenotypic aberrancies in MDS. (The table is adapted from a meeting report on definitions and standards in the diagnosis and treatment of MDS [23, 48, 54, 50]) Common phenotypic abnormalities detected by flow cytometry in MDS Myeloid progenitors Increased, decreased or absent CD45 expression Increased, decreased or absent and/or homogenous CD34 expression Homogenous CD117 expression Asynchronous expression of CD11b and/or CD15 Decreased or absent HLA-DR, CD13 and/or CD33 expression Expression of lymphoid associated antigens: CD2, CD5, CD7, CD19 and/or CD56 Decreased CD38 expression Absolute and relative increase in the number of myeloid progenitors B cell progenitors Absolute and relative (to myeloid progenitors) decrease in B cell progenitors Maturing myeloid cells Decreased CD45 expression Abnormal relationship patterns of myeloid antigens CD11b, CD13 and CD16 Hypogranularity characterized by decreased SSC Decreased or absent CD33 expression Asynchronous expression of CD34 and/or HLA-DR Expression of lymphoid associated antigens: CD5, CD7, CD19 and/or CD56 Maturing monocytes Decreased CD45 expression Abnormal relationship patterns of CD11b and HLA-DR, CD13 and CD16, CD14 and CD33 Increased, decreased or absent CD11b, CD13, CD14, CD16, CD33 and/or HLA-DR Expression of CD34 Expression of lymphoid antigens: CD2, CD5, CD7, CD19 and/or CD56 Absolute and relative increase in the number of monocytes Maturing erythroid cells Abnormal expression of CD34 CD45 and/or CD117 Decreased CD71 expression Absolute and relative increase in the number of immature erythroid cells Persistent low levels of CD71 expression on CD235a positive erythroid precursors The most relevant and commonly described aberrancies that can be detected in MDS are described

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Fig. 8.1↜渀 Aberrant CD7 expression on myeloid progenitors in MDS. To analyze CD7 expression on CD34+ myeloid progenitors it is important to combine the analysis with CD13. Panel a shows CD34+ cells from an healthy individual with a proportion of cells that express CD7. When CD7 is combined with CD13, it is clear that CD7 is expressed on the CD13dim cells. CD7 can be expressed on normal CD13dim and CD34+ cells that are differentiating towards monocytes in the BM of healthy individuals. Therefore, it is important to determine whether CD7 expression is expressed on CD13+ cells. As can be seen in panel b, CD7 is aberrantly expressed on CD13+ and CD34+ cells in the BM of a MDS patient

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Next to qualitative analysis it is also possible to quantify myeloid progenitors by flow cytometry. Flow cytometric studies that analyzed the percentage of myeloid progenitors show that there is a good correlation between the percentage of myeloid progenitors by flow cytometry with the percentage of blasts by morphology [9, 54]. An underestimation of the percentage of myeloid progenitors by flow cytometry might be caused by peripheral blood contamination. Furthermore, the myeloid progenitors as defined by flow cytometry may not correspond to the blast compartment as defined by morphology. This might be explained by a difference in definition of a myeloid progenitor or blasts. The percentage of myeloid progenitors is defined as increased if 5% or more CD34 positive cells were detected. The cut-off level of 5% is also used by morphology to define an increase in blast percentage. However, the cut-off level to define an increase of myeloid progenitors in MDS by flow cytometry might be lower and should probably be 3% or more [35]. There are indications that an increased percentage of CD34 positive cells as defined by a cut-off of 3% or more is indicative for MDS and might be of prognostic relevance in RCMD(RS) patients [34]. In MDS there is a relative decrease of the percentage of B cell progenitors, defined as SSC low, CD45dim, CD34 positive and CD19 positive, Fig.€8.2 [41]. However, in healthy individuals the relative percentage of B cell progenitors also decreases with ageing. It is of importance that the relative percentage of B cell progenitors in MDS BM is compared with the percentage of B cell progenitors in an age-matched control population in order to qualify it as aberrantly low or decreased. The power of flow cytometry over morphology in MDS lies in the qualitative analysis of antigens rather than quantification of myeloid progenitors.

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Fig. 8.2↜渀 Myeloid progenitors and B cell progenitors in MDS. A common finding in MDS is a (relative) decrease or absence of B cell progenitors in the BM. In normal BM, lymphoid progenitors (↜purple arrow) can be identified by lower SSC and CD45 properties compared with myeloid progenitors (↜red arrow). In the figures, CD34+ cells are backgated in a CD45 and SSC plot of an age-matched healthy individual (panel a) and MDS patient (panel b). Panel b shows a MDS patient with decreased B cell progenitors

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Maturing Myeloid Cells In normal BM, neutrophils can be discriminated by intermediate CD45 expression and high SSC properties as compared to lymphocytes. Neutrophils can be distinguished from monocytes that have higher CD45 expression and lower SSC properties compared with neutrophils [51]. By morphology, five stages of neutrophil differentiation can be discerned. The myeloblasts differentiate subsequently from promyelocytes, myelocytes, metamyelocytes, towards band and segmented neutrophils. By flow cytometry, these neutrophil maturation stages can be distinguished by different expression levels of CD11b, CD13 and CD16. Normal neutrophil differentiation patterns are shown in Fig.€8.3. Myeloblasts express CD34, CD117 and a

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Fig. 8.3↜渀 Neutrophil differentiation in MDS. To analyze neutrophil differentiation patterns it is recommended to use the antibody combination CD11b/CD13/CD45/CD16. Panel a shows neutrophil differentiation in the BM of an healthy individual. The arrows indicate the direction of differentiation of the maturing myeloid cells. Panel b shows neutrophil differentiation in the BM of a low risk MDS patient. As can be seen from CD11b/CD13 pattern, in this case CD13 expression is increased on the immature myeloid cells. The CD13/CD16 pattern is more condensed compared with the pattern of the healthy individual in panel a. Panel c shows neutrophil differentiation in a high risk MDS. The CD13/CD16 pattern is compact and the number of immature myeloid cells is increased (↜leftshift). In the CD11b/CD13 plot there is a proportional decrease in CD11bdim cells and mature CD11b+ CD13+ cells. CD11b is also overexpressed as can be seen from the CD11b/ CD13 and CD11b/CD16 patterns

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high levels of CD13 and lack CD11b and CD16 expression. CD11b is absent on promyelocytes, defined by CD117 expression and CD34 absence, and is initially expressed at low levels on myelocytes and is high at the metamyelocyte and segmented neutrophil stage. CD16 follows a similar pattern, but has intermediate expression from the metamyelocyte stage and is highly expressed on mature neutrophils. In contrast, CD13 is present at high levels just before the promyelocyte stage, decreases with differentiation to the myelocyte stage and increases again at the metamyelocyte stage to become highly expressed on segmented neutrophils. The graphic representation of CD11b and CD16, CD11b and CD13, CD16 and CD13 forms three highly conserved patterns for neutrophil differentiation in normal hematopoiesis. The immunophenotypic aberrancies that can be found in MDS in the maturing myeloid or neutrophils are described in Table€8.2. A disturbance in differentiation of neutrophils is reflected by aberrancies in the patterns of myeloid antigens: CD13, CD16 and CD11b, as shown in Fig.€8.3. Hypogranularity, which is reflected by a decreased SSC, is a common finding in MDS (84% of MDS cases) [42]. However, as a single aberrancy it is not specific for MDS. The neutrophil SSC can be assessed by CD45 versus SSC and calculated as a ratio to lymphocyte SSC as an internal reference in the BM. It might be relevant to use the SSC of immature neutrophils compared with mature neutrophils since the mature neutrophil compartment can be influenced by PB contamination [35]. CD33 is expressed dimly on neutrophils in contrast to a bright expression on monocytes. Decreased CD33 expression of both neutrophils and monocytes can be a result of polymorphism and is a common, normal phenomenon. If the SSC of neutrophils is low or decreased and monocytes and neutrophils cannot be separated as distinct populations based on CD45 and SSC properties, CD33 might be discriminatory. CD45 expression may be aberrantly decreased in neutrophils. Abnormalities in the expression of CD13 are also common and might be of diagnostic relevance in MDS [25]. Antigens associated with immaturity such as CD34 and/or HLA-DR can be aberrantly expressed on maturing myeloid cells. Expression of lymphoid antigens can also be detected in the mature myeloid compartment. The expression of a lymphoid antigen on a subset of myelo-monocytic cells might even be of more significance than aberrancies of myelo-monocytic antigens [8].

Monocytic Lineage The added value of flow cytometry in monocytic lineage analysis can be substantial since dyspoiesis in the monocytic lineage is difficult to identify by morphology on the BM level. By morphology, monocytic cells can be identified as myelo/monoblasts, promonocytes and monocytes. Within tissues the monocytes may further differentiate into macrophages. Differentiation from monoblasts to promonocytes is marked by an increase in CD33 expression and loss of CD34 expression with an intermediate expression of CD45. Subsequently, CD45 expression is increased and CD14 and CD11b are gained at the monocytic stage. Furthermore, monocytes retain

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HLA-DR upon differentiation in contrast to the loss of HLA-DR expression during neutrophil maturation [51]. In MDS, the proportion of monocytes relative to lymphocytes or non-erythroid cells can be increased or decreased. The combination of CD14, CD36 and CD64 enables the detection of immature monocytes. CD14 alone can give an underestimation of the proportion of monocytes in the BM because it is expressed on mature monocytes. Abnormalities in the expression patterns of HLA-DR, CD11b, CD13, CD14 and CD33 are common findings. The aberrant increase, decrease or even lack of expression of CD13, CD14, CD16 or CD33 can be found in MDS, Fig.€8.4. Expression of the immature marker CD34 can persist on mature monocytes. Aberrant expression of lymphoid antigens is observed on monocytes with the exception of CD4 which is commonly weakly expressed on monocytes. CD56 can be expressed on normal activated monocytes to a certain extent. Overexpression, defined as 1log increase above normal CD56 expression on monocytes is aberrant and associated with chronic myelomonocytic leukaemia. It can be of relevance for the differential diagnosis of MDS and MDS/MPD such as CMML [21, 43]. The combination of CD56 expression with underexpression of a myeloid marker is reported to be unique for CMML monocytes [56]. Monocytosis with two or more immunophenotypic aberrancies with 20% or more BM monocytes showing moderate CD14 expression diagnosed CMML with 67% sensitivity and 100% specificity [56].

Erythroid and Megakaryocytic Lineage The detection of erythroid dysplasia by flow cytometry in MDS is limited due to lack of markers. Erythroid cells can be identified by the absence of CD45 and low

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scatter properties. The markers that are mainly investigated in the context of dyserythropoiesis in MDS are CD235a and CD71. CD235a or glycophorin A is expressed early upon differentiation at the erythroblast stage of erythroid development and is maintained throughout terminal red cell differentiation. CD71 is the antibody recognizing the transferrin receptor. The transferrin receptor is needed for uptake of iron. Nucleated erythroid cells express CD71 but non-nucleated erythrocytes lack CD71 expression. Erythroid precursor cells express dim CD117 and upon maturation to terminally differentiated erythroid cells CD117 expression is lost [22, 51]. CD105 (endoglin) is a receptor for members of the transforming growth factor beta superfamily (TGF-β) and is specifically expressed on erythroblasts. In combination with CD36 and CD117 the immature erythropoietic cell compartment can be analyzed. Aberrancies that can be found in MDS are abnormal expression of CD117 and CD34, abnormal levels of CD71, asynchronous expression of CD45 versus CD71, asynchronous expression of CD45 versus CD235a and asynchronous expression of CD71 versus CD235a on nucleated red cell precursors. The most frequently observed immunophenotypic aberrancy in the erythroid lineage was persistent or low levels of CD71 expression on glycophorin A-positive erythroid precursors compared with pathologic and healthy BM controls, Fig.€8.5 [27, 42]. A high proportion of immature erythroid cells is also a common finding in MDS patients [27]. Both aberrant CD71 expression and increased immature cells are associated with morphologic features of dyspoiesis. Other markers that are associated with erythroid dysplasia in MDS are the intracellular ferritin subunits, H-ferritin and L-ferritin and mitochondrial ferritin (MtF). b

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Fig. 8.5↜渀 Erythroid differentiation in MDS. Erythroid progenitors lose CD117 and gain CD235a expression upon differentiation. The most mature erythroid cells lose CD71 expression and remain CD235a positive. In healthy individuals there is a ‘gap’ between CD71 positive and negative cells. Panel a shows erythroid differentiation in the BM of an healthy individual. Erythroid progenitors are indicated in dark blue and can be identified by CD117 expression. Panel b shows an MDS patient with aberrant erythroid differentiation. As can be seen from the plot, erythroid progenitors are absent and the ‘gap’ is filled with erythroid cells that have CD71dim expression

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In MDS with ring sideroblasts (RS), erythroid precursor cells express increased iron receptors at the cell surface compared with patients without RS, which is associated with iron storage in the mitochondria. Flow cytometric analyses of proteins of iron metabolism in MDS patients are an indirect way of analyzing erythroid dysplasia. Patients with RS had higher levels of MtF and CD105 than patients without RS. Furthermore, MtF expression is related to presence of RS in BM by morphology [10, 45]. Analysis of dyspoiesis in the megakaryocytic lineage by flow cytometry is limited due to technical aspects. The number of megakaryocytes is relatively too low compared with the other cell lineages in the BM to be analyzed by flow cytometry without extensive enrichment. Furthermore, binding of platelets to non-megakaryocytic cells may result in false positivity for platelet-associated antigens, such as CD36. In a multiparameter setting, CD41a and CD61 have been used to analyze aberrancies in the megakaryocytic lineage [42]. However, characterization of abnormalities in megakaryocytes by flow cytometry was less sensitive than morphology [42].

Pitfalls in Flow Cytometry in MDS Recommendations for sample handling and processing are described in a report from the ELNet working conference for the standardization of flow cytometry in MDS [23]. Basic requirements such as adequate standardization of flow cytometric instruments and procedures should be fulfilled. In MDS sample handling procedures, such as the choice of anti-coagulant, temperature, delays in sample handling can cause considerable changes in antigen expression patterns. It is recommended that BM is collected in heparin-coated tubes, stored at room temperature and analyzed within 24€h after drawing. Delayed processing of samples that are drawn in EDTA-coated tubes may influence antigen expression of antigens such as CD11b [11, 40, 50]. Table€8.3 gives an overview of the pitfalls in flow cytometric analysis of MDS. Although the degree of dysplasia and quantification of cells by flow cytometry correlates with morphology, there are discrepancies between flow cytometric findings and morphologic findings in the BM. When analyzing myeloid progenitors with flow cytometry, it should be kept in mind that the definition of a myeloid blast by morphology can be different than by flow cytometry. Progenitor B cells can also resemble myeloid progenitors. Granular blasts may be excluded from myeloid progenitor analyses and can give an underestimation of myeloid progenitor count by flow cytometry. It is of importance to use a combination of antibodies that defines the myeloid progenitor compartment by flow cytometry and that recognizes contamination with plasmacytoid dendritic cells, basophils and hypogranular neutrophils. The percentage of immature erythroid cells may be underestimated caused by impaired lysing of non-nucleated erythroid cells and loss of a variable amount of nucleated erythroid cells. Therefore, the myeloid to erythroid ratio might be higher in flow cytometry than by morphology. Flow cytometric analysis on BM

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Table 8.3↜渀 Pitfalls in the analysis of BM in MDS by flow cytometry General • Lack of knowledge on normal hematopoiesis and changes in antigen expression that are not specific for MDS • Lack of age-matched (pathologic) controls • Awareness of the presence of a PNH clone Pitfalls in analysis of • Definition by flow cytometry versus definition by morphology myeloid progenitors • Contamination with plasmacytoid dendritic cells, basophils and/or hypogranular neutrophils • Hemodilution • Apoptosis Pitfalls in analysis of maturing myelo• CD33 polymorphism monocytic cells • Hypogranularity • Contamination with eosinophils in the neutrophil gate • Aberrant expression of HLA-DR on mature neutrophils • Aberrant lack of CD10 on mature neutrophils Pitfalls in analysis of • Loss of variable amount of (non)-nucleated erythroid cells durerythroid cells ing the lysing procedure Technical issues • Lack of standardization of flow cytometric instruments and procedures • Inadequate sample handling procedures: choice of anti-coagulant, temperature, delay in sample handling General, patient and (pathologic) control group related points of attention are listed. Pitfalls per subpopulation of hematopoietic cells are described next to basic technical requirements that need to be fulfilled

should preferably be performed on the BM sample from one of the first draws. A discrepancy in blasts count by flow cytometry and morphology may also be caused by hemodilution. The sample that is drawn for morphology smears contains more BM spicules than samples from subsequent draws. Therefore, the BM sample that is analyzed for flow cytometry may be (more) hemodiluted. The degree of PB contamination can be estimated by using the Holdrinet method, which is based on the red blood cell count and leukocyte count in BM and PB [5, 17]. Hemodilution can be corrected by using CD16 expression on mature neutrophils. The percentage of CD16 positive mature neutrophils from disaggregated BM trephine biopsies was determined by flow cytometry and used for normalization of blast counts in BM aspirates [24]. However, the power of flow cytometry is the detection of qualitative aberrancies in the (im)mature myelo-monocytic and erythroid lineage rather than quantification of cells in the BM. Furthermore, in MDS dyspoiesis may cause difficulties in analysis of subpopulations of cells by flow cytometry. Neutrophils in MDS can be hypogranular, resulting in a low SSC in MDS and interfere in monocytes analysis. Eosinophils, defined as CD45 positive and high SSC, can cause a high total neutrophil SSC and can interfere with the differentiation pattern of neutrophils. Therefore, eosinophils should be excluded from neutrophil analysis. Distinction of myelo-monocytic subpopulations in the BM might be hampered due to aberrant expression of HLA-DR or lack of CD10 expression on neutrophils. Next to that, apoptosis can cause loss of antigen expression and can be mistaken for aberrant expression. Knowledge of normal vari-

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ation in expression of antigens at each stage of differentiation of hematopoiesis and polymorphisms such as CD33 is relevant. Moreover, by using an appropriate agematched (pathologic) control group, alterations in antigen expression patterns that are not caused by MDS such as following growth factor treatment and with cell activation can be recognized. An aberrant immunophenotype should be distinguished from variations related to a relative increase of a proportion of BM hematopoietic cells such as a (non-clonal) increase in immature cells, maturation arrest and hemodilution [8]. Furthermore, the presence of a paroxysmal nocturnal hemoglobinuria (PNH) or PNH type cells in the BM of MDS patients is common and might be the cause of aberrant patterns of myelo-monocytic cells [32]. Knowledge of what may cause changes in antigen expression relationships in hematopoiesis that are not attributable to dyspoiesis in MDS and the application of multiparameter analysis can help to overcome some of these pitfalls.

Role in Diagnosis The diagnostic utility of multi-parameter flow cytometry of the myeloid, erythroid and megakaryocytic lineage in the BM for MDS was first described by Stetler-Stevenson et€al. [42]. In a series of 65 patients, a diagnosis of MDS was established by morphology in 45 patients. In these patients, the rate of detection of abnormalities in two or more lineages was 35/40 (88%) by flow cytometry and 37/40 (93%) by morphology. Interestingly, flow cytometry was informative in 15/20 (75%) patients with initial indecisive BM morphology by showing bi- and/or tri-lineage immunophenotypic aberrancies. For the megakaryocytic lineage there were only quantitative findings that were similar with the control group. Therefore, it was concluded that morphology was more sensitive for the detection of megakaryocytic dysplasia. This study showed that flow cytometry could be instrumental in the detection of immunophenotypic aberrancies in cytopenic patients with non-diagnostic BM morphology and cytogenetics. Several studies show that flow cytometric analyses correlate with established diagnostic classification systems, such as French-American-British (FAB) and World Health Organization (WHO) [3, 18, 31, 49, 54]. Table€8.4 gives an overview of the flow cytometric studies that have been performed until now. One of these studies analyzed aberrancies in the immature myeloid and mature myelo-monocytic compartment in a group of 207 MDS patients by flow cytometry [31]. The data analysis was performed by using hierarchal clustering of fluorescent intensities of the antibody expressions. By using the most discriminating markers CD16, CD34, CD36, CD38, CD71 and HLA-DR, the immunophenotypic clusters for myeloid progenitors identified subgroups of patients with RAEB/RAEB-T, CMML and RA(RS). For the neutrophil compartment patients with RA(RS) and CMML, RAEB and RAEB-T clustered together by using CD11b, CD13, CD33, CD36, CD38, CD71 and HLA-DR. Aberrancies in the monocytic compartment did not form discriminatory clusters of MDS patients, which might be caused by an inadequate combina-

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Most important conclusion for the diagnosis and prognosis of MDS Neutrophil Stetler-Steven- Myeloid: 35/40 (88%) MDS patients hypogranularity son et€al. CD11 b/CD16/CD13 were identified by flow Decreased CD71 Erythroid: cytometry [42] expression on CD71/CD235a 15/20 (75%) with inconclusive CD235a+ erythroid Megakaryocytic: morphology were identified CD41a/CD61 as MDS by flow precursors After repeated BM biopsies Immunophenotypic clusters Maynadié et€al. Mature myeloid: [31] CD11b, CD13, based on the most discriminating combination CD33, CD36, of markers correlated with CD38, CD71, FAB classification HLA-DR Myeloid progenitors: CD16, CD34, CD36, CD38, CD71, HLA-DR (Im)mature myelo- Antigen homogeneity FCSS correlates with WHO Wells et€al. 2001 classification Presence of lineage [54] monocytic: infidelity markers Severe pre-transplantation CD13/CD16, FCSS was associated with CD11b/HLA-DR Decreased CD45 post-transplantation relapse expression CD5, CD7, CD19 90% of MDS patients had a CD14, CD33, CD34 positive FCSS Kussick et€al. Myeloid progenitors: Aberrant antigen Flow identified MDS with [20] a sensitivity of 89% and expression in the CD13, CD33, CD38 specificity of 88% myeloid progeniCD117, HLA-DR tor compartment Flow correlated with morphology and cytogenetics A single aberrancy was not able Myeloid progenitors: Increased percentMalcovati to differentiate MDS from age of myeloid CD33, CD34 et€al. [27] non-MDS cases progenitors Myeloid cells: A discriminant analysis identiCD10, CD16, CD33, Presence of CD10fied 87% of MDS patients neutrophils, CD56 expression of Erythroid cells: immature markers CD71/CD235a Increased proportion of erythroid progenitors Lorand-Metze Myeloid progenitors: Myeloid progenitors: A set of parameters, including SSC of CD34 positive cells, SSC CD34 et€al. [25] promyelocytes and metamyMyeloid cells: Myelo-monocytic elocytes was able to classify Number of neutrocells; 87% of MDS patients phils, promyeloCD10, CD11b, cyte SSC CD13, CD16, Monocytes: CD64 Number of Erythroid cells: monocytes CD71/CD235a

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Most important conclusion for the diagnosis and prognosis of MDS Stachurski Myeloid progenitors: Myeloid progenitors: Lineage infidelity marker expression on myeloid Increased CD117 et€al. [40] CD117 progenitors was exclusively expression Prospectively CD2, CD5, CD7, seen in MDS Mature myeloid: CD56 validated Sensitivity to diagnose MDS Hypogranularity, by Truong Myeloid cells: was 98% and specificity aberrant CD11b/CD16/CD13 et€al. [47] 78% CD11b/CD13/CD16 Monocytes: CD56 on monocytes was assopattern CD13 and CD56 ciated with MDS/MPD Monocytes: Aberrant CD13 expression 60% of MDS patients with van de Loos(Im)mature myelo- Presence of lineage transfusion dependency drecht et€al. infidelity marker monocytic: or progressive disease had expression CD13/CD16, [49] myeloid progenitors with CD11b/HLA-DR Decreased percentaberrant CD7 or CD56 age of B cell CD5, CD7, CD19 expression progenitors CD14, CD33, CD34 Neutrophil hypogran- 92% patients with unilineage erythroid dysplasia by ularity, aberrant morphology had multiCD11 b/CD13/ lineage dysplasia by flow CD16 pattern cytometry Increased or decreased number of monocytes The number of patients identiDecrease in relative Ogata et€al. Lymphoid and fied by flow for Japan was number of B cell [35] myeloid 30.8% and for Italy 67.3% progenitors progenitors: Decreased SSC of CD34, CD45 neutrophils Myeloid cells: SSC, CD10 Decreased CD38 expression Goardon et€al. Myeloid progenitors: CD38 expression is identified MDS patients CD34/CD38 decreased in MDS [13] with 95% sensitivity and patients 92% specificity Aberrant immunophenotype of Westers et€al. Myeloid progenitors: Aberrant myeloid myeloid progenitors was a progenitors in CD13, CD33, CD34, [55] biomarker for the prediction 23/46 (50%) of CD45, CD117 of response to growth factor cases CD5, CD7, CD56 treatment in low and int-1 risk MDS patients

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136 Table 8.4╇ (continued) Study Most relevant (com- Most common aberbination of) MoAbs rancies in MDS Matarraz et€al. [30]

Myeloid progenitors: CD34, CD117 Myeloid cells: CD11b/CD13/CD16, CD15, CD33, CD65, MPO Monocytes: CD14, CD36, CD64 CD2, CD56 Erythroid cells: CD36, CD71/ CD235a

Increased number of CD34 positive cells Presence of aberrant CD34 negative myeloid progenitors Decreased number of mature neutrophils CD34 negative erythroid precursors Increased number of CD36 negative/ dim erythroid precursors

Most important conclusion for the diagnosis and prognosis of MDS The number and degree of severity of flow cytometric aberrancies discriminated between MDS and (pathologic) controls High immunophenotypic scores were associated with adverse prognostic factors The immunophenotypic score was an independent prognostic factor for overall survival

The subpopulations of hematopoietic cells in the BM that were analyzed by flow cytometry for each study are given. The most common found aberrancies and the most relevant conclusions for the diagnosis and prognosis of MDS are described in the table for each study. The antibody combinations CD11b/CD16/CD13 and CD71/CD235a were analyzed in combination in order to analyze differentiation of neutrophils and erythrocytes, respectively MPO myeloperoxidase

tion of antibodies or inaccurate gating strategy that was applied in this study to analyze monocytes by flow cytometry. The immunophenotypic clusters correlated with the established FAB classification for the diagnosis of MDS. To make a more simple, numerical display of flow cytometric data, a flow cytometric scoring system (FCSS) was developed [54]. This FCSS showed a good correlation with WHO 2001 classification for MDS [49]. However, flow scores were heterogeneous within WHO subgroups. This indicates that flow cytometry might identify subgroups within existing classification systems and offer a more refined classification with potential prognostic impact. Del Canizo et€al. investigated the immunophenotypic differences between MDS patients and healthy individuals by flow cytometry. The BM of 101 patients MDS patients was analyzed and compared with the BM of 12 healthy volunteers. Four types of aberrant immunophenotypes were identified; CD15 positive, HLA-DR negative, CD34 positive myeloid progenitors, abnormally low CD45 expression on monocytes, CD33 overexpression and/or HLA-DR expression on neutrophils. The presence of three or more aberrancies was more frequent in MDS RAEB patients as compared to lower risk MDS patients [9]. Kussick et€al. validated an antibody panel to analyze the immature myeloid and myelo-monocytic compartment to diagnose MDS in a group of 124 patients with unexplained cytopenias or monocytosis. Five major types of abnormalities were identified: deviations in myeloid antigen intensity, abnormal homogenous antigen expression, asynchronous expression of two myeloid-associated antigens, aber-

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rant expression of non-myeloid antigens on myeloid cells and decreased neutrophil SSC. Subsequently, patients were classified as flow-normal, flow-intermediate and flow-abnormal depending on flow cytometric findings. Antigens that were aberrant in at least 50% or more patients in at least one of the defined flow subgroups were HLA-DR, CD13, CD33, CD38 and CD117 and mainly in the immature myeloid compartment [20]. In a study by Stachurski et€al. a comparable approach was applied in a group of 180 MDS patients. MDS or MDS/MPD patients were correctly classified when flow was positive with a sensitivity of 84% and specificity of 97% [40]. In a later prospective study, 102 cytopenic patients with inconclusive BM morphology and cytogenetics were analyzed using the same flow cytometric approach [47]. Twelve cytopenic patients developed MDS during follow-up and 9 (75%) were identified by flow cytometry. Sixty-one patients had a non-MDS related cytopenia, flow was positive in only 4 (7%) patients. Therefore, the positive predictive power of flow cytometry was 69% and negative predictive value was 95%. Malcovati et€al. used a learning cohort of 103 MDS patients and 46 pathologic and healthy controls and a cohort of 69 MDS patients and 46 pathologic controls to validate a flow cytometric approach for the diagnosis of MDS [27]. A single aberrancy was not able to differentiate MDS from non-MDS cases. Therefore, the erythroid and (im)mature myeloid flow cytometric aberrancies were integrated in a discriminant analysis. In the validation cohort of 69 patients, erythroid and myeloid discriminant analysis correctly diagnosed 60 (87%) MDS patients. In this cohort, conclusive BM morphology was obtained in 44 (64%) MDS patients. Interestingly, the discriminant function analysis identified MDS RA(RS) patients that already had multi-lineage involvement according to flow cytometry but with only erythroid dysplasia by morphology. Previously, multilineage dysplasia as determined by morphology was associated with adverse clinical outcome [28]. A study by van de Loosdrecht et€al. also showed that aberrancies in the myelo-monocytic lineage could be detected in the majority of MDS patients with unilineage erythroid dysplasia (92%, 12/13) according to morphology and WHO classification [49]. To widely apply flow cytometry in laboratories it is necessary to make a diagnostic test that is simple and reproducible, with a high sensitivity and specificity. In a collaborative study between Japan and Italy, a flow cytometric test to diagnose low risk MDS patients was designed based on four cardinal parameters [35]. Bone marrow samples of 134 low-risk MDS patients were analyzed for the percentage of myeloid progenitors, B cell progenitors, CD45 expression of myeloid progenitors and neutrophil SSC. The number of MDS patients that could be identified by flow cytometry was 30.8% for Japan and 67.3% for Italy. The percentages increased to 65.4% and 89.4% for Japan and Italy, respectively, when adjunctive parameters CD11b, CD15 and CD56 expression on myeloid progenitors were added. Until now, most studies focussed on multi-parameter flow cytometry for the diagnosis of MDS. Interestingly, it was reported that CD38 expression on CD34 positive cells might be of value as a single flow cytometric parameter for the diagnosis of MDS [13]. CD38 expression in MDS patients with less than 5% blasts is significantly decreased compared with pathologic controls and even more decreased in MDS patients with more than 5% blasts.

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The majority of flow cytometric studies focussed on the enumeration of phenotypic abnormalities which might provide a measurement for the degree of dysregulation of hematopoiesis or distance from normal. However, some phenotypic aberrancies might be of more significance than others in detecting dysregulation of hematopoiesis. Weighing immunophenotypic aberrancies for their severity may be required for the diagnosis of MDS [23]. Furthermore, developments in flow cytometric analysis software might be helpful in quantification of the distance from normal and discrimination of MDS cases from normal or non-clonal hematopoietic disorders. Flow cytometric analyses of BM of cytopenic patients patients needs to be further developed and validated, also in new disease entities such as idiopathic cytopenia of unknown significance. In conclusion, the current established diagnostic approach for patients with cytopenia and a suspicion of MDS should include morphological examination of BM and PB, BM biopsy and cytogenetic investigations [48]. As can be concluded from flow cytometric studies, immunophenotyping of BM of cytopenic patients might provide complementary diagnostic information.

Role in Prognosis The subgroups in the FAB and WHO 2001 classification system provide prognostic information. MDS patients in FAB RAEB/RAEB-T, WHO RAEB-1 and RAEB-2 have higher risk of progression to an AML and shorter overall survival compared with MDS FAB RA(RS) and WHO 2001 RA(RS)/RCMD(RS). As described above flow cytometric evaluation of BM of MDS patients correlates with the established classification systems FAB and WHO 2001. Within the low risk categories, multi lineage dysplasia is of prognostic relevance. The IPSS and WPSS provide a prognostic scoring system integrating not only morphologic information but also karyotyping and transfusion need, respectively. Several studies show that flow cytometric aberrancies in BM of MDS patients correlate with the prognostic scoring systems IPSS and WPSS (Table€8.4) [25, 27, 31, 36, 49, 54]. In studies that quantified the number of flow cytometric aberrancies in BM of MDS patients, high numbers of aberrancies were associated with (high risk) MDS categories and an adverse clinical outcome [9, 49, 54]. In a series of 77 newly diagnosed MDS patients, immunophenotypic studies were carried out by flow cytometry analyzing the combinations: CD34/CD33/CD38, CD15/CD34/HLADR and HLADR/CD13/CD45 in BM. Overall 90% of patients had immunophenotypic aberrations and 60% showed two or more aberrations. In univariate analysis, the finding of more than three immunophenotypic aberrations had a significant negative influence on survival. In patients lacking cytogenetic information or in which the karyotype was normal, flow cytometry added significant prognostic information in multivariate analysis [2]. Wells et€al. designed a flow cytometric scoring system (FCSS) based on aberrancies in the (im)mature myelo-monocytic compartment. The FCSS comprised the categories normal-mild (0–1 points), moderate (2–3 points) and severe (4 points or more) degrees of dysplasia. In a group of 111 MDS patients that were treated with allogeneic

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hematopoietic stem cell transplantation, flow scores correlated with posttransplantation outcome independent of the IPSS score. The probability of posttransplantation relapse in a MDS patient with a severe pretransplantation FCSS score was 33% (Pâ•›<â•›0.01) with a bad overall survival (36%, Pâ•›<â•›0.01) compared to the normal-mild and moderate FCSS categories. The FCSS was validated in a group of 152 MDS patients. The hazard ratio of MDS patients for relapse after stem cell transplantation in the severe FCSS category was 2.8 (Pâ•›=â•›0.02) compared with MDS patients in the normal-mild category [39]. Interestingly, in the FCSS extra points are given to patients with an increased percentage of myeloid progenitors or aberrant myeloid progenitors when the percentage of myeloid progenitors is not increased in the BM. Myeloid progenitors were abnormal if there was homogenous expression of antigens such as CD34 or CD117 (29% of patients), expression of lymphoid antigens such as CD5, CD7, CD19 or CD56 (23% of patients) and an abnormal decrease in CD45 expression (16% of patients) [54]. There are indications that aberrancies in the (myeloid) progenitor compartment might be of more relevance for prognostication of MDS patients than aberrancies in the mature myelo-monocytic compartment [30, 49, 55]. Low risk MDS patients with less than 5% blasts can already have abnormal blasts by flow cytometry [20, 39, 49, 54, 55]. More importantly, these low risk MDS patients have an adverse clinical outcome with a higher risk for transfusion dependency and/or progressive disease, independent of existing and validated prognostic classification systems [49]. In the group of transfusion dependent patients and those with progressive disease, 60% had myeloid progenitors with CD7 or CD56 expression compared with 9% of the non-transfusion dependent patients. There are indications that myeloblasts with CD7 expression have more aggressive characteristics than myeloblasts without CD7 expression, such as a high proliferative capacity and less apoptosis in vitro [37]. The expression of lymphoid antigens by a significant proportion of myeloid or monocytic cells might carry more weight than altered expression of myeloid or monocytic antigens [8]. Quantification of CD34 positive cells in the PB of MDS patients by flow cytometry might be a relevant tool for the prognostication of MDS patients [6]. Low risk MDS patients with circulating blasts have a comparable prognosis as MDS RAEB1 patients [19]. According to the WHO 2008 classification, a percentage of 2–4% circulating blasts, but less than 5% blasts in the BM defines a patient already as a MDS RAEB-1 [44]. Flow cytometric quantification of circulating CD34 positive cells might be a more sensitive tool than counting by automated differential [1, 6]. At this stage further prospective studies are warranted to validate the prognostic relevance of flow cytometric findings in MDS patients.

Prediction of Response to Therapy by Flow Cytometry An appropriate and active approach to the treatment of MDS patients is important, not only for improvement of the quality of life, but also to increase overall survival [16]. The first line of treatment in IPSS low and intermediate-1 risk MDS patients is

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supportive therapy consisting of transfusions and growth factors such as erythropoietin (Epo) and human recombinant granulocyte-CSF (G-CSF). Response to treatment can be predicted by a model that includes pre-treatment endogenous serum Epo levels and transfusion need [14]. This validated score system distinguishes three patient groups: MDS patients that are likely to respond to growth factor treatment (74%), with intermediate probability (23%) and poor response to treatment (7%) [15]. In a group of 46 IPSS low and intermediate-1 risk MDS patients who were treated with Epo and G-CSF and evaluated by flow cytometry, the presence of immunophenotypically aberrant myeloid progenitors was instrumental in predicting response to growth factor treatment [55]. Aberrancies in the myeloid progenitor compartment included lineage infidelity marker expression (CD5, CD7 or CD56), loss of CD45 expression and loss of CD33 expression. A new predictive model based on flow cytometry and endogenous serum Epo levels was proposed and identified three subgroups of MDS patients. Patients with low serum Epo and normal myeloid progenitors by flow cytometry have a high probability to respond to growth factor treatment (94%) compared with patients with high serum Epo levels and aberrant myeloid progenitors with a low probability to respond to treatment (11%). Concluding from these findings, flow cytometry might add significantly to validated predictive models in the selection of patients that are eligible for Epo/G-CSF treatment. Furthermore, new treatment strategies for high risk MDS patients with the potential to change the natural course of the disease are emerging. These developments require standardized criteria to assess response to treatment. Flow cytometry might be instrumental in predicting and monitoring treatment of MDS patients.

Conclusion Recent developments show that the detection of aberrancies in hematopoietic cells by flow cytometry in MDS might be of diagnostic and prognostic relevance. Furthermore, the detection of aberrant myeloid progenitors might be instrumental in treatment decisions and monitoring. Prospective studies are necessary to validate the previously studied flow cytometric approaches next to validated diagnostic and prognostic classification systems. It is anticipated that flow cytometry will be part of the diagnostic and prognostic work-up of MDS patients next to morphology and cytogenetics in the near future.

References 1. Alhan C et€ al (2009) Do peripheral blasts count in myelodysplastic syndromes? Leuk Res 33:209–211 2. Arroyo JL et€al (2004) Impact of immunophenotype on prognosis of patients with myelodysplastic syndromes. Its value in patients without karyotypic abnormalities. Hematol J 5:227–233

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╇ 3. Bennett JM et€al (1982) Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51:189–199 ╇ 4. Bowen KL, Davis BH (1997) Abnormal patterns of expression of CD16 (Flow cytometryRIII) and CD11b (CRIII) antigens by developing neutrophils in the bone marrow of patients with myelodysplastic syndrome. Lab Hematol 3:292–298 ╇ 5. Brooimans RA et€ al (2008) Flow cytometric differential of leukocyte populations in normal bone marrow: influence of peripheral blood contamination 1. Cytometry B Clin Cytom 76B:18–26 ╇ 6. Cesana C et€al (2008) Prognostic value of circulating CD34+ cells in myelodysplastic syndromes. Leuk Res 32:1715–1723 ╇ 7. Cherian S et€al (2005) Flow-cytometric analysis of peripheral blood neutrophils: a simple, objective, independent and potentially clinically useful assay to facilitate the diagnosis of myelodysplastic syndromes. Am J Hematol 79:243–245 ╇ 8. Craig FE, Foon KA (2008) Flow cytometric immunophenotyping for hematologic neoplasms. Blood 111:3941–3967 ╇ 9. Del Canizo MC et€ al (2003) Immunophenotypic analysis of myelodysplastic syndromes. Haematologica 88:402–407 10. Della Porta MG et€ al (2006) Flow cytometry evaluation of erythroid dysplasia in patients with myelodysplastic syndrome. Leukemia 20:549–555 11. Elghetany MT, Davis BH (2005) Impact of preanalytical variables on granulocytic surface antigen expression: a review. Cytometry B Clin Cytom 65:1–5 12. Feller N et€al (2004) MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukaemia. Leukemia 18:1380–1390 13. Goardon N et€al (2009) Reduced CD38 expression on CD34+ cells as a diagnostic test in myelodysplastic syndromes. Haematologica 94:1160–1163 14. Hellström-Lindberg E et€al (1997) Erythroid response to treatment with G-CSF plus erythropoietin for the anaemia of patients with myelodyplastic syndromes: proposal for a predictive model. Br J Haematol 99:344–351 15. Hellström-Lindberg E et€al (2003) A validated decision model for treating the anaemia of myelodysplastic syndromes with erythropoietinâ•›+â•›granulocyte colony-stimulating factor: significant effects on quality of life. Br J Haematol 120:1037–1046 16. Hellström-Lindberg E, Malcovati L (2008) Supportive care and use of hematopoietic growth factors in myelodysplastic syndromes. Semin Hematol 45:14–22 17. Holdrinet RS et€al (1980) A method for quantification of peripheral blood admixture in bone marrow aspirates. Exp Hematol 8:103–107 18. Jaffe ES et€ al (2001) World Health Organization classification of tumours: pathology and genetics of tumours of haematopoietic and lymphoid tissues. IARC Press, Lyon 19. Knipp S et€al (2008) Presence of peripheral blasts in refractory anemia and refractory cytopenia with multilineage dysplasia predicts an unfavourable outcome. Leuk Res 32:33–37 20. Kussick SJ et€al (2005) Four-color flow cytometry shows strong concordance with bone marrow morphology and cytogenetics in the evaluation for myelodysplasia. Am J Clin Pathol 124:170–181 21. Lacronique-Gazaille C et€al (2007) A simple method for detection of major phenotypic abnormalities in myelodysplastic syndromes: expression of CD56 in CMML. Haematologica 92:859–860 22. Loken MR et€al (1987) Flow cytometric analysis of human bone marrow: I. Normal erythroid development. Blood 69:255–263 23. Loken MR et€al (2008) Flow cytometry in myelodysplastic syndromes: report from a working conference. Leuk Res 32:5–17 24. Loken MR et€al (2008) Normalization of bone marrow aspirates for hemodilution in flow cytometric analyses. Cytometry B Clin Cytom 76B:27–36 25. Lorand-Metze et€al (2007) Detection of hematopoietic maturation abnormalities by flow cytometry in myelodysplastic syndromes and its utility for the differential diagnosis with nonclonal disorders. Leuk Res 31:147–155

142

C. Alhan et al.

26. Lorand-Metze et€al (2008) The prognostic value of maturation-associated phenotypic abnormalities in myelodysplastic syndromes. Leuk Res 32:211–213 27. Malcovati L et€al (2005) Flow cytometry evaluation of erythroid and myeloid dysplasia in patients with myelodysplastic syndrome. Leukemia 19:776–783 28. Malcovati L et€ al (2005) Prognostic factors and life expectancy in myelodysplastic syndromes classified according to WHO criteria: a basis for clinical decision making. J Clin Oncol 23:7594–7603 29. Matarraz S et€al (2008) The immunophenotype of different immature, myeloid and B-cell lineage-committed CD34+ hematopoietic cells allows discrimination between normal/reactive and myelodysplastic syndrome precursors. Leukemia 22:1175–1183 30. Matarraz S et€al (2010) Bone marrow cells from myelodysplastic syndromes show altered immunophenotypic profiles that may contribute to the diagnosis and prognostic stratification of the disease: a pilot study on a series of 56 patients. Cytometry B Clin Cytom 78:154–168 31. Maynadié M et€ al (2002) Immunophenotypic clustering of myelodysplastic syndromes. Blood 100:2349–2356 32. Nakao S et€ al (2006) Clinical significance of a small population of paroxysmal nocturnal hemoglobinuria-type cells in the management of bone marrow failure. Int J Hematol 84: 118–122 33. Ogata K et€al (2002) Clinical significance of phenotypic features of blasts in patients with myelodysplastic syndrome. Blood 100:3887–3896 34. Ogata K et€al (2006) Diagnostic application of flow cytometric characteristics of CD34+ cells in low-grade myelodysplastic syndromes. Blood 108:1037–1044 35. Ogata K et€al (2009) Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study. Haematologica 94:1066–1074 36. Pirruccello SJ et€ al (2006) Myeloblast phenotypic changes in myelodysplasia. CD34 and CD117 expression abnormalities are common. Am J Clin Pathol 125:884–894 37. Satoh C et€ al (2008) Flow cytometric parameters with little interexaminer variability for diagnosing low-grade myelodysplastic syndromes. Leuk Res 32:699–707 38. Satoh C et€al (2009) Aggressive characteristics of myeloblasts expressing CD7 in myelodysplastic syndromes. Leuk Res 33:326–331 39. Scott BL et€ al (2008) Validation of a flow cytometric scoring system as a prognostic indicator for posttransplantation outcome in patients with myelodysplastic syndrome. Blood 112:2681–2686 40. Stachurski D et€ al (2008) Flow cytometric analysis of myelomonocytic cells by a pattern recognition approach is sensitive and specific in diagnosing myelodysplastic syndrome and related diseases: emphasis on a global evaluation and recognition of diagnostic pitfalls. Leuk Res 32:215–224 41. Sternberg A et€al (2005) Evidence for reduced B-cell progenitors in early (low-risk) myelodysplastic syndrome. Blood 106:2982–2991 42. Stetler-Stevenson M et€al (2001) Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome. Blood 98:979–987 43. Subirá D et€al (2008) Immunophenotype in chronic myelomonocytic leukemia: is it closer to myelodysplastic syndromes or myeloproliferative disorders? Trans Res 151:240–245 44. Swerdlow SH (2008) WHO classification of tumours of haematopoietic and lymphoid tissues. WHO Press, Switzerland 45. Tehranchi R et€ al (2005) Aberrant mitochondrial iron distribution and maturation arrest characterize early erythroid precursors in low-risk myelodysplastic syndromes. Blood 106:247–253 46. Terstappen LW et€al (1992) Flow cytometric characterization of acute myeloid leukemia. Part II: phenotypic heterogeneity at diagnosis. Leukemia 6:70–80 47. Truong F et€al (2009) The utility of flow cytometric immunophenotyping in cytopenic patients with a non-diagnostic bone marrow: a prospective study. Leuk Res 33:1039–1046

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143

48. Valent P et€al (2007) Definitions and standards in the diagnosis and treatment of the myelodysplastic syndromes: consensus statements and report from a working conference. Leuke Res 31:727–736 49. van de Loosdrecht AA et€al (2008) Identification of distinct prognostic subgroups in low and intermediate-1 risk myelodysplastic syndromes by flow cytometry. Blood 111:1067–1077 50. van de Loosdrecht AA et€ al (2009) Standardization of flow cytometry in myelodysplastic syndromes: report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromes. Haematologica 94:1124–1134 51. van Lochem EG et€al (2004) Immunophenotypic differentiation patterns of normal hematopoiesis in human bone marrow: reference patterns for age-related and disease-induced shifts. Cytometry B Clin Cytom 60:1–13 52. Wood BL (2007) Myeloid malignancies: myelodysplastic syndromes, myeloproliferative disorders, and acute myeloid leukaemia. Clin Lab Med 27:551–575 53. Wells DA et€al (1998) Multidimensional flow cytometry of marrow can differentiate leukemic from normal lymphoblasts and myeloblasts after chemotherapy and bone marrow transplantation. Am J Clin Pathol 110:84–94 54. Wells DA et€al (2003) Myeloid and monocytic dyspoiesis as determined by flow cytometric scoring in myelodysplastic syndrome correlates with the IPSS and with outcome after hematopoietic stem cell transplantation. Blood 102:394–403 55. Westers TM et€al (2010) Aberrant immunophenotype of blasts in myelodysplastic syndromes is a clinically relevant biomarker in predicting response to growth factor treatment. Blood 115:1779–1784 56. Xu Y et€al (2005) Flow cytometric analysis of monocytes as a tool for distinguishing chronic myelomonocytic leukaemia from reactive monocytosis. Am J Clin Pathol 124:799–806

Chapter 9

MDS as an Autoimmune Process W. Ingram, Y. Kordasti and G. J. Mufti

Introduction The Myelodysplastic Syndromes (MDS) represent a markedly heterogeneous group of haematopoietic stem cell disorders, with a wide diversity in outcome. Whilst some cases of MDS carry an indolent course with prolonged survival, others may progress rapidly to acute myeloid leukaemia (AML) and death. The pathophysiology of MDS remains poorly understood, in part due to the heterogeneity of the disease. Several clonal karyotypic and genetic mutations are reported in MDS and likely to play a key role in its pathogenesis. Moreover, defects in immune responses, both innate and adaptive, together with, or in the absence of, altered cytokine/chemokine profiles are integral to the disease process. It is hypothesised that a combination of environmental factors, genetic background and autoimmune responses play role in the pathogenesis of MDS (Fig.€9.1). This chapter will focus on the immunological aspects of MDS and provide the clinical evidence to support MDS as an autoimmune disorder.

Autoimmune Phenomena in MDS The association between autoimmunity and MDS is well described in the literature with up to 10% of MDS patients reported to have a concomitant autoimmune disorder [1–4]. The first review of which was reported by Hamblin et€al. in 1996 [1]. Several groups have since confirmed the association of MDS with a number of autoimmune disorders such as; vasculitis, arthritis, peripheral neuropathy, pulmonary infiltrates, inflammatory bowel disease, connective tissue disorders, glomeruloneW. Ingram () Department of Haematology, University Hospital of Wales, Heath Park, Cardiff, CF14 4XW, UK Tel.: +44-29-2074-2654 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_9, ©Â€Springer Science+Business Media B.V. 2011

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•Environmental factors •External Agent

•Genetic Background •Age

CD34+ Fate

NK cells

Immune response

Th17

γδ Τ

Th1

Foxp3+ Tregs

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Fig. 9.1↜渀 Interaction of environmental factors, genetic background and immune response in the pathogenesis of bone marrow failure in MDS. The pathophysiology of MDS is multi-factorial; involving genetic, environmental and immunological factors. Genetic factors predispose progenitor cells to an increased susceptibility to environmental triggers, a consequence of which is aberrant expression of cell surface molecules e.g. WT1. The initial immune attack may be directed against these aberrantly expressed proteins on CD34+ progenitor cells which lead to cell death and consequent exposure of self proteins. The self proteins are processed by antigen presenting cells with resulting immune attack against normal CD34+ cells. Several immune subsets then play a role in the immune mediated bone marrow failure in low risk MDS as indicated in the figure

phritis, thyroid abnormalities, sweets syndrome and autoimmune cytopenias [3–7]. The specificity of such autoimmunity is unclear and it remains to be discovered whether there is a relationship between the pathophysiology of autoimmune disorders and the immune mediated bone marrow failure observed in some cases of MDS. Moreover, a high incidence of asymptomatic immunological abnormalities are reported in MDS [8]. One such report describes the presence of autoantibodies, both organ and non-organ specific in 22% of MDS patients [8]. In addition, monoclonal gammopathies were observed in 12% and polyclonal hypergammaglobulinaemia or hypogammaglobulinaemia reported in a further 50% of cases [8]. In keeping with these findings, a further study has shown immunological abnormalities in up to 63% of MDS cases [5]. A summary of the autoimmune manifestations described in MDS patients is shown in Table€9.1. The question as to whether the presence of concurrent immunological disorders adversely affect outcome in MDS has been the subject of investigation by

9╅ MDS as an Autoimmune Process Table 9.1↜渀 Autoimmune manifestations reported in MDS patients

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Autoimmune cytopenias •â•‡ Haemolytic anaemia •â•‡ Thrombocytopenia Connective tissue disorders •â•‡ Systemic lupus erythematosis •â•‡ Raynaud’s phenomena •â•‡ Sjogren’s syndrome •â•‡ Polymyalgia rheumatica •â•‡ Relapsing poychondritis Acute systemic vasculitis Chronic autoimmune manifestations •â•‡ Peripheral neuropathy •â•‡ Skin vasculitis •â•‡ Polyarthritis •â•‡ Glomerulonephritis •â•‡ Vitiligo •â•‡ Chronic inflammatory demyelinating polyneuropathy •â•‡ Pyoderma gangrenosum •â•‡ Ulcerative colitis •â•‡ Hashimoto’s thyroiditis Asymptomatic serological findings •â•‡ Hypogammaglobulinaemia •â•‡ Polyclonal hypergammaglobulinaemia •â•‡ Monoclonal hypergammaglobulinaemia •â•‡ Rheumatoid factor positive •â•‡ Cryoglobulinaemia •â•‡ Anti-neutrophil antibody positive •â•‡ Lupus anticoagulant •â•‡ Coombs test positive

wseveral groups [2, 4, 5]. Although a poorer survival is described in MDS patients with associated autoimmunity, the majority of studies did not take into account the international prognostic scoring system (IPSS). As such, the poorer outcome may just reflect the advanced nature of the underlying disease, rather than a result of the concomitant autoimmune disorder.

Autoimmune Pathogenesis of MDS T cell Clonality and Antigen Specificity Increased CD8+ cytotoxic T lymphocytes (CTLs) displaying an effector phenotype are a feature of MDS [9–11]. A pattern of oligoclonality of such lymphocytes in MDS patients was first demonstrated by X-chromosome inactivation patterns [12]. Spectratyping, a method by which the TCRVβ repertoire is analysed, has confirmed early findings and shown the CD8+ T cells to consist of limited T cell receptors

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(TCRs), termed oligoclonal T cells [9, 10, 13, 14]. Oligoclonal T cells are a feature of several haematological disorders including; MDS, aplastic anaemia, T cell large granulocytic leukaemia and paroxysmal nocturnal haemoglobinuria (PNH) [15–17]. Of interest, in one study of MDS patients a correlation between a positive response to immunosuppressive therapy and a loss of clonal T cells was demonstrated [15]. Clonal expansion of T cells in MDS may indeed represent an autoreactive phenomenon as shown in other disorders such as, rheumatoid arthritis and multiple sclerosis [18, 19]. Although in contrast to the above studies in which the target antigen is well defined, the specificity of immune response in MDS remains unclear. Several hypotheses describing the mechanism by which oligoclonal T cells arise have been proposed. The prevalence of CMV infection increases with age, as such, some researchers speculate that the expansion of CD8+ CTLs could simply be a reflection of CMV infection rather than a genuine autoimmune response. However, other groups report a higher incidence of oligoclonal T cells in MDS patients compared to healthy age matched controls [20]. An alternate hypothesis proposes that effector T cells may be triggered as a result of increased apoptotic cytokines and aberrant expression of oncogenes or fusion genes in haematopoietic stem cells [13]. Whilst another group speculate a viral aetiology, in which chronically infected bone marrow stromal cells or immune cell subsets may lead to changes to the cytokine milieu in the BM microenvironment, which consequently result in a growth advantage for clonal haematopoietic stem cells [21]. In order to provide support for the theory of an antigen driven expansion of T cells in MDS, a search for common antigenic determinants in MDS patients has been performed. The clonotypes have been used as signatures of individual CTL clones, providing a surrogate marker for potential target antigens [16]. In one study, the variable region of the TCR α and β regions in MDS patients and erythroid hypoplasia was examined [14]. Although a limited repertoire of TCRVα regions was reported, with unique sequences in a high proportion of the complementaritydetermining region 3 (CDR3) of the TCR α and β chains of the expanded T-cells, the sequences within the CDR3 were heterogeneous between patients [14]. Similarly, a more recent study demonstrated CTL expansion in over 90% of MDS patients. However, despite the presence of immunodominant CDR3 clonotypes no unique clonotype was identified [15]. Although the studies indirectly support the theory that the CTL expansion in MDS is antigen driven, the specificity of antigen response remains poorly defined. Furthermore, to date, there is no clear evidence to support a unique and universal antigen trigger to the CTL response.

CD4+ T cells and MDS A less clear aspect of the immune biology of MDS is the potential role of CD4+ T cells. There are two important roles for the different subsets/lineages of CD4+ T cells in humans. (1) An indirect induction of the immune response and mainte-

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nance of the necessary inflammatory environment. (2) Regulation of immune response and peripheral tolerance [22]. Based on these two distinctive roles, CD4+ T cells may be grouped into T-helper cells or regulatory T cells (Tregs) respectively. Among different subsets of CD4+ T cells, Tregs and Th17 cells have been shown to have a significant correlation with MDS stage and risk of disease progression [23]. As such, these two subsets of CD4+ T cells are briefly reviewed here.

Regulatory T cells (Tregs) Tregs have been shown to play an important role in the maintenance of self tolerance. Initial studies in the early 1970s demonstrated that T cells were capable of exerting a suppressive function [24, 25]. Interest in the field was subsequently revived following work by Sakaguchi et€al. in the mid 1990s. Sakaguchi reported a population of CD4+ T cells with high expression of CD25, which were able to maintain self tolerance by down-regulating immune responses to self and non-self antigens, in an antigen non-specific manner [26]. Several subsets of T cells with suppressor function have since been identified and include; (1) naturally occurring Tregs; (2) antigen-induced Tr1 and Th3 cells; (3) adaptive Tregs; (4) CD8+ Tregs; (5) certain subsets of NKT cells [27]. Although the development, cytokine profiles and mode of action of the various subsets of regulatory T cells differs considerably, a function central to all is in the suppression of effector T cells [28]. The most widely studied subset being the CD4+CD25+Foxp3+ naturally occurring Tregs. Tregs play an important role in the tumour microenvironment by suppressing anti-tumour immune responses. Increased numbers of CD4+CD25+ Tregs are reported in several haematological and non-haematological malignancies. As previously described, the myelodysplastic syndromes encompass a heterogenous group ranging from cases with low risk disease and minimal blast cell numbers to others who present with advanced disease and rapid disease progression to AML. The pathophysiology of such disease subsets is likely to be quite disparate. Indeed an increased number of CD4+CD25highFoxp3+ Tregs is reported in high risk MDS, whilst no difference was observed on comparison of low risk MDS patients with healthy age matched controls [29]. In this study, high risk MDS was defined as >5% bone marrow blasts or high IPSS. In a further study, the function and trafficking of Tregs in MDS was analysed [30]. Dysfunctional Tregs with impaired bone marrow homing through the CXCL12/CXCR4 axis, as a result of down-regulation of CXCR4 was observed in patients with early stage MDS. In contrast, an increased number of Tregs with normal function and bone marrow homing were reported in cases with advanced MDS [30]. A fall in the number of Tregs was also noted in patients responding to treatment for MDS [30]. These studies support the hypothesis that in early stage disease the lower number of Tregs may facilitate the emergence of autoimmunity, whereas in keeping with other malignancies, the increased number of Tregs in high risk MDS may permit the expansion of leukaemic clones.

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Th17 Cells CD4+ IL-17 producing T cells, which are now widely known as Th17 cells, were recently proposed as the third lineage of T-helper cells in addition to Th1 and Th2 cells [31]. Th17 cells have been shown to play an important role in the immune response to pathogens and in autoimmunity [32–38]. Importantly, CD4+ T-helper cells can be induced to differentiate towards Th1, Th2, Th17 or Tregs depending on the local cytokine environment [39]. Although differences are noted between human and murine studies, IL-17 producing cells which co-express IFN-γ are reported in human studies of autoimmune disease [40, 41]. In light of the knowledge that low risk MDS is associated with a pro-inflammatory environment, the role of IL-17 producing T cells in MDS was examined. Indeed, a significantly higher number of IL-17 producing CD4+ T cells was reported in low risk MDS in comparison with high risk disease [23]. As expected, an inverse relationship between Th17 cells and Tregs was confirmed, with a higher ratio of Th17 cells to Tregs in low risk MDS and a lower ratio in high risk disease. In keeping with the notion that low risk MDS is associated with a pro-inflammatory environment, increased serum levels of IL-17, IL-12, RANTES and IFN-γ are reported in low risk disease. In contrast, an increase in the inhibitory factors IL-10 and soluble IL-2 receptor were observed in high risk MDS patients [23]. Although bone marrow apoptosis was notably higher in low risk disease and correlated with higher numbers of IL-17 producing cells, the mechanism by which IL-17 cells induce apoptosis remains unknown. Together these findings provide evidence to favour the hypothesis that bone marrow failure in low risk MDS may occur as a consequence of increased apoptosis induced by the inflammatory environment, which is created and maintained by Th17 cells, oligoclonal CD8+ T cells and proinflammatory cytokines.

Innate Immune Response Natural killer (NK) cells belong to the innate immune system and are important in the first line of defence against intracellular pathogens and tumour cells. The delicate balance of NK cell activation and inhibitory receptors together with their respective target cell ligands are responsible for regulating NK cell function. The role of NK cells in the pathophysiology of MDS has been explored in a small number of studies. Although a normal number of NK cells is reported in MDS, NK cell function has been shown to be impaired [42, 43]. In particular, NK cell cytotoxicity is significantly impaired in high risk MDS and may be as a result of down-regulation of NK cell activating receptors such as NKG2D [43]. Conversely, NK cell expression of granzyme B is reported to be higher in low and intermediate risk MDS patients with an increase in non-MHC directed killing of autologous haematopoietic precursor cells [44]. These results provide support for a potential role of the innate

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immune response in the pathogenesis of low risk MDS, whereas impaired NK cell function in high risk MDS may facilitate disease progression. Monocytes may also play a role in the immune dysregulation of MDS through the activation of T-helper cells. In a study of low risk MDS the effect of activated monocytes on the activation of T-helper cells through CD40-CD40 ligand (CD154) interactions was examined. Both a higher CD40 expression on monocytes and CD40 ligand expression on T-helper cells was observed [45]. Stimulation of CD40 on monocytes resulted in higher levels of TNF-α in MDS patients. Moreover, coculture of bone marrow mononuclear cells in the presence of CD40 blocking antibody, led to increased colony-forming units, supporting the hypothesis that the CD40-CD40 ligand may play a role in the immune mediated bone marrow failure in MDS [45]. Lastly the role of γδ T cells in the pathophysiology of MDS has recently been explored. γδ T cells play an important role in the rapid response against tumour and non tumour cells and are an important component of lymphoid stress-surveillance response [46–49]. γδ T cells bearing the Vγ9Vδ2 rearrangement constitute a small proportion (up to 10%) of circulating peripheral blood T cells. A lower number of γδ T cells is reported in MDS patients [50]. The results were most profound in MDS patients with associated autoimmune disorders. In a subset of MDS patients the γδ T cells failed to expand in response to bromohalohydrin pyrophosphate and demonstrated limited proliferative potential in response to IL-2, however, the cytolytic function was preserved in those where expansion was observed [50]. Although the importance of such findings to the pathophysiology of MDS remains unclear, the data provides further insight into the complexity of the immune dysregulation in MDS.

Bone Marrow Apoptosis Increased bone marrow apoptosis is a feature of MDS and is thought to contribute towards the ineffective haematopoiesis and consequent peripheral blood cytopenias [51, 52]. The majority of studies demonstrate that increased apoptosis is most prominent during the early stages of disease [53, 54]. Moreover, one study has shown an inverse correlation between the level of apoptosis of CD34+ cells and prognostic stage [55]. Although there remains some controversy over the precise phenotype of the cells which undergo apoptosis, the majority of studies favour involvement of early haematopoietic progenitor cells [53–56]. Disturbances within the bone marrow microenvironment such as the balance of stimulatory and inhibitory cytokines have been shown to play a key role in triggering increased cell death. Increased levels of TNFα and high expression of Fas on CD34+ cells appear to be a consistent finding [57–60]. Increased Fas expression can lead to activation of caspases, in particular caspase 3, which subsequently trigger apoptosis. Several other cytokines which have been studied include; IL-1β, IL-6, IL-8, GM-CSF, TGFβ and stem cell factor but have shown inconsistent results

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[61–63]. Bone marrow stromal cells may also play a role in triggering apoptosis. Studies have shown that stromal cells from MDS patients are able to induce apoptosis of normal CD34+ cells [64]. Furthermore, these stromal cells show decreased or absent growth in long term cultures [64]. Although the precise mechanism by which apoptosis is initiated in MDS is unclear, there is a general consensus that increased apoptosis is central to the pathophysiology of at least the early stages of MDS. Additional studies are nevertheless required to address the contribution of any potential molecular defects in triggering apoptosis and to further examine the role of the bone marrow microenvironment in its ability to support ongoing apoptosis. Importantly, the interplay between haematopoietic stem cells, the bone marrow microenvironment, stromal cells and trafficking of autoreactive T cells requires further study.

Immunosuppressive Therapy The treatment of MDS and the role of haematopoietic stem cell transplantation are described elsewhere within this book. As such, this chapter will focus on providing an overview of the use of immunosuppressive agents in MDS and the evidence generated forthwith to support MDS as an autoimmune disorder. Improvement in peripheral blood cytopenias is reported in just a minority of MDS patients following treatment with steroids [2, 65, 66]. Poor responses together with an increased risk of infection have led to steroids being an unattractive therapeutic option in MDS. Prednisolone and methylprednisolone have nonetheless been used to treat the autoimmune manifestations associated with MDS with variable success [2, 67]. Favourable responses to alternative agents such as cyclophosphamide or azathioprine are also described in a small proportion of MDS patients with associated autoimmune diseases [2]. Antithymocyte globulin (ATG) in combination with cyclosporine A has led to significant haematological responses in MDS [68–74] (Table€ 9.2). Younger age (<60 years), the presence of HLA-DR15 allele and less heavily transfusion dependent patients have been shown to predict response to ATG treatment [75, 76]. A recent study reported an overall response rate of 24% in patients treated with ATG alone, 8% with cyclosporine A and 48% following combination with ATG and cyclosporine A [77]. Transfusion independence and complete haematological response was observed in 31% of all responding patients [77]. Interestingly, age <60 years was the strongest predictor of outcome [77]. In a further study, low IPSS and bone marrow hypocellularity correlated with a favourable response to ATG therapy [78]. Importantly, the removal of CD3+ or CD8+ T cells from bone marrow progenitor cell cultures led to a significant increase in granulocyte-macrophage colony-forming units (CFU-GMs) in patients who responded to ATG, but no difference in nonresponders [79]. Furthermore, in-vitro suppression of CFU-GMs was observed after adding back peripheral blood lymphocytes from patients who responded to ATG. Of particular interest was the finding that the TCRVβ repertoire changed from clonal to

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Table 9.2↜渀 A summary of response rates and predictors of favourable outcome following ATG therapy in MDS Number of patients Response rate Predictors of favourable response median age years Molldrem et€al. [68]

25

Killick et€al. [69]

30 54.5 years (31–73 years) 31 59 years (28–79 years) 61

Yazji et€al. [72] Molldrem et€al. [74]

44% ORR 84% OS at 3 years 50% ORR

NA

16% ORR

NA

34% transfusion independence 47% platelet count increased 29% transfusion independence

Younger age (?what) Lower platelet count (?what)

Saunthararajah et€al. [75, 76]

69

Sloand et€al. [77]

74 ATG alone 42 ATG plus cyclosporine A

24% ORR 48% ORR

Lim et€al. [78]

96 54 years (19–75 years)

42% ORR

NA

HLA-DR15 Younger age Shorter duration red cell dependance PNH clone Younger age HLA-DR 15 IPSS low or intermediate IPSS low Bone marrow hypocellularity

ORR Overall response rate, NA Not available

polyclonal in one responding patient following ATG treatment [79]. A further study confirmed these early findings. Analysis of the TCRVβ spectratype was performed in patients before and after ATG therapy [20]. A loss or diminution in clonal T cell populations was observed in patients responding to ATG treatment. These results provide additional evidence for the role of autoreactive T cells in the pathophysiology of bone marrow suppression in MDS. Allogeneic haematopoietic stem cell transplantation (allo-HSCT) offers the potential for long term survival and even cure, in a subset of MDS patients. The role and outcome of transplantation in MDS is detailed elsewhere within this book. Nonetheless, it is prudent to recall that the benefit of allo-HSCT, in particular with respect to reduced intensity conditioned regimens, is based upon its ability to stimulate an immune mediated rejection of tumour cells. The important role of T cells in this process is highlighted by the reported increased relapse rates and a lower incidence of graft versus host disease (GvHD) following the use of T cell depletion [80, 81]. Moreover, the ability to restore a state of complete remission following administration of donor leukocyte infusions, provides further evidence for the central role of T cells and other potential immune subsets such as NK cells, in the graft versus leukaemia/tumour effect following allo-HSCT [82, 83].

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Concluding Remarks Although there remains no robust evidence to support an autoimmune basis for bone marrow failure in MDS, the observations described within this chapter, in particular skewed T cell subsets, altered cytokine profile, increased apoptosis and the high incidence of concomitant autoimmune disorders, support an immune mediated basis for the pathogenesis of early stage MDS. Moreover, the marked haematological responses following ATG treatment with consequent diminution in T cell clonality provide further evidence to support a key role of T cells in the pathogenesis. Further studies are required to define the potential antigens which stimulate the CTL response and to gain a better understanding of the interplay between the immune subsets and the cytokine/chemokine milieu in the bone marrow microenvironment.

References ╇ 1. Hamblin TJ (1996) Immunological abnormalities in myelodysplastic syndromes. Semin Hematol 33(2):150–162 ╇ 2. Enright H et€al (1995) Paraneoplastic autoimmune phenomena in patients with myelodysplastic syndromes: response to immunosuppressive therapy. Br J Haematol 91(2):403–408 ╇ 3. Castro M et€al (1991) Rheumatic manifestations in myelodysplastic syndromes. J Rheumatol 18(5):721–727 ╇ 4. Billstrom R et€al (1995) Immune-mediated complications in patients with myelodysplastic syndromes—clinical and cytogenetic features. Eur J Haematol 55(1):42–48 ╇ 5. Okamoto T et€al (1997) Correlation between immunological abnormalities and prognosis in myelodysplastic syndrome patients. Int J Hematol 66(3):345–351 ╇ 6. O’Donnell BF, Williams HC, Carr R (1995) Myelodysplastic syndrome presenting as cutaneous vasculitis. Clin Exp Dermatol 20(5):439–442 ╇ 7. Saif MW, Hopkins JL, Gore SD (2002) Autoimmune phenomena in patients with myelodysplastic syndromes and chronic myelomonocytic leukemia. Leuk Lymphoma 43(11):2083– 2092 ╇ 8. Mufti GJ et€al (1986) Immunological abnormalities in myelodysplastic syndromes. I. Serum immunoglobulins and autoantibodies. Br J Haematol 63(1):143–147 ╇ 9. Kook H et€al (2001) Increased cytotoxic T cells with effector phenotype in aplastic anemia and myelodysplasia. Exp Hematol 29(11):1270–1277 10. Melenhorst JJ et€al (2002) Molecular and flow cytometric characterization of the CD4 and CD8 T-cell repertoire in patients with myelodysplastic syndrome. Br J Haematol 119(1):97– 105 11. Epling-Burnette PK et€al (2007) Prevalence and clinical association of clonal T-cell expansions in myelodysplastic syndrome. Leukemia 21(4):659–667 12. Culligan DJ et€al (1992) Clonal lymphocytes are detectable in only some cases of MDS. Br J Haematol 81(3):346–352 13. Epperson DE et€al (2001) Oligoclonal T cell expansion in myelodysplastic syndrome: evidence for an autoimmune process. Leuk Res 25(12):1075–1083 14. Matsutani T et€al (2003) Determination of T-cell receptors of clonal CD8-positive T-cells in myelodysplastic syndrome with erythroid hypoplasia. Leuk Res 27(4):305–312 15. Wlodarski MW et€ al (2006) Molecular strategies for detection and quantitation of clonal cytotoxic T-cell responses in aplastic anemia and myelodysplastic syndrome. Blood 108(8):2632–2641

9â•… MDS as an Autoimmune Process

155

16. Risitano AM et€al (2002) Oligoclonal and polyclonal CD4 and CD8 lymphocytes in aplastic anemia and paroxysmal nocturnal hemoglobinuria measured by V beta CDR3 spectratyping and flow cytometry. Blood 100(1):178–183 17. O’Keefe CL et€al (2004) Molecular analysis of TCR clonotypes in LGL: a clonal model for polyclonal responses. J Immunol 172(3):1960–1969 18. Hong J et€ al (1999) A common TCR V-D-J sequence in V beta 13.1 T cells recognizing an immunodominant peptide of myelin basic protein in multiple sclerosis. J Immunol 163(6):3530–3538 19. Mima T et€al (1999) Dominant and shared T cell receptor beta chain variable regions of T cells inducing synovial hyperplasia in rheumatoid arthritis. Biochem Biophys Res Commun 263(1):172–180 20. Kochenderfer JN et€al (2002) Loss of T-lymphocyte clonal dominance in patients with myelodysplastic syndrome responsive to immunosuppression. Blood 100(10):3639–3645 21. Raza A (1998) Hypothesis: myelodysplastic syndromes may have a viral etiology. Int J Hematol 68(3):245–256 22. Abbas AK (2007) Cellular and molecular immunology, 6th€edn. Saunders, Philadelphia 23. Kordasti SY et€al (2009) IL-17-producing CD4(+) T cells, pro-inflammatory cytokines and apoptosis are increased in low risk myelodysplastic syndrome. Br J Haematol 145(1):64–72 24. Gershon RK, Kondo K (1971) Infectious immunological tolerance. Immunology 21(6):903– 914 25. Berendt MJ, North RJ (1980) T-cell-mediated suppression of anti-tumor immunity. An explanation for progressive growth of an immunogenic tumor. J Exp Med 151(1):69–80 26. Sakaguchi S et€al (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155(3):1151–1164 27. Kennedy R, Celis E (2008) Multiple roles for CD4+ T cells in anti-tumor immune responses. Immunol Rev 222:129–144 28. Vignali DA, Collison LW, Workman CJ (2008) How regulatory T cells work. Nat Rev Immunol 8(7):523–532 29. Kordasti SY et€al (2007) CD4+CD25high Foxp3+ regulatory T cells in myelodysplastic syndrome (MDS). Blood 110(3):847–850 30. Kotsianidis I et€ al (2009) Kinetics, function and bone marrow trafficking of CD4+CD25+FOXP3+ regulatory T cells in myelodysplastic syndromes (MDS). Leukemia 23(3):510–518 31. Dong C (2008) TH17 cells in development: an updated view of their molecular identity and genetic programming. Nat Rev Immunol 8(5):337–348 32. Chen Z, O’Shea JJ (2008) Th17 cells: a new fate for differentiating helper T cells. Immunol Res 41(2):87–102 33. Steinman L (2007) A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nat Med 13(2):139–145 34. Yen D et€al (2006) IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J Clin Invest 116(5):1310–1316 35. Cho ML et€al (2006) STAT3 and NF-kappaB signal pathway is required for IL-23-mediated IL-17 production in spontaneous arthritis animal model IL-1 receptor antagonist-deficient mice. J Immunol 176(9):5652–5661 36. Nakae S et€al (2003) IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist. Proc Natl Acad Sci U S A 100(10):5986–5990 37. Komiyama Y et€al (2006) IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis. J Immunol 177(1):566–573 38. Nakae S et€al (2003) Suppression of immune induction of collagen-induced arthritis in IL-17deficient mice. J Immunol 171(11):6173–6177 39. Afzali B et€al (2007) The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. Clin Exp Immunol 148(1):32–46

156

W. Ingram et al.

40. Annunziato F et€al (2007) Phenotypic and functional features of human Th17 cells. J Exp Med 204(8):1849–1861 41. van Beelen AJ et€al (2007) Stimulation of the intracellular bacterial sensor NOD2 programs dendritic cells to promote interleukin-17 production in human memory T cells. Immunity 27(4):660–669 42. Kiladjian JJ et€al (2006) Cytolytic function and survival of natural killer cells are severely altered in myelodysplastic syndromes. Leukemia 20(3):463–470 43. Epling-Burnette PK et€al (2007) Reduced natural killer (NK) function associated with highrisk myelodysplastic syndrome (MDS) and reduced expression of activating NK receptors. Blood 109(11):4816–4824 44. Chamuleau ME et€al (2009) Immune mediated autologous cytotoxicity against hematopoietic precursor cells in patients with myelodysplastic syndrome. Haematologica 94(4):496–506 45. Meers S et€al (2007) Monocytes are activated in patients with myelodysplastic syndromes and can contribute to bone marrow failure through CD40-CD40L interactions with T helper cells. Leukemia 21(12):2411–2419 46. Hayday AC (2009) Gammadelta T cells and the lymphoid stress-surveillance response. Immunity 31(2):184–196 47. Band H et€al (1990) Recognition of mycobacterial antigens by gamma delta T cells. Res Immunol 141(7):645–651 48. Kunzmann V et€al (2000) Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 96(2):384–392 49. Kunzmann V, Wilhelm M (2005) Anti-lymphoma effect of gammadelta T cells. Leuk Lymphoma 46(5):671–680 50. Kiladjian JJ et€al (2008) Activation of cytotoxic T-cell receptor gammadelta T lymphocytes in response to specific stimulation in myelodysplastic syndromes. Haematologica 93(3):381– 389 51. Yoshida Y (1993) Hypothesis: apoptosis may be the mechanism responsible for the premature intramedullary cell death in the myelodysplastic syndrome. Leukemia 7(1):144–146 52. Raza A et€al (1995) Apoptosis in bone marrow biopsy samples involving stromal and hematopoietic cells in 50 patients with myelodysplastic syndromes. Blood 86(1):268–276 53. Bouscary D et€al (1997) Fas/Apo-1 (CD95) expression and apoptosis in patients with myelodysplastic syndromes. Leukemia 11(6):839–845 54. Rajapaksa R et€al (1996) Altered oncoprotein expression and apoptosis in myelodysplastic syndrome marrow cells. Blood 88(11):4275–4287 55. Parker JE, Mufti GJ (1998) Ineffective haemopoiesis and apoptosis in myelodysplastic syndromes. Br J Haematol 101(2):220–230 56. Mundle S et€al (1999) The relative extent and propensity of CD34+ vs. CD34− cells to undergo apoptosis in myelodysplastic marrows. Int J Hematol 69(3):152–159 57. Lepelley P et€al (1998) Fas/APO-1 (CD95) expression in myelodysplastic syndromes. Leuk Lymphoma 30(3–4):307–312 58. Mundle SD et€al (1999) Correlation of tumor necrosis factor alpha (TNF alpha) with high Caspase 3-like activity in myelodysplastic syndromes. Cancer Lett 140(1–2):201–207 59. Kitagawa M et€al (1998) Localization of Fas and Fas ligand in bone marrow cells demonstrating myelodysplasia. Leukemia 12(4):486–492 60. Sawanobori M et€al (2003) Expression of TNF receptors and related signaling molecules in the bone marrow from patients with myelodysplastic syndromes. Leuk Res 27(7):583–591 61. Maurer AB et€al (1993) Restoration of impaired cytokine secretion from monocytes of patients with myelodysplastic syndromes after in vivo treatment with GM-CSF or IL-3. Leukemia 7(11):1728–1733 62. Bowen D et€al (1993) Serum stem cell factor concentration in patients with myelodysplastic syndromes. Br J Haematol 85(1):63–66 63. Fontenay-Roupie M et€al (1999) Ineffective erythropoiesis in myelodysplastic syndromes: correlation with Fas expression but not with lack of erythropoietin receptor signal transduction. Br J Haematol 106(2):464–473

9â•… MDS as an Autoimmune Process

157

64. Aizawa S et€al (1999) Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro. Leuk Res 23(3):239–246 65. Bagby GC Jr, Gabourel JD, Linman JW (1980) Glucocorticoid therapy in the preleukemic syndrome (hemopoietic dysplasia): identification of responsive patients using in-vitro techniques. Ann Intern Med 92(1):55–58 66. Motoji T et€al (1990) Successful treatment of refractory anemia with high-dose methylprednisolone. Am J Hematol 33(1):8–12. 67. George SW, Newman ED (1992) Seronegative inflammatory arthritis in the myelodysplastic syndromes. Semin Arthritis Rheum 21(6):345–354 68. Molldrem JJ et€ al (1997) Antithymocyte globulin for patients with myelodysplastic syndrome. Br J Haematol 99(3):699–705 69. Killick SB et€al (2003) A pilot study of antithymocyte globulin (ATG) in the treatment of patients with ‘low-risk’ myelodysplasia. Br J Haematol 120(4):679–684 70. Tichelli A et€al (1988) Antilymphocyte globulin for myelodysplastic syndrome. Br J Haematol 68(1):139–140 71. Steensma DP et€al (2003) Antithymocyte globulin has limited efficacy and substantial toxicity in unselected anemic patients with myelodysplastic syndrome. Blood 101(6):2156–2158 72. Yazji S et€al (2003) Antithymocyte globulin (ATG)-based therapy in patients with myelodysplastic syndromes. Leukemia 17(11):2101–2106 73. Aivado M et€al (2002) Favourable response to antithymocyte or antilymphocyte globulin in low-risk myelodysplastic syndrome patients with a ‘non-clonal’ pattern of X-chromosome inactivation in bone marrow cells. Eur J Haematol 68(4):210–216 74. Molldrem JJ et€al (2002) Antithymocyte globulin for treatment of the bone marrow failure associated with myelodysplastic syndromes. Ann Intern Med 137(3):156–163 75. Saunthararajah Y et€al (2003) A simple method to predict response to immunosuppressive therapy in patients with myelodysplastic syndrome. Blood 102(8):3025–3027 76. Saunthararajah Y et€al (2002) HLA-DR15 (DR2) is overrepresented in myelodysplastic syndrome and aplastic anemia and predicts a response to immunosuppression in myelodysplastic syndrome. Blood 100(5):1570–1574 77. Sloand EM et€al (2008) Factors affecting response and survival in patients with myelodysplasia treated with immunosuppressive therapy. J Clin Oncol 26(15):2505–2511 78. Lim ZY et€al (2007) Low IPSS score and bone marrow hypocellularity in MDS patients predict hematological responses to antithymocyte globulin. Leukemia 21(7):1436–1441 79. Molldrem JJ et€ al (1998) Haematological response of patients with myelodysplastic syndrome to antithymocyte globulin is associated with a loss of lymphocyte-mediated inhibition of CFU-GM and alterations in T-cell receptor Vbeta profiles. Br J Haematol 102(5):1314– 1322 80. Remberger M et€ al (2002) Association between pretransplant thymoglobulin and reduced non-relapse mortality rate after marrow transplantation from unrelated donors. Bone Marrow Transplant 29(5):391–397 81. Kottaridis PD et€al (2000) In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation. Blood 96(7):2419–2425 82. Kolb HJ et€al (1995) Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 86(5):2041–2050 83. Depil S et€al (2004) Donor lymphocyte infusion to treat relapse after allogeneic bone marrow transplantation for myelodysplastic syndrome. Bone Marrow Transplant 33(5):531–534

Chapter 10

The Myelodysplastic Overlap Syndromes W. Ingram and G. J. Mufti

Introduction The myelodysplastic overlap syndromes encompass a group of clonal neoplastic bone marrow disorders, which, at the time of presentation, demonstrate clinical, morphological or laboratory findings in keeping with both the myelodysplastic syndromes (MDS) and the myeloproliferative neoplasms (MPN) [1]. Cases present along a continuum, ranging from those with a predominant dysplastic phenotype to others with predominant features of myeloproliferation such as a high white blood cell count (WBC) and/or hepatosplenomegaly. One such disorder is chronic myelomonocytic leukaemia (CMML). The classification of CMML has caused much controversy. Historically, the French-American-British (FAB) classification placed CMML in the MDS subgroup as it did not contain a separate category for such overlap syndromes [2, 3]. However, while some cases of CMML present with a low WBC and overt dysplasia, others present with a marked increase in the WBC count and organomegaly, more in keeping with the MPN. As a result, the 3rd edition of the WHO classification of Tumours of Haematopoietic and Lymphoid Tissues recognised the need to create a separate entity for such disorders. A subgroup termed the myelodysplastic/myeloproliferative neoplasms (MDS/MPN) was created and encompasses disorders with both dysplastic and proliferative features [1, 4]. According to the WHO classification, the MDS/MPN subgroup includes Chronic Myelomonocytic Leukaemia (CMML), atypical Chronic Myeloid Leukaemia (aCML), Juvenille Myelomonocytic Leukaemia (JMML) and the Myelodysplastic/Myeloproliferative neoplasms, unclassifiable (MDS/MPD, U) [1]. Whilst the aetiology of such disorders is unknown, a common finding is the high incidence of RAS/MAPK signalling pathway aberrancies. NRAS/KRAS mutations are detected in approximately a third of cases with CMML and aCML [5, 6]. Mutations of NRAS, KRAS, NF1 or PTPN11 are observed in up to 80% of cases of JMML [7]. W. Ingram () Department of Haematology, University Hospital of Wales, Heath Park, Cardiff, CF14 4XW, UK Tel.: +44-29-2074-2654 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_10, ©Â€Springer Science+Business Media B.V. 2011

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In contrast to the classical BCR-ABL1 negative MPN in which a high incidence of the JAK2 V617F mutation is observed, only a small proportion of CMML and aCML cases carry the mutation. Somatic mutations in TET2 are reported in up to 40% of CMML patients, approximately a third of cases with aCML and a smaller number of unselected cases of MDS or MPN [8–12]. TET2 (Ten-Eleven Translocation-2) is located at chromosome 4q24 and has been thought of as a putative tumour suppressor gene, the function of which is unknown [10]. Within the same family, TET1 encodes an enzyme responsible for the conversion of 5-methylcytosine to 5-hydroxymethylcytosine and thus may have a role in epigenetic regulation [13]. A growing insight into the molecular basis of the MDS and the MPN has highlighted several less well defined disorders such as, refractory anaemia with ring sideroblasts and thrombocytosis (RARS-T) and MDS 5q- with JAK2 V617F mutation. These disorders have the potential to be considered within the same category of overlap syndromes as they display features of both the MDS and the MPN. Such disorders are discussed further within this chapter. An overview of the clinical, laboratory and genetic features of the MDS/MPN overlap syndromes will be provided. JMML is a disorder of childhood and is described in detail elsewhere within this book and therefore will not be discussed further within this chapter.

Chronic Myelomonocytic Leukaemia (CMML) Definition and Presenting Features CMML is characterised by a peripheral blood monocytosis >1â•›×â•›109/l, the morphological features of which are summarised in Table€10.1. The clinical and laboratory features of CMML are highly variable and as such CMML has in the past been divided into myelodysplastic and myeloproliferative subtypes based upon a white blood cell (WBC) count of < or >13â•›×â•›109/l respectively. This classification bears no prognostic significance with the only factor shown to predict survival in CMML being the percentage of peripheral blood (PB) or bone marrow (BM) blasts [1, 14, 15]. The WHO classification therefore divides CMML into two subgroups according to the PB or BM blast count, the blast count including promonocytes. CMML-1 is defined as <5% PB or <10% BM blasts and CMML-2 as 5–19% PB or 10–19% BM blasts, or in the presence of auer rods when the blast count is <20%. A finding of >20% blasts indicates acute myeloid leukaemia (AML). Whilst the majority of patients with CMML present with a high WBC count, a smaller number of patients present with a low WBC count and neutropenia. Constitutional symptoms consisting of general lethargy, weight loss, fevers and night sweats are common. Infections may be increased and bleeding diathesis secondary to thrombocytopenia is frequently observed. Splenomegaly is more commonly observed in patients with leukocytosis, but may be a feature of any subtype of CMML [16]. The enlarged spleen is usually a result of infiltration of the red pulp by leukaemia cells.

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Table 10.1↜渀 Summary of the peripheral blood and bone marrow features observed in the myelodysplastic/myeloproliferative neoplasms Blood Bone marrow Hypercellular BM CMML Persistent monocytosisâ•›>1â•›×â•›109/l Granulocytic proliferation with >10% monocytes Dysplasia ≥1 lineage CMML-1 <5% blasts <10% blasts CMML-2 5–19% blasts 10–19% blasts or presence of auer rods when <20% blasts Atypical CML Hypercellular BM Persistent WBCâ•›>â•›13â•›×â•›109/l Increase in granulocytic precursors Increased neutrophil precursors Prominent dysgranulopoiesis Prominent dysgranulopoiesis Monocytes <10% MDS/MPN, Variable degree of dysplasia and Hypercellular BM unclassifiable myeloproliferation Variable degree of dysplasia and Blastsâ•›<20% myeloproliferation Blastsâ•›<20% Does not fit into any other category of MDS/MPN disorders RARS-Ta Hypercellular BM Variable degree of dysplasia Dysplasia ≥1 lineage Sustained platelet count Ring sideroblasts >15% >450â•›×â•›109/l <5% blasts Large atypical megakaryocytes, sometimes hypolobated MDS with deletion 5q Not as yet defined Hypercellular BM and JAK2 positiveb Usually high platelet count Granulocytic proliferation Megakaryocytes increased, mostly >450â•›×â•›109/l hypolobated or non-lobated Mild leukocytosis present a b

Provisional entity recognised by the WHO classification Currently not recognised as a separate entity by the WHO classification

Epidemiology The median age of CMML at diagnosis is 65–75 years with a slight male predominance. CMML has been poorly defined in epidemiological studies and as such, the true incidence is unknown. It has been estimated at three cases per 100,000 persons over the age of 60 years [3].

Laboratory Features The monocyte count in CMML is by definition >1â•›×â•›109/l, the monocytes usually constituting >10% of peripheral blood leukocytes [1]. The monocytes may show an abnormal chromatin pattern or nuclear lobation, but more often are of normal appearance. Blasts and promonocytes must constitute <20% of total nucleated cells.

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Peripheral blood findings are heterogeneous with both neutrophilia and neutropenia observed. The degree of dysgranulopoiesis can be highly variable with less dysplasia noted in cases presenting with a high WBC count. A marked eosinophilia may be present with a mild anaemia (usually normocytic) and moderate thrombocytopenia frequent findings. The bone marrow is typically hypercellular, although a normocellular or even hypocellular marrow may occur. Granulocytic hyperplasia is often the predominant finding. The degree of dysgranulopoiesis, dyserythropoiesis and dysmegakaryopoiesis is highly variable. In certain cases it may be difficult to distinguish abnormal monocytes from dysplastic granulocytes. In such cases cytochemical stains using a non-specific esterase such as α-naphthyl acetate esterase, may be useful to aid identification of the monocytic lineage. Plasmacytoid dendritic cells can be detected in bone marrow nodules in up to 20% of cases of CMML [1]. With respect to immunophenotyping, the myelomonocytic antigens CD13 and CD33 are usually positive. Expression of the monocytic antigens, CD14, CD64 and CD68 are more variably expressed and therefore less useful in isolation in identification of the monocytic component. Aberrant expression of two or more antigens may be observed. Over expression of CD56, decreased expression of CD14, HLADR, CD13, CD15, CD64 or CD36 and aberrant expression of CD2 may be observed [17–19]. An increase in CD34+ cells may indicate transformation to AML.

Genetics Cytogenetic abnormalities are reported in up to 40% of cases of CMML [16, 20– 22]. Trisomy 8, monosomy 7/del 7q and 12p anomalies are frequently observed [1]. The abnormalities are not specific to CMML and are commonly seen in other myeloid malignancies. The presence of isochromosome 17q may be associated with features suggestive of CMML however the current WHO classification places such cases within the MDS/MPD, U subgroup [1]. RAS mutations are a common finding in the MDS/MPN subgroup with a frequency of approximately 30% in CMML [5, 6]. In contrast to the classical BCRABL1 negative myeloproliferative disorders, the JAK2 V617F mutation is reported in a small proportion (up to 10%) of CMML patients [23–25]. Of interest, a recent study reports an association between the proliferative form of CMML with the JAK2 V617F mutation [26]. Up to 40% of patients with CMML carry mutations in the TET2 gene [8–12, 27]. In addition to CMML, an association between TET2 mutations and monocytosis has been described in patients with systemic mastocytosis, suggesting a role for TET2 in monocyte differentiation [28]. Runt-related transcription factor 1 (RUNX1) plays an important role in normal haematopoiesis and is thought of as a tumour suppressor gene. Mutations of RUNX1 are well described in the literature and known to predispose to leukaemia [29–31]. Mutations in the RUNX1 gene are reported in nearly 40% of patients with CMML [29, 30]. Both the myelodysplastic and my-

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eloproliferative forms of CMML may be affected, unlike RAS mutations which are predominantly observed in patients with myeloproliferative subtypes of CMML [29]. Although the presence of RUNX1 mutations has shown no impact on overall survival, a higher risk of transformation to AML is reported in cases with mutations in the C-terminal region of the gene [30]. In a recent study, 43% of CMML patients exhibited acquired mutations in the ASXL1 gene [32]. Although the function of this gene is unknown, it is thought that the ASXL1 protein may play a role in DNA and or histone modifying complexes [33–35]. Utilisation of single nucleotide polymorphism (SNP) microarrays has revealed the presence of uniparental disomy in 48% of CMML and 38% of patients with MDS/MPN-unclassifiable [36]. The technology has facilitated the search for novel mutations within overlapping regions of UPD, such as CBL mutations within the 11q UPD of patients with MDS/MPNs [36–38]. CBL regulates tyrosine kinase signalling, mutations of which are reported in up to 10% of patients with CMML [36– 38]. A summary of the genetic mutations identified in CMML and the other MDS/ MPN are summarised in Table€10.2.

Table 10.2↜渀 Acquired somatic genetic mutations of the myelodysplastic/myeloproliferative neoplasms Cytogenetic abnormalities Genetic mutations CMMLa RAS mutations 30% Abnormality detected in up to 40% JAK2 V617F mutations 10% cases TET2 mutations 40% Trisomy 8 RUNX1 mutations 40% Monosomy 7/del 7q CBL mutations10% 12p aberrancies ASXL1 mutations 43% RAS mutations 30% Atypical CML Abnormality detected in up to 80% JAK2 V617F mutation rare cases TET2 mutations 30% Trisomy 8 CBL mutations 10% Deletion 20q Absence of BCR-ABL 1 Abnormalities chromosome 12, 13, fusion gene 14, 17 and 19 Absence of Philadelphia chromosome Absence of BCR-ABL 1 MDS/MPN, Isochromosome 17q fusion gene unclassifiable Absence of Philadelphia Absence of PDGFRA, PDGchromosome FRB and FGFR1 Absence of del(5q), t(3; 3)(q21; q26) or inv(3)(q21q26) RARS-T Nil specific JAK2 V617F mutation 60% MPL gene mutations less common TET2 mutations 26% MDS with deletion 5q Deletion 5q JAK2 V617F mutation comand JAK2 positive mon finding CMML with eosinophilia associated with the t(5; 12)(q31-33; p12), ETV6-PDGFRB fusion gene classified as a separate entity by the WHO classification [1]

a

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A small number of cases of CMML may be associated with a marked eosinophilia. In such cases, examination for gene rearrangements of the PDGFRA and PDGFRB genes should be carried out. The t(5; 12)(q31-33; p12) associated with the ETV6-PDGFRB fusion gene is reported in cases of CMML with eosinophilia and in cases previously described as chronic eosinophilic leukaemia (CEL) [39]. As a result of the potential sensitivity of these disorders to the tyrosine kinase inhibitors such as Imatinib, all myeloid and lymphoid disorders associated with abnormalities of the PDGFRA, PDGFRB and FGFR1 gene are classified as a separate entity within the WHO classification [1]. In a small number of cases CMML may present with an eosinophilia >1.5â•›×â•›109/l in the absence of a PDGFR rearrangement. Such cases may still be referred to as CMML with eosinophilia and should be classified as CMML-1 or CMML-2 as described previously [1]. All cases in which the p190 BCR-ABL1 fusion protein is detected should be classified as chronic myeloid leukaemia (CML) [40, 41].

Prognosis and Management The median survival of patients with CMML is reported between 20–40 months with poorer outcome in patients presenting with advanced stage disease [15, 16, 20, 42]. The management of CMML is generally targeted towards ameliorating symptoms related to the underlying disease such as fever, weight loss and splenomegaly. Treatment may be aimed at suppression of the high WBC count in proliferative subtypes of CMML or at improving cytopenias resulting from bone marrow failure or hypersplenism. Therapeutic options include cytoreductive therapy with agents such as hydroxyurea or busulphan, conventional chemotherapy e.g. cytarabine, topotecan, or splenectomy for cases with predominant hypersplenism and consequent cytopenias [43–45]. AML type chemotherapy with or without allogeneic haematopoietic stem cell transplantation (allo-HSCT) may be considered in the small proportion of patients who are fit for such intensive treatment options. The results of allo-HSCT are discussed elsewhere in this book and as such will not be described in detail within this chapter. In summary, the majority of studies which report outcome of CMML patients following allo-HSCT include small numbers of CMML patients thereby making it difficult to accurately predict response rates [46–48]. One of the largest studies conducted by the EBMT, report the outcome of 50 CMML patients using a variety of conditioning regimens [48]. The 5 year overall survival (OS) was reported at 21% with a 5 year disease free survival (DFS) of just 18%. A smaller recent report describes the outcome of 18 CMML patients following RIC allo-HSCT [47]. The 3 year OS was 31%, with a relapse incidence of 47%. Importantly the results show that none of the patients with intermediate or poor risk cytogenetics survived beyond 2 years and a poorer outcome was observed in patients with >5% bone marrow blasts at the time of transplantation. Patient selection is therefore critical in order to identify those who may benefit most from allogeneic HSCT.

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In view of the knowledge that the majority of patients will not be suitable for such intensive treatment options, the role of novel agents such as the hypomethylating agents, 5-azacytidine and decitabine have been explored in MDS and CMML. 5-azacytidine and decitabine are both approved in the US for the treatment of int-2/ high risk MDS. Two pivotal studies report an improved overall survival following treatment with 5-azacytidine when compared with best supportive care in high risk MDS patients [49, 50]. Silverman et€al. report an overall response rate of 60% for patients randomised to 5-azacytidine compared with 5% for best supportive care [49]. Importantly, response was independent of MDS subgroup with comparable responses observed in patients with a diagnosis of RAEB, RAEB-T and CMML when compared with the RA and RARS subgroups. Although encouraging results are reported in CMML following treatment with 5-azacytidine, studies to date have analysed just small numbers of CMML patients and as such, further studies are ongoing to evaluate its role. Reports of the use of decitabine in CMML are also described and show similarly encouraging results [51, 52]. An overall response rate of 69% with 58% complete response is reported in one such study of 19 CMML patients [52]. Ongoing studies to evaluate the role of hypomethylating agents in combination with other novel agents such as the HDAC inhibitors in MDS and CMML are being conducted, the results of which are eagerly awaited.

Atypical Chronic Myeloid Leukaemia (aCML) Definition and Presenting Features Atypical CML is characterised by a leukocytosis >13â•›×â•›109/l consisting of a neutrophilia, in the absence of the Philadelphia chromosome or BCR-ABL 1 fusion gene. An increased number of neutrophil precursors are seen in association with prominent dysgranulopoiesis. The degree of dysgranulopoiesis is often severe which is the main feature distinguishing it from BCR-ABL1 positive CML [1, 4]. The blood and bone marrow features are summarised in Table€10.1. Patients typically present with features similar to that of BCR-ABL1 positive CML, i.e. symptoms of anaemia, occasionally thrombocytopenia or as a result of splenomegaly [1]. In a smaller number of cases a minimal elevation in the WBC >13â•›×â•›109/l more in keeping with that seen in CMML may be the only presenting feature [53].

Epidemiology Atypical CML is a rare disorder, reported at 1–2 cases for every 100 cases of BCRABL 1 positive CML [4, 54]. It typically occurs in the elderly with a median age re-

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ported in the 7th or 8th decade of life however reports of aCML in younger patients are described. The disease is thought to have a slight male predominance [1, 4].

Laboratory Features The WBC count is always >13â•›×â•›109/l but may present at any level with WBC counts of >300â•›×â•›109/l reported [1, 54, 55]. An increase in granulocytic precursors including promyelocytes, myelocytes and metamyelocytes are seen in the PB and may comprise up to 20% of total WBCs. Dysgranulopoiesis is prominent with features such as acquired Pelger-Huet changes, abnormal chromatin clumping or neutrophil hypogranulation. In contrast to CMML, the percentage of monocytes is usually <10% of the total leukocyte count despite an absolute increase in the monocyte count frequently observed. A modest increase in the basophil count may be present but is not a prominent feature, unlike BCR-ABL1 positive CML [1, 4]. The bone marrow is hypercellular with a predominant increase in the granulocytic lineage. The blast count is always less than 20%. Features of dysgranulopoiesis are noted as seen in the blood. The myeloid to erythroid ratio is usually in excess of 10:1 with dysplastic changes in the erythroid lineage. Megakaryocyte number and the degree of dysmegakaryopoiesis are highly variable. Dysplastic changes are generally similar to those seen in MDS. An increase in bone marrow reticulin may be observed at the time of presentation [1, 53]. Atypical CML does not display any specific immunophenotypic characteristics. CD34 staining of bone marrow biopsies may facilitate identification of blast cells and CD14 or CD68R may be useful on immunohistochemical stains to aid identification of monocytes [1].

Genetics Up to 80% of patients with aCML carry a clonal cytogenetic abnormality, the most common being trisomy 8 and deletion (20q) [54, 55]. Other abnormalities of chromosomes 12, 13, 14, 17 and 19 are also reported [4, 54, 55]. Although rare cases with features suggestive of aCML may carry the isochromosome 17q, they are more likely to fit the criteria for CMML or MDS/MPN, unclassifiable as previously discussed. The BCR-ABL1 fusion gene must be absent in order to support the diagnosis of aCML. Rearrangement of the PDGFRA or PDGFRB gene indicates an alternate diagnosis [1]. As observed in CMML, NRAS or KRAS mutations are detected in approximately a third of cases of aCML [56, 57] and CBL mutations in approximately 10% of patients with aCML [36–38]. The JAK2 V617F mutation is rare in aCML, whilst TET2 mutations are reported in up to a third of patients [8–12, 25, 58]. The genetic abnormalities identified in aCML are summarised in Table€10.2.

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Prognosis and Management Patients with aCML generally display an aggressive disease course with a median survival reported between 11–25 months and transformation to AML in up to 40% of cases [4, 54]. The optimal treatment of aCML is unclear. Agents such as hydroxyurea and α-interferon have been used with limited success [59].

MDS/MPN, Unclassifiable Definition and Presenting Features The MDS/MPN, unclassifiable subgroup, enables one to assign a category to a patient who presents with features of both the MDS and the MPN but who does not fit into any of the categories described previously (CMML, aCML, JMML). Such cases may present with any of the symptoms related to MDS or MPN. Patients with a known history of MPN who develop dysplastic features over time should not be included within this category [1]. The blood and bone marrow features are summarised in Table€10.1.

Epidemiology The precise incidence of the MDS/MPN, unclassifiable subgroup is unknown. Reports suggest an incidence of 2% of previously classified MDS cases [4].

Laboratory Features The morphological features of the blood and BM are in keeping with that seen in the MDS and MPN subgroups [56, 60]. The degree of dysplasia is highly variable and proliferation of one or more lineage may be observed with a resultant thrombocytosis >450â•›×â•›109/l or leukocytosis >13â•›×â•›109/l [1]. Blasts always account for <20% of total nucleated cells in the BM or PB. Immunophenotypic, cytochemical and immunohistochemical findings are similar to that described for other MDS/MPNs.

Genetics There are no specific cytogenetic or molecular abnormalities for the MDS/MPN, U subgroup. In order to assign a patient to this category, the Philadelphia chromosome

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and BCR-ABL1 fusion gene must be absent. Cases presenting with gene rearrangements of PDGFRA, PDGFRB or FGFR1 should be classified as a separate entity [1]. Moreover, the presence of isolated deletion (5q), t(3; 3)(q21; q26) or inv(3) (q21q26) generally excludes them from the MDS/MPN, U subgroup, however, emerging data suggests that some cases with del(5q) and JAK2 V617F mutation present with features of both MDS and MPN and may be categorised as an overlap syndrome as discussed later [1, 61].

Prognosis and Management The prognosis of the MDS/MPN, U subgroup is unknown as the group represents a mixed cohort of patients. Improved classification of such disorders will inevitably enhance our understanding of their natural history and provide the ability to explore treatment options further.

 efractory Anaemia with Ring Sideroblasts and R Thrombocytosis (RARS-T) RARS-T is currently acknowledged as a provisional entity within the MDS/MPN category of the WHO classification [1]. Little is known about its epidemiology. The Dusseldorf group report a frequency of 0.7% of all MDS cases [53]. RARS-T differs from RARS in that it is associated with a high platelet count, together with large atypical megakaryocytes, similar to those seen in essential thrombocythaemia (ET) [62–64]. A recently proposed reduction in the defining platelet count from >600â•›×â•›109/l to >450â•›×â•›109/l is in line with the revised criteria for ET [1]. However the lower platelet threshold has led to difficulty in distinguishing some cases of RARS-T from RARS which may also present with a modest elevation in platelet count. With the finding, that up to 60% of RARS-T cases carry the JAK2 V617F mutation, its classification has caused much controversy [65–67]. Some individuals propose that in view of the high frequency of JAK2 mutation, RARS-T should belong to the MPN category, the ring sideroblasts representing an acquired feature in a preexisting MPN. In keeping with this notion, a small number of RARS-T cases demonstrate somatic mutations within the MPL gene (W515L) [65]. Mutations within the juxtamembrane region of the thrombopoietin receptor MPL are reported in the classical MPNs [68–70]. In contrast, a recent study detailing the gene expression profile of RARS and RARS-T highlights similarities between the diseases [65, 71, 72]. Upregulation of ALAS2, a mitochondrial related gene responsible for encoding heme synthesis and

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downregulation of ABCB7, a gene which encodes a protein that is responsible for enabling transport of iron to the cytoplasm were similarly identified in RARS and RARS-T. An additional study demonstrates a high frequency of haemochromatosis associated gene mutations in both RARS and RARS-T [73]. Moreover, the lack of endogenous growth and poor colony formation demonstrated in RARS-T is more in keeping with that observed in MDS rather than the MPN [74]. On the contrary, a recent study reports TET2 mutations in 26% of cases of RARS-T patients, more in keeping with the MPN [75]. Together, these findings support the hypothesis that RARS-T carries features of both the MDS and MPN subgroups, hence the proposal to consider the disorder as an overlap syndrome. Against this proposal is the favourable prognosis of JAK2 positive RARS-T patients when compared with other cases within the MDS/MPN subgroup [53, 76]. Further studies are therefore required to accurately assess the impact of JAK2 V617F mutation on overall survival.

MDS with Deletion 5q and JAK2 Positive The recent finding of JAK2 V617F mutation in cases of MDS with del(5q-) has prompted the notion that these cases may indeed represent a separate entity when compared with those which lack the JAK2 mutation. Such cases generally present with a higher platelet count and in some cases a higher WBC count [61]. One report demonstrates a proliferative bone marrow and granulocytic hyperplasia in patients with MDS del(5q) and the JAK2 V617F mutation [61]. Dysmegakaryopoiesis is observed, with the characteristic hypolobated and mononuclear megakaryocytes as seen in the classical 5q- syndrome. The precise incidence and long term prognosis of such cases is currently unknown. Further studies are required to evaluate the prognostic significance of JAK2 mutation in patients with del(5q) MDS and to assess the role of lenalidomide in such patients.

Concluding Remarks Hitherto, accurate data on the epidemiology and prognosis of patients within the MDS/MPN subgroup has been limited, mainly as a result of such patients frequently being grouped together with other MDS or MPNs and not treated as separate entities. Emerging data on the natural history and prognostic significance of the MDS/MPD overlap syndromes will inevitably lead to further refinement of their classification in the future and is likely to identify further subgroups of patients which belong to this category. An improved understanding of the genetic and epigenetic aberrations in such disorders will facilitate this process and enable the study of novel treatment options including targeted therapies in the future.

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References ╇ 1. Swerdlow SH CE, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (2008) World health organization classification of tumours of haematopoietic and lymphoid tissues, 4th€edn. IARC Press (International Agency for Research on Cancer), Lyon ╇ 2. Bennett JM et€ al (1994) The chronic myeloid leukaemias: guidelines for distinguishing chronic granulocytic, atypical chronic myeloid, and chronic myelomonocytic leukaemia. Proposals by the French-American-British Cooperative Leukaemia Group. Br J Haematol 87(4):746–754 ╇ 3. Bennett JM et€al (1982) Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51(2):189–199 ╇ 4. Jaffe ES, Harris NL, Stein H, Vardiman JW (eds) (2001) World health organization classification of tumours. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. SLH Kleihues P (ed) IARC Press: Lyon ╇ 5. Padua RA et€al (1988) RAS mutations in myelodysplasia detected by amplification, oligonucleotide hybridization, and transformation. Leukemia 2(8):503–510 ╇ 6. Hirsch-Ginsberg C et€al (1990) RAS mutations are rare events in Philadelphia chromosomenegative/bcr gene rearrangement-negative chronic myelogenous leukemia, but are prevalent in chronic myelomonocytic leukemia. Blood 76(6):1214–1219 ╇ 7. Koike K, Matsuda K (2008) Recent advances in the pathogenesis and management of juvenile myelomonocytic leukaemia. Br J Haematol 141(5):567–575 ╇ 8. Delhommeau F et€ al (2009) Mutation in TET2 in myeloid cancers. N Engl J Med 360(22):2289–2301 ╇ 9. Langemeijer SM et€al (2009) Acquired mutations in TET2 are common in myelodysplastic syndromes. Nat Genet 41(7):838–842 10. Tefferi A et€al (2009) Detection of mutant TET2 in myeloid malignancies other than myeloproliferative neoplasms: CMML, MDS, MDS/MPN and AML. Leukemia 23(7):1343–1345 11. Jankowska AM et€al (2009) Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms. Blood 113(25):6403–6410 12. Abdel-Wahab O et€al (2009) Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies. Blood 114(1):144–147 13. Tahiliani M et€ al (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324(5929):930–935 14. Germing U et€ al (2000) Validation of the WHO proposals for a new classification of primary myelodysplastic syndromes: a retrospective analysis of 1600 patients. Leuk Res 24(12): 983–992 15. Germing U et€al (2007) Chronic myelomonocytic leukemia in the light of the WHO proposals. Haematologica 92(7):974–977 16. Germing U et€al (1998) Problems in the classification of CMML—dysplastic versus proliferative type. Leuk Res 22(10):871–878 17. Lacronique-Gazaille C et€al (2007) A simple method for detection of major phenotypic abnormalities in myelodysplastic syndromes: expression of CD56 in CMML. Haematologica 92(6):859–860 18. Xu Y et€al (2005) Flow cytometric analysis of monocytes as a tool for distinguishing chronic myelomonocytic leukemia from reactive monocytosis. Am J Clin Pathol 124(5):799–806 19. Woodlock TJ et€al (1994) Use of cell surface antigen phenotype in guiding therapeutic decisions in chronic myelomonocytic leukemia. Leuk Res 18(3):173–181 20. Fenaux P et€al (1988) Prognostic factors in adult chronic myelomonocytic leukemia: an analysis of 107 cases. J Clin Oncol 6(9):1417–1424 21. Haase D et€al (1995) Cytogenetic findings in 179 patients with myelodysplastic syndromes. Ann Hematol 70(4):171–187 22. Toyama K et€al (1993) Clinical implications of chromosomal abnormalities in 401 patients with myelodysplastic syndromes: a multicentric study in Japan. Leukemia 7(4):499–508

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23. Steensma DP et€al (2005) The JAK2 V617F activating tyrosine kinase mutation is an infrequent event in both “atypical” myeloproliferative disorders and myelodysplastic syndromes. Blood 106(4):1207–1209 24. Jelinek J et€al (2005) JAK2 mutation 1849G>T is rare in acute leukemias but can be found in CMML, Philadelphia chromosome-negative CML, and megakaryocytic leukemia. Blood 106(10):3370–3373 25. Levine RL et€al (2005) The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia. Blood 106(10):3377–3379 26. Pich A et€al (2009) JAK2V617F activating mutation is associated with the myeloproliferative type of chronic myelomonocytic leukaemia. J Clin Pathol 62(9):798–801 27. Kosmider O et€ al (2009) TET2 gene mutation is a frequent and adverse event in chronic myelomonocytic leukemia. Haematologica 94(12):1676–1681 28. Tefferi A et€al (2009) Frequent TET2 mutations in systemic mastocytosis: clinical, KITD816€V and FIP1L1-PDGFRA correlates. Leukemia 23(5):900–904 29. Gelsi-Boyer V et€al (2008) Genome profiling of chronic myelomonocytic leukemia: frequent alterations of RAS and RUNX1 genes. BMC Cancer 8:299 30. Kuo MC et€al (2009) RUNX1 mutations are frequent in chronic myelomonocytic leukemia and mutations at the C-terminal region might predict acute myeloid leukemia transformation. Leukemia 23(8):1426–1431 31. Imai Y et€ al (2000) Mutations of the AML1 gene in myelodysplastic syndrome and their functional implications in leukemogenesis. Blood 96(9):3154–3160 32. Gelsi-Boyer V et€al (2009) Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. Br J Haematol 145(6):788–800 33. Katoh M (2004) Identification and characterization of ASXL3 gene in silico. Int J Oncol 24(6):1617–1622 34. Lee JH, Skalnik DG (2005) CpG-binding protein (CXXC finger protein 1) is a component of the mammalian Set1 histone H3-Lys4 methyltransferase complex, the analogue of the yeast Set1/COMPASS complex. J Biol Chem 280(50):41725–41731 35. Cho YS et€al (2006) Additional sex comb-like 1 (ASXL1), in cooperation with SRC-1, acts as a ligand-dependent coactivator for retinoic acid receptor. J Biol Chem 281(26):17588–17598 36. Dunbar AJ et€al (2008) 250€K single nucleotide polymorphism array karyotyping identifies acquired uniparental disomy and homozygous mutations, including novel missense substitutions of c-Cbl, in myeloid malignancies. Cancer Res 68(24):10349–10357 37. Sanada M et€al (2009) Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms. Nature 460(7257):904–908 38. Grand FH et€ al (2009) Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms. Blood 113(24):6182–6192 39. Drechsler M et€al (2007) Fusion of H4/D10S170 to PDGFRbeta in a patient with chronic myelomonocytic leukemia and long-term responsiveness to imatinib. Ann Hematol 86(5):353– 354 40. Melo JV et€ al (1994) P190BCR-ABL chronic myeloid leukaemia: the missing link with chronic myelomonocytic leukaemia? Leukemia 8(1):208–211 41. Ohsaka A et€al (2002) Philadelphia chromosome-positive chronic myeloid leukemia expressing p190(BCR-ABL). Intern Med 41(12):1183–1187 42. Storniolo AM et€al (1990) Chronic myelomonocytic leukemia. Leukemia 4(11):766–770 43. Bennett JM (2002) Chronic myelomonocytic leukemia. Curr Treat Options Oncol 3(3):221– 223 44. Beran M et€al (1999) Topotecan and cytarabine is an active combination regimen in myelodysplastic syndromes and chronic myelomonocytic leukemia. J Clin Oncol 17(9):2819–2830 45. Beran M et€ al (1996) Topotecan, a topoisomerase I inhibitor, is active in the treatment of myelodysplastic syndrome and chronic myelomonocytic leukemia. Blood 88(7):2473–2479 46. Elliott MA et€al (2006) Allogeneic stem cell transplantation and donor lymphocyte infusions for chronic myelomonocytic leukemia. Bone Marrow Transplant 37(11):1003–1008

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W. Ingram and G. J. Mufti

47. Krishnamurthy P et€al (2010) Allogeneic haematopoietic SCT for chronic myelomonocytic leukaemia: a single-centre experience. Bone Marrow Transplant 45:1502–1507 48. Kroger N et€al (2002) Allogeneic stem cell transplantation of adult chronic myelomonocytic leukaemia. A report on behalf of the chronic leukaemia working party of the European Group for Blood and Marrow Transplantation (EBMT). Br J Haematol 118(1):67–73 49. Silverman LR et€ al (2002) Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B. J Clin Oncol 20(10):2429–2440 50. Fenaux P et€al (2009) Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 10(3):223–232 51. Kantarjian H et€al (2007) Results of a randomized study of 3 schedules of low-dose decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic leukemia. Blood 109(1):52–57 52. Aribi A et€al (2007) Activity of decitabine, a hypomethylating agent, in chronic myelomonocytic leukemia. Cancer 109(4):713–717 53. Orazi A, Germing U (2008) The myelodysplastic/myeloproliferative neoplasms: myeloproliferative diseases with dysplastic features. Leukemia 22(7):1308–1319 54. Breccia M et€al (2006) Identification of risk factors in atypical chronic myeloid leukemia. Haematologica 91(11):1566–1568 55. Hernandez JM et€al (2000) Clinical, hematological and cytogenetic characteristics of atypical chronic myeloid leukemia. Ann Oncol 11(4):441–444 56. Vardiman JW (2004) Myelodysplastic/myeloproliferative diseases. Cancer Treat Res 121:13–43 57. Reiter A et€al (2009) Molecular basis of myelodysplastic/myeloproliferative neoplasms. Haematologica 94(12):1634–1638 58. Jones AV et€al (2005) Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 106(6):2162–2168 59. Kurzrock R et€al (2001) BCR rearrangement-negative chronic myelogenous leukemia revisited. J Clin Oncol 19(11):2915–2926 60. Neuwirtova R et€al (1996) Mixed myelodysplastic and myeloproliferative syndromes. Leuk Res 20(9):717–726 61. Ingram W et€al (2006) The JAK2 V617F mutation identifies a subgroup of MDS patients with isolated deletion 5q and a proliferative bone marrow. Leukemia 20(7):1319–1321 62. Gupta R, Abdalla SH, Bain BJ (1999) Thrombocytosis with sideroblastic erythropoiesis: a mixed myeloproliferative myelodysplastic syndrome. Leuk Lymphoma 34(5–6):615–619 63. Streeter RR, Presant CA, Reinhard E (1977) Prognostic significance of thrombocytosis in idiopathic sideroblastic anemia. Blood 50(3):427–432 64. Shaw GR (2005) Ringed sideroblasts with thrombocytosis: an uncommon mixed myelodysplastic/myeloproliferative disease of older adults. Br J Haematol 131(2):180–184 65. Malcovati L et€ al (2009) Molecular and clinical features of refractory anemia with ringed sideroblasts associated with marked thrombocytosis. Blood 114(17):3538–3545 66. Ceesay MM et€al (2006) The JAK2 V617F mutation is rare in RARS but common in RARST. Leukemia 20(11):2060–2061 67. Remacha AF et€al (2006) Occurrence of the JAK2 V617F mutation in the WHO provisional entity: myelodysplastic/myeloproliferative disease, unclassifiable-refractory anemia with ringed sideroblasts associated with marked thrombocytosis. Haematologica 91(5):719–720 68. Beer PA et€al (2008) MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort. Blood 112(1):141–149 69. Pikman Y et€al (2006) MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med 3(7):e270 70. Pardanani AD et€al (2006) MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood 108(10):3472–3476

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71. Pellagatti A et€al (2006) Gene expression profiles of CD34+ cells in myelodysplastic syndromes: involvement of interferon-stimulated genes and correlation to FAB subtype and karyotype. Blood 108(1):337–345 72. Boultwood J et€al (2008) The role of the iron transporter ABCB7 in refractory anemia with ring sideroblasts. PLoS One 3(4):e1970 73. Nearman ZP et€al (2007) Hemochromatosis-associated gene mutations in patients with myelodysplastic syndromes with refractory anemia with ringed sideroblasts. Am J Hematol 82(12):1076–1079 74. Vardiman JW et€al (2009) The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 114(5):937–951 75. Flach J et€al (2010) Mutations of JAK2 and TET2, but not CBL are detectable in a high portion of patients with refractory anemia with ring sideroblasts and thrombocytosis. Haematologica 95(3):518–519 76. Atallah E et€al (2008) Prognostic interaction between thrombocytosis and JAK2 V617F mutation in the WHO subcategories of myelodysplastic/myeloproliferative disease-unclassifiable and refractory anemia with ringed sideroblasts and marked thrombocytosis. Leukemia 22(6):1295–1298

Chapter 11

Iron and Copper Metabolism in the Myelodysplastic Syndromes Judit Várkonyi, Gabriella Bekő, Zoltán Prohászka and István Karádi

Introduction Iron (Fe) and Copper (Cu) metabolism is closely related throughout the entire process of erythropoiesis. An overwiev is provided on this relation in Section Iron (Fe) and Copper (Cu) Metabolism is Closely Related Throughout the Entire Process of Erythropoiesis. The role of copper deficiency in dysplastic erythropoiesis is discussed in Section Copper Deficiency-Related Alterations in Erythropoiesis. Section Different Aspects of Iron Overload Related to MDS is dedicated to the special issue of iron overload in MDS. Lastly, Section Precedent Study Results offers a short summary of a recent study preceding the material presented in this chapter.

Iron (Fe) and Copper (Cu) Metabolism is Closely Related Throughout the Entire Process of Erythropoiesis Copper (Cu) and iron (Fe) are integral and functional components of proteins and enzymes that take part in the metabolic pathways closely related to hemoglobin synthesis and erythrocyte turnover. Fe and Cu are essential for human beings and they may cause disease through deficiency or toxicity. The daily Fe uptake normally is only 2€mg; keeping balance with the 2€mg daily loss by desquamating cells in the stool. The majority of metabolic iron turnover in the body is accounted for by the continuous synthesis and destruction of erythrocytes. The typical lifespan of an erythrocyte is 120 days. After this time period, senescent erythrocytes are engulfed by tissue macrophages residing in the spleen and the liver. Fe liberated from hemoglobin enters a new cycle or forms stores depending upon body requirements. In normal bone marrow upon erythropoietin (EPO) stimuli induced by hypoxia and/ J. Várkonyi () 3rd Department of Internal Medicine, Semmelweis University, Kútvölgyi út 4, 1125 Budapest, Hungary e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_11, ©Â€Springer Science+Business Media B.V. 2011

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or anemia there is a significant increase in Fe absorption from the gut followed by mobilization of iron stores from the liver. It is worth noting that there is no physiological mechanism for iron excretion and therefore regulation of iron metabolism takes place at the level of absorption. Cu is an essential element needed for normal development. Deficiency of Cu is uncommon since normal food covers the requirement that is 2–6€mg/d. Most mammals when kept on a Cu deficient diet, develop anemia. The principal difference between Fe or Cu deficiency is that the later exhibits an iron overload in storage organs such as the liver or spleen [1]. Elvehjem and Sherman were among the firsts to notice that Cu deficiency increases hepatic Fe [2]. Conversely, Fe is known to interfere with Cu utilization [3, 4]. Iron overload itself can cause Cu deficiency as it was shown in animal experiments when dietary Fe was kept high. When Cu was added, tissue Fe content decreased [5]. Higher dietary Fe decreased not only Cu but ceruloplasmin (CP) levels too and adversely increased cardiac weight, and hepatic iron which could be minimized by adequate Cu nutrition. It seems likely that the higher amount of Cu prevented some of the ill effects of higher Fe. At the lower amount of dietary Cu, the mean CP may be slightly lower than normal and higher dietary Fe decreased this value by 80% [6]. More than 93% of the serum Cu is CP bound, therefore hypocuprinaemia is correlated with hypocoeruloplasminaemia [7, 8]. Cu is an essential cofactor for numerous redox enzymes such as hephaestin, cyclooxigenase, cytochrome C oxidase, ferroxidase, superoxide dismutase (SOD) and coeruloplasmin (CP). Hephaestin is a ferroxidase CP homologue expressed in the duodenum and with CP participates in Fe charge modification. Hephaestin works in concert with ferroportin to permit Fe efflux from enterocytes to load transferrin. Dimetal transferase 1 (DMT1) is the duodenal Fe transporter. Expression of DMT1 and ferroportin both are regulated by the availability of Cu [9, 10]. Cu absorption occurs by the ATP7A protein on the basolateral surface of enterocytes but other mechanisms of uptake are also known involving the intracellular ligand metallothionein (MTO) as well. MTO has high affinitiy for transitional metals that interfere with each other for uptake [11, 12]. Absorbed Cu is then associated in over 90% with CP. CP is a multicopper ferroxidase and it is essential for the mobilization of Fe from storage tissues. Thus in Cu deficiency the mobilization and utilization of Fe is not possible. Aceruloplasminaemia is an autosomal recessive disorder of iron metabolism [13]. Affected individuals sustain an insidious, long-term accumulation of parenchymal iron that eventually manifests the consequences of iron overload clinically characterized by diabetes, retinal degeneration and neurological symptoms- the latter in relation to iron depostition in the basal ganglia. Harris et€al. were successful in generating a murine model of CP homozygous null mice [14]. This model provided clearcut evidence that CP plays an essential role in storage iron efflux from reticuloendothelial cells and hepatocytes. Serum CP and ferroxidase activity are decreased in HFE C282Y homozygote male IO patients and normal when Fe depleted- suggested that Fe may modulate the CP gene expression [15]. Hereditary Hemochromatosis (HH) in its classical type

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1 form is one of a group of inherited iron overload (IO) conditions resulting from cystein/tyrosin replacement at position 282, a common mutation affecting mostly caucasians [16]. As it was proposed regarding this mutation there is an uncontrolled iron absorbtion from the gut. Besides C282Y there is an H63D mutation that has significant impact on iron metabolism especially in homozygotes or in compound heterozygote form together with C282Y although its clinical expression is milder than the C282Y mutation.

Copper Deficiency-Related Alterations in Erythropoiesis In red blood cells Cu is bound to SOD. SOD level is low in Cu deficiency. In such a situation the life span of erythrocytes is shorter—as the elimination of superoxides being insufficient leading to cell membrane injury [17]. The formation of ringed sideroblasts—that is the main characteristic for an MDS subtype called sideroblastic anemia- can be attributed to SOD or cytochrome-c-oxidase deficiency as well [18]. In Cu deficiency there is a maturation arrest at proerythroblast stage and vacuolization of bone marrow precursor cells often seen with increased storage Fe. Cu deficiency causes microcytic anemia, neutropenia, and thrombocytopenia masquerading as MDS [19, 20].

Different Aspects of Iron Overload Related to MDS Hepcidin, the key regulator of iron metabolism, is a peptide formed in the liver. Hepcidin inhibits iron absorption from duodenal enterocytes and iron influx from macrophages that recycle iron from senescent red blood cells and from hepatocytes that store iron. Hepcidin acts by causing internalization and further degradation of ferroportin, the single iron exporter. Hepcidin production depends on iron–transferrin availability and is increased by the inflammatory cytokine IL-6. In inherited iron overload conditions hepcidin production is low due to dysregulation of participating proteins e.g. inactivating mutations in the hepcidin gene itself. In anemia of chronic disease due to high levels of IL-6, hepcidin is high, but in ineffective hemopoiesis such as in MDS hepcidin level depends on opposing influences [21]. In aplastic anemia for example resulted from inflammatory reactions hepcidin is high and EPO is high. In these cases iron overload rarely occurs (personal observation). In RARS being another form of ineffective erythropoiesis that frequently coexists with HFE gene mutations hepcidin is low and EPO level is low [22]. According to authors observations, these are the types of MDS at high risk of iron overload. However no direct effect of EPO has been found on hepcidin. There was a putative erythropoietic suppressing factor on hepcidin synthesis hypothetized—as in ineffective erythropoiesis of thalassemia—where hepcidin is found to be low in spite of high transferrin saturation and ferritin levels [23]. Growth differentiation factor-15

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(GDF-15) was identified as a candidate for one of the erythroid factors suppressing hepcidin [24]. In a study of 40 MDS patients, the authors found high GDF-15 levels in correlation with sTfR levels and ineffective erythropoiesis. Soluble transferrin receptor (sTfR) is a marker of iron uptake of erythrocytes-as transferrin by getting rid of iron in the cytosol, appears on the red cell surface and than entering the plasma by shedding from the red cell membrane. Those patients who reached erythroid response by EPO treatment, GDF-15 and sTfR level decreased in parallel increase in hepcidin levels [25]. At the cellular level iron toxicity is mediated via generation of reactive oxigen species (ROS). It has been shown in animal models that accumulation of ROS leads to senescence of haemopoitic stem cells and that ROS causes DNA damage and promotes the development of malignancy. These effects of ROS may be particularly important in MDS, in which haematopoiesis is already severely compromised and genetic instability is a striking feature [26]. Plasma from patients with IO resulting either from hereditary IO disorders (hemochromatosis) or due to regular blood transfusions contains high amounts of non transferrin bound iron (NTBI) [27]. There is not such NTBI iron in normal control plasma from healthy individuals. NTBI is the iron that exceeds the iron binding capacity of transferrin in HH that leads to tissue deposition and is highly correlated with serum ferritin levels. NTBI can accelerate lipid peroxidation by free radical generation: LOOH (lipid peroxide)â•›+â•›Fe (III)â•›→â•›LO2 (peroxil radicals)â•›+â•›H+â•›+â•›Fe (II) [28]. Following maintenance venesection therapy, NTBI might disappear from plasma or significantly reduced as shown in a limited number of HH cases [29]. MDS patients surprisingly might benefit more than expected from iron chelation therapy (ICT) as by enhancing Fe depletion from stores resulting in a decreased blood transfusion need as well, that is a critical point in MDS patients determining quality of life and survival and even transfusion independence might be achieved [30–35]. Fe deprivation leads to prolongation of life by preventing leukaemic transformation and contributing to long survival after bone marrow transplantation [36]. The beneficial effects of chelation therapy in MDS will be discussed in details in the next chapter (by C. Hershko).

Precedent Study Results It has been shown that the HFE gene mutations occur with a relatively high frequency in MDS patients of caucasian origin [37]. Subsequently the same study group found one third in a cohort of 33 consecutive MDS patients having either of the C282Y or H63D mutations. Patients with the inherited gene mutations had in general higher Tfsat and lower Cu levels in comparison to those in the HFE wt group [38]. When data were interpreted according to how many units of blood had been previously transfused—the differences between the two groups became more pronounced. Even in the HFE wt group however by transfusional escalation—the tendency towards decreasing Cu and raising trsat was well documented (Table€11.2).

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The Present Study The present study has been designed to test the reproducibility of the previous findings in a much larger group of participating patients. In addition we were interested to see how copper or iron level changes would be affected by blood transfusions and by the absence or presence of HFE gene mutations.

Materials and Methods 58 consecutive newly diagnosed MDS patients were studied at the 3rd Department of Internal Medicine at Semmelweis University for two years from November 2007 to December 2009. MDS subtype distribution was as follows: RA: 10, RAS/RARS: 16, RAMCD: 8, 5q-: 4, RAEB: 13, Hypoplastic: 5, MPS/MDS: 1 secondary MDS: 1. Serum Fe, transferrin and serum Cu concentrations were measured simultaneously—Fe by quanidin/Ferrozine method (code: 20737585); transferrin by immunoturbidometric assay (code: 03015050) and both by Roche Integra-800, that also presented Tfsat results. The estimated normal levels for serum Fe in females was: 6.6–26.0€μmol/l and in males was 11.0–28.0€μmol/l. Normal upper level for Tfsat was: 45% and for ferritin: 400€μmol/l, for both sexes. Cu was determined by the direct colorimetric assay according to Abe et€al. [39]. The estimated normal level for serum Cu in males was 11–22€μmol/l and in females was 12.6–24.4€μmol/l. Roche Tina—quant coeruloplasmin (CP) test was in use on Hitachi Modular automat. Serum CP level was determined according to earlier descriptions [40]. Normal serum CP level was found to be in the range of 0.20–0.60€g/l in both sexes. The authors included in the study the dataset of 11 patients with Hereditary Hemochromatosis serving as a parallel group since 19/58 MDS patients had HFE gene mutation as well. MDS HFE mutants were: H63D heterozygote: 15; compound heterozygote: 1; C282Y heterozygote: 3. 3/11 HH patients were compound heterozygotes H63D/C282Y and nine were C282Y homozygotes. HFE gene determination was carried out by the method of Feder et€al. [16]. Informed consent was obtained from all patients. The study had been approved by the local ethical committee No. 12236-45/2004-1018EKU TUKEB and granted by ETT 016/2009.

Results 19 out of the 58 consecutive MDS patients had a mutation in the HFE gene. The present study—where twice as many patients had been recruited than in the previous historical one- suggests (Tables€11.1 and 11.2) that more HFE mutant

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Table 11.1↜渀 Iron-copper parameters in the group of MDS patients in relation to the HFE gene status and preceded by less than 10 or more than 20 units of blood transfusion therapya Se Cu (μmol/l) Tf sat (%) Se Cu (μmol/l) Tf sat (%) HFE wt HFE mutant (nâ•›=â•›22) (nâ•›=â•›11) 47.8 (27–80) BTTâ•›<â•›10 units 16.8 (10.4–26.9) 39.9 (14–92) BTTâ•›<â•›10 units 15.18 (12.3–19.1) BTTâ•›>â•›20 units 6.35 (4.0–8.5) 78.2 (39–116) BTTâ•›>â•›20 units 5.86 (5.1–7.1) 97.6 (95–101) All 13.1 (4.0–26.9) 52.7 (14–116) All 11.1 (5.1–19.1) 64.4 (27–101) Marking gene status: homozygote: 2, heterozygote: 1, wt: 0, 1/1 means compound heterozygote a HFE gene mutation distribution was as follows: 1/1: 1, C282Y-1: 2 and H63D-1: 8 persons. There wasn’t any relevant difference between the two groups regarding MDS subgroup distribution. Normal upper level for Tfsat: 45% [38]

Table 11.2↜渀 Iron overload (IO) in MDS patients in relation to their HFE and copper status and the amount of previous blood transfusions All MDS patientsa Low Cu/CP Normal Cu/ High tr sat/ferritin Normal tr sat/ferritin (n: 58) CP HFE mutantsb (n: 19) 8 (35%) 11 (65%) 12 (63%) (after 0–10 7 (37%) units of blood had been transfused) 9 (23%) 30 (77%) 21 (52%) (after 10–20 18 (48%) HFE wt (n: 39) units of blood had been transfused) Normal values for serum Cu in males was 11–22€μmol/l and in females was 12.6–24.4€μmol/l. Normal serum CP level for both sexes 0.20–0.60€g/l. Normal upper level for Tfsat: 45% and for ferritin: 400€μmol/l in both sexes a MDS subtype distribution: RA: 10, RAS/RARS: 16, RAMCD: 8, 5q-: 4, RAEB: 13, Hypoplastic: 5, MPS/MDS: 1 secondary MDS: 1 b HFE mutants were: H63D heterozygote: 15, compound heterozygote: 1, C282Y heterozygote: 3

MDS patients develop IO even at a lower blood transfusion threshold than their HFE wt counterparts. Also more of them develop Cu/CP deficiency as well. In the parallel study 6/11 HH patients had upfront either low copper or CP levels. Those who had already been treated by phlebotomies, the Cu/CP values were in the normal range (Table€11.3).

Discussion According to the results shown in Table€11.1 that 19/58 MDS patients had mutation in the HFE gene. They are more likely to develop iron overload and Cu deficiency earlier in their disease course in contrast to MDS patients with the wt HFE gene. In the parallel study 6/11 HH patients had upfront Cu/CP deficiency (Table€11.3).

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Table 11.3↜渀 Copper and coeruloplasmin parameters of HH patients tested upfront or after venesections Patient Genetic Ferritin Cu CP U/Upfront alteration V/Vena section 400.5 10.8 0.17 U No. 1 +â•›−/+â•›− No. 2 +â•›−/+â•›− 15.9 0.21 V >1910 No. 3 351 12.2 0.16 U +â•›−/+â•›− No. 4 +â•›+/−â•›− 1812.9 14.1 0.26 U No. 5 +â•›+/−â•›− 1652 14.3 0.18 U No. 6 +â•›+/−â•›− 1223 16.9 0.22 U No. 7 +â•›+/−â•›− 1227 18.7 0.22 U No. 8 +â•›+/−â•›− 93.1 11.6 0.20 U +â•›+/−â•›− No. 9 962 12.9 0.19 U 1273 16.0 0.22 V No. 10 +â•›+/−â•›− No. 11 +â•›+/−â•›− 81.5 13.8 0.17 U +â•›−/+â•›−: compound heterozygote, +â•›+/−â•›−: C282Y homozygote

It is proposed therefore that Cu deficiency in MDS patients might be partly related to, or aggravated by the presence of HFE gene mutations. The tendency to develop decreasing Cu and raising transferrin saturation (trsat) by transfusional escalation even in those who have wt HFE gene had already been documented (38). In the actual study 21/39 HFE wt MDS patients developed high iron, Trsat and ferritin parameters and 9/39 copper deficiency related to blood transfusions or iron load itself. Results found in the small cohort of HH patients indicate that while HFE gene mutations favours iron absorption, copper absorption by contrast is insufficient in almost half the cases. It might be also noted that copper/CP level seems to be independent from ferritin concentrations. It probable depends on other signals arriving to the duodenal site where absorption is taking place. It is hypothetized that the 2 HH patients who had already regular venesections—and by this they forwarded intensive stimuli to erythropoiesis—have normal Cu levels, by an erythropoiesis driven enhanced duodenal Cu absorption mechanism that might overcome the effects of genetic mutation related preferencial iron uptake. In polytransfused MDS patients with wt HFE the uptake of both metals is inhibited– in accordance with normal regulatory machinery. On the contrary, in HFE gene mutant MDS patients copper deficiency develops due to a preferential iron uptake by the common duodenal metal transporters or through other yet not fully known mechanisms probably in relation to disturbed sensing of hypoxia, anemia, iron—separately or both. CP oxidizes Fe into it’s trivalent form (Fe3+) that in turn is able to complex with transferrin. As in Cu deficiency CP levels are decreased, CP cannot perform its function as effectively as at normal concentrations. This will result in an increase in non-transferrin bound iron (NTBI) that further confers oxidative harm to tissues. Correction of copper deficiency seems to be therefore of great importance in iron overload conditions to stop this vicious circle.

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This is in line with the findings of Klevay LM et€al. showing in an animal model and later by Laine F et€al. in humans claiming that people with iron overload might benefit from copper supplementation [6, 41]. In MDS serum Fe concentration, ferritin and Tfsat are often found increased even prior to BTT, these parameters well reflecting those pathogenetic mechanisms that have been here reviewed. According to the analysis of hundreds of EPO levels in our laboratory arriving from different sources, the highest EPO levels were encountered in Aplastic Anemia (data not shown). These patients are almost always HFE wt and have low/ normal iron, tr sat for reasons such as increased IL-6 induced high hepcidin levels that would accompany the underlying inflammatory process. The high EPO levels found in anemic patients in general reflect normal hypoxia signal regulation. There are however cases when EPO levels are not elevated unless anemia is present. In such cases the hypoxia-to-EPO signal transduction pathway must be impaired. What is then the difference between those with low or high EPO MDS patients? Let us speculate then what the characteristics for those MDS patients with low EPO level should be?—They must have mutant HFE, higher trsat and ferritin level, unless blood transfusion therapy would result in rise of their hepcidin level. In MDS however hepcidin level is low especially in RARS and this is explained by the hepcidin suppressor GDF-15 that is liberated from the early erythropoietic cells of ineffective erythropoiesis. Our results show that their low hepcidin level almost in 1/3- or 1/2 of all the cases are partly related- and it cannot be excluded—to the presence of mutant HFE gene. Effective EPO therapy for MDS patients with low EPO levels had already been confirmed and is highly recommended for the purpose of preventing transfusion related IO as well [42]. By summarizing all the above findings and taking them into consideration, the authors recommend their recently published guide to identify MDS patients who are at the highest risk for iron overload and who should be treated with iron chelation as early in the course of disease as possible (Table€11.4). In conclusion, the present study indicates that in MDS, iron overload develops for several reasons due to ineffective erythropoiesis itself/and to BTT and/or to coexistent HFE gene mutation and/or to Cu deficiency/or to a combination of all these factors. Our data confirm that increasing the amount of BTT causes a decrease in Cu level in relation to the extent of IO even in HFE wt MDS patients, where there is a negative feedback to inhibit the uptake of both metals. It is of concern that chronic deficiency states might open alternate metabolic pathways in cell metabolism as a Table 11.4↜渀 Parameters of significance to decide on upfront iron chelation therapy (ICT) in MDS EPO HFE C282Y/H63D Ringa sideroblasts A/MDS at immediate risk for IO Low/Normal Mutant Present B/MDS not at immediate risk for IO High wt Non IO iron overload, ICT iron chelation therapy, EPO erythropoietin, HFE hemochromatosis gene, wt wild type gene a Patients of low Epo/high hepcidin-those like of anemia of chronic disease might benefit from EPO therapy that is preventing and can overcome IO [22]

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compensatory mechanism for cell survival, eventually resulting in more primitive cell lines that might overgrow and start to dominate thus contributing to or initiating the process of malignant transformation [43]. It is still to be determined how CP and Cu levels change while on iron chelation therapy (ICT) in HH patients and how MDS patients might benefit from Cu supplementation with or without ICT.

References ╇ 1. Undritz E (1964) Iron metabolism. Springer-Verlag, Berlin ╇ 2. Elvehjem CA, Sherman WC (1932) The action of copper in iron metabolism. J Biol Chem 98:309–319 ╇ 3. Davis GK, Mertz W (1986) Copper. In: Walter Mertz (ed) Trace elements in human and animal nutrition, 5th edn. Academic Press, San Diego ╇ 4. Owen CA (1982) Physiological aspects of copper. Noyes Publications, Park Ridge ╇ 5. Klevay LM (2001) Iron overload can induce mild copper deficiency. J Trace Elements Med Biol 14:237–240 ╇ 6. Klevay LM, Saari JT (1993) Comparative responses of rats to different copper intakes and modes of supplementation. Proc Soc Exp Biol Med 203:214–220 ╇ 7. Holmberg CG, Laurell CB (1947) Investigation in serum copper. Acta Chem Scand 1:945 ╇ 8. Lahey ME, Gubler CJ, Chase MS, Cartwright GE, Wintrobe MM (1952) Studies on copper metabolism. III. The metabolism of iron in copper deficient. Blood 7:1053 ╇ 9. Sharp P (2004) The molecular basis of copper and iron interactions. Proc Nutr Soc 63:563– 569 10. Chung J, Haile DJ, Wessling-Resnick M (2004) Copper-induced ferroportin-1 exprerssion in J774 macrophages in associated with increased iron efflux. Proc Natl Sci, U S A 101:2700– 2705 11. Llanos RM, Mercet JF (2002) The molecular basis of copper homeostasis copper related disorders. DNA Cell Biol 21:259–270 12. Willis MS, Monaghan SA, Miller ML, McKenna RW, Perkins WD, Levinson BS, Bhushan V, Knoff SM (2005) Zinc—induced copper deficiency. Am J Clin Pathol 123:125–131 13. Gitlin JD (1998) Aceruloplasminemia. Pediatr Res 44:271–276 14. Harris ZL, Durley AP, Man Tsz K, Gitlin JD (1999) Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux. Proc Natl U S A 96:10812–10817 15. Laine F, Ropert M, Le Lan C, Loreal O, Bellissant E, Jard C, Pourchard M, Le Treut A, Brissot P (2002) Serum ceruloplasmin and ferroxidase activity are decreased in HFE C282Y homozygote male iron-overload patients. J Hepatol 36:60–65 16. Feder JN, Gnirke A, Thomas W, Tsushiashi Z, Ruddy DA, Basava A et€al (1996) A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 13:399–408 17. Hirase N, Abe Y, Sadamura S, Yufu Y, Muta K, Umemura T et€al (1992) Anemia and neutropenia in a case of copper deficiency: role of copper in normal hematopoiesis. Acta Hematol 87:195–197 18. Williams DM, Loukopoulos D, Lee GR, Cartwright GE (1976) Role of copper in mitochondrial iron metabolism. Blood 48:77–85 19. Gregg XT, Reddy V, Prchal JT (2002) Copper deficiency masquerading as myelodysplastic syndrome. Blood 100:1493–1495 20. Koca E, Buyukasik Y, Cetiner D, Yilmaz R, Sayinalp N, Yasavul U, Uner A (2008) Copper deficiency with increased hematogones mimicking refractory anemia with excess blasts. Leuk Res 32:495–499 21. Nemeth E (2008) Iron regulation and erythropoiesis. Curr Opin Hematol 15(3):169–175

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22. Nearman ZP, Szpruka H, Serio B, Warshawsky I, Theil K, Lichtin A, Sekeres MA, Maciejewski JP (2007) Hemochromatosis associated gene mutations in patients with myelodysplastic syndromes with refractory anemia with ringed sideroblasts. Am J Hematol 82:1076–1079 23. Kattamis A, Papassotiriou I, Palaiologou D, Apostolakou F, Galani A, Ladis V, Sakellaropoulos N, Papanikolaou G (2006) The effects of erythropoetic activity and iron burden on hepcidin expression in patients with thalassemia major. Haematologica 91(6):809–812 24. Tanno T, Bhanu NV, Oneal PA, Goh SH, Staker P, Lee PY, Moroney JW, Reed CH, Luban NLC, Wang RH, Eling TE, Childs R, Ganz T, Leitman SF, Fucharoen S, Miller JL (2007) High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med 13:1096–1101 25. Messa E, Maffe C, Volpe G, Campostrini M, Gioia D, Carturan S, Zanone C, Levis A, Cilloni D, Girelli D, Camaschella C, Saglio G (2009) Analysis of iron homeostasis and erythroid activity in a cohort of low-risk myelodysplastic patients. Leukemia Research. International Symposium on Myelodysplastic Syndromes (MDS), Patras, Greece P087, S108 26. Chan L, Buckstein R, Reis M, Chesney A, Lam A, Cheung M, Piliots E, Gu L, Wells R (2009) Iron overload and haematopoiesis in MDS: Does blood transfusion promote progression to AML? Leukemia Research. 10th International Symposium on Myelodysplastic Syndromes (MDS), Patras, Greece P092, S112 27. Hesrhko C, Peto T (1980) Non-transferrin plasma iron. Br J Haematol 66:149 28. Breuer W, Ronson A, Slotki IN, Abramov A, Hershko C, Cabantchik ZL (2000) The assessment of serum nontransferrin-bound iron in chelation therapy and iron supplementation. Blood 95:2975–2982 29. Aruoma OI, Bomford A, Polson RJ, Halliwell B (1988) Nontransferrin-bound iron in plasma from hemochromatosis patients: effect of phlebotomy therapy. Blood 72:1416–1419 30. Malcovati L (2007) Impact of transfusion dependency and secondary iron overload on the survival of patients with myelodysplastic syndromes. Lek Res 31(Suppl 3):S2–S6 31. Malcovati L (2009) Red blood cell transfusion therapy and iron chelation in patients with myelodysplastic syndromes. Clin Lymph Myeloma 9(Suppl 3):S305–S311 32. Leitch HA (2007) Improving clinical outcome in patients with myelodysplastic syndrome and iron overload using iron chelation therapy. Leuk Res 31(Suppl 3):S7–S9 33. Jensen PD, Heckendorff L, Pedersen B, Bendix-Hansen K, Jensen FT, Christensen T, Boesen AM, Ellegaard J (1996) The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload. Br J Haematol 94:288–299 34. Kersten MJ, Lange R, Smeets ME, Vregdenhil G, Roozendaal KJ, Lameijer W et€al (1996) Long- term treatment of transfusional iron overload with the oral iron chelator deferiprone (L1): a Dutch multicenter trial. Ann Hematol 73:247–252 35. Várkonyi J, Tarkocács G, Benedek Sz, Demeter J, Varga F, Fekete S, Andrikovics H, Tordai A (2001) HFE gene mutations and the effect of deferoxamine therapy in myelodysplastic syndrome. Blood(Abstr) 98(11):4844 36. Kallinapur AR (2005) Genomic screening and complications of hematopoietic stem cell transplantation: has the time come? Bone Marrow Transplant 35:1–16 37. Várkonyi J, Tarkovács G, Karádi I, Andrikovics H, Varga F, Varga F, Demeter J, Tordai A (2003) High incidence of hemochromatosis gene mutations in the myelodysplastic syndrome: the Budapest study on 50 patients. Acta Haematol 109:64–67 38. Várkonyi J, Szabó T, Sebestyén P, Tordai A, Andrikovics H, Kollai G, Karádi I (2006) New aspects of copper and iron metabolism in the myelodysplastic syndromes. Chemotherapy 52:66–68 39. Abe A, Yamashia S, Noma A (1989) Sensitive, direct colorimetric assay for copper in serum. Clin Chem 35:552–554 40. Parker MM, Humoller FL, Mahler DJ (1967) Determination of copper and Zinc in biological material. Clin Chem 13:40–48 41. Laine F, Ropert M, Le Lan C, Loreál O, Bellissant E, Jard C, Pouchard M, Le Treut A, Brissot P (2002) Serum ceruloplasmin and ferroxidase activity are decreased in HFE C282Y homozygote male iron-overload patients. J Hepatol 36:60–65

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42. Greenberg PL, Sun Z, Miller KB, Bennett JM, Tallman MS, Dewald G, Paietta E, Van der Jagt R, Houston J, Thomas ML, Cella D, Rowe JM (2009) Treatment of myelodysplastic syndrome patients with erythropoietin with or without granulocyte colony-stimulating factor: results of a prospective randomized phase 3 trial by the Eastern Cooperative Oncology Group (E1996). Blood 114:2393–2400 43. Varkonyi J (2008) Chronic deficiency states—initially reversible metabolic changes resulting in true myelodysplasia. Hungarian Med J 2(2):329–330

Chapter 12

Pathogenesis and Management   of Iron Overload in MDS Chaim Hershko

Iron is one of the most common elements in nature and, as a transition metal, is essential for the functioning of proteins involved in oxidative energy production, oxygen transport, mitochondrial respiration, inactivation of harmful oxygen radicals and DNA synthesis. Because of its poor solubility, living organisms developed efficient mechanisms for the acquisition, transport and storage of iron, but there is no natural mechanism for the excretion of excess iron [1]. Normal iron homeostasis fails to prevent the harmful accumulation of iron in two major disease categories: in inherited hemochromatosis syndromes where abnormal hepcidin regulation results in increased intestinal iron absorption and; in iron loading anemias such as the myelodysplastic syndrome (MDS) or the inherited hemoglobinopathies such as thalassemia major (TM) and sickle cell anemia where transfusional iron overload is aggravated by increased iron absorption, caused by increased rates of ineffective erythropoiesis [2, 3] (Fig.€12.1). The rate of intestinal iron absorption in iron-loading anemias is variable, depending on the underlying hematologic disorder but is generally only a fraction of the rate associated with transfusional iron loading. The outpouring of catabolic iron derived from senescent erythrocytes may exceed the iron-carrying capacity of transferrin, resulting in the emergence of non-transferrin-bound iron (NTBI). NTBI is cleared preferentially by the liver, myocardium and other internal organs at a rate exceeding 200 times that of transferrin iron. NTBI catalyzes the formation of free radicals, resulting in oxidative stress and damage to mitochondria, lysosomes, lipid membranes, proteins and DNA [4] (Table€12.1). Humans cannot increase iron excretion to compensate for iron overload. Consequently, excess iron must be removed therapeutically. The most simple and effective method of removing, or preventing the accumulation of excess iron in hereditary hemochromatosis is by repeated phlebotomies. However, in iron loading anemias, phlebotomies are impractical and alternative methods, such as the use of iron chelators enhancing urinary and/or fecal iron excretion, have been developed. C. Hershko () Department of Hematology, Shaare Zedek Medical Center, PO Box 3235, Jerusalem, Israel Tel.: +972-2-533-20-51 Fax: +972-2-570-06-93 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_12, ©Â€Springer Science+Business Media B.V. 2011

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Transfusion

Erythron

Gut

Liver NTBI

RE cells

Endocrines

Heart

Fig. 12.1↜渀 Iron balance in MDS. Iron overload is the joint product of repeated blood transfusions and inappropriately increased iron absorption caused by ineffective erythropoiesis and anemia. Excess iron acquired by hyperabsorption is filtered through the liver via the portal system, leading to predominant hepatic parenchymal siderosis typical of all conditions with increased iron absorption such as hereditary hemochromatosis and untransfused MDS. The first station in the accumulation of transfusional hemosiderosis is the RE system (spleen). Subsequently, RE iron is recycled to parenchymatous organs. With increasing severity of iron overload, the iron binding capacity of circulating transferrin is exceeded, resulting in the emergence of NTBI. The rate of NTBI iron uptake by the heart, liver and other organs is many times faster than of transferrin iron, resulting in accelerated rates of iron deposition and cellular damage. Green arrows: transferrin iron, Brown arrows: NTBI

Table 12.1↜渀 Damage caused by reactive oxygen species (ROS). [4] Lipid peroxidation Oxidation of amino acid side chains (especially cysteine) Formation of protein-protein cross-links Oxidation of polypeptide backbones leading to protein fragmentation DNA damage, and DNA strand breaks

Methods for Evaluating Iron Overload in MDS Monitoring Transfusional Burden With regular blood transfusions, iron stores increase to many times normal unless chelation treatment is given. Approximately 200€mg of iron is present in each unit

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of donated blood or about 1.08€mg of iron per 1€ml of pure red blood cells. The total amount of iron transfused can be calculated by the volume of each unit transfused and the sum of blood transfusions. Mean transfusional loading in transfusion-dependent anemias is about 0.4€mg/kg/day but this varies considerably. In adults, the number of transfusions received is an important predictor of myocardial siderosis. In the landmark studies of Buja and Roberts and of Schafer et€al. [5, 6] important myocardial siderosis emerged only after the transfusion of about 100 units of blood. Of particular significance are the observations of Schafer indicating, that in their small group of 15 patients with a mean of 120 units of blood transfused, iron loading has been present after less than 4 years in 14 of 15 patients and, that impaired left ventricular function was only present in the most heavily transfused patients or in those with coexisting coronary disease. The important takehome message of these early studies is that it is possible to predict the severity of cardiac siderosis from the number of transfusions received, that in adults significant heart disease may develop after 4 years of heavy transfusions and, that patients with coexisting coronary disease are at particular risk.

Monitoring Serum Ferritin Serum ferritin is an inexpensive and widely available method for estimating body iron. In subjects without other comorbidities, ferritin is a useful indicator of iron stores, with each unit of serum ferritin ng/ml indicating about 8€mg of storage iron. These relations have been validated by following the rate of ferritin decrease in subjects with hereditary hemochromatosis subjected to phlebotomy treatment. However, variations in body iron stores account for only 57% of the variability in serum ferritin [7]. Inappropriately increased serum ferritin values are encountered in the presence of inflammation and hepatocellular damage. Conversely, ascorbate depletion decreases serum ferritin values. Nevertheless, control of serum ferritin has been shown to have prognostic significance in thalassaemia major patients on long-term chelation therapy [8, 9]. Clinical correlations in thalassemia showed that patients with serum ferritin levels exceeding 2,500€ng/ml are at increased risk of cardiac complications [8]. However, elderly MDS patients often suffer from additional pathologies associated with nonspecific increase in serum ferritin [10] and their siderotic cardiac complications are difficult to distinguish from coronary and other common causes of heart disease. Moreover, in hematologic malignancies such as AML and the blastic phase of CML, serum ferritin levels are exceptionally high [11]. Observations on the adverse prognostic significance of increased pre-transplant serum ferritin in MDS and AML [12, 13] and the adverse prognostic significance of increased serum ferritin in transfusion dependent MDS patients [14] should be interpreted with extreme caution to avoid misinterpretation of the cause and effect relation between serum ferritin, survival, and progression of MDS to acute leukemia. In spite of these concerns, regular periodic ferritin measurements in stable polytransfused MDS patients are still

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useful in following trends in iron accumulation and response to chelating therapy. However, serum ferritin measurements alone should not be considered as a reliable indicator for iron chelation therapy unless confirmed by other, independent criteria such as measurements of liver or myocardial iron concentrations [15].

Assessment of Hepatic and Myocardial Siderosis Historically, measurement of liver iron concentrations (LIC) required the use of liver biopsy samples. However, efficient non-invasive methods are now widely available. Current MRI techniques for measuring LIC [16, 17] rely on the principle that tissue iron exerts a paramagnetic effect on surrounding tissues that affects the relaxation time of molecules excited by the application of a magnetic field. One such method is now registered in the EU and US and can utilize widely available MRI equipment with little extra training of local staff [16]. It offers measurements that approximates linearity over a clinically useful range. Body iron stores can be accurately predicted from the liver iron concentration (LIC) using the formula: Total Body Iron stores in mg/kgâ•›=â•›10.6â•›×â•›the LIC (in mg/g dry wt) [18]. Normal LIC values are less than 1.8€mg/g dry wt but levels up to 7€mg/g dry wt may be encountered without evidence of harmful effect [19]. LIC values above 15–20€mg/g dry wt are associated with adverse prognosis [20, 21], progressive liver fibrosis [22] and abnormal liver function [23]. In unchelated patients, high LIC values predict an increased risk of myocardial iron deposition [5, 6] but once chelation therapy has been initiated, this simple relationship no longer exists because iron is cleared faster from the liver than from the heart [24, 25]. The development of MRI techniques for estimating myocardial iron [23, 24] allowed better understanding of the factors influencing myocardial iron deposition and removal, and the risks associated with such deposition. The method most extensively used to estimate myocardial iron is the T2* technique, where the T2* in ms is inversely correlated with tissue iron concentration. The risk of developing a clinically relevant fall in left ventricular function increases as the T2* falls [25, 26]. Cardiac iron measurements by T2* have become the current standard for estimating the risk of siderotic heart disease. The proportion of patients developing cardiac failure with a T2* of less than six is over 50% within one year, whereas in patients with a normal T2* of 20<, iron-associated heart disease is non-existent. Consequently it is now possible to identify MDS patients with life-threatening siderosis by quantitating both their hepatic and cardiac iron. Although these correlations have been established in patients with transfusion-dependent hereditary anemias, extrapolation of these rules to the management of MDS-associated siderosis appears to be legitimate. Moreover, it is sensible to introduce iron chelation therapy even before reaching critical concentrations of tissue iron if the rate of such iron accumulation can be predicted from the rate of blood transfusions, and the rate of increase in tissue iron concentrations.

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Plasma Non-Transferrin Bound Iron Measurements A number of assays are available for NTBI measurements that yield variable reference ranges but generally correlate with each other [27]. An assay measuring a labile subfraction, the component capable of accelerating oxidation of a fluorophore, termed LPI assay [28] is convenient for measuring iron in the presence of chelators. Progressive removal of this subfraction has been seen with deferasirox [29], consistent with the notion that continuous chelation minimises exposure to NTBI species [30]. Recent studies in thalassemic patients indicate, that the presence or absence of NTBI in polytransfused patients may be a useful indicator of increased risk of siderotic myocardial disease [31].

Goals of Iron Chelation in MDS The primary objective of iron chelation therapy (ICT) is to maintain body iron at safe levels at all times. Iron stored as ferritin or hemosiderin is not chelated directly at clinically useful rates so that once accumulated, iron removal is slow and inefficient, relying on the tiny fraction of ‘labile iron’ that is available for chelation at any moment. In thalassemia, chelation with DFO has traditionally been started only after 2–3 years of transfusion or when ferritin exceeds 1000€ng/ml, to avoid unwanted over-chelation at low levels of body iron. While the slow process of decreasing tissue iron to safe levels is being achieved, a second goal is to make iron as safe as possible by binding the toxic labile iron pools responsible for causing tissue damage. Plasma non transferin iron and labile iron rebound rapidly after a chelator is cleared from plasma [28, 30] so that in principle, the continuous presence of a chelator is desirable. Continuous chelation therapy also has the potential to minimise the uptake of NTBI species into organs such as heart and endocrine tissues. With the introduction of orally effective iron chelators that are easy to take and suitable for use in elderly patients, there is increasing interest in the use of ICT in MDS [32–38]. Ideally, evidence supporting the beneficial effects of iron chelation in MDS should rest on controlled studies indicating improved life expectancy and, in exceptional cases, reversal of myocardial disease. However, such evidence is presently unavailable. Nevertheless, it is possible to rely on the extensive experience accumulated in thalassemia and employ well defined indicators of increased risks of life-threatening complications to identify patients who may benefit from iron chelation therapy.

Selection of MDS Patients for Chelation Therapy The choice of MDS patients who may benefit from chelation therapy has been a topic of continued interest. Within the last decade a number of National Society guidelines, proceedings of Consensus meetings and NCCN guideline updates have

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been published [39, 40] and the majority of these publications have been analyzed in a review by Gattermann [41]. Despite some differences in detail, the principles underlying these recommendations are remarkably similar. First, it is recognized that life-limiting transfusional iron overload is only relevant for MDS patients with a relatively favourable prognosis. It is estimated that about 35–55% of MDS patients can be classified within this category including patients with dyserythropoiesis only, low risk refractory cytopenia with multilineage dysplasia, and possibly intermediate–I risk refractory cytopenia with multilineage dysplasia. Second, the magnitude and rate of iron accumulation are critical. Clinical correlations have shown that significant cardiac siderosis may only be expected after the transfusion of about 100 units of blood [5, 6, 23, 42–44] (Table€12.2) which, with a transfusion rate of 2–4 units per month may be reached after about 2–4 years of treatment. Recommendations to introduce ICT following a transfusional load of 20–50 units should be modified by also considering the rate of transfusions predicting the accumulation of 100 or more blood units within 4 years or less. Last, because of coexistent morbidities, in MDS serum ferritin is a poor indicator of iron overload. With the availability of reliable non-invasive technology permitting accurate estimation of iron accumulation in vital organs, the proposed threshold ferritin values levels ranging from 1,000 to 2,500€ng/dL should be replaced by estimates based on state-of-the-art MRI methodology. Whenever such technology is not available, employing the threshold of a transfusion load approaching or exceeding 100 units of blood is a simple, and practical way of identifying MDS patients at serious risk of life-threatening siderosis.

Table 12.2↜渀 Relation of cardiac siderosis to transfusional iron burden in non-thalassemic polytransfused adults Buja et€al. 131 transfusion dependent adults. Significant myocardial iron accumulation encountered in 60% of patients after 101–200 and 100% of patients after 201–300 blood transfusions [5] Schafer et€al. 15 transfusion dependent adults after 60–210 transfusions. Left ventricular cardiac function was impaired in only the most heavily transfused patients [6] Jensen et€al. 14 transfusion dependent adults. Abnormal myocardial iron accumulation was shown in patients with a threshold liver iron concentration above 400€µM/g (22.3€mg/g dry weight)a [23] Di Tucci et€al. 27 transfusion dependent adults. Cardiac T2* correlated with transfusion burden. No patient who had received less than 290€ml/kg of packed red blood cells (101 unitsâ•›=â•›20€g of iron) had a pathological cardiac T2* value (<20€ms) [42] Chacko J et€al. 11 transfusion dependent MDS patients. Five unchelated and six chelated patients with median transfusion burdens of 63 and 112 red cell units respectively. Myocardial T2* indicated absent iron loading in 10/11 patients and borderline-normal in one patient [43] Konen E et€al. 11 transfusion dependent MDS patients with a median transfusion burden of 90 blood units. None of the patients showed evidence of abnormal cardiac iron by cardiovascular T2 magnetic resonance imaging [44] a Employing the Angelucci formula (18) and assuming a mean body weight of 75€kg, this is the equivalent of 1.773€g of elemental iron representing over 89 units of transfused blood

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The importance of ICT in MDS is not universally acknowledged [45–47]. Although the favourable effects of ICT in thalassemia including reversal of cardiac arrhythmias, improvement in left ventricular ejection fraction, arrest of hepatic fibrosis, and reduction of glucose intolerance is well documented, it is not yet clear whether these specific outcomes are directly applicable to MDS.

Choice of Iron Chelators in MDS Iron(III) has six coordination sites that can be accommodated by one large molecule such as deferoxamine (DFO) (hexadentate chelation, Fig.€ 12.2). Such molecules tend to have high affinity to iron(III) but it has not been possible to design hexadentate chelators that are small enough to allow efficient oral absorption. Smaller molecules can be absorbed from the gut more effectively and can bind iron(III) in either a 2:1 ratio with each molecule providing three binding sites (tridentate chelation, e.g. deferasirox, DFS) or a 3:1 ratio with each molecule providing two sites (bidentate chelation, e.g. deferiprone, DFP).

Fig. 12.2↜渀 The three leading iron chelating drugs for clinical use. Deferoxamine is a hexadentate drug and a single molecule is able to cover all six coordination sites of iron. By comparison, deferiprone is bidentate requiring three molecules and deferasirox is tridentate, requiring two molecules to cover all six coordination sites

194 Table 12.3↜渀 Comparison of the three leading iron chelating drugs. [78] Compound Deferoxamine Deferiprone Molecular weight 657 139 (daltons) Chelating properties Hexadentate Bidentate Iron binding affinity (pM) 26.6 19.9 Chelation efficiency (%) 13 7 Recommended dose 30–50 75–100 mg/kg/day Oral three times daily Delivery s.c. or i.v. 8–12€h 5 days/week Half-life 20–30€min 3–4€h Lipid solubility Low Intermediate Excretion Urinary and fecal Urinary 90–450 Peak plasma levels (µM) 5–10 Gastrointestinal Adverse effects Ocular, auditory upset, arthralgia, toxicity, growth agranulocytosis/ retardation, neutropenia local reactions, allergy

C. Hershko

Deferasirox 373 Tridentate 22.5 27 20–40 Oral, once daily 12–16€h High Fecal 80 Gastrointestinal upset, rash, ocular, auditory toxicity, mild reversible increase in creatinine

A comparison of molecular weights, chelating properties, recommended daily dose, method of delivery and other properties of the three leading chelators deferoxamine (DFO), deferiprone (DFP) and deferasirox (DFS) is presented in Table€12.3.

Deferoxamine Because of its proven efficacy and the extensive experience with its long-term use in many thousands of thalassemic patients, deferoxamine (DFO) is still considered the gold standard of iron chelation therapy. DFO is infused via a thin s.c. needle inserted to the arm or abdomen nightly, connected to a portable pump over 8–12€h, five to seven times per week at a daily dose of 20–60€mg/kg. In most patients, a urinary iron excretion of 0.5€mg/kg/d is usually sufficient to ensure negative iron balance. The introduction of DFO for iron chelation therapy of transfusional siderosis has changed the life expectancy and life quality of patients with thalassemia major. Its long-term efficacy has been extensively documented in large multicenter trials in Italy and elsewhere. Only 70% of patients born before 1970 and hence prior to the modern era of iron chelation survived to age 20 year compared with 89% of patients born after 1970 and therefore receiving effective chelation from an early age [48]. In a report on thalassemic patients treated by DFO at a single institution, survival at 40 years was 83% and in compliant patients born after 1975 survival at 25 years

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was 100% [49]. The cohort-of-birth related improvement in survival was reflected in an inverse, mirror-like decrease in cardiac mortality, supporting the assumption that prevention of cardiac mortality is the most important beneficial effect of DFO therapy. The strongest direct evidence supporting the beneficial effects of DFO on hemosiderotic heart disease is the reversal of established myocardiopathy in some far-advanced cases. Compliance with the rigorous requirements of daily subcutaneous DFO infusions is still a serious limiting factor in treatment outcome and all patients noncompliant with DFO treatment remain at risk of lethal cardiac complications [49, 50]. In practice, for an elderly MDS population the inconvenience of long term subcutaneaous DFO administration makes such treatment unacceptable for the vast majority of patients.

Deferiprone Urinary iron excretion with DFP is roughly equal to DFO and is probably derived from the same chelatable pool with one exception: because of its smaller size (m.w. 139 versus 560) and higher lipophilicity compared with DFO deferiprone readily enters cells [51]. Thus DFP may access intracellular chelatable iron more effectively than DFO. On the other hand DFP, unlike DFO does not enhance biliary iron excretion, probably because of its rapid glucuronidation and inactivation within hepatocytes. The cardioproective effect of DFP is the most remarkable advantage of this compound. Long-term, prospective trials are now available comparing the ability of chelation therapy with either deferoxamine or deferiprone to prevent heart disease. In a study involving all thalassemic patients treated at seven Italian hospitals [52] the estimated hazard of a cardiac event on DFP was less than one-tenth that in patients on DFO. Further evidence supporting the cardioprotective effect of DFP was produced in a study of 61 thalassemic patients with moderate cardiac siderosis (T2* 8–20€ms) [53] randomized to continue on DFO 43€mg/kg/d or DFP 92€mg/kg/d. After one year of treatment, the improvement in T2* and increase in left ventricular ejection fraction were significantly greater for DFP than for DFO. The authors concluded that DFP monotherapy was significantly more effective than DFO in improving asymptomatic myocardial siderosis in beta-thalassemia major. Because of the risk of drug induced agranulocytosis, regular weekly WBC counts are recommended for the early detection of neutropenia. This limitation complicates the management of iron chelation treatment in MDS patients with disease-related neutropenia.

Deferasirox DFS is a synthetic chelator with a plasma half-life of 11–19€h allowing its use by once-daily oral dosing. The ability of DFS to eliminate NTBI from the circulation is outstanding [29, 54].

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In a randomised propective study involving 586 thalassemic patients in whom the long term effects of DFS were compared with DFO [55] both drugs were equally effective as judged by LIC and ferritin measurements at DFS dose ranges of 20– 30€mg/kg/d These results agree very well with the results of previous studies. A question of vital importance is the effect of DFS on cardiac siderosis and the prevention of cardiac complications. The efficacy of deferasirox in reducing or preventing cardiac iron overload was assessed in a recent study in 192 patients with beta-thalassemia in a 1-year prospective, multicenter trial [56]. 114 of these patients had abnormal myocardial T2* ranging from 5 to 20€ms indicating cardiac siderosis, and serum ferritin more than 2,500€ng/ml. Myocardial T2* improved from a baseline of 11.2€ms to 12.9€ms (Pâ•›<â•›0.001) By comparison, in the prevention arm consisting of patients with normal T2*, baseline myocardial T2* remained unchanged from 32.0 to 32.5€ms (Pâ•›=â•›0.57). This prospective study suggests that deferasirox is effective in removing and preventing myocardial iron accumulation. In general, DFS is well tolerated [55, 57]. Adverse events are generally mild, including transient gastrointestinal events in 15%, skin rash in 11% and mild, dosedependent increases in serum creatinine in 38% of patients which generally remained within the normal range and did not exceed two times the upper limit of normal. Increase in creatinine typically occurred within a few weeks of starting or increasing therapy, was not progressive and was reversible or stabilised after dose adjustment when necessary. Follow up data in the five core phase II/III studies are now at a median of 3.5 years with no evidence of new or progressive toxicities. Because DFS treatment involves once-daily oral administration, it is particularly suitable for the management of elderly people who need minimal inconvenience of treatment. At present, it is too early to allow any statements on the impact of DFS on survival. Recommended patient monitoring includes monthly creatinine and liver function and annual auditory and ophthalmic examinations, including slit-lamp examination and fundoscopy.

Combined Chelation In patients failing to respond to ICT monotherapy, combined chelation is a reasonable approach. Considerable experience has been accumulated using combined DFO and DFP treatment in thalassemic patients. However, in MDS there is presently insufficient information on the potential risks and benefits of such treatment.

The Impact of ICT on Survival in MDS At present, only a limited number of uncontrolled studies are available regarding the impact of iron chelation therapy on survival in MDS.

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Raptis et€al. [58] reviewed the medical records of patients diagnosed with MDS and examined the survival of ICT-eligible patients defined as having received over 20 units of blood or serum ferritin over 1,000: Among 78 ICT-eligible patients with lower-risk MDS, 32 (41%) received ICT. Median overall survival for all ICT-eligible patients was significantly longer in ICT-treated patients than in untreated patients (8.7 years versus 4.7 years), and the authors concluded that receipt of ICT was associated with significantly longer survival. In another retrospective study, Leitch et€al. [59] examined the effect of ICT on survival in patients with transfusion dependent myelofibrosis. Of 25 transfusion dependent patients, 10 received ICT for a median of 18 months. Five year overall survival for transfusion dependent patients on ICT was 89% compared with 34% with no ICT. In another study in MDS by the same author [60] significantly more patients receiving ICT survived to 4 years (80% versus 44%; p╛<╛0.03), suggesting that MDS patients with iron overload might benefit from ICT. The most recent and extensive study on ICT in MDS [61] involved 97 low or intermediate 1 IPSS regularly transfused patients, followed for 2.5 years. 44 (45%) were not chelated and 53 (55%) received ICT, mainly deferoxamine. The median duration of chelation was 36 months. During the follow-up period, 66 of the 97 patients died, including 51% and 73% of chelated and non-chelated patients. Median overall survival in non-chelated and in chelated patients was 53 and 124 months respectively (p╛<╛0.0003). However, a number of observations argue against the assumption that the observed difference in survival in this study can be attributed to ICT: (a) Causes of death did not significantly differ between the two groups and there was no excess of deaths attributed to iron overload in the non-chelated patients. (b) The end-of study mean serum ferritins in chelated and non-chelated patients was identical. (c) Non chelated patients were older and had somewhat higher IPSS. (d) The higher rate of progression to AML in the non-chelated group could reflect an a priori worse prognosis. The major limitation of retrospective or non-randomized survival analysis in MDS patients receiving ICT is the initial selection of patients. The decision to use or avoid ICT in MDS rests on clinical evaluation of individual patients. Among subjects conforming with current consensus recommendations, some patients are more likely to receive long term ICT because they are expected to live long enough to benefit from ICT. This, in turn, would appear later as a favourable effect of ICT on survival. Although all authors of the above publications emphasize the need for randomized prospective future studies, the likelihood that such studies will ever be undertaken or that they will yield meaningful results is low. Elderly MDS patients have low compliance with ICT and have an increased risk of adverse effects such as neutropenia, impaired renal function and other complications leading to early discontinuation of treatment. Moreover, the high rate of competing causes of morbidity and mortality may render all possible beneficial effects of ICT on specific ironrelated damage invisible, as was indeed found in the study of Rose et€al. [61]. On the other hand, it should be noted that even the universally acknowledged beneficial effects of deferoxamine on survival in thalassemia have never been proven by prospective randomized studies. In view of these considerations, justification for the

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implementation of long-term ICT in MDS patients will continue to rest on thoughtful initial evaluation of individual patients, taking in consideration the most relevant clinical criteria namely IPSS category, the likelihood of living long enough to require 100 or more units of blood within the next 2–4 years, and periodic evaluation of the severity and distribution of siderosis employing state-of-the-art methodology.

Effects of ICT on Hemopoiesis in MDS Improvement in red cell production following ICT in MDS patients was first reported by Jensen et€al. [62, 63]. In patients on iron chelation treatment with deferoxamine for up to 60 months. Reduction of transfusion requirement was seen in 7/11 (64%) patients. Five patients (46%) became transfusion independent. All patients in whom iron chelation was highly effective showed improvement of erythropoietic output accompanied by an increase in serum transferrin receptor concentrations. Improved red cell production was also observed in patients with myelofibrosis and transfusional iron overload following deferiprone therapy [64]. More recently, deferasirox therapy was claimed to result in improved hemoglobin levels and a reduction in transfusion dependence in three patients with MDS and several patients with myelofibrosis [65, 66]. Collectively, these observations indicate that an improvement in red cell production may be expected in some MDS patients on ICT, and that contrary to previous claims, this effect is not limited to any specific iron chelator in clinical use. The mechanism responsible for the effect of iron chelation on hemoglobin production is presently unknown. In a patient with homozygous glutaredoxin 5 (GRLX5) mutation, the human counterpart of the zebrafish shiraz mutation, with sideroblastlike microcytic anemia and iron overload, deferoxamine treatment resulted in improved heme synthesis and the partial correction of anemia [67]. In this case, the proposed mechanism of improved hemoglobin synthesis was redistribution of mitochondrial iron into the cytosol following ICT, relieving the repression of ALA-synthase2 translation. Obviously, such an effect may only be expected in a minority of MDS patients with excessive mitochondrial iron deposition. Understanding the mechanism of improved anemia following ICT may have practical implications, allowing the prediction of improved red cell production in selected patients.

 laims for Protection from Other Harmful Effects   C of Iron in MDS In a recent review, Pullarkat has suggested that prevention of end-organ damage in iron overload associated with MDS may not be the most important target of ICT and that the removal of labile plasma iron or NTBI which, in turn, may be responsible for increased rates of infection, accelerated leukemic transformation and

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life-threatening complications of stem cell transplantation, may all be improved by the depletion of the labile iron pool [68]. Although the role of iron in the generation of toxic oxygen derivatives and free radicals through the Fenton or Haber-Weiss reaction is well established [69] and, in theory, such radicals may have a role in the above clinical complications, a closer look at the evidence supporting these claims leaves much to be desired. The lower rates of leukemic transformation in chelated MDS patients were all observed in non-randomized [61] or retrospective studies and they may simply indicate biased patient selection for ICT in a disease where leukemic transformation is a leading cause of mortality. The adverse effect of increased ferritin values on transplant outcome is even more difficult to interpret. In all probability, increased pre-transplant serum ferritin is a reflection of co-morbidities such as inflammation, infection and leukemic burden [10, 11]. There are no studies in MDS in which the use of pre-transplant serum ferritin as an indicator of iron overload severity has been validated by MRI measurements. Even if they were, increased iron burden could only indicate the increased risk of allotransplant complications in polytransfused patients and not the risk of an associated iron burden. Infection may, under specific circumstances be aggravated by excess iron [70]. Unbound transferrin is recognized as a natural resistance factor [71] and some microorganisms such as candida are particularly affected by the presence of NTBI [72, 73]. While infectious disease does not appear to represent a major threat in hereditary hemochromatosis and in stable low risk MDS, it is not unlikely that in the context of gross myelosuppression associated with conditioning for BMT, NTBI may aggravate infectious complications. However, ICT is not an innocuous intervention during myeloablative therapy because iron chelators inhibit ribonucleotide reductase, preventing new DNA synthesis and hence may prevent cell proliferation and engraftment [74]. It is not surprising therefore, that despite evidence for the bactericidal effects of deferoxamine, transplantationists have been reluctant to introduce iron chelators for preventing transplant associated mortality. In leukemic patients, deferoxamine treatment following allogeneic hematopoietic stem cell transplantation appeared to improve relapse incidence and disease free survival in a retrospective analysis [75]. However, in the only prospective controlled study evaluating the effect of intravenous DFO therapy during BMT in patients transplanted for thalassemia major, ICT did not affect the incidence of infections, engraftment parameters, or GVHD [76]. In view of these considerations, the call for clinical trials of ICT that would include patients with all grades of MDS to evaluate the benefit of iron chelation therapy [68] appears to be unjustified. Retrospective studies in MDS suggesting a protective effect of ICT against AML transformation and iron-related mortality [58–61] need close scrutiny. As emphasized in a recent review [77], ICT is potentially toxic, cumbersome, and costly. Since most MDS patients eventually require RBC transfusions, the public health implications both of transfusion dependence and ICT in MDS are considerable.

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Conclusions Iron overload in MDS is the joint outcome of multiple blood transfusions and inappropriately increased iron absorption associated with ineffective erythropoiesis. The long-term consequences of iron toxicity including cirrhosis, myocardiopathy and endocrine disorders are preventable and mostly reversible by effective iron chelation therapy (ICT). With the introduction of orally effective iron chelators that are easy to take and suitable for use in elderly patients, there is increasing interest in introducing ICT for patients with MDS. Ideally, evidence supporting the beneficial effects of iron chelation in MDS should rest on improved life expectancy and in exceptional cases, reversal of myocardial disease. However, such evidence is presently unavailable. The selection of MDS patients who may benefit from ICT involves a number of considerations: First, it is recognized that life-limiting transfusional iron overload is only relevant for MDS patients with a relatively favourable prognosis. Second, significant cardiac siderosis may only be expected after the transfusion of about 100 units of blood requiring several years of intensive transfusion therapy. Last, because of coexistent morbidities, serum ferritin in MDS is a poor indicator of iron overload and recommendations for ICT based on threshold ferritin values should be replaced by state-of-the-art MRI methodology. Retrospective studies in MDS suggest a protective effect of ICT against ironrelated mortality, decreased transfusion requirements and possibly decreased rates of leukemic transformation. However, additional studies are required to document the beneficial effects of long term ICT in MDS in order to justify the cost, potential drug toxicity, and other public health implications of such treatment in transfusion dependent MDS patients. Acknowledgment╇ The author was the recipient of travel support and honoraria from Novartis Basel Switzerland and Apotex Toronto Canada.

References 1.╇ Andrews NC (2002) A genetic view of iron homeostasis. Semin Hematol 39:227–234 2.╇ Finch CA, Deubelbeiss K, Cook JD et€al (1970) Ferrokinetics in man. Medicine (Baltimore) 49:17–53 3.╇ Tanno T, Bhanu NV, Oneal PA et€ al (2007) High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med 13:1096–1101 4.╇ Hershko C (2007) Mechanism of iron toxicity. Food Nutr Bull 28(4 Suppl):S500–S509 5.╇ Buja LM, Roberts WC (1971) Iron in the heart. Etiology and clinical significance. Am J Med 51:209–221 6.╇ Schafer AI, Cheron RG, Dluhy R et€al (1981) Clinical consequences of acquired transfusional iron overload in adults. N Engl J Med 5304:319–324 7.╇ Brittenham GM, Cohen AR, McLaren CE et€ al (1993) Hepatic iron stores and plasma ferritin concentration in patients with sickle cell anemia and thalassemia major. Am J Hematol 42:81–85

12â•… Pathogenesis and Management of Iron Overload in MDS

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╇ 8. Olivieri NF, Nathan DG, MacMillan JH et€al (1994) Survival in medically treated patients with homozygous beta-thalassemia. N Engl J Med 331:574–578 ╇ 9. Gabutti V, Piga A (1996) Results of long-term iron-chelating therapy. Acta Haematol 95: 26–36 10. Lee MH, Means RT Jr (1995) Extremely elevated serum ferritin levels in a university hospital: associated diseases and clinical significance. Am J Med 98:566–571 11. Matzner Y, Konijn AM, Hershko C (1980) Serum ferritin in hematologic malignancies. Am J Hematol 9:13–22 12. Armand P, Kim HT, Cutler CS et€ al (2007) Prognostic impact of elevated pretransplantation serum ferritin in patients undergoing myeloablative stem cell transplantation. Blood 109:4586–4588 13. Lim ZY, Fiaccadori V, Gandhi S et€al (2010) Impact of pre-transplant serum ferritin on outcomes of patients with myelodysplastic syndromes or secondary acute myeloid leukaemia receiving reduced intensity conditioning allogeneic haematopoietic stem cell transplantation. Leuk Res 34:723–727 14. Malcovati L (2009) Red blood cell transfusion therapy and iron chelation in patients with myelodysplastic syndromes. Clin Lymphoma Myeloma 3(9 Suppl):S305–S311 15. Malcovati L (2007) Impact of transfusion dependency and secondary iron overload on the survival of patients with myelodysplastic syndromes. Lek Res 31(3 Suppl):S2–S6 16. St Pierre TG, Clark PR, Chua-anusorn W et€al (2005) Noninvasive measurement and imaging of liver iron concentrations using proton magnetic resonance. Blood 105:855–861 17. Gandon Y, Olivie D, Guyader D et€al (2004) Non-invasive assessment of hepatic iron stores by MRI. Lancet 363:357–362 18. Angelucci E, Brittenham GM, McLaren CE et€al (2000) Hepatic iron concentration and total body iron stores in thalassemia major. N Engl J Med 343:327–331 19. Cartwright GE, Edwards CQ, Kravitz K et€al (1979) Hereditary hemochromatosis. Phenotypic expression of the disease. N Engl J Med 301:175–179 20. Brittenham GM, Griffith PM, Nienhuis AW et€ al (1994) Efficacy of deferoxamine in preventing complications of iron overload in patients with thalassemia major. N Engl J Med 331:567–573 21. Telfer PT, Prestcott E, Holden S et€al (2000) Hepatic iron concentration combined with longterm monitoring of serum ferritin to predict complications of iron overload in thalassaemia major. Br J Haematol 110:971–977 22. Angelucci E, Muretto P, Nicolucci A et€al (2002) Effects of iron overload and hepatitis C virus positivity in determining progression of liver fibrosis in thalassemia following bone marrow transplantation. Blood 100:17–21 23. Jensen PD, Jensen FT, Christensen T et€al (2003) Evaluation of myocardial iron by magnetic resonance imaging during iron chelation therapy with deferrioxamine: indication of close relation between myocardial iron content and chelatable iron pool. Blood 101:4632–4639 24. Anderson LJ, Holden S, Davis B et€al (2001) Cardiovascular T2-star (T2*) magnetic resonance for the early diagnosis of myocardial iron overload. Eur Heart J 22:2171–2179 25. Anderson LJ, Westwood MA, Holden S et€al (2004) Myocardial iron clearance during reversal of siderotic cardiomyopathy with intravenous desferrioxamine: a prospective study using T2* cardiovascular magnetic resonance. Br J Haematol 127:348–355 26. Davis BA, O’Sullivan C, Jarritt PH, Porter JB (2004) Value of sequential monitoring of left ventricular ejection fraction in the management of thalassemia major. Blood 104:263–269 27. Jacobs EM, Hendriks JC, van Tits BL et€al (2005) Results of an international round robin for the quantification of serum non-transferrin-bound iron: need for defining standardization and a clinically relevant isoform. Anal Biochem 341:241–250 28. Cabantchik ZI, Breuer W, Zanninelli G, Cianciulli P (2005) LPI-labile plasma iron in iron overload. Best Pract Res Clin Haematol 18:277–287 29. Daar S, Pathare A, Nick H et€al (2009) Reduction in labile plasma iron during treatment with deferasirox, a once-daily oral iron chelator, in heavily iron-overloaded patients with betathalassaemia. Eur J Haematol 82:454–457

202

C. Hershko

30. Porter JB, Abeysinghe RD, Marshal L et€al (1996) Kinetics of removal and reappearance of non-transferrin-bound plasma iron with deferoxamine therapy. Blood 88:705–713 31. Piga A, Longo F, Duca L et€al (2009) High nontransferrin bound iron levels and heart disease in thalassemia major. Am J Hematol 84:29–33 32. Gattermann N (2009) The treatment of secondary hemochromatosis. Dtsch Arztebl Int 106:499–504 33. Remacha AF, Arrizabalaga B, Del Cañizo C et€al (2010) Iron overload and chelation therapy in patients with low-risk myelodysplastic syndromes with transfusion requirements. Ann Hematol 89:147–154 34. Jabbour E, Garcia-Manero G, Taher A, Kantarjian HM (2009) Managing iron overload in patients with myelodysplastic syndromes with oral deferasirox therapy. Oncologist 14:489–496 35. Dreyfus F (2008) The deleterious effects of iron overload in patients with myelodysplastic syndromes. Blood Rev 22(2 Suppl):S29–S34 36. Metzgeroth G, Dinter D, Schultheis B et€al (2009). Deferasirox in MDS patients with transfusion-caused iron overload—a phase-II study. Ann Hematol 88:301–310 37. Fenaux P, Rose C (2009) Impact of iron overload in myelodysplastic syndromes. Blood Rev 23(1 Suppl):S15–S19 38. Schmid M (2009) Iron chelation therapy in MDS: what have we learnt recently? Blood Rev 23(1 Suppl):S21–S25 39. Bennett JM (2008) MDS foundation’s working group on transfusional iron overload. Consensus statement on iron overload in myelodysplastic syndromes. Am J Hematol 83:858–861 40. Mittelman M, Lugassy G, Merkel D et€al (2008) Iron chelation therapy in patients with myelodysplastic syndromes: consensus conference guidelines. Isr Med Assoc J 10:374–376 41. Gattermann N (2007) Guidelines on iron chelation therapy in patients with myelodysplastic syndromes and transfusional iron overload. Leuk Res 31(3 Suppl):S10–S15 42. Di Tucci AA, Matta G, Deplano S et€al (2008) Myocardial iron overload assessment by T2* magnetic resonance imaging in adult transfusion dependent patients with acquired anemias. Haematologica 93:1385–1388 43. Chacko J, Pennell DJ, Tanner MA et€ al (2007) Myocardial iron loading by magnetic resonance imaging T2* in good prognostic myelodysplastic syndrome patients on long-term blood transfusions. Br J Haematol 138:587–593 44. Konen E, Ghoti H, Goitein O et€al (2007) No evidence for myocardial iron overload in multitransfused patients with myelodysplastic syndrome using cardiac magnetic resonance T2 technique. Am J Hematol 82:1013–1016 45. Chee CE, Steensma DP, Wu W et€al (2008) Neither serum ferritin nor the number of red blood cell transfusions affect overall survival in refractory anemia with ringed sideroblasts. Am J Hematol 83:611–613 46. Tefferi A, Vardiman JW (2009) Myelodysplastic syndromes. N Engl J Med 361(19):1872–1885 47. Steensma DP. (2009) Myelodysplasia paranoia: iron as the new radon. Leuk Res 33: 1158–1163 48. Gabutti V, Borgna-Pignatti C (1994) Clinical manifestations and therapy of transfusional haemosiderosis. Bailliere’s Clin Haematol 7:919–940 49. Davis BA, O’Sullivan C, Jarritt PH et€al (2004) Value of sequential monitoring of left ventricular ejection fraction in the management of thalassemia major. Blood 104:263–269 50. Modell B, Khan M, Darlison M (2000) Survival in beta-thalassaemia major in the UK: data from the UK Thalassaemia register. Lancet 355:2051–2052 51. Hershko C, Link G, Pinson A et€al (1991) Iron mobilization from myocardial cells by 3-hydroxypyridin-4-one chelators: studies in rat heart cells in culture. Blood 77:2049–2053 52. Borgna-Pignatti C, Cappellini MD, De Stefano P et€ al. (2006). Cardiac morbidity and mortality in deferoxamine- or deferiprone-treated patients with thalassemia major. Blood 107:3733–3737 53. Pennell DJ, Berdoukas V, Karagiorga M et€ al (2006) Randomized controlled trial of deferiprone or deferoxamine in beta-thalassemia major patients with asymptomatic myocardial siderosis. Blood 107:3738–3744

12â•… Pathogenesis and Management of Iron Overload in MDS

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54. Zanninelli G, Breuer W, Cabancthik ZI (2009) Daily labile plasma iron as an indicator of chelator activity in Thalassaemia major patients. Br J Haematol 147:744–751 55. Cappellini MD, Cohen A, Piga A et€al (2006) A phase 3 study of deferasirox (ICL670), a once-daily oral iron chelator, in patients with beta-thalassemia. Blood 107:3455–3462 56. Pennell DJ, Porter JB, Cappellini MD et€al (2010) Efficacy of deferasirox in reducing and preventing cardiac iron overload in beta-thalassemia. Blood 115:2364–2371 57. Elalfy MS, El-Beshlawi A, Taher A et€al (2009) Efficacy and safety of deferasirox, an oral iron chelator, in heavily iron-loaded patients with beta-thalassemia: the ESCALATOR study. Eur J Haematol 82:458–465 58. Raptis A, Duh MS, Wang ST et€al (2010) Treatment of transfusional iron overload in patients with myelodysplastic syndrome or severe anemia: data from multicenter clinical practices. Transfusion 50:190–199 59. Leitch HA, Chase JM, Goodman TA et€al (2009) Improved survival in red blood cell transfusion dependent patients with primary myelofibrosis (PMF) receiving iron chelation therapy. Hematol Oncol 28:40–48 60. Leitch HA (2007) Improving clinical outcome in patients with myelodysplastic syndrome and iron overload using iron chelation therapy. Leuk Res 31(3 Suppl):S7–S9 61. Rose C, Brechignac S, Vassilief D et€al (2010) GFM (Groupe Francophone des Myélodysplasies). Does iron chelation therapy improve survival in regularly transfused lower risk MDS patients? A multicenter study by the GFM. Leuk Res 34(7):864–870 62. Jensen PD, Heickendorff L, Pedersen B et€al (1996) The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload. Br J Haematol 94:288–299 63. Jensen PD, Jensen IM, Ellegaard J (1992) Desferrioxamine treatment reduces blood transfusion requirements in patients with myelodysplastic syndrome. Br J Haematol 80:121–124 64. Smeets ME, Vreugdenhil G, Holdrinet RS (1996) Improvement of erythropoiesis during treatment with deferiprone in a patient with myelofibrosis and transfusional hemosiderosis. Am J Hematol 51:243–244 65. Messa E, Cilloni D, Messa F et€al (2008) Deferasirox treatment improved the hemoglobin level and decreased transfusion requirements in four patients with the myelodysplastic syndrome and primary myelofibrosis. Acta Haematol 120:70–74 66. Di Tucci AA, Murru R, Alberti D et€al (2007) Correction of anemia in a transfusion-dependent patient with primary myelofibrosis receiving iron chelation therapy with deferasirox (Exjade, ICL670). Eur J Haematol 78:540–542 67. Camaschella C, Campanella A, De Falco L et€al (2007) The human counterpart of zebrafish shiraz shows sideroblastic-like microcytic anemia and iron overload. Blood 110:1353–1358 68. Pullarkat V (2009) Objectives of iron chelation therapy in myelodysplastic syndromes: more than meets the eye? Blood 114:5251–5255 69. Halliwell B (2007) Biochemistry of oxidative stress. Biochem Soc Trans 35:1147–1150 70. Hershko C, Peto TE, Weatherall DJ (1988) Iron and infection. Br Med J 296:660–664 71. Bullen JJ, Rogers HJ, Spalding PB, Ward CG (2006) Natural resistance, iron and infection: a challenge for clinical medicine. J Med Microbiol 55:251–258 72. Caroline L, Rosner F, Kozinn PJ (1969) Elevated serum iron, low unbound transferrin and candidiasis in acute leukemia. Blood 34:441–451 73. Karp JE, Merz WG (1986) Association of reduced total iron binding capacity and fungal infections in leukemic granulocytopenic patients. J Clin Oncol 4:216–220 74. Cooper CE, Lynagh GR, Hoyes KP et€al (1996) The relationship of intracellular iron chelation to the inhibition and regeneration of human ribonucleotide reductase. J Biol Chem 271:20291–20299 75. Kaloyannidis P, Yannaki E, Sakellari I et€al (2010) The impact of desferrioxamine postallogeneic hematopoietic cell transplantation in relapse incidence and disease-free survival: a retrospective analysis. Transplantation 89:472–479 76. Gaziev D, Giardini C, Angelucci E et€al (1995) Intravenous chelation therapy during transplantation for thalassemia. Haematologica 80:300–304

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C. Hershko

77. Leitch HA, Vickars LM (2009) Supportive care and chelation therapy in MDS: are we saving lives or just lowering iron? Hematology 2009:664–672 (Am Soc Hematol Educ Program) 78. Hershko C (2006) Oral iron chelators: new opportunities and new dilemmas. Haematologica 91:1307–1312

Chapter 13

Cytokines in MDS: Abnormalities   and Treatment Howard S. Oster, Drorit Neumann and Moshe Mittelman

Erythroid Series Erythropoietin (Erythroid Stimulating Agents) Biology:╇ Most patients with myelodysplastic syndromes (MDS) suffer from anemia, related to defective erythropoiesis in the bone marrow (BM) [1–5]. Although the pathogenesis, as well as the basic defects in the BM, has not been fully elucidated, research has shed some light on it [6–15]. MDS has been characterized by ineffective erythropoiesis, erythroid apoptosis, mitochondrial release of cytochrome C and dysregulated suppressive cytokines, including tumor necrosis factor, transforming growth factor, interleukin-1, interferons and vascular endothelial growth factors. Endogenous serum erythropoietin (EPO) level has been found to be relatively low [16], i.e., higher than the normal range yet lower than expected based on the known feedback mechanisms for anemia (see below). Red blood cells (RBC) transfusion has been (and still is) for years the major treatment for symptomatic anemia, in the absence of response to other therapeutic regimens [1–3, 8, 17, 18]. However, recurrent RBC transfusions are associated with risks, including biological transmissions [8], and iron overload [19, 20]. EPO is a ~30-kd glycoprotein hormone produced by the kidneys in response to hypoxia, and serves as both a mitogen and survival factor for the erythroid lineage [21–24]. The cloning of the EPO gene [25], and the introduction of recombinant human EPO (rHuEPO), or erythroid stimulating agents (ESAs), into clinical practice has provided a new therapeutic hope for many anemia patients, including MDS-related anemia. We now can summarize a two-decade clinical experience with rHuEPO. M. Mittelman () Department of Medicine, Tel Aviv Sourasky Medical Center, 6 Weizmann St., 64239 Tel Aviv, Israel Fax: +972-3-6974855 e-mail: [email protected] Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_13, ©Â€Springer Science+Business Media B.V. 2011

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The symptomatic anemia in MDS, the complications related to RBC transfusions, the pathogenesis, as well as the relatively low endogenous serum EPO levels, all prompted the application of the new recombinant product in the treatment of MDS. The success achieved with anemia of end-stage renal disease [26], was encouraging. rHuEPO in the Treatment of MDS-Related Anemia:╇ Since the early 1990s dozens of reports and clinical trials have demonstrated varying degrees of success with ESAs for treatment of the anemia of MDS. Initially erythroid response (ER) rate of 20–25% was achieved [7, 8, 27–29]. Subsequently, using better regimens, optimizing EPO dose, adding iron, and a better selection of patients, resulted in higher response rates, within the 50% range [30–36]. Recent meta-analyses have confirmed the advantage of ESAs over placebo, as well as the higher ER rate in anemic MDS patients [37–40]. This has made ESAs a standard therapeutic approach in MDS-related anemia [1, 3, 10, 41]. A successful ER is characterized by a rise in the Hemoglobin (Hb) concentration, the hematocrit (HCT) level and the RBC count, a reduction in the blood transfusion requirements and an improved quality of life (QoL) [7, 8, 31, 38, 42–45]. How Do We Define an ER?╇ Since many authors used various definitions, it was mandatory to set standard criteria. The International Working Group (IWG) has initially [46] proposed such criteria, and later [47] has updated them. These MDS response criteria have been well accepted and currently serve as the basis for interpretation of all trials in MDS. According to the present criteria, complete response is defined by Hb level exceeding the 11€g/dl threshold, lasting for at least 4 weeks, and an ER is defined if Hb rises by more than 1.5€g/dl or RBC transfusion requirements are reduced, lasing 8 weeks or more. The Mechanism of Action of rHuEPO:╇ has not been fully clarified, but it is assumed that the hormone inhibits apoptosis, counteracts cytokines and stimulates erythroid proliferation and differentiation [7, 11–15, 24]. Prediction of Response to ESAs:╇ Several parameters might predict ER in rHuEPO-treated anemic MDS patients. Endogenous serum EPO level appears to be the major predicting factor [28, 31]. According to the Hellström-Lindberg predictive model [31], the combination of low serum EPO (<500€mU/ml) and low RBC transfusion needs (<2€µm/ml) predicts an ER of 74%, while if both criteria are not met, the expected ER rate is only 7%. The intermediate state is associated with an ER of 23%. This model has been confirmed by others [29, 38, 39, 48–50]. Other predicting parameters have gained less consensus [12, 30, 31, 38–40, 51–53]: These include having a more favorable MDS form, such as RA and RARS or IPSS low/ Int1, having only unilineage dysplasia, a 7 days reticulocyte count [54], different gene expression profile [55] and shorter time from diagnosis to treatment [39, 56]. ER Duration:╇ The Nordic group reported [33], that responding patients benefited from the recombinant hormone for a period of 3–116 months, with a median of 23 months. Most authors’ experience is consistent with about 2 years of benefit [39].

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EPO Resistance:╇ What are the reasons for resistance to rHuEPO or the loss of response? Several factors have been mentioned [7, 8, 39, 57–60]. Iron deficiency, or probably functional deficiency (related to inflammatory processes?) appears to be a major cause. It has been suggested to apply a routine practice of adding oral or intravenous iron, to rHuEPO treated MDS patients, even without evidence of decreasing ferritin [61]. Other factors are progressive disease or leukemic transformation, active inflammation or infection, pure red cell aplasia [62], down regulation of EPO receptors (“exhaustion of erythroid progenitors”?) and other types of anemia. ESA Products:╇ Three commercial EPO products, or ESAs, are used in clinical practice: Epoetin-alfa, Epoetin-beta and Darbopoetin (DA) alfa. Another long-acting agent, CERA has not been sufficiently evaluated in the hematological setup to allow discussion here. Epoetin(s) alfa are known as Epogen for renal patients in the USA, and Procrit for other anemic indications in the USA and Eprex in Europe. Epoetin beta, also known as Recormon is used in Europe. DA is a long acting ESA. Dosing and Regimens:╇ The classical rHuEPO dose for MDS-related anemia is 150€u/kg, about 10,000€µ per injection, administered subcutaneously (SC) or intravenously (IV) three times per week [7, 8, 60]. Since the onset of ER is within 4–8 weeks, it is advised to monitor the patient during that period. If no ER is observed— a doubling of the dose is reasonable. If no response is achieved with the double dose following 8–12 weeks of treatment, the patient is apparently resistant to the hormone and additional such treatment is not warranted, unless the cause for resistance is identified and overcome [7, 35, 60]. It has been suggested that an improved response can be achieved with a higher rHuEPO dose [40, 63]. Later, we learned that a single weekly dose of 30,000–40,000€ µ (or 60,000–80,000€ µ) is similarly effective [49, 54, 64–66]. DA dose is 2.25€ µ/kgâ•›×â•›3/week, mounting to a total of ~150€µg/week (the dose can be doubled), or 500€µg/3€week [60]. Maintenance:╇ Once response is achieved, the patient should be carefully followed and monitored with adjusting the ESA dose to maintain Hb level, using 12€g/dl as an upper limit, according to the ASH/ASCO and European guidelines [60, 67]. This is important to avoid undesired effects of thrombotic complications related to a high Hb, associated with increased blood viscosity (see also below). Adverse Effects (AE):╇ The common AE are local skin irritation and cutaneous reactions, flu-like syndrome, and elevation of blood pressure, which are easily controlled. [7, 8, 38, 59, 60, 68]. A rare complication of production of neutralizing anti-EPO antibodies, causing pure red cell aplasia (PRCA) was reported [62], but was found to be related to the production process which was subsequently repaired. Thromboembolic complications were reported to be more common among ESAs treated patients (RR 1.7). However, these can be minimized if the guidelines are maintained and the Hb is not allowed to rise to undesired levels [60]. Are ESAs Safe?╇ For years we believed that indeed this is the case. This assumption was supported by the following arguments: (1) Anemia has been shown to be a poor prognostic factor [5]. (2) The need for RBC transfusions has been shown to

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be associated with poor prognosis [69]. (3) The common (50%) reason for death in low-risk MDS patients (according to the IPSS [5] was found to be cardiac [70]. This is believed to be related, at least partially to iron overload due to multiple blood transfusions [19, 20]. Also, correcting the anemia may improve the cardiac function [71, 72]. (4) Better oxygenation by rHuEPO has been shown to render tumors to be more sensitive to radiation therapy [73]. (5) Thousands of MDS patients have been treated with ESAs and no safety issue has been raised. Grey Clouds over the “Red” Hormone:╇ Concerns have been raised in the early 1990s when several groups have reported the results of randomized trials demonstrating a shorter survival in cancer patients treated with ESAs compared with nonEPO treated patients, suggesting that EPO might promote tumor growth [74–82]. Several mechanisms have been proposed: (a) Stimulation of EPO receptors occasionally present on some tumor cells [76, 83]. (b) High Hb and Hct rendering the blood too viscous [76]. (c) Pro-angiogenic activity. These reports prompted the FDA Oncology Drug Advisory Committee (ODAC) to a series of actions, including adding a black-box warning in the package insert of the drugs. Also, ASCO and ASH adjusted the guidelines for the use of ESAs [60], and a similar action was taken in Europe [67]. However, from a critical review of the reports and the literature one can draw several conclusions: (1) Methodological problems and defects in studies’ design can be detected in almost each report [84]: (a) ESAs were administered for nonapproved indications, such as patients with no anemia or who were not treated with chemotherapy. (b) Many patients were allowed to increase their Hb levels to a risky target of 17–18€g/dl [76]. (c) The study groups were unbalanced [74, 75]. (d) Early termination of the study [74, 75, 77]. (2) Even when EPO receptors are present on tumor cells they are often non-functional [84–86]. (3) All (!) these patients suffered from solid tumors—no negative impact on survival has been described on patients with hematological neoplasms such as MDS or multiple myeloma. (4) In contrast with the above-mentioned reports, others describe a different outcome (see below). “Positive” EPO Outcomes:╇ We [87, 88], as well as others [89–91], have suggested possible survival advantage with rHuEPO in hematological and non-hematology neoplasms, although not in a randomized study. Other randomized trials [92] have failed to detect a survival difference. Meta-analyses confirmed the no- or minimal difference between treated and non-treated rHuEPO patients with regard to survival [68, 93, 94]. In MDS, although no prospective randomized trial has been performed, reports suggest increased survival in rHuEPO-treated MDS patients [48]. The Nordic and the Pavia teams have found a survival advantage in 121 Nordic patients treated with EPO and G-CSF, compared with 237 untreated Italian patients, with a Hazard Ratio (HR) of 0.61 [95]. The French GFM group has compared their 284 treated MDS patients with 225 untreated MDS patients from IMRAW data, served for the original IPSS classification [5], and found a superior overall survival (64% vs 39%, HR 0.4) [39]. Additional EPO Effects:╇ One should always keep in mind that rHuEPO, by treating anemia, improves patients’ quality of life. Also, better mobilization of iron has

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been reported [96]. Additional effects have been described with this hormone which is considered a pleotrophic agent, including neuroprotection, Immune and oxygenizing effects [87, 88, 97–99]. We have recently reported several stimulating immunomodulating effects of the hormone both in vitro and in vivo in murine models [88, 98–101], as well as in human subjects [102]. We have also found improved immune functions in MDS patients treated with rHuEPO [103]. In summary, although concerns have been raised and caution is needed, the use of ESAs in MDS-related anemia appears to be safe and beneficial, as long as the guidelines are maintained [60]. Briefly, one can emphasize the 4Ts (TTTT): T1: Treat chemotherapy-related anemia (refers to solid tumors but not MDS). T2: Treat when Hb drops below 10 (Ten)€g/dl. T3: Target Hb should not exceed 12 (twelve)€g/ dl. T4: Be aware of the risk of thromboembolism. rHuEPO Combinations:╇ In an attempt to improve the ER, other cytokines have been tested together with rHuEPO. The agent that has been introduced into clinical practice is G-CSF that was found to have a synergistic effect with ESAs, increasing the ER by 30–40% [39, 43, 50, 51, 104].

The Myeloid Lineage Granulopoietic Agents: G/GM-CSF Biology:╇ Neutropenia affects 50–60% of MDS patients [1, 105]. In addition MDS patients often suffer from impaired function of the leukocytes [106]. Despite the near normal or sometimes increased number of marrow precursor cells, there are peripheral cytopenias implying that cell death, apoptosis, may play a significant role in the bone marrow [9]. An elevated apoptotic index in MDS compared with that of normal bone marrow (based on morphological and ultrastructural changes) suggests that apoptosis may be a dominant factor. Granulocyte-macrophage progenitor (colony forming unit-gm) assays provided evidence of abnormal growth characteristics, which apparently contribute to ineffective granulopoiesis [107]. The data have been inconclusive as the rates of apoptosis can be variable. Studies by Raza et€al. [9] and Dar et€al. [108] have suggested that there is more apoptosis in those MDS patients with more-advanced disease, while others have shown more apoptosis in earlier stages [109]. The studies that have demonstrated increased apoptosis in late-stage disease have shown that the actual cells undergoing apoptosis are not the blasts, but the differentiated cells of all three cell lines as well as cells of stromal origin [9, 108] This may suggest that the leukemic blasts produce factors that induce apoptosis in the surrounding cells [9]. There are cytokines involved as well, for example, TNF-α [108]. Some studies have found elevated TNF in early disease, and others in late-stage disease, and its role in MDS in general, and apoptosis in particular is controversial [105].

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Treatment:╇ Practical questions raised are 1) whether the counts are sufficiently low to warrant treatment and 2) whether there are too many blasts to treat the patient safely with cytokines therapy [110]. Stimulation by GM-CSF or by G-CSF could possibly induce a malignant clone to proliferate. As such, any study that examines the efficacy of these drugs, must also evaluate its safety. In a phase II trial of GM-CSF in MDS, the benefits were modest [111]. The patients had therapy-related MDS which already has a worse course and prognosis than primary MDS. In this study, only 2 of 14 patients doubled their neutrophil count, and any increases seen were not dose-related and were not sustained longer than a few days after the 14-day treatment ceased. Other studies with primary MDS had a better response that was dose-related. These studies were small and of limited time, so that the full long-term effect could not be observed [112]. In other studies, GM-CSF was associated with an increased neutrophil count and a reduced rate of infection. The increase was not sustained even in those patients who continued maintenance therapy. All patients who developed acute leukemia had had more than 14% blasts [113]. Two phase I/II trials showed that G-CSF is well tolerated and effective for improving neutropenia. The first was a trial for 6–8 weeks in which 10 of the 12 participants sustained 2- to 10-fold increase in leukocyte count and 5- to 40-fold increase in neurtrophil count [114]. There were no cases of acute transformation to acute leukemia during that time period (note that seven were classified as RAEB, and three as RAEB-T). While the results were promising, they were short lived: After 2–4 weeks, the counts returned to baseline. In the second trial, therefore, the responders were given maintenance therapy [115]. In this trial, 10 of the 11 patients given continued treatment had a persistent response for 3–16 months. Nine of the patients had had more than 14% blasts and two of them converted to AML; one other patient, who had less than 14% blasts initially, also converted. It is difficult to determine in this type of study whether the conversion was due to the natural history of the disease or was enhanced by the G-CSF treatment. Their retrospective analysis determined that there were fewer episodes of infection during the time when the ANC was greater than 1,500. In two phase III trials [116, 117], neutrophil count did improve with the addition of G-CSF. While treatment with G-CSF is promising in terms of increased WBC and neutrophil counts, reduced infections and apparent safety in early disease (less than 14% blasts), more data are required to determine the indications and contraindications for its use.

The Megakaryocytic Lineage Thrombopoietic Agents Biology:╇ One of the most common abnormalities in MDS is thrombocytopenia, affecting 40–60% of the MDS patients [1–3, 105]. Thrombocytopenia-associated

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bleeding is a major cause of death [118, 119]. There is very little known about the pathophysiology of thrombocytopenia, but a deficiency both in the number and the function of megakaryocytic as measured by aggregometry [118, 119], has been shown. This may be caused in part by platelet clumping. Treatment:╇ Of all the studies that have been performed, none to date have shown that cytokine therapy has improved survival, or has significantly reduced morbidity. The classic treatment for thrombocytopenia is platelet transfusion, but repeated administration can cause alloimmunization in 20–85% of patients [119]. This phenomenon can be reduced, however, with filtration of leukocytes and irradiation [120]. Treatment of thrombocytopenia began to shift in the 1990s when the gene for thrombopoietin was cloned, and a recombinant form was produced [121]. It works by inducing growth of megakaryocyte progenitor cells [119]. The difficulty with this treatment is that it can cause the formation of anti-thrombopoietin antibodies which subsequently cause thrombocytopenia [122]. Other treatments have included IL-11, IL-6, and IL-3. Interleukin IL-11 has been examined in a study where it was administered subcutaneously. IL-11 has pleotropic effects stimulating proliferation of hematopoietic stem cells and megakaryocyte progenitors and inducing maturation of megakaryocytes [123]. Five of 11 patients with MDS responded with response duration of 12 to more than 30 weeks [124]. In another study of MDS and aplastic anemia patients, 6 of 14 MDS patients responded with a median response duration of 3 months. Three of these patients demonstrated a multi-lineage response [123]. IL-11 is known as a thrombopoietic cytokine that promotes growth of hematopoietic stem cells and megakaryocytic progenitors and induces megakaryocyte differentiation. Interleukin IL-6 can promote thrombopoiesis in MDS but in a phase I trial only 3 of 22 patients fulfilled criteria for response (five others had partial, but clinically significant responses), and all patients experienced at least some level of toxicity [125]. Interleukin IL-3 has also been studied, with modest results [126]. In a phase I/II trial IL-3 was associated with an increase in the myeloid line in all 9 patients. It also had an effect of increasing the platelet count in 2 of the 4 patients with profound initial thrombocytopenia, but was associated with the development of thrombocytopenia in two patients with normal initial platelet counts. It had little or no effect on the erythroid line. Most recently, treatment with thrombopoietin receptor agonists Romiplostim and Eltrombopag have been studied. In a phase I/II trial of Romiplostim, 44 patients received the drug weekly for three weeks by subcutaneous injection and continued for 41 of these patients for up to one year in the extension phase. Forty-six percent of these patients maintained a durable platelet response [127]. There were no antibodies formed either to Robiplostim or to the endogenous thrombopoietin. Eltrombopag is another such drug, approved for treatment of chronic ITP. In a study examining the effects of the drug on bone marrow cells from patients with MDS, there was an increase in megakaryocytic differentiation and formation of normal megakaryocytic colonies. There was no increase in the number of malignant mononuclear cells [128]. Further studies need to be performed on both these drugs,

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but the fact that they improve platelet counts in ITP, work without the formation of neutralizing antibodies, and do not induce or enhance the growth of malignant clones make these drugs very promising candidates for clinical implementation.

References ╇ 1. DeAngelo DJ, Stone RM (2005) Myelodysplastic syndromes: biology and treatment. In: Hoffman R et€al (eds) Hematology—basic principles and practice, 4th€edn. Elsevier, Philadelphia, pp€1195–1208 ╇ 2. Mittelman M (1990) The myelodysplastic syndromes—1990. Isr J Med Sci 26:468–478 ╇ 3. Mittelman M, Lessin LS (1991) Myelodysplasia (preleukemia). In: Moossa AR, Schimpff SC, Robson MC (eds) Comprehensive textbook of oncology, 2nd€edn. Williams & Wilkins, Baltimore, pp€1232–1244 ╇ 4. Bennett JM, Catovski D, Daniel MT et€al (1982) Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51:189–199 ╇ 5. Greenberg P, Cox C, LeBeau MM et€al (1997) International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 89:2079–2098 ╇ 6. Cazzola M, Beguin Y (1992) New tools for clinical evaluation of erythron function in man. Br J Haematol 80:278–284 ╇ 7. Mittelman M (1993) Recombinant erythropoietin in myelodysplastic syndromes: whom to treat an how? More questions than answers. Acta Haematol 90:53–57 ╇ 8. Mittelman M, Lessin LS (1994) Clinical application of recombinant erythropoietin in myelodysplasia. Hematol/Oncol Clin NAm 8:993–1009 ╇ 9. Raza A, Gezer S, Mundle S et€al (1995) Apoptosis in bone marrow biopsy sample involving stromal and hematopoietic cells in 50 patients with myelodysplastic syndromes. Blood 86:268–276 10. Bowen DT, Hellström-Lindberg E (2002) Treatment of anemia in myelodysplastic syndromes. In: Bennett JM (ed) The myelodysplastic syndromes—pathobiology and clinical management. Marcel Dekker, New York, pp€345–372 11. Gardin C, Fenaux P (2004) Supportive care including growth factors in myelodysplastic syndromes. Rev Clin Exp Hematol 8:E3 12. Hellström-Lindberg E (2005) Update on supportive care and new therapies: immunomodulatory drugs, growth factors and epigenetic-acting agents. Hematol—Am Soc Hematol—Edu 2005:161–166 13. Katsoulidis E, Li Y, Yoon P et€al (2005) Role of the p38 mitogen-activated protein kinase pathway in cytokine-mediated hematopoietic suppression in myelodysplastic syndromes. Cancer Res 65:9029–9037 14. Tehranchi R, Fadeel B, Forsblom AM et€ al (2003) Granulocyte colony-stimulating factor inhibits spontaneous cytochrome c release and mitochondria-dependent apoptosis of myelodysplastic syndrome hematopoietic progenitors. Blood 101:1080–1086 15. Tehranchi R, Invernizzi R, Grandien A et€ al (2005) Aberrant mitochondrial iron distribution and maturation arrest characterize early erythroid precursors in low-risk myelodysplastic syndromes. Blood 106:247–253 16. Miller C, Miller CB, Jones RJ et€al (1990) Decreased erythropoietin response in patients with the anemia of cancer. N Engl J Med 322:1689–1692 17. Bowen D, Culligan D, Jowitt S et€al (2003) Guidelines for the diagnosis and therapy of adult myelodysplastic syndromes. Br J Haematol 120:187–2000 18. Tefferi A, Vardiman JW (2009) Myelodysplastic syndromes. N Engl J Med 361:1872–1885

13â•… Cytokines in MDS: Abnormalities and Treatment

213

19. Mittelman M, Lugassy G, Merkel D et€al (2008) Iron chelation therapy in patients with myelodysplastic syndromes—consensus conference guidelines (For the MDS Israel Group and The Israel Society of Hematology). Isr Med Assoc J 10:374–376 20. Bennett JM (2008) Consensus statement on iron overload in myelodysplastic syndromes. Am J Hematol 83:859–861 21. Erslev AJ (1991) Erythropoietin. N Engl J Med 324:1339–1344 22. Krantz S (1991) Erythropoietin. Blood 77:419–434 23. Spivak JL (1986) The mechanism of action of erythropoietin. Int J Cell Cloning 4:139–166 24. Spivak JL, Pham T, Isaacs M, Hankins DW (1991) Erythropoietin is both a mitogen and a survival factor. Blood 77:1228–1233 25. Lin F-K, Suggs S, Lin CH et€al (1985) Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A 82:7580–7584 26. Eschbach JW, Kelly MR, Haley NR et€al (1987) Treatment of anemia of progressive renal failure with recombinant human erythropoietin. N Engl J Med 321:158–163 27. Hellström E, Birgegard G, Helmers C et€al (1991) Treatment of myelodysplastic syndromes with recombinant human erythropoietin. Eur J Haematol 47:355–360 28. Mittelman M, Floru S, Djaldetti M (1992) Subcutaneous erythropoietin for treatment of refractory anemia in hematologic disorders. Blood 80:841–842 29. Rose EH, Abels RI, Nelson RA et€al (1995) The use of r-HuEpo in the treatment of anaemia related to myelodysplasia (MDS). Br J Haematol 89:831–837 30. Hellström-Lindberg E, Negrin R, Stein R et€al (1997) Erythroid response to treatment with GCSF plus erythropoietin for the anemia of patients with myelodysplastic syndrome: proposal for a predictive model. Br J Haematol 99:344–351 31. Hellström-Lindberg E, Gulbrandsen N, Lindberg G et€al (2003) A validated decision model for treating the anaemia of myelodysplastic syndromes with erythropoietin + granulocyte colony-stimulating factor: significant effects of on quality of life. Br J Haematol 120:1037– 1046 32. Patton JF, Sullivan T, Mun Y et€al (2005) A retrospective cohort study to assess the impact of therapeutic substitution of darbepoetin alfa for epoetin alfa in anemic patients with myelodysplastic syndrome. J Support Oncol 3:419–426 33. Jadersten M, Montgomery SM, Dybedal I et€al (2005) Long-term outcome of treatment of anemia in MDS with erythropoietin and G-CSF. Blood 106:803–811 34. Mannone L, Gardin C, Quarre MC et€al (2006) High-dose darbepoetin alpha in the treatment of anaemia of lower risk myelodysplastic syndrome results of a phase II study. Br J Haematol 133:513–519 35. Giraldo P, Nomdedeu B, Loscertales J et€al (2006) Darbepoetin alpha for the treatment of anemia in patients with myelodysplastic syndromes. Cancer 107:2807–2816 36. Gabrilove J, Paquette R, Lyons RM et€ al (2008) Phase 2, single-arm trial to evaluate the effectiveness of darbepoetin alfa for correcting anaemia in patients with myelodysplastic syndromes. Br J Haematol 142:379–393 37. Hellström-Lindberg E (1995) Efficacy of erythropoietin in the myelodysplastic syndromes: a meta-analysis of 205 patients from 17 studies. Br J Haematol 89(1):67–71 38. Ross SD, Allen IE, Probst CA et€al (2007) Efficacy and safety of erythropoiesis-stimulating proteins in myelodysplastic syndrome: a systematic review and meta-analysis. Oncologist 12:1264–1273 39. Park S, Grabar S, Kelaidi C et€al (2008) Predictive factors of response and survival in myelodysplastic syndrome treated with erythropoietin and G-CSF: the GFM experience. Blood 111:574–582 40. Moyo V, Lefebvre P, Duh MS et€al (2008) Erythropoiesis-stimulating agents in the treatment of anemia in myelodysplastic syndromes: a meta-analysis. Ann Hematol 87:527–536 41. Mufti GJ (2004) Emerging therapeutic and supportive care approaches for the myelodysplastic syndromes. Rev Clin Exp Hematol 8:E4

214

H. S. Oster et al.

42. Stasi R, Abruzzese E, Lanzetta G et€ al (2005) Darbepoetin alfa for the treatment of anemic patients with low- and intermediate-1-risk myelodysplastic syndromes. Ann Oncol 16(12):1921–1927 43. Balleari E, Rossi E, Clavio M et€ al (2006) Erythropoietin plus granulocyte colony-stimulating factor is better than erythropoietin alone to treat anemia in low-risk myelodysplastic syndromes: results from a randomized single-centre study. Ann Hematol 85:174–180 44. Spiriti MA, Latagliata R, Niscola P et€al (2005) Impact of a new dosing regimen of epoetin alfa on quality of life and anemia in patients with low-risk myelodysplastic syndrome. Ann Hematol 84:167–176 45. Marsh JC, Ganser A, Stadler M (2007) Hematopoietic growth factors in the treatment of acquired bone marrow failure states. Semin Hematol 44:138–147 46. Cheson BD, Bennett JM, Kantarjian H et€al (2000) Report of an international working group to standardize response criteria for myelodysplastic syndromes. Blood 96:3671–3674 47. Cheson BD, Greenberg PL, Bennett JM et€al (2006) Clinical application and proposal for modification of the International Working Group (IWG) response criteria in myelodysplasia. Blood 108:419–425 48. Walvik J, Stenke L, Bernell P et€al (2002) Serum erythropoietin (EPO) levels correlate with survival and independently predict response to EPO treatment in patients with myelodysplastic syndromes. Eur J Haematol 68:180–185 49. Musto P, Falcone A, Sanpaolo G et€al (2003) Efficacy of a single, weekly dose of recombinant erythropoietin in myelodysplastic syndromes. Br J Haematol 122:269–271 50. Gotlib J, Lavori P, Quesada S et€ al (2009) A phase II intra-patient dose-escalation trial of weight-bases darbepoetin alfa with or without granulocyte-colony stimulating factor in myelodysplastic syndromes. Am J Hematol 84:15–20 51. Hellström-Lindberg E, Ahlgren T, Beguin Y et€ al (1998) Treatment of anemia in myelodysplastic syndromes with granulocyte colony-stimulating factor plus erythropoietin: results from a randomized phase II study and long-term follow-up of 71 patients. Blood 92:68–75 52. Musto P, Lanza F, Balleari E et€al (2005) Darbepoetin alpha for the treatment of anaemia in low-intermediate risk myelodysplastic syndromes. Br J Haematol 128:204–209 53. Sekeres MA, Fu AZ, Maciejewski JP, Golshayan AR et€ al (2007) A Decision analysis to determine the appropriate treatment for low-risk myelodysplastic syndromes. Cancer 109:1125–1132 54. Bowen D, Hyslop A, Keenan N et€al (2006) Predicting erythroid response to recombinant erythropoietin plus granulocyte colony-stimulating factor therapy following a single subcutaneous bolus in patients with myelodysplasia. Haematologica 91:709–710 55. Cortelezzi A, Colombo G, Pellegrini C et€al (2008) Bone marrow glycophorin-positive erythroid cells of myelodysplastic patients responding to high-dose rHuEPO therapy have a different gene expression pattern from those of nonresponders. Am J Hematol 83:531–539. 56. Di Raimondo F, Longo G, Cacciola E Jr et€al (1996) A good response rate to recombinant erythropoietin alone may be expected in selected myelodysplastic patients. A preliminary clinical study. Eur J Haematol 56:7–11 57. Hedenus M, Birgegård G, Näsman P et€al (2007) Addition of intravenous iron to epoetin beta increases hemoglobin response and decreases epoetin dose requirement in anemic patients with lymphoproliferative malignancies: a randomized multicenter study. Leukemia 21:627– 632 58. Bastit L, Vandebroek A, Altintas S et€ al (2008) Randomized, multicenter, controlled trial comparing the efficacy and safety of darbepoetin alpha administered every 3 weeks with or without intravenous iron in patients with chemotherapy-induced anemia. J Clin Oncol 26:1611–1618 59. Jädersten M, Hellström-Lindberg E (2009) Myelodysplastic syndromes: biology and treatment. J Intern Med 265:307–328 60. Rizzo JD, Somerfield MR, Hagerty KL et€al (2008) Use of epoetin and darbepoetin in patients with cancer: 2007 American Society of Clinical Oncology/American Society of Hematology clinical practice guideline update. J Clin Oncol 26:132–149

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61. Glaspy JA (2009) Erythropoietin in cancer patients. Annu Rev Med 60:181–192 62. Casadevall N, Dupuy E, Molho-Sabatier P et€al (1996) Autoantibodies against erythropoietin in a patient with pure red-cell aplasia. N Engl J Med 334:630–633 63. Mundle S, Lefebvre P, Vekeman F et€al (2009) An assessment of erythroid response to epoetin alpha as a single agent versus in combination with granulocyte- or granulocyte-macrophage-colony-stimulating factor in myelodysplastic syndromes using a meta-analysis approach. Cancer 115:706–715 64. Garypidou V, Verrou E, Vakalopoulou S et€al (2003) Efficacy of a single, weekly dose of recombinant erythropoietin in myelodysplastic syndromes. Br J Haematol 123:958 65. Gabrilove JL, Cleeland CS, Livingston RB et€al (2001) Clinical evaluation of once-weekly dosing of epoetin alfa in chemotherapy patients: improvements in hemoglobin and quality of life are similar to three-times-weekly dosing. J Clin Oncol 19:2875–2882 66. Cazzola M, Beguin Y, Kloczko J et€al (2003) Once-weekly epoetin beta is highly effective in treating anaemic patients with lymphoproliferative malignancy and defective endogenous erythropoietin production. Br J Haematol 122:386–393 67. Bokemeyer C, Aapro MS, Courdi A et€al (2004) EORTC guidelines for the use of erythropoietic proteins in anaemic patients with cancer. Eur J Cancer 40:2201–2216 68. Bohlius J, Wilson J, Seidenfeld J (2006) Recombinant human erythropoietins and cancer patients: updated meta-analysis of 57 studies including 9353 patients. J Natl Cancer Inst 98(10):708–714 69. Malcovati L, Della Porta MG, Cazzola M (2006) Predicting survival and leukemic evolution in patients with myelodysplastic syndrome. Haematologica 91:1588–1590 70. Malcovati L, de La Porta MG, Pascutto C et€al (2005) Prognostic factors and life expectancy in myelodysplastic syndromes classified according to WHO criteria: a basis for clinical decision making. J Clin Oncol 2005 23:7594–7603 71. Silverberg DS, Wexler D, Sheps D et€al (2001) The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study. J Am Coll Cardiol 37:1775–1780 72. Zeidman A, Fradin Z, Blecher A, Oster HS, Avrahami Y, Mittelman M (2004) Anemia as a risk factor for ischemic heart disease. Isr Med Assoc J 6:16–18 73. Vaupel P, Dunst J, Engert A et€al (2005) Effects of recombinant human erythropoietin (rHuEPO) on tumor control in patients with cancer-induced anemia. Onkologie 28:216–221 74. Leyland-Jones B (2003) BEST Investigators and study group. Breast cancer trial with erythropoietin terminated unexpectedly. Lancet Oncol 4:459–460 75. Leyland-Jones B, Semiglazov V, Pawlicki M et€al (2005) Maintaining normal hemoglobin levels with epoetin alfa in mainly nonanemic patients with metastatic breast cancer receiving first-line chemotherapy: a survival study. J Clin Oncol 23:5960–5972 76. Henke M, Laszig R, Rübe C et€al (2003) Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial. Lancet 362:1255–1260 77. Wright JR, Ung YC, Julian JA et€al (2007) Randomized, double-blind, placebo-controlled trial of erythropoietin in non-small-cell lung cancer with disease-related anemia. J Clin Oncol 25:1027–1032 78. Smith RE Jr, Aapro MS, Ludwig H et€al (2008) Darbepoetin alpha for the treatment of anemia in patients with active cancer not receiving chemotherapy or radiotherapy: results of a phase III, multicenter, randomized, double-blind, placebo-controlled study. J Clin Oncol 26:1040–1050 79. Overgaard J, Hoff CM, Hansen HS, et€al (2009) Randomized study of darbepoetin alfa as modifier of radiotherapy in patients with primary squamous cell carcinoma of the head and neck (HNSCC): Final outcome of the DAHANCA 10 trial. J Clin Oncol 27:15s, (suppl; abstr 6007) 80. Hedenus M, Adriansson M, San Miguel J et€ al (2003) Efficacy and safety of darbepoetin alfa in anaemic patients with lymphoproliferative malignancies: a randomized, double-blind, placebo-controlled study. Br J Haematol 122:394–403

216

H. S. Oster et al.

81. Thomas G, Ali S, Hoebers FJ et€al (2008) Phase III trial to evaluate the efficacy of maintaining hemoglobin levels above 12.0 g/dL with erythropoietin vs above 10.0 g/dL without erythropoietin in anemic patients receiving concurrent radiation and cisplatin for cervical cancer. Gynecol Oncol 108:317–325 82. Untch M, Fasching PA, Bauerfeind I et€al (2008) PREPARE trial. A randomized phase III trial comparing preoperative, dose-dense, dose-intensified chemotherapy with epirubicin, paclitaxel and CMF with a standard dosed epirubicin/cyclophosphamide followed by paclitaxel ± darbepoetin alfa in primary breast cancer: A preplanned interim analysis of efficacy at surgery. J Clin Oncol 26:(May 20 suppl; abstr 517) 83. Takeshita A, Shinjo K, Higuchi M et€ al (2000) Quantitative expression of erythropoietin receptor (EPO-R) on acute leukaemia cells: relationships between the amount of EPO-R and CD phenotypes, in vitro proliferative response, the amount of other cytokine receptors and clinical prognosis. Japan Adult Leukaemia Study Group. Br J Haematol 108:55–63 84. Nowrousian MR, Dunst J, Vaupel P (2008) Erythropoiesis-stimulating agents: favorable safety profile when used as indicated. Strahlenther Onkol 184:121–136 85. Longmore GD (2007) Do cancer cells express functional erythropoietin receptors? N Engl J Med 356:2447 86. Jelkman W (2010) Erythropoietin: back to basics. Blood 115:4151–4152 87. Mittelman M, Zeidman A, Kanter P, Katz O, Oster H, Rund D, Neumann D (2004) Erythropoietin has an anti-myeloma effect—a clinical observation supported by animal studies. Eur J Haematol 72:155–165 88. Mittelman M, Neumann D, Peled A, Kanter P, Haran-Ghera N (2001) Erythropoietin induces tumor regression and anti-tumor immune responses in murine myeloma models. Proc Natl Acad Sci U S A 98:5181–5186 89. Dunst J (2001) The use of epoetin alfa to increase and maintain hemoglobin levels during radiotherapy. Semin Oncol 28(2 Suppl 8):42–48 90. Littlewood TJ, Bajetta E, Nortier JW et€al (2001) Effects of epoetin alfa on hematologic parameters and quality of life in cancer patients receiving nonplatinum chemotherapy: results of a randomized, double-blind, placebo-controlled trial. J Clin Oncol 19:2865–2874 91. Baz R, Walker E, Choueiri TK et€ al (2007) Recombinant human erythropoietin is associated with increased overall survival in patients with multiple myeloma. Acta Haematol 117: 162–167 92. Aapro M, Scherhag A, Burger HU (2008) Effect of treatment with epoetin-beta on survival, tumour progression and thromboembolic events in patients with cancer: an updated meta-analysis of 12 randomised controlled studies including 2301 patients. Br J Cancer 99:14–22 93. Bohlius J, Langensiepen S, Schwarzer G et€al (2005) Recombinant human erythropoietin and overall survival in cancer patients: results of a comprehensive meta-analysis. J Natl Cancer Inst 97:489–498 94. Bohlius J, Schmidlin K, Brillant C et€al (2009) Recombinant human erythropoiesis-stimulating agents and mortality in patients with cancer: a meta-analysis of randomised trials. Lancet 373:1532–1542 95. Jadersten M, Malcovati L, Dybedal I et€ al (2008) Erythropoietin and granulocyte-colony stimulating factor treatment associated with improved survival in myelodysplastic syndrome. J Clin Oncol 26:3607–3613 96. Cermák J (2006) Erythropoietin administration may potentiate mobilization of storage iron in patients on oral iron chelation therapy. Hemoglobin 30:105–112 97. Brines ML, Ghezzi P, Keenan S et€al (2000) Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Natl Acad Sci U S A 97:10526–10531 98. Katz O, Barzilay E, Skaat A et€al (2005) Erythropoietin induced tumour mass reduction in murine lymphoproliferative models. Acta Haematol 114:177–179 99. Prutchi Sagiv S, Lifshitz L, Orkin R et€al (2008) Erythropoietin effects on dendritic cells: potential mediators in its function as an immunomodulator? Exp Hematol 36:1682–1690

13â•… Cytokines in MDS: Abnormalities and Treatment

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100. Katz O, Gil L, Lifshitz L et€al (2007) Erythropoietin enhances immune responses in mice. Eur J Immunol 37:1584–1593 101. Lifshitz L, Tabak G, Mittelman M, Gassmann M, Neumann D (2010) Macrophages as novel targets for erythropoietin. Haematologica 95(11):1823–1831 102. Prutchi-Sagiv S, Golishevsky N, Oster HS, Katz O, Cohen A, Naparstek E, Neumann D, Mittelman M (2006) Erythropoietin treatment in advanced multiple myeloma is associated with improved immunological functions: could it be beneficial in early disease? Br J Haematol 135:660–672 103. Prutchi-Sagiv S, Golishevski N, Katz O, Oster HS, Naparstek E, Hoffman M, Neumann D, Mittelman M (2006) T-cell abnormalities in patients with myelodysplastic syndromes: improved immunological functions in patients treated with recombinant erythropoietin. Blood 108:756a (Abstr No. 2675) 104. Casadevall N, Durieux P, Dubois S et€al (2004) Health, economic, and quality-of-life effects of erythropoietin and granulocyte colony-stimulating factor for the treatment of myelodysplastic syndromes: a randomized, controlled trial. Blood 104:321–327 105. Greenberg PL, Young NS, Gattermann N (2002) Myelodysplastic syndromes. Hematology 2002:136–161 (educational book) 106. Martin S, Baldock SC, Ghoneim AT et€al (1983) Defective neutrophil function and microbicidal mechanisms in the myelodysplastic disorders. J Clin Pathol 36:1120–1128 107. Ohmori M, Ohmori S, Ueda Y (1991) Ineffective hemopoiesis in the myelodysplastic syndromes (MDS) as studied by daily in situ observation of colony-cluster formation. Int J Cell Cloning 9:521–530 108. Dar S, Mundle S, Andric T et€al (1999) Biological characteristics of myelodysplastic syndrome patients who demonstrated high versus no intramedullary apoptosis. Eur J Haematol 62:90–94 109. Rajapaska R, Ginzton N, Rott LS et€al (1996) Altered oncoprotein expression and apoptosis in myelodysplastic syndrome marrow cells. Blood 88:4275–4287 110. Nimer SD (2008) Myelodysplastic syndromes. Blood 111(10):4841–4851 111. Gradishar WJ, Le Beau MM, O’Laughlin R et€al (1992) Clinical and cytogenetic responses to granulocyte-macrophage colony-stimulating factor in therapy-related myelodysplasia. Blood 80(10):2463–2470 112. Vadhan-Raj S, Keating M, LeMaistre A et€al (1987) Effects of recombinant human granulocyte-macrophage colony-stimulating factor in patients with myelodysplastic syndromes. N Engl J Med 317:1545–1552 113. Lieschke GJ, Ramenghi U, O’Connor MP et€al (1992) Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. N Engl J Med 327:99–106 114. Negrin RS, Haeuber DH, Nagler A et€al (1989) Treatment of myelodysplastic syndromes with recombinant human granulocyte colony-stimulating factor: a phase I-II trial. Ann Intern Med 110:976–984 115. Negrin RS, Haeuber DH, Nagler A et€al (1990) Maintenance treatment of patients with myelodysplastic syndromes using recombinant human granulocyte colony-stimulating factor. Blood 76:36–43 116. Greenberg P, Taylor K, Larson R et€ al (1993) Phase III randomized multicenter trial of G-CSF vs. observation for myelodysplastic syndromes (MDS). Blood (Abstr) 82(Suppl 1):196a 117. Chuncharunee S, Intragumtornchai T, Chaimongkol B et€ al (2001) Treatment of myelodysplastic syndrome with low-dose human granulocyte colony–stimulating factor: a multicenter study. Int J Hematol 74:144–146 118. Zeidman A, Sokolover N, Fradin Z, Cohen A, Redlich O, Mittelman M (2004) Platelet function and its clinical significance in the myelodysplastic syndromes. Hematol J 5:234–238 119. Bryan J, Jabbour E, Prescott H et€al (2010) Thrombocytopenia in patients with myelodysplastic syndromes. Semin Hematol 47:274–280

218

H. S. Oster et al.

120. TRAP study group (1997) Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. N Engl J Med 337:1861–1869 121. Kaushansky K (2008) Historical review: megakaryopoiesis and thrombopoiesis. Blood. 111:981–986 122. Li J, Yang C, Xia Y et€al (2001) Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood 98:3241–3248 123. Tsimberidou AM, Giles FJ, Khouri I et€al (2005) Low-dose interleukin-11 in patients with bone marrow failure: update of the MD Anderson cancer center experience. Ann Oncol 16:139–145 124. Kurzrock R, Cortes J, Thomas DA et€al (2001) Pilot study of low-dose interleukin-11 in patients with bone marrow failure. J Clin Oncol 19:4165–4172 125. Gordon MS, Nemunaitis J, Hoffman R et€al (1995) A phase I trial of recombinant human interleukin-6 in patients with myelodysplastic syndromes and thrombocytopenia. Blood 85:3066–3076 126. Ganser A, Seipelt G, Lindemann A et€al (1990) Effects of recombinant human interleukin-3 in patients with myelodysplastic syndromes. Blood 76(3):455–462 127. Kantarjian H, Fenaux P, Sekeres MA et€al (2009) Safety and efficacy of Romiplostim in patients with lower-risk myelodysplastic syndrome and thrombocytopenia. J Clin Oncol 28:437–444 128. Will B, Kawahara M, Luciano JP et€ al (2009) Effect of the nonpeptide thrombopoietin receptor agonist Eltrombopag on bone marrow cells from patients with acute myeloid leukemia and myelodysplastic syndrome. Blood 114:3899–3908

Chapter 14

Therapeutic Modalities and New Molecular Targets in MDS Guillermo Garcia-Manero

Introduction In general, patients with MDS are classified based on the IPSS classification [1]. Although newer more refine classifications have been recently developed [2–4], the IPSS still remains the most commonly used prognostic score in the US. Although in principle the IPSS should be restricted to a very specific subset of patients (those with de novo disease that have not received prior therapy), it is commonly used in patients with MDS that do not meet this criteria. With the IPSS classification patients are divided into those with low, intermediate-1 (INT), INT-2 and high-risk disease [1]. These four subgroups have distinct expectations in terms of survival and risk of transformation to acute myelogenous leukemia (AML) [1]. Patients in the low and INT-1 subsets (usually referred as lower-risk disease) have a more indolent course of disease and therefore the use of interventions with early risk of mortality is usually not recommended. In contrast, patients with higher-risk disease (those with INT-2 and high-risk disease) have a natural course similar to those of patients with AML. For these patients certain degree of induction mortality is acceptable. Standard treatment interventions for patients with MDS are summarized in Table€14.1. In this chapter, I will review these different therapeutic interventions. I will exclude the use of growth factors and stem cell transplantation (SCT) because they are discussed in other chapters. I will also review specific clinical situations, for instance the problem of patients that relapsed, or do not benefit, from hypomethylating agent based therapy, and the emerging role of incorporating different cytogenetic and molecular alterations in the decision making involved in selecting therapy for patients with MDS.

G. Garcia-Manero () Section of Myelodysplastic Syndromes, Department of Leukemia, MD Anderson Cancer Center, University of Texas, 1515 Holcombe Blvd, PO Box 428, Houston, TX 77025, USA Fax: +1-713-794-4297 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_14, ©Â€Springer Science+Business Media B.V. 2011

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220 Table 14.1↜渀 Treatment options for patients with MDS

G. Garcia-Manero Risk Lower

Intervention Transfusion support Prophylactic antibiotics Growth factors Iron chelation Lenalidomide Hypomethylating agents Immune modulation Stem cell transplantation Investigational clinical trials

Higher

Hypomethylating agents AML-like therapy Stem cell transplantation Investigational clinical trials

Standard Treatment of Patients with Lower Risk MDS Identification of Patients with Lower Risk Disease but Poor Prognosis: Can We Treat Earlier? Before I start discussing the different therapies for lower risk MDS, I am going to introduce the concept that one of the main limitations of the IPSS is that it is a poor predictor of outcome in patients with lower risk disease [1, 4]. This is particularly important as it is estimated that close to two thirds of patients with MDS are on this category [5]. The reported long survival of these patients and the advanced median age of patients with MDS have resulted in the common practice of a “watch and wait” approach for patients with lower risk MDS. Based on the data to be presented below, I will argue that a significant fraction of patients with lower risk disease should be considered for earlier intervention. That said, this concept will need to be tested in specific clinical trials and some of the new classifications need to be validated internationally. For the last decade, investigators at MD Anderson Cancer Center (MDACC) have followed the IPSS score to stratify patients for therapy. They also developed an easy rule based on percent of bone marrow blasts (more or less than 10%) that allows to separate patients with higher versus lower risk disease. Patients with >10% blasts being candidates for more intensive therapy and those with less being candidates for observation. Obviously this was before the advent of some of the therapies outlined in Table€14.1, particularly the hypomethylating agents. Based on this practice, it was our perception that a subset of patients with lower risk disease had a worse natural history than predicted and decided to study the outcome of this group of patients [4]. To do this, we analyzed 856 patients with lower risk MDS referred to MDACC from 1976 to 2005. After evaluating a large number of patient characteristics, we developed a multivariate analysis model (Table€14.2) that allowed us to predict the survival of patients based on character-

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Table 14.2↜渀 Prognosis of patients with lower risk MDS using the MDACC model. (Adapted from [4]) A. Characteristics and score associated with prognosis in lower risk MDS Adverse factor Coefficient P value Assigned score 1 Unfavorable cytogeneticsa 0.203 <â•›0.0001 0.348 2 <â•›0.0001 Ageâ•›≥â•›60 years 0.216 1 Hgbâ•›<â•›10 (g/dl) <â•›0.0001 2 0.498 Pltâ•›<â•›50â•›×â•›109/l <0.0001 1 0.277 0.0001 50â•›−â•›200â•›×â•›109/l 0.195 0.0001 1 BM blastsâ•›≥â•›4% B. Estimated survival outcome within each score range and proposed risk categories Score

No. of patients Median (month)

4-year survival (%) Category

0 1 2 3 4 5 6 7

11 58 113 185 223 166 86 13

78 82 51 40 27 9 7 NA

NR 83 51 36 22 14 16 9

1

2 3

Hgb hemoglobin, Plt platelets, NA not available In this analysis, diploid and 5q- only were favorable cytogenetic, all others were considered as unfavorable cytogenetics

a

istics that included age, hemoglobin, platelet count, cytogenetics and percentage of bone marrow blasts. Based on this score, patients with lower risk MDS could have from 0 to 7 points on the score and with an estimated survival range of not reached to 9 months (Table€14.2). For simplicity, we divided patients into three categories: 1, 2 and 3. Of importance, close to two thirds of patients referred to MDACC were in categories 2 and 3 with a median survival of 27 and 14 months respectively. The implication of these data is that potentially patients with a high score and lower risk disease could be candidates for more aggressive therapies and earlier incorporation of therapy. The following are two potential scenarios where this type of tool could be of importance: selecting patients with lower risk disease for SCT and considering therapy in patients that are transfusion independent. Traditionally SCT is not offered to patients with lower risk disease [6]. This is based on data from Cutler et€al. that indicated that early transplantation in these patients was not associated with improvement of survival [6]. Cutler has applied the MDACC lower risk model [4] to this patient population has presented data that patients with a higher-risk score benefit from SCT in terms of survival (Cutler, unpublished). The second scenario is that of the patient with poor prognosis but that is transfusion independent. Transfusion dependency is usually the trigger use to initiate therapy in MDS. Based on the MDACC lower risk model, a patient could be transfusion independent but with the following characteristics: older age, hemoglobin less than 10€g/dl but not yet requiring transfusions, platelets less than 50â•›×â•›109/l but above 10, blasts 5% and intermediate risk cytogenetics. That would give the patient seven points and a pre-

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dicted median survival of 9 months. The question would be whether a “watch and wait” approach is optimal for this patient and whether earlier intervention, including SCT or others, could be indicated. I would argue that the later is the most logical approach. This is further supported by the fact that only 10–20% of patients with lower-risk MDS transform to AML [4] and that the cause of death in this subset of patients is most frequent intrinsic to MDS and not to transformation to AML [7]. I emphasize here that this is an evolving concept and that currently this information is being used for clinical trial development in this group of patients.

“Supportive Care” Measures in Lower Risk MDS There are three main supportive care measures commonly used in patients with MDS. As shown in Table€14.1, these include transfusion support, the use of prophylactic antibiotics and growth factor support. It should be noted that growth factor is considered by several investigators as disease modifying [8]. Transfusional support is the most common measure used in patients with MDS. It is estimated that the vast majority of patients will required red cell and/or platelets transfusions. Thresholds for transfusion vary from region to region. In our center, we use a cut off of platelet count for transfusion of less 10â•›×â•›109/l, and a hemoglobin level of less than 8€g/dl. If patients are symptomatic (dyspnea, angina) or actively bleeding these thresholds are obviously modified. The source of blood product, number and type of units transfused depend on center volume and availability. There is also limited data in terms of the role of prophylactic antibiotics in MDS. Isolated severe neutropenia is rare in MDS and in our experience rarely associated with severe infection. Prophylactic treatment with antibacterial, antiviral and antifungal therapy is common practice at MDACC but not standard of care. The cost and role of this approach in inducing drug resistance have not been evaluated in detail yet. Prophylactic antimicrobial therapy could be tailored to the type of therapy and the chances that such therapy could induce mucosal damage.

What Is the Role of Iron Chelation? Because red cell transfusion occurs in over 50% of patients with MDS, there is clear evidence of iron accumulation in these patients. What is not clear at the present time is the impact of this process on organ function. Furthermore, it is currently not known whether improvement of iron deposition has an impact on the natural history of MDS. In principle and extrapolating from the data in hemoglobinopathies, the principal targets of iron accumulation are the myocardium and the liver parenchyma. Therefore the current concept is that excess iron accumulation can result in heart dysfunction or liver cirrhosis. The data in this regard is scant in MDS. A series of 292 patients evaluated in a transfusion center in Japan indicated that 24% of patients developed evidence of severe CHF and 7% liver failure [9]. This data implies therefore that

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end organ damage should be an important cause of death in this patient population particularly in those with lower risk disease red cell transfusion dependent. I do not observe this in my own clinical experience. It is very rare for us to find patients with evidence of CHF or liver cirrhosis. Indeed, we performed a study of cause of death in patients with lower risk disease. The most common causes of death in our series were infection followed by bleeding [7]. CHF and liver cirrhosis were rare events in our patient population [7]. There is an obvious referral difference in the group of patients evaluated at MDACC but not to explain such a difference. The next question is whether chelating iron has an impact on the natural history of MDS. At the present time there is no data from randomized clinical trials to suggest such an effect. Studies from Canada and France have suggested that those patients receiving chelation have longer survival than those that did not receive chelation or suboptimal chelation [10, 11]. A multicenter randomized study is now ongoing to evaluate the role of chelation therapy with the oral compound desferoxamine on the natural history of MDS. This study should clarify this topic. A recent report from the Spanish MDS group evaluated the impact of red cell transfusion in the survival and transformation to AML of patients with MDS [12]. In this analysis of 2,994 patients, 835 were red cell transfusion dependent at initial presentation and 525 became transfusion dependent during follow up. Red cell transfusion dependent patients had a significant worse survival but importantly also had a significantly increased risk of transformation to AML. Transformation cannot be explained by liver or heart toxicity and implies that iron accumulation has a direct toxic effect of the hematopoietic stem cell. Data indicating that chelation results in improvement of hematopoietic function has not been validated [13]. Finally another alternative that could explain potential beneficial effect of chelation consists on decreasing risk of fungal infection in patients with MDS. Although it has not been studied systematically in MDS, it is known that certain subtypes of fungus need iron for cell proliferation [14]. This data could explain why outcome of patients with iron overload that receive SCT is worse and why chelation in the peritransplant setting may improve this [15]. At the present time there are no clear guidelines for initiation of iron chelation. The NCCN panel recommended a ferritin level of 2000 for initiation of chelation whereas other groups recommend lower thresholds [16]. That said without evidence of clinical benefit and in view of the cost of chelation approaches, one could make the point that chelation is not indicated at the present time.

The Role of Lenalidomide in Patients with Alteration of Chromosome 5 Immune modulatory agents (IMIDs) such as thalidomide have been extensively studied in MDS [17]. Initial studies with lenalidomide in lower risk MDS indicated that a subset of patients with anemia and alteration of chromosome 5 had significant benefit

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with lenalidomide [18]. These results were confirmed in a follow up study restricted to patients with alterations of chromosome 5 [19]. Responses with lenalidomide occurred in 76% of 148 patients. Sixty-seven percent of the patients became transfusion independent by week 24. Lenalidomide induced responses fast, with a median time to response of 4.6 weeks. Approximately 60% of responders maintained response for at least 1 year. Furthermore cytogenetic responses were documented in 45% of evaluable patients. Of importance, List et€al. also identified prognostic characteristics of response. This included presence of a platelet count over 100â•›×â•›103€╛Ku/l and recent onset of anemia [19]. It should be noted that patients with neutropenia or severe thrombocytopenia (platelets less than 50â•›×â•›109/l) were excluded from the study [19]. A phase III randomized study of lenalidomide versus placebo was reported at the 2009 American Society of Hematology (ASH) meeting in New Orleans [20]. In that study, lenalidomide was used at two different dose levels (5€mg orally daily or 10€mg orally daily). The data presented clearly demonstrated that lenalidomide at a dose of 10€mg daily is superior to placebo and potentially 5€mg dosing. Lenalidomide is the standard for patients with anemia lower risk MDS and with an alteration of chromosome 5. An issue that has been argued is the potential for lenalidomide to increase the risk of transformation to AML. In the randomized study of lenalidomide the risk of transformation was 1% for patients receiving 10€mg, 6% for those receiving 5 and 1% in the placebo. We had evaluated the outcome of patients with chromosome 5 in relation to transformation and found that outside the setting of 5q- syndrome, alterations of chromosome 5 have a relatively poor prognosis and are frequently associated with other alterations such as chromosome 7 [21]. Therefore, we believe that the rate of transformation observed represents the natural history of the disease. One of the key questions that has been triggered by the selective activity of lenalidomide in patients with del5q is the discovery of genes involved in response to lenalidomide. At the present time, no such biomarker exist. Expression of a gene known as SPARC [22] and an erythroid response [23] have been associated with response to lenalidomide. Because the critically deleted region in chromosome 5 is relatively short (40–43 genes), this work has also resulted in the identification of RPS14 [24] and miRNAs 145 and 146b [25] as being involved in the pathogenesis of del5q- MDS. Dysfunction of RPS14 seems to be related to anemia in these patients and the miRNA to platelet biogenesis [24, 25]. Recently, List et€al. has also presented data involving two phosphatases located in 5q31, Cdc25c and PP2A, that could have a role in cytotoxic effect of lenalidomide in patients with del5q [26] and potentially with resistance to the drug.

The Role of Lenalidomide in Patients Without Alteration of Chromosome 5 or with Higher Risk Disease Another area of interest is whether lenalidomide has activity in patients without alteration of chromosome 5 and those with higher risk disease or AML. Initial data in

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patients without an alteration of chromosome 5 and lower risk MDS indicated that red cell responses are significantly lower (26%) and most importantly that their duration is also significantly shorter (41 weeks) compared to patients with alteration of chromosome 5 [27]. That said, the data from this studies indicate that a fraction of patients without del5q could derived significant benefit from lenalidomide. This is being investigated now in an ongoing randomized clinical trial. At the present I do not recommend the use of lenalidomide in patients without an alteration of chromosome 5, particularly if they also have significant thrombocytopenia or increased transfusion needs. The other issue is whether lenalidomide has a role in patients with higher-risk disease or AML with or without an alteration of chromosome 5. Two studies have been reported in patients with higher risk disease. In a French study [28], 47 patients with higher risk disease were treated with standard dose lenalidomide. The reported response rate was 27% (13 patients) including seven patients with a complete remission (CR). Median response remission was 6.5 months. CR were almost exclusively documented in patients with documented isolated chromosome 5 alterations. Responses were also more frequently observed in patients with platelet count over 100€k 109â•›×â•›l. A study performed at MDACC showed less significant results (Borthakur, in preparation). These results clearly suggest that lenalidomide has clinical activity as single agent in this patient population but should be considered either in combination or using different dose or schedules. Recently, the group at Washington University have reported data using a high dose schedule of lenalidomide (50€mg orally a day) in elderly patients with AML without an alteration of chromosome 5. Response rate was 30% but of short duration [29]. This data indicate that lenalidomide should have a role in combination strategies in patients with higher risk MDS and AML. For instance studies with the combination of 5-azacitidine and lenalidomide have been reported with promising results [30].

Use of Hypomethylating Agents in Lower Risk MDS There are two hypomethylating agents approved in the US: decitabine [31] and 5-azacitidine [32]. 5-azacitidine is approved for all patients with MDS [32] whereas decitabine excludes patients with low risk disease [31]. In Europe the only compound approved is 5-azacitidine and is for patients with int-2 or high risk disease [33]. At the present time, there is significantly less data with the role of the hypomethylating agents in low or int-1 disease. The main concern being the risk of myelosuppresion in this patient population whose main problem is pancytopenia but have otherwise a relatively low burden of disease (% of marrow blasts). Most of the data comes from “registry” studies that are not well controlled. A study performed in the community setting in the US comparing a 5 day schedule of 5-azacitidine versus a 5-2-2 and a 10 schedule indicated that a 5-day schedule in lower risk patients is associated with activity and is safe in this patient population [34]. No specific clinical trial for patients with low or int-1 disease has been performed so far with 5-azacitidine.

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A study with a very low dose schedule of decitabine was recently presented [35]. This was a multicenter study comparing decitabine 20€mg subcutaneous either dailyâ•›×â•›3 or weeklyâ•›×â•›3 every 28 days in patients with low or int-1 MDS. Patients transfusion independent but with poor prognosis were allowed on this study. The data presented indicated that although complete response rate was less than 10%, patients treated in the dailyâ•›×â•›3 arm had significant benefit compared to those treated on the weekly arm and that trilineage responses were observed in close to 70% of patients. No induction mortality was documented and transformation to AML was restricted to patients in the weeklyâ•›×â•›3 arm. This study is still ongoing but suggests that this type of low intensity program of decitabine may have a role in patients with lower risk disease.

Immune Modulation in MDS A significant fraction of patients with MDS have features that overlap with those of processes related to immune deregulation such as autoimmune diseases, large granular lymphocytosis, aplastic anemia and proximal nocturnal hemoglobinuria (PNH) [36]. This observation together with evidence of deregulation of immune function in MDS has suggested that immune-therapy could have a role in patients with MDS [37]. The group at the NHLBI lead by Young et€al. have published results that therapy with ATG with or without cyclosporine [38] results in responses in 30% of patients with MDS. These investigators have also developed an algorithm that includes HLA-DR15 haplotype, younger age and recent need of red cell transfusions that predicts for response to this type of therapy [39]. Using this algorithm, the same investigators have reported a high percentage of response using a low schedule of alemtuzumab [40]. These results have not been reproduced by other groups such as those of Mufti [41] and the MDACC (Ravandi, unpublished) where the strongest characteristic associated with response is the presence of marrow hypocellularity. Recently the group at Moffitt Cancer Center has published data that the CD4/CD8 ratio could be used to predict response to ATG based therapy [42].

Standard Treatment of Patients with Higher Risk MDS The treatment of patients with higher risk MDS has improved significantly over the last 5 years. The advent of two hypomethylating agents (decitabine and 5-azacitidine) has represented a revolution in the treatment of these patients. Despite their effect in modifying the natural history of the disease, these drugs do not represent a curative approach for most patients. Therefore multiple questions exist when approaching these patients. For instance: (1) who is a candidate for intensive AMLlike therapy; (2) who is a candidate for hypomethylating based therapy; (3) are there age limits for this therapy; (4) when should SCT be performed in this patient popu-

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lation; (5) is there an optimal therapy for patients that “fail” hypomethylating based therapy. I will try to answer some of these questions in the following paragraphs. The reader should be aware that some of these questions have not really been studied in clinical trials and that some of the suggestions are personal approaches based on my experience.

Decitabine Decitabine or 5-aza-2′-deoxycitidine is a nucleoside analogue with hypomethylating activity [43]. In this review, I will not discuss the role of aberrant DNA methylation [44] in MDS [45]. That said, at the present time there is no clear evidence that the clinical responses observed with this class of agent are related to induction of global or gene specific hypomethylation [46, 47]. Decitabine has been studied in MDS and AML for over two decades using different doses and schedules. The most recent experience has focused on using this agent at low doses. Using this approach, decitabine has an excellent safety and activity profile. Decitabine is approved in the US to be used following a 3-day and a 5-day schedule. The dose of the 3-day schedule is 15€mg/m2 over 3€hr every 8€hr. European investigators have pioneered the use of this type of schedule [48]. The rationale behind it is that the three day schedule with the drug administered over 3€hr three times a day resembles as much as possible a continuous infusion approach. The hypothesis is that continuous infusions are potentially the most effective inducing hypomethylation [49]. With the three day schedule, the reported CR rate in the phase III US study was 9% and overall response rate of 17%. Duration of response was 10 months. Two randomized studies of this schedule of decitabine have been conducted: one in the US [31] and the other in Europe. In the US study there was no effect on overall survival [31] but an effect on trend towards longer time to progression to AML [31]. Because European Union required a positive survival study for the approval of decitabine, a second study was conducted in Europe. The schedule was the same with the drug administered every 4–6 weeks. These results were presented at the ASH 2008 meeting and a final manuscript is expected this year. In this study, Lubbert et€al. reported that decitabine failed to achieve the primary endpoint of the trial that was survival (unpublished). The 5-day schedule of decitabine was developed by the MDACC group [50]. A phase I trial had determined that decitabine infused over 1€hr at doses of 10–20€mg/ m2 daily for 5–20 days was safe [50]. Furthermore a schedule of 15€mg/m2 dailyâ•›×â•›10 was associated with significant activity in patients with advanced MDS and AML (65% response rate). This led to a three arm phase II Bayesian randomized study of decitabine where the drug was administered either at a dose of 20€mg/m2 IVâ•›×â•›5, 10€ mg/m2 IVâ•›×â•›10 days or 20€ mg/m2 SCâ•›×â•›5 [51]. In this study 95 patients were treated including 18 patients with CMML. The 20 mg/m2 IVâ•›×â•›5 arm was found to be superior to the other two arms. The CR rate in this arm was 39% compared to 24 and 21% with the other two arms. To further confirm this experience, a second 5-day decitabine schedule study (known as the ADOPT trial) was performed [52].

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In this study, 99 patients with MDS were treated. The overall response rate was 32% including17 CRs for an overall improvement rate of 51%. Most responses were observed by cycle 2. The response rate of the ADOPT trial was lower than the MDACC experience group but probably better and as safe as the 3-day schedule [31, 51]. Based on this data, the 5-day schedule was recently approved in the US. No randomized survival study has been performed with the 5-day schedule in MDS so far. A study focusing on AML is to be reported at some point in 2010. More recently, investigators at Ohio State University have published the results of a 10-day schedule of decitabine in AML. In this phase II study, decitabine at 20€mg/m2 dailyâ•›×â•›10 was administered to older patients with previously untreated AML [53]. Fifty-three patients were treated with 36% with antecedent hematologic disorder or therapy-related disease and 16 with poor risk karyotypes. With this schedule, CR rate was 47%. Median time to response was three cycles of therapy. Induction death was very low. A relationship was observed between higher levels of miR-29b and clinical response. The importance of this study is the discovery by these investigators of a miRNA29b as predictor of response and the fact that prolonged schedules of therapy are safe and associated with significant activity in AML. These results are in line with those of the initial phase I trial at MDACC [50, 53]. In summary, decitabine is a safe and active agent in higher-risk MDS but no study so far has shown a significant benefit in terms of overall survival in MDS.

5-azacitidine 5-azacitidine is another hypomethylating agent [43]. Structurally is similar to decitabine although in vitro has more modest hypomethylating activity. As with decitabine, 5-azacitidine has been used for several decades in both solid tumors and leukemias using different dose and schedules [54]. Several sequential studies of 5-azacitidine performed by the CALGB have demonstrated the activity and safety of this compound [54]. The most frequent dose and schedule used in MDS has been 75€mg/m2 subcutaneous (SC) dailyâ•›×â•›7 days every 28 days. This schedule was first studied in trial CALGB9221 led by Dr Silverman et€al. [32]. This was a randomized cross-over study comparing 5-azacitidine at the dose schedule shown above with best supportive care (BSC) of 191 patients. Hematological response was documented in 60% of patients on the 5-azacitidine arm including 7% CRs. This compared to 5% in the supportive care arm (Pâ•›<â•›0.001). Median time to leukemic transformation or death was also improved in the 5-azacitidine arm: 21 months versus 13 months for supportive care (Pâ•›=â•›0.007). Transformation to AML occurred in15% of patients on the 5-azacitidine arm versus 38% in the supportive care arm (Pâ•›=â•›0.001) [32]. Quality-of-life was improved in patients treated with 5-azacitidine compared to control [55]. Although the cross-over design did not allow to detect a significant difference in survival, in a landmark analysis, survival was superior in the 5-azacitdine arm. This study led to the approval of 5-azacitidine in US for patients with MDS. Because of the same issues with European regulators discussed

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above for decitabine, a randomized study, this time without cross over, was performed in Europe comparing 5-azacitidine with a menu of options that could include best supportive care (BSC), low dose ara-C and intensive therapy following a 7â•›+â•›3 schema [33]. The primary endpoint of the study was overall survival. In this study (known as AZA-001), 358 patients were randomly assigned to receive 5-azacitidine (nâ•›=â•›179) or conventional care regimens (nâ•›=â•›179). Median overall survival was 24.5 months (9.9-not reached) for the 5-azacitidine group versus 15.0 months (5.6–24.1) for the conventional care group. The hazard ratio was 0.58 (95% CI 0.43–0.77; pâ•›=â•›0.000,1). This study therefore demonstrates that 5-azacitidine improved survival of patients with higher risk MDS [33]. This study is also now being analyzed for further patient subset characterization. First is the question of the treatment of older patients (median age 70 years) with 5-azacitidine [56]. In a recently published study, 113 patients were considered as elderly, 55 of them received 5-azacitidine. Median OS for 5-azacitidine-treated patients was 24.5 months compared with 16.0 months for CCR-treated patients (hazard ratioâ•›=â•›0.47; 95% CI, 0.28–0.79; Pâ•›=â•›0.005), and the 2-year OS rates were 50% and 16% (Pâ•›=â•›0.001). Of interest, 5-azacitidine administration was associated with fewer total days in hospital than conventional therapy. Based on this data, there is no reason not to treat older individuals with MDS with this type of approach. Another issue is when to stop therapy in patients treated with 5-azacitidine. Time to response in most current studies of 5-azacitidine is 6 to 9 cycles of therapy. Therefore a significant fraction of therapeutic time is spent in non-responding patients. This may be difficult to accept by both patients and physicians. It is now standard of care that therapy with 5-azacitidine, and also decitabine, should be continued for as long as possible in patients not achieving a CR but stable disease without excess toxicity or evidence of disease progression. This is further supported by an analysis of the AZA-001 study that indicated that survival was improved in a subset of patients that had not achieved a CR [33]. This is a major paradigm change in that not achieving a CR can still be associated with significant clinical improvement. Finally, patients that achieved a CR should continue on therapy for as long as possible. This is based on data from MDACC from two studies of combination epigenetic therapy [46, 47]. Patients that achieved a CR in those studies were maintained for 24 months. At that time per protocol therapy was stopped. Relapse was universal, approximately after 2 months of stopping therapy, and outcome of these patients was very poor [57].

Does Intensive Chemotherapy Still Have a Role in the Treatment of Higher Risk MDS? In this manuscript I am not going to review in detail results of AML therapy in MDS. By en large these have been poor and its use limited to younger patients. CR rates with this type of AML therapy are lower than in patients with de novo AML, mortality during induction phase can be as high as 30–40% and response rates are not

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long lasting with most patients relapsing in the first 12 months. Based on this and in contrast with the data shown above with the hypomethylating agents, it is difficult to justify widespread use of AML therapy in higher risk MDS, but there are some exceptions. First in the AZA-001 by multivariate analysis presence of alterations of chromosome 7 were associated with better outcome with 5-azacitidine therapy [33]. Based on this, I tend not use intensive therapy in this subset of patients that already have a very poor prognosis with AML therapy. That said in a patient with diploid karyotype, particularly if the patient is young and there is the possibility to perform an SCT early, intensifying therapy and taking the patient to transplant as soon as possible maybe the best option for the patient. This has not been tested in clinical trials.

 ew Targets and New Drugs in MDS: Investigational N Approaches The main problem in MDS is the heterogeneity of the disease and the lack of molecular targets in a significant fraction of patients. The discovery of such targets is fundamental as demonstrated by the significant activity of lenalidomide in patients with del5q MDS [19]. At the present time, we do not have other significant molecular targets in MDS. This is particularly problematic for the hypomethylating agents. Despite their clear role in the disease, we do not have currently a predictive biomarker of response (unless the data with miRNA29b is reproduced [53]). Table 14.3 shows a list of ongoing investigational approaches in MDS and their potential targets if known. Some of these are discussed below. Table 14.3↜渀 Examples of investigational approaches and potential targets in MDS Agent Target Indication Clofarabine Nucleoside analogue Excludes low risk MDS Sapacitaine Nucleoside analogue Excludes low risk MDS Oral azacitidine Hypomethylating agent All risks HDAC inhibitors HDAC Lower risk disease P38MAPK inhibitors P38 MAPK Lower risk disease TLK Gluthatione pathway Lower risk disease Higher dose lenalidomide IMID Higher risk MDS Combination epigenetic DNA methylation, histone Higher risk MDS therapies acetylation DNA topoisomerase I Topo I Higher risk disease inhibitors Flt-3 inhibitors Flt-3 As per target JAK2 inhibitors JAK2 As per target MEKK inhibitors MEKK As per target Aurora kinase inhibitors Aurora kinase As per target Combinations of Higher risk disease lenalidomide ON1910 Higher risk disease

Phase II II I/II I/II I II II II II I I I I I II/III

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Nucleoside Analogues: Clofarabine and Sapacitabine Nucleoside analogues have significant clinical activity in leukemia. A number of studies have been performed with clofarabine in AML and more recently in patients with higher risk MDS [58, 59]. Both an IV and an oral formulation are available for research. The most common schedule investigated in MDS is that of 5 consecutive days. In a recent report of oral clofarabine 32 patients were treated including 20 patients that had received prior hypomethylating agents. Three doses of clofarabine were studied: 40, 30 and 20€mg/m2 daily for 5 days. CR was documented in 25% of patients for an overall response rate of 43%. No induction mortality was documented but renal failure was documented in four patients. The most common problem was myelosuppression. As expected, toxicity was better with lower doses. Sapacitabine is an oral compound with nucleoside analogue activity and also with the capacity to induce single strand DNA breaks [60]. Although earlier in its development compared to clofarabine, sapacitabine has been reported to have a 20–30% response rate in patients with relapsed MDS with an excellent toxicity profile [61].

Oral Formulation of 5-azacitidine As discussed earlier, the hypomethylating agents require chronic long term use. Therefore an oral formulation will be a significant advantage for patients with MDS. An oral formulation of 5-azacitidine is currently being investigated in MDS and AML [62]. In a study reported at ASH 2009 [63], 41 patients were treated. Response rate in MDS was 34%, including 17% CR, most in patients with previously untreated disease. Dose limiting toxicities were diarrhea observed at a dose of 500€mg orally for 7 days. At lower doses no toxicities were documented. The diarrhea was attributed to the mannitol contained in the formulation. No induction related mortality has been observed using this schedule. Of significant interest, the pharmacokinetic and dynamic characteristics of oral 5-azacitidine were significantly lower than that of the parenteral version with reported exposure less than 20% of that of the parenteral one. The fact that responses were observed, albeit the first cycle of therapy on this trial was with parenteral azacitidine, implies that perhaps low concentration schedules of 5-azacitidine could be as effective as standard ones but with less toxicity. Prolonged schedules of this formulation at 14 and 21 days are currently being investigated.

Histone Deacetylase (HDAC) Inhibitors HDAC inhibitors are a heterogeneous group of drugs with the capacity to induce histone acetylation and potentially reverse epigenetic gene silencing [64]. These compounds are considered to be complementary to the hypomethylating agents and have synergistic activity in vitro [65, 66]. A number of these compounds are being

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studied in MDS and AML including vorinostat [67], MGCD0103 [68], panabinostat [69], JNJ-26481585 [70] among several others. The activity of these compounds as single agents, particularly in lower risk MDS is low, and most approaches using these compounds include combination therapies discussed below. Recently, it has been proposed that reversal of reactive oxygen species is important for the activity of this class of compounds [71].

Combination Strategies As discussed through the text, a number of single agent compounds with clinical activity are currently available for patients with MDS. It is logical to combine these agents mainly in view of their low single agent toxicity profiles. Two major lines of combination approaches are currently being investigated. These include the so called “epigenetic” combinations and more recently combination using lenalidomide. The rationale for the use of combinations of a hypomethylating agents and an HDAC inhibitor (epigenetic combinations) stands from the observation in vitro that this type of combination have synergistic capacity to reactivate epigenetically silent genes and also by the observation that they also have synergistic antileukemia activity [65, 66]. This concept has led to a number of phase I/II trials using either decitabine or 5-azacitidine with different HDAC inhibitors such a valproic acid, MGCD0103, vorinostat and ongoing studies with panabinostat [72]. With this type of approach response rates in previously untreated patients with AML have been reported to be close to 50%. Of importance, rapidity of response is accelerated with median time of response being 1 to 2 cycles of therapy [46]. A number of phase II studies are comparing single agent hypomethylating therapy versus the combination. Studies with valproic acid have indicated that those patients with the highest valproic acid levels in blood tended to have a higher response rate [46, 47]. This indicates that ongoing studies with potent third generation HDAC inhibitors may result in significant clinical benefit. More recently, there has been significant interest in the development of combinations of lenalidomide with hypomethylating agents. In the initial study of Sekeres et€al. the combination of 5-azacitidine and lenalidome, using both compounds at standard doses and schedules, was shown to be safe in MDS and to be associated with significant clinical activity [30]. Studies are ongoing evaluating combination with higher doses of lenalidomide and with decitabine.

Phase I Trials in MDS A number of agents are being developed in MDS. Examples include, for instance, the use of p38MAPK inhibitors [73] are being studied in lower risk MDS. Ongoing studies indicate that these compounds are safe and have potential activity in this setting. GSTpi inhibitors with a role in detoxifying pathways are also being

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studied. As a single agent this type oral compound has been shown to have clinical activity in patients with lower risk MDS [74]. Currently this compound is being studied in combination with lenalidomide also in lower risk MDS. Following the lead in AML, a number of targeted approaches are also being investigated in MDS. Although the frequency of Flt3 mutated or JAK2 positive patients is relatively low in MDS, specific inhibitors for these alterations are being developed. Finally mutations of Ras oncogenes occur in close to 20% of patients with MDS and at a higher rate in CMML. Although the use of farnesyl inhibitors, that in principle inhibit the Ras pathway, has been relatively disappointing more recently inhibitors of MEKK have been shown to have potential activity in patients with these mutations.

Therapy for Patients that Have Been Exposed to Hypomethylating Based Therapy One of the most relevant clinical problems in MDS is the development of treatment alternatives for patients that either do not respond or lose response to hypomethylating agent based therapy. At the present time the mechanism to resistance and failure to hypomethylating agents is not understood and therefore it is difficult to develop targeted interventions for these patients. What is known is that the outcome of these patients is very poor with median survival of less than 5 months and that responses to cytarabine or another hypomethylating agents are sporadic [75]. Currently these patients are treated on some of the studies described above.

MDS

Low-risk (IPSS low, INT-1) (BM blasts < 10%)

High-risk (IPSS INT-2, high) (BM blasts ≥ 10%) Age < 60 Intensive Chemotherapy Hypomethylating agents Clinical trial

Standard: Any age Iron chelation Growth factors Hypomethylating agents Lenalidomide (5q-) Immunemodulation Clinical trial

Age ≥ 60 Hypomethylating agents Intensive chemotherapy1 Clinical trial

re

lu Fai

Failure/Progression

e2

r ilu

Fa

SCT

Fig. 14.1↜渀 A proposed treatment algorithm for patients with MDS. [76]

1 Consider in younger patients with diploid cytogenetics 2 Consider earlier in younger patients

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A Treatment Algorithm for Patients with MDS A number of guidelines are in place to recommend treatment interventions for patients with MDS [16]. It is not the intention of this paragraph to substitute them but to put in perspective, based on some the data summarized above, some of these interventions. These are shown in Fig.€14.1. In general patients are divided based on their risk: lower versus higher risk disease. Therapies can be considered either “supportive”, standard or investigational. Because response rates are low with current approaches, and very few of them are known to change the natural history of the disease, in my opinion almost all patients with MDS are candidates for investigational clinical trials.

References ╇ 1. Greenberg P, Cox C, LeBeau MM et€al (1997) International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 89:2079–2088 ╇ 2. Malcovati L, Germing U, Kuendgen A et€al (2007) Time-dependent prognostic scoring system for predicting survival and leukemic evolution in myelodysplastic syndromes. J Clin Oncol 25:3503–3510 ╇ 3. Kantarjian H, O’Brien S, Ravandi F et€al (2008) Proposal for a new risk model in myelodysplastic syndrome that accounts for events not considered in the original international prognostic scoring system. Cancer 113:1351–1361 ╇ 4. Garcia-Manero G, Shan J, Faderl S et€al (2008) A prognostic score for patients with lower risk myelodysplastic syndrome. Leukemia 22:538–543 ╇ 5. Rollison DE, Howlader N, Smith MT et€ al (2008) Epidemiology of myelodysplastic syndromes and chronic myeloproliferative disorders in the United States, 2001–2004, using data from the NAACCR and SEER programs. Blood 112:45–52 ╇ 6. Cutler CS, Lee SJ, Greenberg P et€al (2004) A decision analysis of allogeneic bone marrow transplantation for the myelodysplastic syndromes: delayed transplantation for low-risk myelodysplasia is associated with improved outcome. Blood 104:579–585 ╇ 7. Dayyani F, Conley AP, Strom SS et€al (2010) Cause of death in patients with lower-risk myelodysplastic syndrome. Cancer 116:2174–2179 ╇ 8. Sekeres MA, Steensma DP (2009) Defining prior therapy in myelodysplastic syndromes and criteria for relapsed and refractory disease: implications for clinical trial design and enrollment. Blood 114:2575–2580 ╇ 9. Takatoku M, Uchiyama T, Okamoto S et€al (2007) Retrospective nationwide survey of Japanese patients with transfusion-dependent MDS and aplastic anemia highlights the negative impact of iron overload on morbidity/mortality. Eur J Haematol 78(6):487–494 10. Rose C, Brechignac S, Vassilief D et€al (2007) Positive impact of iron chelation therapy (CT) on survival in regularly transfused MDS patients. A prospective analysis by the GFM. Blood 110:249 11. Leitch H, Goodman T, Wong K et€al (2006) Improved survival in patients with myelodysplastic synsdromes (MDS) receiving iron chelation therapy. Blood 118:249 (Abstract) 12. Sanz G, Nomdedeu B, Such E et€al (2008) Independent impact of iron overload and transfusion dependency on survival and leukemic evolution in patients with myelodysplastic syndrome. Blood 112:640 13. Jensen PD, Heickendorff L, Pedersen B et€al (1996) The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload. Br J Haematol 94:288–299

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14. Ribes JA, Vanover-Sams CL, Baker DJ (2000) Zygomycetes in human disease. Clin Microbiol Rev 13:236–301 15. Armand P, Kim HT, Cutler CS et€ al (2007) Prognostic impact of elevated pretransplantation serum ferritin in patients undergoing myeloablative stem cell transplantation. Blood 109:4586–4588 16. Greenberg PL, Attar E, Battiwalla M et€al (2008) Myelodysplastic syndromes. J Natl Compr Cancer Netw 6:902–926 17. Musto P (2004) Thalidomide therapy for myelodysplastic syndromes: current status and future perspectives. Leuk Res 28:325–332 18. List A, Kurtin S, Roe DJ et€al (2005) Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J Med 352:549–557 19. List A, Dewald G, Bennett J et€al (2006) Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion. N Engl J Med 355:1456–1465 20. Fenaux P, Giagounidis A, Selleslag D et€al (2009) RBC transfusion independence and safety profile of lenalidomide 5 or 10€mg in Pts with low- or Int-1-risk MDS with Del5q: results from a randomized phase III trial (MDS-004). Blood 114:944 21. Kantarjian H, O’Brien S, Ravandi F et€al (2009) The heterogeneous prognosis of patients with myelodysplastic syndrome and chromosome 5 abnormalities: how does it relate to the original lenalidomide experience in MDS? Cancer 115:5202–5209 22. Pellagatti A, Jadersten M, Forsblom AM et€al (2007) Lenalidomide inhibits the malignant clone and up-regulates the SPARC gene mapping to the commonly deleted region in 5q- syndrome patients. Proc Natl Acad Sci U S A 104:11406–11411 23. Ebert BL, Galili N, Tamayo P et€ al (2008) An erythroid differentiation signature predicts response to lenalidomide in myelodysplastic syndrome. PLoS Med 5:e35 24. Ebert BL, Pretz J, Bosco J et€al (2008) Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature 451:335–339 25. Starczynowski DT, Kuchenbauer F, Argiropoulos B et€al (2009) Identification of miR-145 and miR-146a as mediators of the 5q- syndrome phenotype. Nat Med 16(1):49–58 26. Wei S, Chen X, Rocha K et€ al (2009) A critical role for phosphatase haplodeficiency in the selective suppression of deletion 5q MDS by lenalidomide. Proc Natl Acad Sci U S A 106(31):12974–12979 27. Raza A, Reeves JA, Feldman EJ et€al (2008) Phase 2 study of lenalidomide in transfusiondependent, low-risk, and intermediate-1 risk myelodysplastic syndromes with karyotypes other than deletion 5q. Blood 111:86–93 28. Ades L, Boehrer S, Prebet T et€al (2009) Efficacy and safety of lenalidomide in intermediate-2 or high-risk myelodysplastic syndromes with 5q deletion: results of a phase 2 study. Blood 113:3947–3952 29. Vij R, Nelson A, Uy G et€ al (2009) A phase II study of high dose lenalidomide as initial therapy for acute myeloid leukemia in patients >60 years old. Blood 114:842 30. Sekeres MA, List AF, Cuthbertson D et€ al (2010) Phase I combination trial of lenalidomide and azacitidine in patients with higher-risk myelodysplastic syndromes. J Clin Oncol 28:2253–2258 31. Kantarjian H, Issa JP, Rosenfeld CS et€al (2006) Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study. Cancer 106:1794–1803 32. Silverman LR, Demakos EP, Peterson BL et€al (2002) Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B. J Clin Oncol 20:2429–2440 33. Fenaux P, Mufti GJ, Hellström-Lindberg E et€ al (2009) Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 10:223–232 34. Lyons RM, Cosgriff TM, Modi SS et€ al (2009) Hematologic response to three alternative dosing schedules of azacitidine in patients with myelodysplastic syndromes. J Clin Oncol 27:1850–1856

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35. Garcia-Manero G, Couriel D, Tambaro F et€al (2009) A phase II randomized bayesian study of very low dose subcutaneous decitabine administered daily or weekly times three in patients with lower risk myelodysplastic syndrome (MDS). Blood 114:119 36. Young NS, Calado RT, Scheinberg P (2006) Current concepts in the pathophysiology and treatment of aplastic anemia. Blood 108:2509–2519 37. Kordasti SY, Ingram W, Hayden J et€al (2007) CD4+CD25high Foxp3+ regulatory T cells in myelodysplastic syndrome (MDS). Blood 110:847–850 38. Sloand EM, Wu CO, Greenberg P, Young N, Barrett J (2008) Factors affecting response and survival in patients with myelodysplasia treated with immunosuppressive therapy. J Clin Oncol 26:2505–2511 39. Saunthararajah Y, Nakamura R, Wesley R, Wang QJ, Barrett AJ (2003) A simple method to predict response to immunosuppressive therapy in patients with myelodysplastic syndrome. Blood 102:3025–3027 40. Sloand E, Olnes M, Weinstein B, Scheinberg P, Young N (2009) Alemtuzumab treatment of intermediate-1 (INT-1) myelodysplasia patients is associated with sustained improvement in blood counts and cytogenetic remissions. Blood 114:116 41. Lim ZY, Killick S, Germing U et€al (2007) Low IPSS score and bone marrow hypocellularity in MDS patients predict hematological responses to antithymocyte globulin. Leukemia 21:1436–1441 42. Zou JX, Rollison DE, Boulware D et€al (2009) Altered naive and memory CD4+ T-cell homeostasis and immunosenescence characterize younger patients with myelodysplastic syndrome. Leukemia 23:1288–1296 43. Quintas-Cardama A, Santos FP, Garcia-Manero G (2010) Therapy with azanucleosides for myelodysplastic syndromes. Nat Rev Clin Oncol 7:433–444 44. Herman JG, Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349:2042–2054 45. Shen L, Kantarjian H, Guo Y et€al (2010) DNA methylation predicts survival and response to therapy in patients with myelodysplastic syndromes. J Clin Oncol 28:605–613 46. Garcia-Manero G, Kantarjian HM, Sanchez-Gonzalez B et€al (2006) Phase 1/2 study of the combination of 5-aza-2′-deoxycytidine with valproic acid in patients with leukemia. Blood 108:3271–3279 47. Soriano AO, Yang H, Faderl S et€al (2007) Safety and clinical activity of the combination of 5-azacytidine, valproic acid, and all-trans retinoic acid in acute myeloid leukemia and myelodysplastic syndrome. Blood 110:2302–2308 48. Lubbert M, Wijermans P, Kunzmann R et€al (2001) Cytogenetic responses in high-risk myelodysplastic syndrome following low-dose treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine. Br J Haematol 114:349–357 49. Aparicio A, Eads CA, Leong LA et€al (2003) Phase I trial of continuous infusion 5-aza-2′deoxycytidine. Cancer Chemother Pharmacol 51:231–239 50. Issa JP, Garcia-Manero G, Giles FJ et€al (2004) Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2′-deoxycytidine (decitabine) in hematopoietic malignancies. Blood 103:1635–1640 51. Kantarjian H, Oki Y, Garcia-Manero G et€al (2007) Results of a randomized study of 3 schedules of low-dose decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic leukemia. Blood 109:52–57 52. Steensma DP, Baer MR, Slack JL et€al (2009) Multicenter study of decitabine administered daily for 5 days every 4 weeks to adults with myelodysplastic syndromes: the alternative dosing for outpatient treatment (ADOPT) trial. J Clin Oncol 27(23):3842–3848 53. Blum W, Garzon R, Klisovic RB et€al (2010) Clinical response and miR-29b predictive significance in older AML patients treated with a 10-day schedule of decitabine. Proc Natl Acad Sci U S A 107:7473–7478 54. Silverman LR, McKenzie DR, Peterson BL et€al (2006) Further analysis of trials with azacitidine in patients with myelodysplastic syndrome: studies 8421, 8921, and 9221 by the Cancer and Leukemia Group B. J Clin Oncol 24:3895–3903

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55. Kornblith AB, Herndon JE II, Silverman LR et€al (2002) Impact of azacytidine on the quality of life of patients with myelodysplastic syndrome treated in a randomized phase III trial: a Cancer and Leukemia Group B study. J Clin Oncol 20:2441–2452 56. Fenaux P, Mufti GJ, Hellström-Lindberg E et€al (2010) Azacitidine prolongs overall survival compared with conventional care regimens in elderly patients with low bone marrow blast count acute myeloid leukemia. J Clin Oncol 28:562–569 57. Kadia T, Estrov Z, Ravandi F et€al (2009) Long term followup and patterns of failure in patients with acute myeloid leukemia (AML) and high risk myelodysplastic syndrome (MDS) treated on studies combining a hypomethylating agent and the histone deacetylase inhibitor (HDACi) valproic acid. Blood 114:2074 58. Kantarjian HM, Erba HP, Claxton D et€al (2010) Phase II study of clofarabine monotherapy in previously untreated older adults with acute myeloid leukemia and unfavorable prognostic factors. J Clin Oncol 28:549–555 59. Faderl S, Garcia-Manero G, Estrov Z et€al (2010) Oral clofarabine in the treatment of patients with higher-risk myelodysplastic syndrome. J Clin Oncol 28:2755–2760 60. Serova M, Galmarini CM, Ghoul A et€ al (2007) Antiproliferative effects of sapacitabine (CYC682), a novel 2′-deoxycytidine-derivative, in human cancer cells. Br J Cancer 97:628– 636 61. Garcia-Manero G, Luger S, Venugopal P et€al (2009) A randomized phase 2 study of sapacitabine, an oral nucleoside analogue, in older patients with myelodysplastic syndrome (MDS) refractory to hypomethylating agents. Blood 114:1758 62. Garcia-Manero G, Stoltz M, Ward M, Kantarjian H, Sharma S (2008) A pilot pharmacokinetic study of oral azacitidine. Leukemia 22:1680–1684 63. Garcia-Manero G, Gore S, Skikne B et€ al (2009) A phase 1, open-label, dose-escalation study to evaluate the safety, pharmacokinetics, and pharmacodynamics of oral azacitidine in patients with myelodysplastic syndromes (MDS) or acute myelogenous leukemia (AML). Blood 114:117 64. Garcia-Manero G, Issa JP (2005) Histone deacetylase inhibitors: a review of their clinical status as antineoplastic agents. Cancer Invest 23:635–642 65. Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB (1999) Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 21:103–107 66. Yang H, Hoshino K, Sanchez-Gonzalez B, Kantarjian H, Garcia-Manero G (2005) Antileukemia activity of the combination of 5-aza-2′-deoxycytidine with valproic acid. Leuk Res 29:739–748 67. Garcia-Manero G, Yang H, Bueso-Ramos C et€al (2008) Phase 1 study of the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid [SAHA]) in patients with advanced leukemias and myelodysplastic syndromes. Blood 111:1060–1066 68. Garcia-Manero G, Assouline S, Cortes J et€al (2008) Phase 1 study of the oral isotype specific histone deacetylase inhibitor MGCD0103 in leukemia. Blood 112:981–989 69. Giles F, Fischer T, Cortes J et€al (2006) A phase I study of intravenous LBH589, a novel cinnamic hydroxamic acid analogue histone deacetylase inhibitor, in patients with refractory hematologic malignancies. Clin Cancer Res 12:4628–4635 70. Tong WG, Wei Y, Stevenson W et€ al (2010) Preclinical antileukemia activity of JNJ26481585, a potent second-generation histone deacetylase inhibitor. Leuk Res 34:221–228 71. Hu Y, Lu W, Chen G et€al Overcoming resistance to histone deacetylase inhibitors in human leukemia with the redox modulating compound {beta}-phenylethyl isothiocyanate. Blood 116(15):2732–2741 72. Gore SD (2005) Combination therapy with DNA methyltransferase inhibitors in hematologic malignancies. Nat Clin Pract Oncol 2:(Suppl 1):S30–S35 73. Verma A, List AF (2005) Cytokine targets in the treatment of myelodysplastic syndromes. Curr Hematol Rep 4:429–435

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74. Raza A, Galili N, Smith S et€al (2009) Phase 1 multicenter dose-escalation study of ezatiostat hydrochloride (TLK199 tablets), a novel glutathione analog prodrug, in patients with myelodysplastic syndrome. Blood 113:6533–6540 75. Jabbour E, Garcia-Manero G, Batty N et€al Outcome of patients with myelodysplastic syndrome after failure of decitabine therapy. Cancer 116(16):3830–3834 76. Atallah E, Garcia-Manero G (2008) Treatment strategies in myelodysplastic syndromes. Cancer Invest 26:208–216

Chapter 15

Haematopoietic Stem Cell Transplantation   in MDS for Adults Tamás Masszi

Introduction Myelodysplastic syndromes comprise a heterogeneous group of stem cell diseases. Different classification systems like the French American and British (FAB), the World Health Organization (WHO) and the International Prognostic Scoring System (IPSS) tried to differentiate the specific entities and envisage the prognosis. The spectrum is wide, including indolent diseases with a probability of survival over 10 years but on the other end patients rapidly progress to acute myeloid leukaemia and will die within a few months. However, most studies include different entities making difficult the clear interpretation of results. Most of the MDS patients are elder than 60 years. As life expectancy is increasing in the developed world the prevalence of MDS is increasing as well. Allogeneic haematopoietic stem cell transplantation is considered to be the only curative treatment for MDS but most patients are too old for myeloablative transplant. Moreover, patients over the age of sixty with the worst IPSS scores have the shortest life expectancy with standard care urging more effective treatment modalities. Younger patients with better IPSS scores may survive 5–10 years even with supportive treatment but these younger patients are those, who have the best transplant results and can be cured too. Unfortunately, with standard conditioning even these younger patients have a high probability of transplant related mortality of approximately 30–40%. Finally, it must be emphasized, that there are no large prospective studies comparing stem cell transplantation versus standard chemotherapy or supportive care in MDS. In consequence, the therapeutic decision making on stem cell transplantation is very difficult and debates are ongoing in the field.

T. Masszi () Department of Haematology and Stem Cell Transplantation, St. István and St. László Hospital of Budapest, St. László Campus, Gyáli út 5-7, 1097 Budapest, Hungary Tel.: +36-1-455-8218 Fax: +36-1-455-8252 e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_15, ©Â€Springer Science+Business Media B.V. 2011

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I ndication and Timing of Haematopoietic Stem Cell Transplantation The largest retrospective series published comparing a non-transplant approach with stem cell transplantation came from the United States [1]. Cutler et€al. analyzed the treatment outcome according to IPSS of 260 MDS patients receiving HLA-identical sibling donor HSCT versus 184 non-transplant patients treated with supportive care only. This study showed that high risk MDS patients (IPSS intermediate-2 and IPSS high) benefited from immediate HSCT with myeloablative conditioning. On the contrary, patients with IPSS low and intermediate-1 risk score had better overall survival if the transplant was delayed until progression, meaning the development of a new cytogenetic abnormality, the appearance of clinically relevant cytopenia, or the increase of the percentage of marrow blasts (Figs.€15.1 and 15.2). On the basis of this study—although the cohorts’ median ages were between 40 and 50 years, and exclusively IPSS score was the analyzed parameter—it has been generally accepted by most transplant physicians to delay the transplant in low and intermediate-1 IPSS patients, and offer immediate transplant only for patients with intermediate-2 and high IPSS score. Important to note that IPSS does not include all risk parameters determining the probability of survival of MDS patients receiving different kinds of therapy. A new risk model of MDS proposed by the MD Anderson Cancer Center included other parameters like poor performance status, age, and previous transfusions as well [2]. In a recent study of 365 MDS patients transfusion dependence was associated with reduced survival, and increased transplant related mortality. The negative effect of transfusion dependence on TRM and survival was also true for the patients with less than 5% bone marrow blast counts [3]. Therefore, it seems to be prudent to consider early transplant even in patients with low IPSS if they are transfusion dependent.

MDS with < 60–70 years with donor 1. Intermediate –2 or high IPSS 2. Secondary MDS 3. Transfusion dependence 4. MDS with marrow fibrosis with neutropenia/thrombocytopenia

Fig. 15.1↜渀 Timing of HSCT in MDS

YES for any of the points

NO for all points

Immediate transplant

Delay transplant until progression

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MDS < 60–70 years old No donor

Donor (family/MUD/CB)

Blast count > 5%

1. Sorror (HCT-CI) score >2 2. Older than 55 y

No

Both No

Yes

Stem cell collection Chemo/hypomathylating agent and Auto-SCT*

CR

Yes

RIC HSCT

Myeloablative HSCT Blast count >10% No

No

Any yes

BMT or PBSCT

Yes PBSCT

Blast count >10% No

Yes

PBSCT chemo/hypomethylating agent

*recommended only in clinical trials

PBSCT

Fig. 15.2↜渀 Decision making for HSCT in MDS

Two other important bad prognostic features that IPSS also ignores are secondary or therapy related MDS and MDS with marrow fibrosis. Recent publications suggest that patients with secondary MDS or MDS with marrow fibrosis may benefit from earlier transplant [4, 5].

Patient Age, Co-Morbidities and Conditioning Regimen Since three quarters of MDS patients are over the age of 60 and most studies included patients younger than 60 years old, age is a critical issue at decision making when planning stem cell transplantation. There are only a few studies evaluated the outcome after HSCT in older patients. Wallen et€al. published a study on 52 patients over the age of 60 (60–68 year median age 63 year) who received myeloablative conditioning and experienced 27% non relapse mortality at 100 days and 43% at 3 years [6]. Another study of 215 elderly pts (median age 57 year) resulted in a day 100 transplant related mortality of 13% and 30% at 1 year for early disease and 21% and 49% for advanced disease respectively [7]. These results suggest that myeloablative conditioning might be feasible in selected elderly patient populations. However, the relatively high incidence of transplant related mortality gave preference for the application of reduced intensity conditioning (RIC) regimens over the age of 55 year and in those with comorbidities.

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Comorbid conditions in HSCT can be best assessed with the haematopoietic cell transplantation specific comorbidity index (HCT-CI) described by Sorror et€al. [8]. With the help of his score Sorror et€al. reported 186 MDS patients undergoing myeloablative or reduced intensity conditioning HSCT. High comorbidity index was the strongest determinant of overall survival, disease free survival and non-relapse mortality, but unfortunately correlated to a higher relapse risk too. Even in patients younger than 50 year old with high comorbidity index the non relapse mortality of standard conditioning was unacceptably high advocating that those patients should rather be transplanted with reduced intensity conditioning. On the other hand Sorror’s results suggest that patients with low comorbidity index have similar outcome after myeloablative and reduced intensity HSCT as well [9]. No randomized prospective studies compared reduced intensity conditioning versus myeloablative transplants so far. The largest retrospective trial was done by the European Group for Blood and Marrow Transplantation (EBMT) and compared the transplant results of 215 MDS and secondary AML patients who received reduced intensity conditioning (RIC) to that of 621 MDS and secondary AML patients transplanted with standard conditioning regimens [10]. The median age of the reduced intensity group was 56 year, and 45 year for the full conditioning group. The 3-year transplant related mortality rate was 22% for the RIC and 32% for the high dose conditioning cohort (pâ•›=â•›0.04) while the overall survival did not differ significantly (41% for the RIC and 45% for the myeloablative group). In multivariate analysis disease type, disease status, and age significantly influenced overall and progression free survival. Although the currently existing data nor do provide strong evidence for favouring reduced intensity conditioning for patients over the age of 55 years neither are there evidences supporting the opposite, standard conditioning. Graft versus host disease occurs more frequently in elderly patients and RIC transplants generally result in lower incidence of acute GVHD compared to myeloablative conditioning. This may be a reason to choose RIC along with the aim to avoid higher incidence of early transplant related mortality. Weather patients should receive intensive chemotherapy before allo-transplantation is not clear. A large retrospective study did not find benefit of intensive chemotherapy before myeloablative HSCT [3]. However, this is probably not the case with RIC the curative effect of which is primarily based on the antitumour effect of the graft, consequently needs minimal tumour mass at the time of transplant. The results of Martino’s study suggest that RIC results are equal to full conditioning transplants only if patients are transplanted in CR [11]. A prospective randomized study has been launched by the EBMT (www.ebmt. org/5WorkingParties/CLWP/clwp8.html) addressing the question of remission induction in MDS before HSCT. A novel approach to reach CR before transplant in advanced MDS patients is the use of hypomethylating agents instead of intensive chemotherapy. This would especially fit to the RIC HSCT strategy of elderly patients with comorbidities.

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 tem Cell Sources—Bone Marrow (BM) Versus Peripheral S Blood (PBSC)—for HSCT in MDS There are two meta-analyses comparing BM to PBSC grafts in myeloablative HSCT [12, 13]. PBSC grafts resulted in faster neutrophil engraftment and platelet recovery and less relapse rates at 3 years. Regarding non relapse mortality, there was no difference in between the two arms. Both studies described increased risk of acute and chronic GVHD in PBSC transplants. These studies consisted of less than 10% MDS patients, consequently no direct conclusions can be drawn for specific MDS transplants. Dey et€al. compared RIC transplants in a retrospective study according to stem cell source. Faster neutrophil recovery, decreased need of transfusions, and increased risk of chronic GVHD was found with PBSC grafts [14]. A retrospective EBMT trial focused on 234 MDS patients who received HLA matched sibling transplants comparing PBSC (102) and BM (132) grafts. The 2 years event free survival was 50% in the PBSC and 39% in the BM group in spite of a higher percentage of low-risk MDS patients in the BM group. Neutrophil recovery was significantly faster in the PBSC arm [15]. In conclusion, PBSC as stem cell source for allogeneic HSCT especially for advanced MDS seems to be better compared to bone marrow graft. The possibly increased risk of acute and chronic GVHD will accompany with decreased risk of relapse [16].

Autologous Transplantation When achieving complete remission after chemotherapy, autologous haematopoietic stem cell transplantation has been considered in MDS as well. The aim of this kind of post-remission intensification if patients are missing an HLA identical donor is the prevention of relapse, similarly to acute leukaemia. As a matter of fact the therapeutic approach is limited because of the low complete remission rates with sole induction chemotherapy, and because of the difficulties of stem cell collection. In the sole prospective randomized trial comparing allogeneic stem cell transplantation versus autologous HSCT and chemotherapy, stem cell mobilization was successful in only 45% of the patients in the recovery phase following chemotherapy with G-CSF [17]. This is probably the low number of normal residual stem cells and/or the damaged bone marrow stroma in MDS that results in the relatively frequent insufficient stem cell collection product making the transplantation impossible. In this study the 4 year survival of the 65 patients randomized between autologous transplant and chemotherapy was 37% and 27% respectively. There were a few EBMT studies addressing autologous haematopoietic stem cell transplantation in MDS. All of them resulted in an approximately 30–40% survival rate at 3 years with an 60–70% relapse rate while transplant related mortality was around 15% (see Table 15.1).

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Table 15.1↜渀 Autologous HCT in MDS for adults Author N Relapse risk Ducastelle et€al. [18] 53 75% relapsed 336 61% at 3 years de Witte et€al. [19]

Overall DFS survival 68% at 1 year 38% at 1 year 34% at 3 years 24% at 3 years

de Witte et€al. [20] Kroger et€al. [21] de Witte et€al. [22]

35 54.5% at 4 years (all) 33% at 4 years 27% at 4 years 65 58% 35% at 3 years 32% at 3 years 173 58% at 3 years 32% at 3 years 30% at 3 years

Al-Ali et€al. [23]

290 41% at 3 years

41% at 3 years 28%

TRM 9.4% 13% at 3 years 11.4% 12% 29% at 3 years 17%

DFS disease-free survival, TRM transplant related mortality

Ducastelle et€al. published the long term outcome of 53 patients autografted in first complete remission with MDS or secondary AML preceded by MDS. They experienced a relapse rate of 75% with most of the relapses occurring in the first 2 years after the transplant. Approximately 15% of their patients reached long term survival with some of them having continuous complete remission 50–119 months after transplantation. These patients are probably definitively cured [18]. Although a small percentage of patients achieving CR with chemotherapy may benefit from auto HSCT and provide long term disease free survival, in case of an HLA identical donor allogeneic transplantation is generally preferred because of the high relapse rate. According to the final results of the prospective randomized European Intergroup Trial autologous transplantation did not provide longer survival than intensive chemotherapy. Therefore, autologous transplantation is recommended only in clinical trials. This is also clear that better mobilisation approaches are necessary to establish auto-transplantation in MDS for a wider patient population.

Allogenic Transplantation Allogeneic Transplantation with Family Donor Although the results of allogeneic transplantation have been improving over time, age is still a limiting factor. Even if the upper age limit for myeloablative allogeneic HSCT have increased from 50 to 60 years recently due to improvement of transplant technique and supportive care, only a minority of MDS patients will receive this treatment being approximately 75% of patients older than 60 years at diagnosis. In spite of these limitations, in a retrospective analysis the EBMT published the results of 885 transplants with matched siblings. With a follow up of 3 years the overall survival, the disease free survival and the transplant related mortality rates were 41%, 36% and 43% respectively [22].

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The International Bone Marrow Transplant Registry (IBMTR) reported on 452 matched family donor transplants similar results with 42% overall survival, 40% disease free survival and 37% transplant related mortality at 3 years [24]. Conditioning regimen in the myeloablative setting generally consisted of total body irradiation (TBI) and cyclophosphamide (CY) or Busulphan (BU) plus CY. The addition of Thiotepa has been explored in a small cohort of patients with the favourable finding of 75% survival for low risk and 57% for high risk patients [25]. Deeg et€al. reported encouraging results with BU-CY adjusting the busulphan dosage to targeted blood levels. This approach resulted in 28% non-relapse mortality with family donors in their study [26]. The only prospective randomized trial comparing the results of matched related allogeneic and autologous transplantation on an intent to treat basis according to having or not having a donor showed a trend of better 4 year survival (54%) of patients younger than 55 years with a donor compared to those without a donor (41%). Chromosomal characteristics proved to be important prognostic factor while IPSS did not influence the outcome in this study due to an absent impact of the marrow blast percentage as patients were randomized in CR. The study concludes that allogeneic SCT (with family donors) may be the treatment of choice for young MDS patients (younger 55 year), characterized by poor risk or intermediate risk cytogenetics [17].

Allogeneic Transplantation with Matched Unrelated Donor Although several reports have demonstrated the feasibility of the application of matched unrelated donors (MUD) for the treatment of MDS patients the early reports were disappointing. The National Marrow Donor Program (NMDP) in the United States reported 510 patients with MDS transplanted between 1988 and 1998. Disease free survival was 29%, transplant related mortality was 54% at 2 years [27]. Donor and recipient age, HLA mismatching, patient CMV seropositivity, and grades II–IV acute GVHD were the most import factors adversely affected transplant related mortality. Factors associated with better DFS included younger patient age, the diagnosis of refractory anaemia, grade 0 or grade I acute GVHD, higher cell dose, recipient CMV negativity, less than 9 month time interval from diagnosis to transplant and transplantation during the last 4 years of the study. In the targeted busulphan study published by Deeg et al., the results were also much better for the 64 MUD patients resulting 59% relapse free survival and 30% non relapse mortality at 3 years [26]. The above results confirm that MUD transplantation with myeloablative conditioning is able to cure selected MDS patients, but being the transplant related mortality very high there is abundant room for improvement.

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One possible approach is reducing the toxicity of the preparative regimen. Several reports appeared presenting the results of RIC transplantation most of them mixing MUD and family donors. Many of these trials have short follow up and small sample size. In summary, low non relapse mortality, high relapse rate, and low engraftment failure are demonstrated in these trials confirming the feasibility of RIC transplantation in the MUD setting as well. Prospective randomized studies are warranted for comparing RIC regiments with standard conditioning. Results of some representative studies on allogeneic transplantation in MDS for adults published with myeloablative or reduced intensity conditioning are summarized in Tables 15.2 and 15.3 respectively.

Allogeneic Transplantation with Cord Blood Experience with cord blood transplantation is very limited especially in adults. A retrospective analysis of 450 bone marrow and 150 umbilical cord blood transplantation in the US included 27 MDS patients who received bone marrow and 10 who received cord blood. Although subset analysis for MDS patients was not performed overall mortality and treatment failure for the entire population were similar for mismatched bone marrow and umbilical cord blood transplants [38]. The largest single centre study comes from Japan. In the period between 1998 and 2009, 33 advanced MDS patients (7 RAEB and 22 MDS-related secondary AML) received unrelated cord blood transplantation following myeloablative conditioning regimen. The median age was 42 years; the median weight of the patients was 55€kg. The cumulative incidences of neutrophil recovery at day 50 and platelet recovery at day 200 were 91% and 88% respectively. The cumulative incidence of grade II–IV acute GVHD was 67% and 34% of extensive chronic GVHD. The probability of event free survival at 5 years was 70%, transplant related mortality and relapse were 14 and 16% respectively [39]. Majhail et€ al. published results on reduced intensity conditioning umbilical cord blood (UCB) transplantation [40]. They compared 47 related matched family donor transplants to 43 umbilical cord blood transplants. All patients were older than 55 years, 50% having AML/MDS diagnosis. 23% of the cord blood transplants had MDS. The 3-year probabilities of progression free survival (30% versus 34%) and overall survival (43% versus 34%) were similar for matched related donor and cord blood transplant recipients. The cumulative incidence of grade II–IV acute GVHD (42% versus 49%) and transplant related mortality at 6 months (23% versus 28%) were comparable, but UCB recipients had a lower incidence of chronic GVHD at 1 year (40% versus 17%, Pâ•›=â•›0.02). UCB transplant seems to be a possible option in MDS even for elderly patients with reduced intensity conditioning.

DFS/PFS/EFS/RFS 28% at 2 years EFS 29% at 2 years DFS 40% at 3 years DFS 30% at 2 years PFS 41% at 3 years PFS 44% at 3 years PFS 55.6% at 2 years EFS 50% at 4 years DFS (ITT)

Overall survival 30% at 2 years 30% at 2 years 42% at 3 years 27% at 2 years 45% at 3 years 48% at 3 years 62.9% at 2 years 52% at 4 years (ITT)

37% at 3 years TRM 31% at 1 year NRM 32% at 3 years NRM 32%NRM 25% at 2 yearsTRM 19% at 4 years NRM

49% at 2 years TRM 54% at 2 years TRM

TRM/NRM

49 months 31.6 months 50 months 27 months 34.6 months 52 months

7.9 years 24 months

Follow up

DFS disease-free survival, EFS event free survival, F family donor, ITT intent-to treat, MUD matched unrelated donor, NRM nonrelapse mortality, PFS progression free survival, RFS relapse free survival, Syn syngeneic, TRM transplant related mortality

Table 15.2↜渀 Myeloablative HCT in MDS for adults Author N Median age Donor (years) Yakoub-Agha et€al. [28] 70 37 F, MUD, SYN Castro-Malaspina et€al. 510 38 MUD [27] 452 Sierra et€al. [24] 38 F 137 52 F Kojima et€al. [29] 621 45 F Martino et€al. [10] Scott et€al. [30] 112 53 F, MUD 36.7 F, MUD Yakoub-Agha et€al. [31] 236 40 42 F Martino et€al. [11]

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Overall survival 34% at 3 years 37% at 2 years 41% at 3 years 43% at 3 years 41% at 3 years 28% at 3 years 59% at 2 years 27% at 3 years 49% at 4 years (ITT) 45% at 4 years

Donor

F, MUD F, MUD F, MUD MUD F, MUD F, MUD F, MUD F, MUD F F

27% at 3 years PFS 56% at 2 years PFS 34% at 3 years DFS (MDS) 41% at 3 years DFS 33% at 3 years PFS 27% at 3 years PFS 57% at 2 years DFS 27% at 3 years RFS 43% at 4 years DFS (ITT) 43% at 4 years DFS

DFS/PFS/EFS/RFS

30% at 1 year NRM 15% at 1 year NRM 19% at 1 year TRM 30% at 3 years TRM 22% at 3 years NRM 39% NRM 21% at 2 years TRM 32% at 3 years NRM 20% at 4 years NRM 21% at 4 years NRM

TRM/NRM

40 months 20.3 months 36 months 34 months 38 months 20 months 23 months 47 months 50 months 43 months

Follow up

DFS disease-free survival, EFS event free survival, F family donor, ITT intent-to treat, MUD matched unrelated donor, NRM nonrelapse mortality, PFS progression free survival, RFS relapse free survival, Syn syngeneic, TRM transplant related mortality

Table 15.3↜渀 RIC HSCT for adults in MDS Author N Median age (years) De Lima et€al. [32] 94 61/54 Kojima et€al. [29] 70 57 76 Tauro et€al. [33] 52 75 52 Lim et€al. [34] Martino et a1. [10] 215 56 38 Scott et€al. [30] 62 53 110 Lim et€al. [35] Laport et€al. [36] 148 59 59 Martino et€al. [11] 47 53 Valcarcel et€al. [37] 93

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Conclusions and Future Directions Allogeneic haematopoietic stem cell transplantation is the treatment of choice for younger MDS patients who have a histocompatible (sibling, unrelated or umbilical cord blood) donor because in contrast to all other current approaches a subset of patients with MDS may be cured. Patient advising and decision of the transplant should be based on prudent calculation of risks including IPSS, transfusion dependence, marrow blastosis and fibrosis, cytopenia, risks of infection, and comorbidities. Albeit there is a clear chance for cure following allogeneic HSCT there is also a high risk of transplant related mortality and similarly high risk of relapse. Therefore, balanced consulting with the patient is crucial. The discussion should contain description of chances with other therapeutic options including supportive care too. Autologous transplantation should be considered in the frame of clinical trials for those patients who gained complete remission following chemotherapy and do not have an HLA identical donor. Future prospective studies are warranted regarding the use of MUD, and umbilical cord blood transplants comparing to transplantation with family donors. The question is still open, whether remission induction therapy (with cytostatic chemotherapy or with hypomethylating agents or maybe with histone deacetylase inhibitors) will improve the outcome of allogeneic transplant either in the myeloablative or in the reduced intensity conditioning setting. Many of the studies performed so far included mixed patient population of de novo MDS, therapy related secondary MDS and secondary AML arising from MDS. Since these different entities have different prognostic value on transplant outcome future studies should be based on pure patient populations.

References 1. Cutler CS, Lee SJ, Greenberg P et€al (2004) A decision analysis of allogeneic bone marrow transplantation for the myelodysplastic syndromes: delayed transplantation for low-risk myelodysplasia is associated with improved outcome. Blood 104(2):579–585 2. Kantarjian H, O’Brien S, Ravandi F et€al (2008) Proposal for a new risk model in myelodysplastic syndrome that accounts for events not considered in the original International Prognostic Scoring System. Cancer 113(6):1351–1361 3. Alessandrino EP, Della Porta MG, Bacigalupo A et€al (2008) WHO classification and WPSS predict post transplant outcome in patients with myelodysplastic syndrome: a study from the GITMO (gruppo italiano trapianto di midollo osseo). Blood 112:895–902 4. Chang CK, Storer BE, Scott BL et€al (2007) Hematopoietic cell transplantation in patients with myelodysplastic syndrome or acute myeloid leukemia arising from myelodysplastic syndrome: similar outcomes in patients with de novodisease and disease following prior therapy or antecedent hematologic disorders. Blood 110:1379–1387 5. Scott BL, Storer BE, Greene JE et€al (2007) Marrow fibrosis as a risk factor for posttransplantation outcome in patients with advanced myelodysplastic syndrome or acute myeloid leukemia with multilineage dysplasia. Biol Blood Marrow Transplant 13:345–354

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╇ 6. Wallen H, Gooley TA, Deeg HJ et€al (2005) Ablative allogeneic hematopoietic cell transplantation in adults 60 years of age and older. J Clin Oncol 23:3439–3446 ╇ 7. Ditschkowski M, Elmaagaeli AH, Trenschel R et€al (2006) Myeloablative allogeneic hematopoietic stem cell transplantation in elderly patients. Clin Transplant 20:127–131 ╇ 8. Sorror ML, Maris MB, Storb R et€ al (2005) Hematopoietic cell transplantation (HCT)specific comorbidity index: a new tool for risk assessment before allogeneic HCT. Blood 106:2912–2919 ╇ 9. Sorror ML, Sandmaier BM, Storer BE et€al (2007) Comorbidity and disease status based risk stratification of outcomes among patients with acute myeloid leukemia or myelodysplasia receiving allogeneic hematopoietic cell transplantation. J Clin Oncol 25:4246–4254 10. Martino R, Iacobelli S, Brand R et€al (2006) Retrospective comparison of reduced-intensity conditioning and conventional high-dose conditioning for allogeneic hematopoietic stem cell transplantation using HLA-identical sibling donors in myelodysplastic syndromes. Blood 108:836–846 11. Martino R, Valcarcel D, Brunet S et€al (2008) Comparable non-relapse mortality and survival after HLA-identical sibling blood stem cell transplantation with reduced or conventionalintensity preparative regimens for high-risk myelodysplasia or acute myeloid leukemia in first remission. Bone Marrow Transplant 41:33–38 12. Stem Cell Trialists’ Collaborative Group (2005) Allogeneic peripheral blood stem cell compared with bone marrow transplantation in the management of hematologic maligfnancies: an individual patient data meta-analysis of nine randomized trials. J Clin Oncol 23:5074– 5087 13. Cutler C, Giri S, Jeyapalan S et€al (2001) Acute and chronic graft-versus-host disease after allogeneic peripheral-blood stem cell and bone marrow transplantation: a meta-analysis. J Clin Oncol 19:3685–3691 14. Dey BR, Shaffer J, Yee AJ et€ al (2007) Comparison of outcomes after transplantation of peripheral blood stem cells versus bone marrow following an identical nonmyeloablative conditioning regimen. Bone Marrow Transplant 40:19–27 15. Guardiola P, Runde V, Bacigalupo A et€al (2002) Retrospective comparison of bone marrow and granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells for allogeneic stem cell transplantation using HLA identical sibling donors in myelodysplastic syndromes. Blood 99:4370–4378 16. del Canizo MC, Martinez C, Conde E et€al (2003) Peripheral blood is safer than bone marrow as a source of hematopoietic progenitors in patients with myelodysplastic syndromes who receive an allogeneic transplantation. Results from the Spanish registry. Bone Marrow Transplant 32:987–992 17. de Witte T, Hagemeijer A, Suciu S et€al (2010) Value of allogeneic stem cell transplantation (SCT) versus autologous SCT and chemotherapy in patients with myelodysplastic syndromes and secondary acute myeloid leukemia. Final results of a prospective randomized European Intergoup Trial. Hematologica. doi:103324/haematol.2009.019182 18. Ducastelle S, Ades L, Gardin C et€al (2006) Long-term follow up of autologous stem cell transplantation after intensive chemotherapy in patients with myelodysplastic syndrome or secondary acute myeloid leukemia. Haematologica 91:373–376 19. de Witte T, Brand R, van Biezen A et€al (2006) The role of stem cell source in autologous hematopoietic stem cell transplantation for patients with myelodysplastic syndromes. Haematologica 91:750–756 20. de Witte T, Suciu S, Verhoef G et€al (2001) Intensive chemotherapy followed by allogeneic or autologous stem cell transplantation for patients with myelodysplastic syndromes (MDSs) and acute myeloid leukemia following MDS. Blood 98:2326–2331 21. Kroger N, Brand R, van Biezen A et€al (2006) Autologous stem cell transplantation for therapy-related acute myeloid leukemia and myelodysplastic syndrome. Bone Marrow Transplant 37:183–189 22. De Witte T, Hermans J, Vossen J et€al (2000) Haematopoietic stem cell transplantation for patients with myelodysplastic syndromes and secondary acute myeloid leukaemias: a report

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23.

24. 25. 26. 27. 28.

29. 30. 31.

32.

33.

34. 35.

36. 37.

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on behalf of the chronic leukemia working party of the European Group for Blood and Marrow Transplantation. Br J Haematol 110:620–630 Al-Ali HK, Brand R, van Biezen A et€al (2007) A retrospective comparison of autologous and unrelated donor hematopoietic cell transplantation in myelodysplastic syndrome and secondary acute myeloid leukemia: a report on behalf of the Chronic Leukemia Working Party of the European Group for Blood and Mararow Transplantation (EBMT). Leukemia 21:1945–1951 Sierra J, Perez WS, Rozman C et€al (2002) Bone marrow transplantation from HLA-identical siblings as treatment for myelodysplasia. Blood 100:1997–2004 Fujimaki K, Taguchi J, Fujita H et€al (2004) Thiotepa/cyclophosphamide/TBI as a conditioning regimen for allogeneic hematopoietic stem cell transplantation in patients with myelodysplastic syndrome. Bone Marrow Transplant 33:789 Deeg HJ, Storer B, Slattery J et€al (2002) Conditioning with targeted busulfan and cyclophsophamide for hemopoietic stem cell transplantation from related and unrelated donors in patients with myelodysplastic syndrome. Blood 100:1201–1207 Castro-Malaspina H, Harris RE, Gajewski J et€al (2002) Unrelated donor marrow transplantation for myelodysplastic syndromes: outcome analysis in 510 transplants facilitated by the National Marrow Donor Program. Blood 99:1943–1951 Yakoub-Agha I, de La Salmoniere P, Ribaud P et€al (2000) Allogeneic bone marrow transplantation for therapy-related myelodysplastic syndrome and acute myeloid leukemia: a long-term study of 70 patients—Report of the French Society of Bone Marrow Transplantation. J Clin Oncol 18:963–971 Kojima R, Kami M, Kanda Y et€al (2005) Comparison between reduced intensity and conventional myeloablative allogeneic stem-cell transplantation in patients with hematologic malignancies aged between 50 and 59 years. Bone Marrow Transplant 36:667–674 Scott BL, Sandmaier BM, Storer B et€ al (2006) Myeloablative vs nonmyeloablative allogeneic transplantation for patients with myelodysplastic syndrome or acute myelogenous leukemia with multilineage dysplasia: a retrospective analysis. Leukemia 20:128–135 Yakoub Agha I, Mesnil F, Kuentz M et€al (2006) Allogeneic marrow stem cell transplantation from human leukocyte antigen-identical siblings versus human leukocyte antigen-allelicmatched unrelated donors (10/10) in patients with standard-risk hematologic malignancy: a prospective study from the French Society of Bone Marrow Transplantation and Cell Therapy. J Clin Oncol 24:5695–5702 de Lima M, Anagnostopoulos A, Munsell M et€al (2004) Nonablative versus reduced-intensity conditioning regimens in the treatment of acute myeloid leukemia and high-risk myelodysplastic syndrome: dose is relevant for long-term disease control after allogeneic hematopoietic stem cell transplantation. Blood 104:865–872 Tauro S, Craddock C, Peggs K et€al (2005) Allogeneic stem cell transplantation used a reduced intensity conditioning regimen has the capacity to produce durable remission and longterm disease free survival in patient with high-risk acute myeloid leukemia and myelodysplasia. J Clin Oncol 23:9387–9393 Lim ZY, Ho AYL, Ingram W et€al (2006) Outcomes of alemtuzumab-based reduced intensity conditioning stem cell transplantation using unrelated donors for myelodysplastic syndromes. Br J Haematol 135:201–209 Lim ZY, Pearce L, HO AYL et€al (2007) Delayed attainment of full donor chimaerism following alemtuzumab-based reduced-intensity conditioning haematopoeitic stem cell transplantation for acute myeloid leukaemia and myelodysplastic syndromes i8s associated with improved outcomes. Br J Haematol 138:517–526 Laport GG, Sandmaier BM, Storer BE et€al (2008) Reduced-intensity conditioning followed by allogeneic hematopoietic cell transplantation for adult patients with myelodysplastic syndrome and myeloproliferative disorders. Biol Blood Marrow Transplant 14:246–255 Valcarcel D, Martino R, Caballero D et€al (2008) Sustained remission of high-risk acute myeloid leukemia and myelodysplastic syndrome after reduced intensity conditioning allogeneic hematopoietic transplantation. Chronic graft-versus-host disease is the strongest factor improving survival. J Clin Oncol 26:577–584

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T. Masszi

38. Laughlin M, Eapen M, Rubinstein M et€ al (2004) Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med 351:2265–2275 39. Sato A, Ooi J, Takahashi S et€al (2010) Unrelated cord blood transplantation after myeloablative conditioning in adults with advanced myeloblastic syndromes. Bone Marrow Transplant. [Epub ahead of print] (9 April 2010; doi: 10.1038/bmt.2010.9). 40. Majhal N, Brunstein C, Toblyn M et€ al (2008) Reduced-intensity allogeneic transplant in patients older than 55 years: unrelated umbilical cord blood is safe and effective for patients without a matched related donor. Biol Blood Marrow Transplant 14(3):282–289

Chapter 16

JMML and Myelodysplastic Syndrome   in Children Henrik Hasle

Introduction Myelodysplastic and myeloproliferative disorders are much rarer in children than in adults. The need for a pediatric approach to the diagnosis and management has emerged over the last two decades [1] and was integrated in the latest WHO classification [2] diving the diseases into three main groups; myelodysplastic syndrome (MDS), juvenile myelomonocytic leukemia (JMML), and the myeloid leukemias of Down syndrome (ML-DS). The JMML like myeloproliferative disorder in children with Noonan syndrome was recently recognized as a specific entity [3]. MDS is the only of the main subgroups in children that has a partial overlap with the spectrum of disease in adults. However, there are significant differences between MDS in children and adults (Table€16.1). There is no sharp age-distinction between adult and childhood MDS and young adults with MDS share many of the features of childhood MDS and may benefit from similar management. Many children have associated abnormalities e.g. preexisting BM failure or congenital abnormalities. Cure is the therapeutic aim in children with MDS which is often not realistic in adults. The rarity of MDS in children and the lack of definite morphologic and cytogenetic markers have contributed to the paucity of MDS in the pediatric literature and very few protocol-based studies on pediatric MDS. Most studies on MDS and JMML have been performed by the European Working Group on MDS in childhood (EWOG-MDS) (www.ewog-mds.org) [4–9].

H. Hasle () Department of Pediatrics, Skejby Hospital, Aarhus University, 8200 Aarhus N, Denmark e-mail: [email protected] J. Várkonyi (ed.), The Myelodysplastic Syndromes, DOI 10.1007/978-94-007-0440-4_16, ©Â€Springer Science+Business Media B.V. 2011

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254 Table 16.1↜渀 Major differences between MDS in children Incidence/year/million and adults RA with ringed sideroblasts Constitutional abnormalities Cytogenetic aberrations -7/del(7q) -5/del(5q) Mutation of NRAS Hypermethylation Main aim of treatment

H. Hasle Children 1–2 <2% 30% 50% 30% 1–2% Rare >50% Curative

Adults >30 25% <5% 40% 10% 20% Common >50% Palliative

Myelodysplastic Syndrome (MDS) Classification The first classification of childhood malignancies including MDS appeared in 2005 [10]. The term preleukemia was predominant in the 1970s but misleading since it gave the impression that the patient has yet to develop leukemia. MDS is a clonal malignant disease in itself and not a precursor of a malignant condition. Furthermore, MDS has confusingly been described together with the transient pancytopenia that may precede acute lymphoblastic leukemia (ALL) [11]. JMML was previously termed juvenile chronic myeloid leukemia (JCML), or chronic myelomonocytic leukemia (CMML). Monosomy 7 in children was included as a separate entity in the early classifications of childhood MDS but complete loss of chromosome 7 occurs in all MDS subgroups and there is no evidence to consider monosomy 7 as a distinct entity [4, 12]. The FAB classification from 1982 has prognostic impact in children [4, 13] but lacked to address the specific diseases in children. The WHO classification from 2001 [14] was also based upon review of adult cases and although JMML was recognized as a separate entity the classification of MDS did not acknowledge the special features of MDS in children. A pediatric approach to the WHO classification separated myelodysplastic and myeloproliferative disorders in children into three main groups; JMML, MDS, and ML-DS (Table€16.2) [1]. MDS is subdivided into refractory cytopenia of childhood (RCC), RAEB and RAEB-T. The change in nomenclature from RA to RCC reflects that anemia is not a prerequisite for the diagnosis [5]. The pediatric modification of the WHO classification allows unambiguous classification of more than 95% of the patients [15]. The revised WHO classification from 2008 [16] keeps JMML separate and re� cognizes ML-DS and RCC as unique groups. Children with more than 2% blasts in the peripheral blood (PB) or 5% in the bone marrow (BM) are classified as RAEB using the same criteria as in adults. RAEB-T is kept for selected patients but it is emphasized that the diagnosis cannot rely on a single blast count but must be a comprehensive evaluation of clinical features, natural course, morphology, immunophenotype, and cytogenetics.

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Table 16.2↜渀 Diagnostic categories of myelodysplastic and myeloproliferative diseases in children according to the pediatric approach to the WHO classification [1] and the revised WHO classification [16] and newer data Myelodysplastic/myeloproliferative disease • Juvenile myelomonocytic leukemia (JMML) clinical diagnosis of NF1 or somatic mutation in PTPN11, RAS, or CBL in 85% • JMML-like myeloproliferative disorder in Noonan syndrome Myeloid proliferations related to Down syndrome (DS) • Transient abnormal myelopoiesis (TAM)a • Myeloid leukemia of Down syndrome (ML-DS)a Myelodysplastic syndrome (MDS) • Refractory cytopenia (RCC) (PB blasts <2% and BM blasts <5%) • Refractory anemia with excess blasts (RAEB) (PB blasts 2–19% or BM blasts 5–19%) • RAEB in transformation (RAEB-T) (PB or BM blasts 20–29%)/AML with myelodysplasiarelated changes (PB or BM blastsâ•›>20%) a

Characterized by GATA1 mutations

Primary and Secondary MDS MDS usually occurs in a previously healthy child and is conformingly named “de novo” or “primary”. MDS may also develop in a child with a known predisposing condition and referred to as “secondary”. Secondary MDS is seen after chemoor radiation therapy (therapy-related MDS), in patients with inherited BM failure disorders or acquired aplastic anemia, and in the context of familial MDS. It is to be recognized, however, that children with so-called “primary” MDS may have an underlying yet unknown genetic defect predisposing them to MDS. Therefore, the distinction between primary and secondary disease may be arbitrary. Myeloid neoplasia in patients with predisposing conditions often shares the biologic characteristics of MDS regardless of the presenting blast count.

Epidemiology Incidence, Sex, Age and Subtype Distribution Combined population based data from Denmark and British Columbia (BC) in Canada showed an annual incidence of MDS of 1.8 and of JMML of 1.2 per million children corresponding to a total of 6% of all hematological malignancies in children (Table€16.3) [17–19]. Data from the UK suggest a lower incidence of MDS of 0.8/million (Table€16.3) [20]. The male/female distribution in pediatric MDS is equal with a median age at presentation of 6.8 years [17, 18, 20–22].

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Table 16.3↜渀 Annual incidence of hematological malignancies in children 0–14 years. Combined data from Denmark 1980–1991 and British Columbia 1982–1996 [17, 18] and data from UK 1990–1999 [20] Denmark and BC UK N % Incidence per million Incidence per million ALL 815 79 38.5 Nd AMLa 115 11 5.4 5.8 MDSa 38 4 1.8 0.8 Myeloid leukemia of DS 19 2 0.9 0.6 JMML 25 2 1.2 0.6 CML 13 1 0.6 0.5 PV/ET 3 0 0.1 Nd Unclassified 3 0 0.1 Total 1,030 100 48.7 – PV polycythemia vera, ET essential thrombocythemia a Excluding Down syndrome (DS)

Associated Abnormalities Constitutional abnormalities are present in up to 30% of childhood MDS [17, 18, 20–23]. Down syndrome has been reported in 25% of those with a morphologic diagnosis of MDS but is no longer included in series of MDS significantly changing the distribution of MDS subtypes [24]. MDS has been reported in a number of constitutional cytogenetic abnormalities other than trisomy 21, but there is only solid evidence for an association between trisomy 8 mosaicism and MDS [25] where trisomy 8 may be constitutional in 15–20% of patients with +8 in the malignant cells [26]. Sporadic reports of MDS and AML in Klinefelter and Turner syndrome have appeared but no increased risk has been documented in larger cohort studies [27]. Inherited Bone Marrow Failure Inherited BM failure disorders have an increased risk of MDS/AML (Table€16.4). The risk varies greatly and is highest in Fanconi anemia, dyskeratosis congenita, Table 16.4↜渀 Inherited bone marrow failures associated with myeloid neoplasia Constitutional conditions Genes involved Lifelong risk of Bone marrow failure syndromes myeloid neoplasia Fanconi anemia 50% 12 autosomal, 1 X-linked Severe congenital neutropenia 20–40% HAX1, ELA2, GFI1, WASP Shwachman-Diamond syndrome 30% SBDS Blackfan-Diamond anemia 2% RPS19, RPS24, RPS17 Dyskeratosis congenita DKC1, TERC, TERT, NOP10, NHP2 10% Amegakaryocytic MPL <10% thrombocytopenia Familial thrombocytopenia No data RUNX1

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and severe congenital neutropeni (SCN). Myeloid leukemia develops in a large fraction of patients with Fanconi anemia during childhood or early adult life. The risk varies according to genetic subgroup and associated abnormalities [28]. The traits of Fanconi anemia may be subtle and the diagnosis should always be considered even in adults. Diagnosing low grade MDS in a patient with Fanconi anemia is a challenge because cytopenia and dysplasia may be part of the BM failure of Fanconi anemia and cytogenetic aberrations may be temporary [29]. Large studies from the International SCN Register show a stable 15-year cumulated risk of MDS of 15% [30]. There is no direct cause-and-effect relationship between the development of MDS and G-CSF therapy but the risk of MDS is highest in patients with a poor response to G-CSF. MDS develops in 30% of those with Shwachman-Diamond syndrome [31] and is often associated with chromosome 7 abnormalities of which isochromosome 7q may represent a separate entity with a long stable clinical course and a low risk of MDS [32]. The acquisition of clonal chromosome abnormalities is more frequent with age reflecting the karyotype instability of the syndrome [33]. Screening 120 children diagnosed with primary RCC identified only one patient with a SBDS gene mutation [34]. The cumulative incidence of MDS/AML in dyskeratosis congenita may be as high as reported in Fanconi anemia [35]. Most patients with dyskeratosis congenita carry mutations in genes encoding components of the telomerase complex; DKC1, TERC or TERT impairing telomerase activity, causing excessive telomere shortening and eventually inducing cellular senescence and apoptosis. Screening of telomere length and telomerase mutations in sibling donors is advised to prevent potentially fatal graft failure [36]. Two large studies identified only three unrecognized TERC mutations among 217 children with MDS [37, 38]. MDS/AML has occasionally been described in patients with Diamond-Blackfan anemia [39], familial platelet disorder [40], congenital amegakaryocytic thrombocytopenia [41] but no reliable estimates are available of the excess risk.

Acquired Aplastic Anemia MDS develops in 10–15% of those patients with aplastic anemia not treated with hematopoietic stem cell transplantation (HSCT) [42]. The risk of MDS is higher among those diagnosed as non-severe aplastic anemia [43], suggesting that some cases of RCC are misdiagnosed as aplastic anemia. A high risk of MDS is particularly noted in those with a poor response to G-CSF [44].

Familial MDS Families with several members affected with MDS often show monosomy 7 or deletion 7q [4, 13, 45]. It is uncertain whether -7 per se increases the risk for familial

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cases. Germline mutations in RUNX1 and CEBPA may cause familial MDS/AML but the genetic cause remains obscure in most reported pedigrees [45]. Some families with several members with MDS show underlying dyskeratosis congenita. It should therefore be considered screening potential sibling donors for telomere length and telomerase mutations to avoid major transplantation complications.

Pathophysiology MDS is a clonal disease arising in a progenitor cell restricted to myelopoiesis, erythropoiesis and megakaryopoiesis but the initiating events have remained obscure, in children like in adults. Mutation in the tumor-suppressor gene TET2 was recently identified in 20% of adult patients with various myeloid disorders including MDS [46]. However, TET2 mutations are not seen in JMML [47] and have not yet been studied in pediatric MDS. Inherited disorders with DNA repair defects like Fanconi anemia or acquired mutations in genes maintaining genetic stability may result in a mutator phenotype predisposing to MDS [48]. Subsequent events, e.g. mutations in proto-oncogenes like RAS, TP53, or WT1, and karyotypic changes like monosomy 7, may be part of a final common pathway of disease progression [49, 50].

Clinical and Laboratory Features The presenting features in almost all cases of MDS are those of pancytopenia. Single lineage cytopenia may occasionally be the presenting characteristic. In a few cases the cytopenia is an incidental finding during a routine work-up. Not all children with RCC have anemia, but macrocytosis (elevated MCV) is a characteristic finding [5]. Fetal hemoglobin (HbF) is frequently moderately elevated. WBC is low to normal. Leukocytosis is generally not a feature of MDS and in the case of increased WBC the diagnosis should be reconsidered (Fig.€16.1). Some patients present with moderate hepatosplenomegaly but most have no organomegaly.

Bone Marrow Features The BM cellularity varies but in contrast to adults hypocellular RCC is the most common finding [5]. Both PB and BM display characteristic dysplastic features with megaloblastic erythropoiesis, bizarre small or unusual large megakaryocytes, and dysgranulopoiesis [51]. The characteristic dysplastic features are suggestive of

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AML

t(8;21)(q22;q22) inv(16)(p13q22) t(15;17)(q22;q12) t(9:11)(p22;q23) t(1;22)(p13;q13)

PB/BM Blasts >30%

–7

BM blasts

WBC >15 –20

<20%

Organomegaly

Repeat BM after 2 weeks

MDS

Fig. 16.1↜渀 Algorithm for distinguishing MDS from AML

MDS but not diagnostic [1]. Interobserver variation in the evaluation of dysplasia exists [52] and centralized review is recommended [51].

Cytogenetics An abnormal karyotype is found in 55% of children with advanced primary MDS and in 76% with secondary advances MDS [53]. Monosomy 7 is the most common cytogenetic abnormality in childhood MDS seen in 25% of the patients [53]. Trisomy 8 and trisomy 21 are the most common numerical abnormalities after monosomy 7. Constitutional trisomy 21 is clinically obvious when present, whereas constitutional trisomy 8 mosaicism may be clinically silent and should be tested for when trisomy 8 is found in the BM [25]. Monosomy 7 as the only cytogenetic aberration is not an unfavorable feature in childhood MDS [4, 12, 21, 54] whereas structural complex abnormalities are associated with a very poor outcome [53]. Monosomy 7 is associated with a shorter time to progression in children with RCC [5]. Favorable cytogenetic aberrations identified in adults as -Y, 20q- and 5q- are so infrequent in children that they are of no practical importance [6]. AML specific translocations, e.g. t(8;21)(q22;q22), t(15;17)(q22;q12), or inv(16) (p13q22) should be considered as AML regardless of the blast count [16].

Immunophenotype Flow cytometry immunophenotyping has not the same diagnostic yield in MDS as in acute leukemia. Only few data on immunophenotype characteristics of MDS in children have been reported [55]. No consensus is available on standard protocols and techniques of flow cytometry in childhood MDS.

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Differential Diagnosis The two main diagnostic challenges are to distinguish MDS with a low blast count from aplastic anemia and other non-clonal disorders including inherited BM failure syndromes and to differentiate MDS with excess of blasts from AML. Refractory Cytopenia Versus Aplastic Anemia BM cellularity is decreased in most cases of RCC [5, 56]. A trephine biopsy is fundamental for the evaluation of a child with suspected aplastic anemia or MDS. Hypoplastic MDS may be difficult to discriminate from aplastic anemia but careful sequential morphologic studies will almost always establish the correct diagnosis. The biopsy in hypoplastic MDS shows scarcely scattered granulopoiesis, patchy islands of immature erythropoiesis and micromegakaryocytes [2, 57]. Immunohistochemcal studies may be helpful demonstrating a high expression of p53 and low expression of survivin in MDS compared with patients with non-clonal BM failures [58]. MDS Versus Non-Clonal Disorders BM dysplasia may be present in a variety of disorders of very different etiologies, e.g. infection, drug therapy and chronic disease. RCC is a diagnosis of exclusion after ruling out infectious diseases like parvovirus [59, 60], herpes virus 6 [61], HIV [62], and visceral leishmaniasis [63]. Vitamin B12 deficiency [64], copper deficiency [65], drug therapy [66], rheumatoid arthritis [67], metabolic disorders [68], and other causes of cytopenia and dysplasia [69, 70]. RARS is extremely rare in children and the finding of sideroblastic anemia should prompt investigation for possible mitochondrial cytopathy like Pearson syndrome or disorders of heme synthesis [71, 72]. It may be difficult to diagnose MDS in children who have a low blast cell count and no clonal marker. The minimal diagnostic criteria listed in Table€16.5 may help in this situation [1]. Dysplasia may be observed in many reactive conditions in children. Since hematopoiesis is often dysplastic in patients with inherited BM failure disorders, MDS should only be diagnosed if the BM blast count is increased, a Table 16.5↜渀 Minimal diagnostic criteria for MDS At least two of the following • Sustained unexplained cytopenia (neutropenia, thrombocytopenia, or anemia) • At least bilineage morphologic myelodysplasia • Acquired clonal cytogenetic abnormality in hematopoietic cells • Increased blasts (≥5%)

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persistent clonal chromosomal abnormality is present, or the BM becomes hypercellular in the presence of persistent PB cytopenia [1]. Separating MDS from AML AML is the major differential diagnosis of advanced MDS. There are significant differences in clinical features, cytogenetics and in response to therapy between MDS and AML [73] reflecting fundamental biologic differences [74] thus making the morphologically based classification a surrogate marker for the distinction between biological entities. A British study suggested a better outcome following AML therapy in patients with RAEB-T compared with RAEB [75] reflecting that the morphologically defined RAEB-T group is heterogeneous and blast count in a single specimen is insufficient to differentiate MDS from AML. Biological features rather than any arbitrary cut-off in blast count may be more important in distinguishing MDS from (chemosensitive) AML [76]. An algorithm to facilitate the distinction between MDS and AML is presented in Fig.€16.1. Monosomy 7 is strongly suggestive of MDS [4] and patients presenting with monosomy 7 and a blast count above 30% may share many features with MDS rather than with true de novo AML [77]. It should be emphasized that most children with myeloid malignancies have clear-cut AML, some have MDS with low blast count and only a few percentages have borderline features.

Prognosis and Natural Course Children with RCC and RAEB or even RAEB-T may show a long and stable clinical course without treatment. Blood transfusions may only be required infrequently and severe infections are rarely seen. The condition may smolder with unchanged cytopenia for months or even years. In a series of 67 children with primary RCC; four died from complications of pancytopenia prior to therapy or progression and 20 progressed to more advanced MDS at a median of 1.7 years from presentation [5]. RCC with monosomy 7 is associated with a higher risk of progression, once progression has occurred the outcome is inferior even after HSCT [5]. The International Prognostic Scoring System (IPSS) for MDS weighted data on BM blasts count, cytopenia and cytogenetics and separated patients into four prognostic groups. Children show more poor risk features than adults (Table€16.6). Only thrombocytopenia and BM blasts >5% correlate with poor survival in children [6]. Overall the IPSS provides little diagnostic information in children but identifies a very small group (7%) of the patients with low-risk disease and a very favorable outcome [6]. Adolescence and complex cytogenetics are associated with a poorer outcome [53].

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Table 16.6↜渀 Distribution and overall survival of children [6] and adults [155] with MDS in the four groups of the international prognostic scoring system (IPSS) IPSS group Children Adults Median survival Median survival N╛=╛142 (%) N╛=╛816 (%) (years) (years) Low 7 33 5.7 >10 Intermediate 1 47 9.7 38 3.5 Intermediate 2 25 4.5 22 1.2 High 21 2.2 7 0.4

Treatment Myeloablative therapy is the only treatment option with a realistic curative potential. Different therapy strategies like hematopoietic growth factors, differentiating agents, amifostine, anti-angiogenic drugs, immune modulation, low dose cytotoxic drugs, or hypomethylating therapy have been investigated in adults not candidates for HSCT. During recent years lenalidomide and hypomethylation therapy have shown promising results with prolonged survival in adults. Lenalidomide is especially effective in patients with del(5q) and unlike to be of benefit in children. Hypomethylating therapy has not yet been properly tested in children. Immunosuppressive therapy with antithymocyte globulin and cyclosporine was given to 39 children with hypoplastic RCC from EWOG-MDS and Japan. Complete or partial response was noted in 70% and the overall and failure-free survival rates at 3 years were 90% and 60%, respectively [78, 79]. The long-term outcome of immunosuppressive therapy in MDS is not known. DNA methyltransferase inhibitors, azacitidine and decitabine, have shown clinical efficacy in adults with MDS [80]. Hypermethylation occur at a similar frequency in children and adults [81, 82] thus making children potential candidates for hypomethylating therapy, however, so far treatment results from pediatrics are lacking. AML Type Chemotherapy Conventional intensive chemotherapy without HSCT is unlikely to eradicate the primitive pluripotent cells involved in MDS. Induction chemotherapy is associated with significant morbidity and mortality with a complete remission rate less than 60%, treatment related mortality rate between 10 and 30%, many relapses, and overall survival less than 30% [12, 22, 73, 75]. However, a few studies have reported outcomes in MDS patients not significantly different from that in AML especially in patients with RAEB-T or AML following MDS [12, 75, 83]. The results reflect the heterogeneous nature of RAEB-T and emphasis that a single morphologically evaluation is insufficient for relevant treatment stratification [1]. Children with monosomy 7 diagnosed as AML have a poor response to induction chemotherapy as in MDS patients but in contrast to MDS those who responded well to chemotherapy had an outcome similar to other AML patients [77].

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Autologous SCT has only been reported infrequently in children. Two studies included eight children with only one long term survivor [12, 75].

Hematopoietic Allogeneic Stem Cell Transplantation HSCT is the therapy of choice for virtually all forms of MDS in childhood. Myeloablative therapy with busulfan, cyclophosphamide, and melphalan, have cured more than half of children with MDS both after matched family donor (MFD) and matched unrelated donor (MUD) HSCT [84, 85]. Total body irradiation (TBI) can generally be omitted since it has no superior anti-leukemic effect compared with busulfan and is associated with more long-term effects in children [86]. Stage of disease has a significant effect on relapse and outcome following HSCT with a very low relapse rate in RCC. In children with RCC and absence of profound cytopenia postponement of HSCT with a watch and wait strategy may be justified especially in patients with a normal karyotype [5]. A fludarabine based reducedintensity conditioning regimen in 19 children with RCC and normal karyotype resulted in an overall survival and DFS at 3 years of 84% and 74%, respectively [87], comparable to those of patients treated with myeloablative HSCT [86]. It remains unknown whether AML-type induction chemotherapy prior to HSCT for advanced MDS can reduce relapse and thus improve DFS. Data from EWOGMDS on children with primary advanced MDS showed no benefit of intensive AML-type therapy preceding HSCT [85]. Small series of patients transplanted as first line therapy have shown survival of 65–70% [73, 88]. Considering the significant morbidity and mortality of induction chemotherapy and the high rate of TRM following HSCT, highest in adolescents [85], most children with MDS may benefit from HSCT as first line therapy sparing the toxicity related to induction chemotherapy. Children without a matched donor and progressive disease should be considered for haploidentical HSCT [89]. Relapse following HSCT is associated with a very grave outcome. Successful withdrawal of immunosuppressive therapy and donor leukocyte infusions in early relapse have occasionally been reported [90, 91]. Close analyses of chimerism status post HSCT may allow initiation of pre-emptive immunotherapy [92].

Myeloid Leukemia and Down Syndrome Individuals with Down syndrome (DS) have a more than 150-fold increased risk of myeloid leukemia during the first five years of life [93]. The recognition of the unique biological features of the GATA1 mutated myeloid malignancy has resulted in consensus about the term myeloid leukemia of Down syndrome (ML-DS) [1, 16] and it is no longer relevant to talk about MDS or AML in young children with DS. Only in the rare case of a DS patient older than 4 years of age without GATA1 mutation may MDS be considered [94].

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Transient Abnormal Myelopoiesis Increased WBC with circulating megakaryoblasts often accompanied by anemia and thrombocytopenia may be seen in up to 10% of newborns with DS. [95]. The percentage of blasts is often higher in blood than in BM. The condition is referred to as transient abnormal myelopoiesis (TAM), transient leukemic reaction, or transient myeloproliferative disorder. The presentation is indistinguishable from leukemia and some have therefore favored the name transient leukemia [95, 96]. Life-threatening complications, mainly progressive hepatic dysfunction, may occur in 10–20% of the patients with TAM, but spontaneous remission appears in the majority within one to three months [96]. Generally no chemotherapy is indicated in TAM, however, in those with progressive hepatic or pulmonary problems or a very high WBC a short course of low-dose cytarabine may be very effective [97]. ML-DS develops 1–3 years later in about 20% of those who have recovered from TAM [95, 96]. The development of ML-DS is predicted with acquired clonal cytogenetic abnormalities [96] and persistently elevated WT1 expression [98].

Myeloid Leukemia of Down syndrome The unique myeloid malignancy in young children with DS is now labeled myeloid leukemia of Down syndrome (ML-DS) [1]. ML-DS is preferred to acute megakaryoblastic leukemia (AKML) because other phenotypes may be seen sharing the same biologic and clinical characteristics. It is no longer appropriate to use the terms MDS or AML (AKML) in young children with DS. Myeloid leukemia in older DS children (4 years or older) tend to be GATA1 negative and has a higher risk of relapse [94, 99]. Such patients may represent spontaneous AML not fulfilling the criteria for ML-DS.

Pathobiology Leukemia in children with trisomy 21 mosaicism selectively involves the trisomic cells pointing at the etiological role of the additional chromosome 21 as the first hit in the multistep process leading to leukemia. Patients with TAM and ML-DS have an acquired mutation in the GATA1 gene [100]. The GATA1 gene encodes a transcription factor essential for the normal erythroid and megakaryocytic differentiation in accordance with the selective involvement of these two lineages in ML-DS [101]. A model of the pathogenic steps in myeloid leukemia of DS is presented in Fig.€16.2. Trisomy 21 is the first event that may predispose the cells to a proliferative advantage or further mutations. GATA1 mutation found in TAM and may be

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2nd event

3rd event

TAM

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20%

Myeloid leukemia

WBC high GATA1 mutation

Trisomy 21

WBC low-normal 5%

? 1%

Normal Liver hematopoiesis Conception

Myeloid leukemia

Bone marrow hematopoiesis Birth

Age 1–3 years

Fig. 16.2↜渀 Pathogenesis of myeloid leukemia in Down syndrome

present in 3–4% of newborns with DS and normal hematology [102]. The mechanisms of the spontaneous regression of TAM remain unexplained but may be associated with the natural switch of hematopoiesis from fetal liver to BM [103]. A large proportion of those with TAM and about 1% (>100 fold increased risk) of DS without abnormal hematology in the newborn period develop myeloid leukemia [93].

Epidemiology Myeloid leukemia develops in 1% of the children with DS [93] corresponding to an annual incidence of 0.6–1.0 per million children [17, 18, 20] (Table€16.3). The age distribution is very unusual with 49% being one year of age at diagnosis, 34% two years of age, and only 2% more than four years of age [104]. Only very few presented before one year of age and there appears to be no age-overlap between TAM and myeloid leukemia of DS [105–107].

Clinical and Laboratory Features Isolated thrombocytopenia is often the presenting feature of ML-DS. Platelet count and WBC are lower at diagnosis than in non-DS patients in contrast to the very high WBC seen in TAM. The blast cells have in most cases morphologic and antigen features of megakaryoblasts, although other morphological variants may occur. Many patients have a relatively indolent course characterized by a period of thrombocytopenia and dysplasia with relatively few blasts in the BM.

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Cytogenetics Numerical aberrations, mainly trisomy 8 and an extra chromosome 21 (tetrasomy 21), are the most common acquired cytogenetic abnormalities [108]. The recurrent structural aberrations seen in AML are not found in ML-DS.

Treatment In contrast to TAM, ML-DS is fatal if untreated but responds well to AML treatment with a very favorable outcome. Several groups have reported long-term survival in DS patients well above 80% [105–107, 109, 110]. DS children are at a low risk for relapse and due to the high risk for treatment related toxicity they benefit from less time-intensive therapy allowing recovery prior to initiation of the next chemotherapeutic course [106, 111]. HSCT is associated with excess toxicity without therapeutic gain and is not indicated in the DS child in first remission [111, 112]. DS myeloblasts are 10 fold more sensitive to cytarabine in vitro than non-DS cells [113, 114]. Further studies of the molecular mechanism of the increased sensitivity to chemotherapy in DS may lead to new approaches in the treatment of AML.

Juvenile Myelomonocytic Leukemia (JMML) JMML is a unique pediatric disorder, pediatric equivalent of what the FAB group termed CMML. JMML was previously named juvenile chronic myeloid leukemia (JCML) recognizing the distinction from CML occurring in older children and adults.

Epidemiology The reported incidence of JMML varies from 0.6 to 1.2/million children per year (Table€ 16.3) [17, 18, 20]. The median age at presentation is 1.8 years, 35% are below one year of age at presentation and only 4% more than 5 years of age [115]. JMML displays a male predominance with a male:female ratio of 2:1 [21, 115]. Neurofibromatosis type 1 (NF1) is associated with a more than 200 fold increased risk of JMML [116]. NF1 is known clinically in 10–15% of the children with JMML; relative more frequent in children diagnosed after 5 years of age [115]. Noonan Syndrome╇ Infants with Noonan syndrome (NS) may show a JMML like myeloproliferative disorder (NS/MPD) with spontaneous regression (Table€16.7). NS/MPD is diagnosed during the first few months of life, often during the first weeks in contrast to the median age of 1.8 years at diagnosis of non-NS JMML

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[115]. In the majority the hematological abnormalities gradually resolve but normalization may take several months or even years especially the monocytosis and splenomegaly which may persist for several years [117, 118]. NS/MPD has striking parallels with the transient leukemia/TAM of newborns with Down syndrome. Unlike the GATA1 mutation in TAM the NS/MPD has no somatic molecular marker and there is no documented effective therapy in those NS patients with an aggressive course. Following the identification of PTPN11 germline mutation in 50% of patients with NS studies in non-NS JMML showed somatic PTPN11 mutations in 35% [119]. The PTPN11 mutations found in JMML have a stronger SHP-2 activation than the mutations in NS, whereas the mutations in NS/MPD have an intermediate gain of function effect [7, 120]. It is presumed that the strong activation resulting from the PTPN11 mutation in JMML is incompatible with life when occurring as a germline mutation.

Clinical and Laboratory Features Patients present with pallor, fever, infection, bleeding or symptoms from the organomegaly [115]. Hepatomegaly, splenomegaly, generalized lymphadenopathy, and skin rash may be the first signs leading to medical attention. Elevated WBC with absolute monocytosis, anemia, and thrombocytopenia are almost universal. WBC at presentation exceeds 50â•›×â•›109/l in 30% and is above 100â•›×â•›109/l in 7% [115]. Increased fetal hemoglobin (HbF) is a main characteristic of JMML with the notable exception of those with monosomy 7 who almost all have normal HbF for age [115].

Table 16.7↜渀 Characteristic findings of JMML-like myeloproliferative disorder (NS/MPD) in children with Noonan syndrome and JMML without Noonan (non-NS) JMML-like JMML Noonan Non-NS Incidence No data 1.2/106/year Age at onset Median age 1.8 years <2 months Leukocytosis ++ ++ Monocytosis ++ ++ Hepatosplenomegaly ++ ++ Myelopoiesis Polyclonal Clonal Cytogenetics Normal Abnormal in 35% 90% (germline) 35% (somatic) PTPN11 mutation Most common substitution T73I E76K Biological effect Moderate GoF Strong GoF Outcome Spontaneous regression Fatal without transplantation GoF gain of function

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A macular-papular skin rash is seen in 35% of the patients [115]. Diabetes insipidus has been reported as the presenting feature in a few cases with JMML and monosomy 7 [4].

Cytogenetics Monosomy 7 (mostly as the sole abnormality) is present in 25–30% of JMML, 10% have other aberrations, and 60% show a normal karyotype [115]. Data from the EWOG-MDS did not show any major clinical differences between JMML in patients with and without -7 [115] and monosomy 7 syndrome is no longer consider a diagnostic entity [4].

Differential Diagnoses JMML may mimic e.g. infections and immunodeficiency delaying the diagnosis. On the other hand infections, inborn errors of metabolism and immunodeficiency may cause monocytosis and organomegaly and represent diagnostic pitfalls. A diagnosis of JMML, especially in infants, should therefore be made with caution [121]. A period of observation is recommended in cases without clear-cut features. Several viral infections mimicking JMML have been reported; Epstein-Barr virus [122], cytomegalovirus [123], herpes virus-6 [124] and parvovirus [125]. Immunodeficiencies like Wiskott-Aldrich syndrome [126] and leukocyte adhesion defect may also mimic JMML [121]. The international consensus on current diagnostic criteria of JMML includes molecular genetics as a mandatory part of the work-up as incorporated in the EWOGMDS 2006 protocol (www.ewog-mds.org). Blood film appearance is characteristic and often more helpful in diagnostics than BM morphology where monocytosis often is much more discrete.

Pathophysiology JMML is a clonal disorder that arises from a pluripotent stem cell [127]. The mononuclear cells of peripheral blood and BM show spontaneous proliferation when cultured in semisolid systems display GM-CSF hypersensitivity providing clues to the molecular aberrancies underlying JMML [128–131]. The hypothesis that a specific defect in the GM-CSF signal transduction pathway plays a major role in the pathogenesis of JMML led to studies of the Ras signal transduction pathway downstream of the receptor (Fig.€16.3). Members of the Ras family of signaling proteins regulate cellular proliferation by cycling between an active guanosin triphosphate (GTP-)-

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Grb2 JAK STAT

Shc

SOS

269

Ras-GDP Neurofibromin

Gab2

SHP-2 Ras-GTP CBL

Ras pathway mutations in JMML somatic NRAS or KRAS 25% PTPN11/SHP-2 35% germline Neurofibromin(NF1) 15% CBL

10–15%

Fig. 16.3↜渀 Aberrations in the Ras signaling pathway leading to excessive proliferation

bound state (Ras-GTP) and an inactive guanosine diphosphate (Ras-GDP)-bound state. Ras activation is a crucial component of the proliferative response to growth factors. Ras point mutations that cause high constitutive Ras-GTP levels are noted in 25% of JMML patients [132–134]. Children with neurofibromatosis type 1 (NF1), have an increased risk of malignant myeloid disorders, especially JMML [116]. About 15% of children with JMML carry the clinical diagnosis of NF1 [115]. The NF1 gene functions as a tumor-suppressor gene, and loss of the normal NF1 allele was noted in leukemic cells of NF-1 patients [135, 136]. As expected, leukemic cells showed an elevated percentage of Ras in the GTP-bound state [137]. NF1 and Ras mutations are mutually exclusive in JMML patients indicating that one abnormality is sufficient to activate Ras. A recent study of CBL in a subset of JMML patients without mutations in RAS or PTPN11 identified mutations in 40% corresponding to 10–15% of JMML patients overall and no CBL mutations in the JMML samples with known mutations in RAS or PTPN11 [138]. The identification of homozygous CBL mutations in JMML suggests that CBL is a new tumor suppressor gene and indicates that CBL may have a role in deregulating the Ras pathway in JMML. CBL mutations are often germline and associated with developmental delay [139]. With somatic PTPN11 mutations in 35%, Ras gene mutations in 25%, and germline NF1 gene mutation in 10–15% and CBL mutation in another 10–15% mutually exclusive abnormalities of the Ras signaling pathway are identified in 85% of the JMML patients (Fig.€16.3).

Natural Course and Prognostic Factors JMML is a rapidly fatal disorder if left untreated. Low platelet count, age above 2 years, high hemoglobin F, and high BM blast count at diagnosis are the main factors predicting a short survival [6, 13, 115]. Multivariate analysis demonstrates a presenting low platelet count as the strongest factor predicting a poor survival. Non-

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transplanted children presenting with a platelet count <33â•›×â•›109/l have an almost 100% mortality within the first year from diagnosis [115, 140]. Blastic transformation is infrequent with JMML and most untreated patients die from organ failure due to infiltration of the leukemic cells. Specific RAS mutations may show spontaneously regression of JMML [141, 142] but the prognostic value of genetic subtype is not yet established [143]. Gene expression may separate JMML into subgroups with distinct prognosis [144].

Treatment Intensive chemotherapy is mostly unsuccessful in JMML because of an increased risk of treatment related death, a low rate of true remissions and long-term survival less than 10% [73, 115, 140]. Isoretinoin (13-cis retinoic acid) has an inhibitory effect on spontaneous growth in vitro and may induce temporary remission in at least a portion of JMML patients [145]. The evaluation of the efficacy of JMML therapy is hampered by the lack of uniform criteria of response and divergent responses in hepatosplenomegaly, white cell, and platelet count as well as the fact that about 20% of patients observed without therapy show response [146]. Purine analogs, etoposide, and cytarabine as single agents are associated with the best response rates for white cell count and spleen size [146]. Reduction in methylation and clearance of the cytogenetic abnormal cells was reported in a patient with JMML and monosomy 7 on azacitidine therapy [147]. The potential for demethylating therapy in JMML is unknown. Allogeneic SCT is the only curative approach for JMML resulting in overall survival in more than half the patients after both family and unrelated donor HSCT [8, 148–150] or cord blood [151]. Younger age at HSCT and male sex predict for improved survival [8]. Busulfan-based myeloablative therapy offers a greater anti-leukemic efficacy than TBI [8]. Disease recurrence remains the major cause of treatment failure. Reduced intensity and duration of GvHD prophylaxis may significantly contribute to successful leukemia control [8] and both acute and chronic GvHD is associated with a lower risk of relapse [8, 148, 149]. Older age, female sex, increased percentage of HbF, and blast percentage in the BM above 20% predicted the occurrence of relapse in univariate analysis [8]. Monosomy 7 is associated with an outcome comparable to or even better than that of patients with normal karyotype [8, 20]. Relapse occurs early at a median of 2–4 months from transplantation [8, 149] and generally within the first year. Early detection of donor cells by increasing mixed chimerism may be successfully eradicated by reducing ongoing immunosuppressive therapy [152]. Donor lymphocyte infusion (DLI) in JMML relapse is largely unsuccessful [153]. A second or even a third transplantation gives a relatively high chance of survival [9, 154].

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References ╇ 1. Hasle H, Niemeyer CM, Chessells JM et€al (2003) A pediatric approach to the WHO classification of myelodysplastic and myeloproliferative diseases. Leukemia 17:277–282 ╇ 2. Baumann I, Niemeyer CM, Bennett JM, Shannon K (2008) Childhood myelodysplastic syndrome. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (eds) WHO classification of tumours of haematopoietic and lymphoid tissues. IARC, Lyon, pp€104–107 ╇ 3. Hasle H (2009) Malignant diseases in Noonan syndrome and related disorders. Horm Res 72(Suppl 2):8–14 ╇ 4. Hasle H, Aricò M, Basso G et€al (1999) Myelodysplastic syndrome, juvenile myelomonocytic leukemia, and acute myeloid leukemia associated with complete or partial monosomy 7. Leukemia 13:376–385 ╇ 5. Kardos G, Baumann I, Passmore SJ et€al (2003) Refractory anemia in childhood: a retrospective analysis of 67 patients with particular reference to monosomy 7. Blood 102:1997–2003 ╇ 6. Hasle H, Baumann I, Bergsträsser E et€al (2004) The International Prognostic Scoring System (IPSS) for childhood myelodysplastic syndrome (MDS) and juvenile myelomonocytic leukemia (JMML). Leukemia 18:2008–2014 ╇ 7. Kratz CP, Niemeyer CM, Castleberry RP et€al (2005) The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan Syndrome/myeloproliferative disease. Blood 106:2183–2185 ╇ 8. Locatelli F, Nollke P, Zecca M et€al (2005) Hematopoietic stem cell transplantation (HSCT) in children with juvenile myelomonocytic leukemia (JMML): results of the EWOG-MDS/ EBMT trial. Blood 105:410–419 ╇ 9. Yoshimi A, Mohamed M, Bierings M et€ al (2007) Second allogeneic hematopoietic stem cell transplantation (HSCT) results in outcome similar to that of first HSCT for patients with juvenile myelomonocytic leukemia. Leukemia 21:556–560 10. Steliarova-Foucher E, Stiller C, Lacour B, Kaatsch P (2005) International classification of childhood cancer, 3rd edn. Cancer 103:1457–1467 11. Hasle H, Heim S, Schroeder H, Schmiegelow K, Østergaard E, Kerndrup G (1995) Transient pancytopenia preceding acute lymphoblastic leukemia (pre-ALL). Leukemia 9:605–608 12. Woods WG, Barnard DR, Alonzo TA et€al (2002) Prospective study of 90 children requiring treatment for juvenile myelomonocytic leukemia or myelodysplastic syndrome: a report from the Children’s Cancer Group. J Clin Oncol 20:434–440 13. Passmore SJ, Hann IM, Stiller CA et€al (1995) Pediatric myelodysplasia: A study of 68 children and a new prognostic scoring system. Blood 85:1742–1750 14. Jaffe ES, Harris NL, Stein H, Vardiman JW (2001) (eds) World Health Organization classification of tumours. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. IARC, Lyon 15. Elghetany MT (2007) Myelodysplastic syndromes in children: a critical review of issues in the diagnosis and classification of 887 cases from 13 published series. Arch Pathol Lab Med 131:1110–1116 16. Swerdlow SH, Campo E, Harris NL et€al (2008) WHO classification of tumours of haematopoietic and lymphoid tissues. IARC, Lyon 17. Hasle H, Kerndrup G, Jacobsen BB (1995) Childhood myelodysplastic syndrome in Denmark: incidence and predisposing conditions. Leukemia 9:1569–1572 18. Hasle H, Wadsworth LD, Massing BG, McBride M, Schultz KR (1999) A population-based study of childhood myelodysplastic syndrome in British Columbia, Canada. Br J Haematol 106:1027–1032 19. Hasle H (2000) Incidence of essential thrombocythaemia in children. Br J Haematol 110:751 20. Passmore SJ, Chessells JM, Kempski H, Hann IM, Brownbill PA, Stiller CA (2003) Paediatric MDS and JMML in the UK: a population based study of incidence and survival. Br J Haematol 121:758–767

272

H. Hasle

21. Luna-Fineman S, Shannon KM, Atwater SK et€al (1999) Myelodysplastic and myeloproliferative disorders of childhood: a study of 167 patients. Blood 93:459–466 22. Sasaki H, Manabe A, Kojima S et€al (2001) Myelodysplastic syndrome in childhood: a retrospective study of 189 patients in Japan. Leukemia 15:1713–1720 23. Bader-Meunier B, Mielot F, Tchernia G et€al (1996) Myelodysplastic syndrome in childhood: report of 49 patients from a French multicenter study. Br J Haematol 92:344–350 24. Stary J, Baumann I, Creutzig U, Harbott J, Michalova K, Niemeyer C (2008) Getting the numbers straight in pediatric MDS: distribution of subtypes after exclusion of down syndrome. Pediatr Blood Cancer 50:435–436 25. Hasle H, Clausen N, Pedersen B, Bendix-Hansen K (1995) Myelodysplastic syndrome in a child with constitutional trisomy 8 mosaicism and normal phenotype. Cancer Genet Cytogenet 79:79–81 26. Maserati E, Aprili F, Vinante F et€al (2002) Trisomy 8 in myelodysplasia and acute leukemia is constitutional in 15–20% of cases. Genes Chromosom Cancer 33:93–97 27. Hasle H, Olsen JH, Nielsen J, Hansen J, Friedrich U, Tommerup N (1996) Occurrence of cancer in women with Turner syndrome. Br J Cancer 73:1156–1159 28. Rosenberg PS, Huang Y, Alter BP (2004) Individualized risks of first adverse events in patients with Fanconi anemia. Blood 104:350–355 29. Cioc AM, Wagner JE, MacMillan ML, Defor T, Hirsch B (2010) Diagnosis of myelodysplastic syndrome among a cohort of 119 patients with fanconi anemia: morphologic and cytogenetic characteristics. Am J Clin Pathol 133:92–100 30. Rosenberg PS, Zeidler C, Bolyard AA et€ al (2010) Stable long-term risk of leukaemia in patients with severe congenital neutropenia maintained on G-CSF therapy. Br J Haematol 150:196–199 31. Smith OP (2002) Shwachman-Diamond syndrome. Semin Hematol 39:95–102 32. Minelli A, Maserati E, Nicolis E et€ al (2009) The isochromosome i(7)(q10) carrying c.258â•›+â•›2t>c mutation of the SBDS gene does not promote development of myeloid malignancies in patients with Shwachman syndrome. Leukemia 23:708–711 33. Maserati E, Pressato B, Valli R et€al (2009) The route to development of myelodysplastic syndrome/acute myeloid leukaemia in Shwachman-Diamond syndrome: the role of ageing, karyotype instability, and acquired chromosome anomalies. Br J Haematol 145:190–197 34. Karow A, Flotho C, Schneider M, Fliegauf M, Niemeyer CM (2010) Mutations of the Shwachman-Bodian-Diamond syndrome (SBDS) gene in patients presenting with refractory cytopenia—do we have to screen? Haematologica 95:689–690 35. Alter BP, Giri N, Savage SA et€ al (2010) Malignancies and survival patterns in the National Cancer Institute inherited bone marrow failure syndromes cohort study. Br J Haematol 150:179–188 36. Calado RT, Young NS (2009) Telomere diseases. N Engl J Med 361:2353–2365 37. Field JJ, Mason PJ, An P et€al (2006) Low frequency of telomerase RNA mutations among children with aplastic anemia or myelodysplastic syndrome. J Pediatr Hematol Oncol 28:450–453 38. Ortmann CA, Niemeyer CM, Wawer A, Ebell W, Baumann I, Kratz CP (2006) TERC mutations in children with refractory cytopenia. Haematologica 91:707–708 39. Vlachos A, Ball S, Dahl N et€al (2008) Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference. Br J Haematol 142:859–876 40. Ganly P, Walker LC, Morris CM (2004) Familial mutations of the transcription factor RUNX1 (AML1, CBFA2) predispose to acute myeloid leukemia. Leuk Lymphoma 45:1–10 41. Maserati E, Panarello C, Morerio C et€ al (2008) Clonal chromosome anomalies and propensity to myeloid malignancies in congenital amegakaryocytic thrombocytopenia (OMIM 604498). Haematologica 93:1271–1273 42. Ohara A, Kojima S, Hamajima N et€al (1997) Myelodysplastic syndrome and acute myelogenous leukemia as a late clonal complication in children with acquired aplastic anemia. Blood 90:1009–1013

16â•… JMML and Myelodysplastic Syndrome in Children

273

43. Führer M, Rampf U, Baumann I et€al (2005) Immunosuppressive therapy for aplastic anemia in children: a more severe disease predicts better survival. Blood 106:2102–2104 44. Kojima S, Ohara A, Tsuchida M et€al (2002) Risk factors for evolution of acquired aplastic anemia into myelodysplastic syndrome and acute myeloid leukemia after immunosuppressive therapy in children. Blood 100:786–790 45. Owen C, Barnett M, Fitzgibbon J (2008) Familial myelodysplasia and acute myeloid leukaemia—a review. Br J Haematol 140:123–132 46. Delhommeau F, Dupont S, Della VV et€al (2009) Mutation in TET2 in myeloid cancers. N Engl J Med 360:2289–2301 47. Muramatsu H, Makishima H, Jankowska AM et€al (2010) Mutations of E3 ubiquitin ligase Cbl family members but not TET2 mutations are pathogenic in juvenile myelomonocytic leukemia. Blood 115:1069–1975 48. Maserati E, Minelli A, Pressato B et€al (2006) Shwachman syndrome as mutator phenotype responsible for myeloid dysplasia/neoplasia through karyotype instability and chromosomes 7 and 20 anomalies. Genes Chromosomes Cancer 45:375–382 49. Luna-Fineman S, Shannon KM, Lange BJ (1995) Childhood monosomy 7: epidemiology, biology, and mechanistic implications. Blood 85:1985–1999 50. Tamaki H, Ogawa H, Ohyashiki K et€al (1999) The Wilms’ tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes. Leukemia 13:393–399 51. Cantù-Rajnoldi A, Fenu S, Kerndrup G, van Wering ER, Niemeyer CM, Baumann I (2005) Evaluation of dysplastic features in myelodysplastic syndromes: experience from the morphology group of the European Working Group of MDS in Childhood (EWOG-MDS). Ann Hematol 84:429–433 52. Barnard DR, Kalousek DK, Wiersma SR et€al (1996) Morphologic, immunologic, and cytogenetic classification of acute myeloid leukemia and myelodysplastic syndrome in childhood: a report from the Childrens Cancer Group. Leukemia 10:5–12 53. Gohring G, Michalova K, Beverloo HB et€al (2010) Complex karyotype newly defined: the strongest prognostic factor in advanced childhood MDS. Blood 116:3766–3769 54. Woods WG, Neudorf S, Gold S et€al (2001) A comparison of allogeneic bone marrow transplantation, autologous bone marrow transplantation, and aggressive chemotherapy in children with acute myeloid leukemia in remission. Blood 97:56–62 55. Veltroni M, Sainati L, Zecca M et€al (2009) Advanced pediatric myelodysplastic syndromes: can immunophenotypic characterization of blast cells be a diagnostic and prognostic tool? Pediatr Blood Cancer 52:357–363 56. Goyal R, Varma N, Marwaha RK (2005) Myelodysplastic syndrome with erythroid hypoplasia. J Clin Pathol 58:320–321 57. Elghetany MT, Hudnall SD, Gardner FH (1997) Peripheral blood picture in primary hypocellular refractory anemia and idiopathic acquired aplastic anemia: an additional tool for differential diagnosis. Haematologica 82:21–24 58. Al-Rahawan MM, Alter BP, Bryant BJ, Elghetany MT (2008) Bone marrow cell cycle markers in inherited bone marrow failure syndromes. Leuk Res 32:1793–1799 59. Hasle H, Kerndrup G, Jacobsen BB, Heegaard ED, Hornsleth A, Lillevang ST (1994) Chronic parvovirus infection mimicking myelodysplastic syndrome in a child with subclinical immunodeficiency. Am J Pediatr Hematol Oncol 16:329–333 60. Yarali N, Duru F, Sipahi T, Kara A, Tezic T (2000) Parvovirus B19 infection reminiscent of myelodysplastic syndrome in three children with chronic hemolytic anemia. Pediatr Hematol Oncol 17:475–482 61. Kagialis-Girard S, Durand B, Mialou V et€al (2006) Human herpesvirus 6 infection and transient acquired myelodysplasia in children. Pediatr Blood Cancer 47:543–548 62. Mueller BU, Tannenbaum S, Pizzo PA (1996) Bone marrow aspirates and biopsies in children with human immunodeficiency virus infection. J Pediatr Hematol Oncol 18:266–271 63. Yarali N, Fisgin T, Duru F, Kara A (2002) Myelodysplastic features in visceral leishmaniasis. Am J Hematol 71:191–195

274

H. Hasle

64. Wollman MR, Penchansky L, Shekhter Levin S (1996) Transient 7q- in association with megaloblastic anemia due to dietary folate and vitamin B12 deficiency. J Pediatr Hematol Oncol 18:162–165 65. Angotti LB, Post GR, Robinson NS, Lewis JA, Hudspeth MP, Lazarchick J (2008) Pancytopenia with myelodysplasia due to copper deficiency. Pediatr Blood Cancer 51:693–695 66. Gesundheit B, Kirby M, Lau W, Koren G, Abdelhaleem M (2002) Thrombocytopenia and megakaryocyte dysplasia: an adverse effect of valproic acid treatment. J Pediatr Hematol Oncol 24:589–590 67. Yetgin S, Ozen S, Saatci U, Bakkaloglu A, Besbas N, Kirel B (1997) Myelodysplastic features in juvenile rheumatoid arthritis. Am J Hematol 54:166–169 68. Hinson DD, Rogers ZR, Hoffman GF et€ al (1998) Hematological abnormalities and cholestatic liver disease in two patients with mevalonate kinase deficiency. Am J Med Genet 78:408–412 69. Bader-Meunier B, Rieux-Laucat F, Croisille L et€al (2000) Dyserythropoiesis associated with a fas-deficient condition in childhood. Br J Haematol 108:300–304 70. Kratz CP, Rogge T, Kopp M, Baumann I, Niemeyer CM (2005) Myelodysplastic features in an infant with cystic fibrosis presenting with anaemia, oedema and failure to thrive. Eur J Pediatr 164:56–57 71. Finsterer J (2007) Hematological manifestations of primary mitochondrial disorders. Acta Haematol 118:88–98 72. Atale A, Bonneau-Amati P, Rotig A et€al (2009) Tubulopathy and pancytopaenia with normal pancreatic function: a variant of Pearson syndrome. Eur J Med Genet 52:23–26 73. Hasle H, Kerndrup G, Yssing M et€al (1996) Intensive chemotherapy in childhood myelodysplastic syndrome. A comparison with results in acute myeloid leukemia. Leukemia 10:1269– 1273 74. Albitar M, Manshouri T, Shen Y et€al (2002) Myelodysplastic syndrome is not merely “preleukemia”. Blood 100:791–798 75. Webb DKH, Passmore SJ, Hann IM, Harrison G, Wheatley K, Chessells JM (2002) Results of treatment of children with refractory anaemia with excess blasts (RAEB) and RAEB in transformation (RAEBt) in Great Britain 1990–1999. Br J Haematol 117:33–39 76. Vardiman JW, Harris NL, Brunning RD (2002) The World Health Organization (WHO) classification of the myeloid neoplasms. Blood 100:2292–2302 77. Hasle H, Alonzo TA, Auvrignon A et€ al (2007) Monosomy 7 and deletion 7q in children and adolescents with acute myeloid leukemia: an international retrospective study. Blood 109:4641–4647 78. Yoshimi A, Baumann I, Fuhrer M et€al (2007) Immunosuppressive therapy with anti-thymocyte globulin and cyclosporine A in selected children with hypoplastic refractory cytopenia. Haematologica 92:397–400 79. Hasegawa D, Manabe A, Yagasaki H et€ al (2009) Treatment of children with refractory anemia: the Japanese Childhood MDS Study Group trial (MDS99). Pediatr Blood Cancer 53:1011–1015 80. Fenaux P, Mufti GJ, Hellstrom-Lindberg E et€ al (2009) Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 10:223–232 81. Vidal DO, Paixao VA, Brait M et€al (2007) Aberrant methylation in pediatric myelodysplastic syndrome. Leuk Res 31:175–181 82. Hasegawa D, Manabe A, Kubota T et€al (2005) Methylation status of the p15 and p16 genes in paediatric myelodysplastic syndrome and juvenile myelomonocytic leukaemia. Br J Haematol 128:805–812 83. Creutzig U, Bender-Götze C, Ritter J et€al (1998) The role of intensive AML-specific therapy in treatment of children with RAEB and RAEB-T. Leukemia 12:652–659 84. Castro-Malaspina H, Harris RE, Gajewski J et€al (2002) Unrelated donor marrow transplantation for myelodysplastic syndromes: outcome analysis in 510 transplants facilitated by the National Marrow Donor Program. Blood 99:1943–1951

16â•… JMML and Myelodysplastic Syndrome in Children

275

╇ 85. Strahm B, Nollke P, Zecca M et€al (2010) Hematopoietic stem cell transplantation for advanced MDS in children: results of the EWOG-MDS98 study. Leukemia (in press) ╇ 86. Locatelli F, Noellke P, Fischer A et€ al (2007) Hematopoietic stem cell transplantation (HSCT) after a myeloablative conditioning regimen in children with refractory cytopenia (RC): results of a retrospective analysis from the EWOG-MDS Group. Blood (Abstract) 110:251 ╇ 87. Strahm B, Locatelli F, Bader P et€ al (2007) Reduced intensity conditioning in unrelated donor transplantation for refractory cytopenia in childhood. Bone Marrow Transpl 40:329– 333 ╇ 88. Nagatoshi Y, Okamura J, Ikuno Y, Akamatsu M, Tasaka H (1997) Therapeutic trial of intensified conditioning regimen with high- dose cytosine arabinoside, cyclophosphamide and either total body irradiation or busulfan followed by allogeneic bone marrow transplantation for myelodysplastic syndrome in children. Int J Hematol 65:269–275 ╇ 89. Kalwak K, Wojcik D, Gorczynska E et€al (2004) Allogeneic hematopoietic cell transplantation from alternative donors in children with myelodysplastic syndrome: is that an alternative? Transpl Proc 36:1574–1577 ╇ 90. Tamura K, Kanazawa T, Suzuki M, Koitabashi M, Ogawa C, Morikawa A (2006) Successful rapid discontinuation of immunosuppressive therapy at molecular relapse after allogeneic bone marrow transplantation in a pediatric patient with myelodysplastic syndrome. Am J Hematol 81:139–141 ╇ 91. Skinner R, Velangi M, Bown N (2008) Donor lymphocyte infusions for post-transplant relapse of refractory anemia with excess blasts and monosomy 7. Pediatr Blood Cancer 50:670–672 ╇ 92. Bader P, Niemeyer C, Willasch A et€ al (2005) Children with myelodysplastic syndrome (MDS) and increasing mixed chimaerism after allogeneic stem cell transplantation have a poor outcome which can be improved by pre-emptive immunotherapy. Br J Haematol 128:649–658 ╇ 93. Hasle H, Clemmensen IH, Mikkelsen M (2000) Risks of leukaemia and solid tumours in individuals with Down’s syndrome. Lancet 355:165–169 ╇ 94. Hasle H, Abrahamsson J, Arola M et€al (2008) Myeloid leukemia in children 4 years or older with Down syndrome often lacks GATA1 mutation and cytogenetics and risk of relapse are more akin to sporadic AML. Leukemia 22:1428–1430 ╇ 95. Zipursky A, Brown E, Christensen H, Sutherland R, Doyle J (1997) Leukemia and/or myeloproliferative syndrome in neonates with Down syndrome. Semin Perinatol 21:97–101 ╇ 96. Massey G, Zipursky A, Doyle JJ et€ al (2002) A prospective study of the natural history of transient leukemia (TL) in neonates with Down syndrome (DS): a Pediatric Oncology Group (POG) study. Blood 100:87a ╇ 97. Klusmann JH, Creutzig U, Zimmermann M et€al (2008) Treatment and prognostic impact of transient leukemia in neonates with Down syndrome. Blood 111:2991–2998 ╇ 98. Hasle H, Lund B, Nyvold CG, Hokland P, Østergaard M (2006) WT1 gene expression in children with Down syndrome and transient myeloproliferative disorder. Leuk Res 30:543– 546 ╇ 99. Gamis AS, Alonzo TE, Lange B, Woods WG, Smith FO (2001) Acute myelogenous leukemia (AML) in Downs syndrome (DS) patients: outcome, toxicities, and prognostic factors from the CCG 2891 trial. Blood 98:720a 100. Wechsler J, Greene M, McDevitt MA et€ al (2002) Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome. Nat Genet 32:148–152 101. Zipursky A, Wang H, Brown EJ, Squire J (1994) Interphase cytogenetic analysis of in vivo differentiation in the myelodysplasia of Down syndrome. Blood 84:2278–2282 102. Pine SR, Guo Q, Yin C, Jayabose S, Druschel CM, Sandoval C (2007) Incidence and clinical implications of GATA1 mutations in newborns with Down syndrome. Blood 110:2128– 2131

276

H. Hasle

103. Roy A, Roberts I, Norton A, Vyas P (2009) Acute megakaryoblastic leukaemia (AMKL) and transient myeloproliferative disorder (TMD) in Down syndrome: a multi-step model of myeloid leukaemogenesis. Br J Haematol 147:3–12 104. Hasle H (2001) Pattern of malignant disorders in individuals with Down’s syndrome. Lancet Oncol 2:429–436 105. Creutzig U, Reinhardt D, Diekamp S, Dworzak M, Stary J, Zimmermann M (2005) AML patients with Down syndrome have a high cure rate with AML-BFM therapy with reduced dose intensity. Leukemia 19:1355–1360 106. Abildgaard L, Ellebæk E, Gustafsson G et€al (2006) Optimal treatment intensity in children with Down syndrome and myeloid leukaemia: data from 56 children treated on NOPHOAML protocols and a review of the literature. Ann Hematol 85:275–280 107. Rao A, Hills RK, Stiller C et€ al (2006) Treatment for myeloid leukaemia of Down syndrome: population-based experience in the UK and results from the Medical Research Council AML 10 and AML 12 trials. Br J Haematol 132:576–583 108. Forestier E, Izraeli S, Beverloo B et€al (2008) Cytogenetic features of acute lymphoblastic and myeloid leukemias in pediatric patients with Down syndrome—an iBFM-SG study. Blood 111:1575–1583 109. Gamis AS, Woods WG, Alonzo TA et€al (2003) Increased age at diagnosis has a significantly negative effect on outcome in children with Down syndrome and acute myeloid leukemia: a report from the Children’s Cancer Group Study 2891. J Clin Oncol 21:3415–3422 110. Kudo K, Kojima S, Tabuchi K et€al (2007) Prospective study of a pirarubicin, intermediatedose cytarabine, and etoposide regimen in children with Down syndrome and acute myeloid leukemia: the Japanese Childhood AML Cooperative Study Group. J Clin Oncol 25:5442– 5447 111. Lange BJ, Kobrinsky N, Barnard DR et€ al (1998) Distinctive demography, biology, and outcome of acute myeloid leukemia and myelodysplastic syndrome in children with Down syndrome: Children’s Cancer Group Studies 2861 and 2891. Blood 91:608–615 112. Creutzig U, Ritter J, Vormoor J et€al (1996) Myelodysplasia and acute myelogenous leukemia in Down’s syndrome. A report of 40 children of the AML-BFM Study Group. Leukemia 10:1677–1686 113. Taub JW, Huang X, Matherly LH et€ al (1999) Expression of chromosome 21-localized genes in acute myeloid leukemia: differences between Down syndrome and non-Down syndrome blast cells and relationship to in vitro sensitivity to cytosine arabinoside and daunorubicin. Blood 94:1393–1400 114. Zwaan CM, Kaspers GJ, Pieters R et€al (2002) Different drug sensitivity profiles of acute myeloid and lymphoblastic leukemia and normal peripheral blood mononuclear cells in children with and without Down syndrome. Blood 99:245–251 115. Niemeyer CM, Aricò M, Basso G et€al (1997) Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases. Blood 89:3534–3543 116. Stiller CA, Chessells JM, Fitchett M (1994) Neurofibromatosis and childhood leukaemia/ lymphoma: a population based UKCCSG study. Br J Cancer 70:969–972 117. Bader-Meunier B, Tchernia G, Mielot F et€al (1997) Occurrence of myeloproliferative disorders in patients with Noonan syndrome. J Pediatr 130:885–889 118. Niihori T, Aoki Y, Ohashi H et€al (2005) Functional analysis of PTPN11/SHP-2 mutants identified in Noonan syndrome and childhood leukemia. J Hum Genet 50:192–202 119. Tartaglia M, Niemeyer CM, Fragale A et€al (2003) Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 34:148–150 120. Loh ML, Vattikuti S, Schubbert S et€al (2004) Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis. Blood 103:2325–2331 121. Karow A, Baumann I, Niemeyer CM (2009) Morphologic differential diagnosis of juvenile myelomonocytic leukemia—pitfalls apart from viral infection. J Pediatr Hematol Oncol 31:380

16â•… JMML and Myelodysplastic Syndrome in Children

277

122. Herrod HG, Dow LW, Sullivan JL (1983) Persistent Epstein-Barr virus infection mimicking juvenile chronic myelogenous leukemia: immunologic and hematologic studies. Blood 61:1098–1104 123. Kirby MA, Weitzman S, Freedman MH (1990) Juvenile chronic myelogenous leukemia: differentiation from infantile cytomegalovirus infection. Am J Pediatr Hematol Oncol 12:292–296 124. Lorenzana A, Lyons H, Sawaf H, Higgins M, Carrigan D, Emanuel PD (1997) Human herpes virus-6 (HHV-6) infection in an infant mimicking juvenile chronic myelogenous leukemia. J Pediatr Hematol Oncol 19:370 125. Gupta N, Gupta R, Bakhshi S (2009) Transient myeloproliferation mimicking JMML associated with parvovirus infection of infancy. Pediatr Blood Cancer 52:411–413 126. Watanabe N, Yoshimi A, Kamachi Y et€al (2007) Wiskott-Aldrich syndrome is an important differential diagnosis in male infants with juvenile myelomonocytic leukemialike features. J Pediatr Hematol Oncol 29:836–838 127. Busque L, Gilliland DG, Prchal JT et€al (1995) Clonality in juvenile chronic myelogenous leukemia. Blood 85:21–30 128. Niemeyer CM, Kratz CP (2008) Paediatric myelodysplastic syndromes and juvenile myelomonocytic leukaemia: molecular classification and treatment options. Br J Haematol 140:610–624 129. Koike K, Matsuda K (2008) Recent advances in the pathogenesis and management of juvenile myelomonocytic leukaemia. Br J Haematol 141:567–575 130. Emanuel PD (2008) Juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia. Leukemia 22:1335–1342 131. Chan RJ, Cooper T, Kratz CP, Weiss B, Loh ML (2009) Juvenile myelomonocytic leukemia: a report from the 2nd International JMML Symposium. Leuk Res 33:355–362 132. Miyauchi J, Asada M, Sasaki M, Tsunematsu Y, Kojima S, Mizutani S (1994) Mutations of the N-ras gene in juvenile chronic myelogenous leukemia. Blood 83:2248–2254 133. Kalra R, Paderanga DC, Olson K, Shannon KM (1994) Genetic analysis is consistent with the hypothesis that NF1 limits myeloid cell growth through p21ras. Blood 84:3435–3439 134. Flotho C, Valcamonica S, Mach-Pascual S et€al (1999) RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML). Leukemia 13:32–37 135. Shannon KM, O’Connell P, Martin GA et€al (1994) Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders. N Engl J Med 330:597–601 136. Side L, Taylor B, Cayouette M et€al (1997) Homozygous inactivation of the NF1 gene in bone marrow cells from children with neurofibromatosis type 1 and malignant myeloid disorders. N Engl J Med 336:1713–1720 137. Bollag G, Clapp DW, Shih S et€al (1996) Loss of NF1 results in activation of the Ras signaling pathway and leads to aberrant growth in haematopoietic cells. Nat Genet 12:144–148 138. Loh ML, Sakai DS, Flotho C et€al (2009) Mutations in CBL occur frequently in juvenile myelomonocytic leukemia. Blood 114:1859–1863 139. Niemeyer CM, Kang MW, Shin DH et€ al (2010) Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia. Nat Genet 42(9):794–800 140. Lutz P, Zix-Kieffer I, Souillet G et€al (1996) Juvenile myelomonocytic leukemia: analyses of treatment results in the EORTC Children’s Leukemia Cooperative Group (CLCG). Bone Marrow Transpl 18:1111–1116 141. Matsuda K, Shimada A, Yoshida N et€al (2007) Spontaneous improvement of hematologic abnormalities in patients having juvenile myelomonocytic leukemia with specific RAS mutations. Blood 109:5477–5480 142. Flotho C, Kratz CP, Bergstrasser E et€al (2008) Genotype-phenotype correlation in cases of juvenile myelomonocytic leukemia with clonal RAS mutations. Blood 111:966–967

278

H. Hasle

143. Yoshida N, Yagasaki H, Xu Y et€ al (2009) Correlation of clinical features with the mutational status of GM-CSF signaling pathway-related genes in juvenile myelomonocytic leukemia. Pediatr Res 65:334–340 144. Bresolin S, Zecca M, Flotho C et€al (2010) Gene expression-based classification as an independent predictor of clinical outcome in juvenile myelomonocytic leukemia. J Clin Oncol 28:1919–1927 145. Castleberry RP, Emanuel PD, Zuckerman KS et€al (1994) A pilot study of isotretinoin in the treatment of juvenile chronic myelogenous leukemia. N Engl J Med 331:1680–1984 146. Bergstraesser E, Hasle H, Rogge T et€al (2007) Non-hematopoietic stem cell transplantation treatment of juvenile myelomonocytic leukemia: a retrospective analysis and definition of response criteria. Pediatr Blood Cancer 49:629–633 147. Furlan I, Batz C, Flotho C et€al (2009) Intriguing response to azacitidine in a patient with juvenile myelomonocytic leukemia and monosomy 7. Blood 113:2867–2868 148. Manabe A, Okamura J, Yumura-Yagi K et€ al (2002) Allogeneic hematopoietic stem cell transplantation for 27 children with juvenile myelomonocytic leukemia diagnosed based on the criteria of the International JMML Working Group. Leukemia 16:645–649 149. Smith FO, King R, Nelson G et€al (2002) Unrelated donor bone marrow transplantation for children with juvenile myelomonocytic leukaemia. Br J Haematol 116:716–724 150. Yusuf U, Frangoul HA, Gooley TA et€al (2004) Allogeneic bone marrow transplantation in children with myelodysplastic syndrome or juvenile myelomonocytic leukemia: the Seattle experience. Bone Marrow Transpl 33:805–814 151. de Vries AC, Bredius RG, Lankester AC et€al (2009) HLA-identical umbilical cord blood transplantation from a sibling donor in juvenile myelomonocytic leukemia. Haematologica 94(2):302–304 152. Yoshimi A, Niemeyer CM, Bohmer V et€al (2005) Chimaerism analyses and subsequent immunological intervention after stem cell transplantation in patients with juvenile myelomonocytic leukaemia. Br J Haematol 129:542–549 153. Yoshimi A, Bader P, Matthes-Martin S et€al (2005) Donor leukocyte infusion after hematopoietic stem cell transplantation in patients with juvenile myelomonocytic leukemia. Leukemia 19:971–977 154. Faraci M, Micalizzi C, Lanino E et€al (2005) Three consecutive related bone marrow transplants for juvenile myelomonocytic leukaemia. Pediatr Transpl 9:797–800 155. Greenberg P, Cox C, Le Beau MM et€al (1997) International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 89:2079–2088

Appendix

For cytological evaluation of peripheral blood smear and bone marrow aspirate here are a few characteristic slides provided.

PB Alterations

Eosinophil dysgranulation and hypogranulated segment

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Nuclear abnormality in segments and atypical thrombocytes

Hypersegmentation of granulocytes

Appendix

Appendix Pseudo Pelger Huet anomaly

BM Alterations

5q- bone marrow

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282 5q- mononuclear megakaryocyte

Dyserythropoiesis

Dyshamopoiesis

Appendix

Appendix Dyserythropoiesis: pronounced nuclear abnormalities in macronormoblasts

Bone marrow macro erythrocytes with irregular shaped nuclei of dyserythropoiesis

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Appendix

Bone marrow ring sideroblasts with Prussian blue staining of iron

For quality check of bone marrow aspirate Orazi A. Pathobiology (2007) 74: 97–114, for the definition of myeloblasts and ring sideroblasts Mufti G et€al. Haematologica (2008) 93(11): 1712–1717; for the diagnosis of atypical MDS monocytes: Vardiman JW et€al. Introduction and overview of the classification of myeloid neoplasms. In: WHO classification of haemopoietic and lymphoid tissues. IARC: Lyon 2008. Figures€3.1, 3.2, 3.3, and 3.4 (p.€21) are recommended.

Index

5q- syndrome, 49, 107, 169, 224 A aberrant phenotype, 45 aCGH, 64, 91, 92 acute GVHD, 242, 245, 246 Allogenic transplantation, 244 array based techniques, 87 Autologous transplantation, 243–245, 249 B blast cells, 25–27, 33, 35, 36, 38, 39, 45, 48, 51, 166, 265 C Cell cycle, 12, 63, 90, 91 Children, 253–255, 257–261, 263–266, 269, 270 Chromosomal banding analysis, 67 Classification, 2, 25, 28, 39, 43, 48, 49, 51, 69, 71, 75, 89, 96, 97, 103, 106, 107, 109–112, 114, 115, 133, 136–140, 159, 160, 162, 164, 168, 169, 208, 219, 220, 239, 253, 254, 261, 284 CMV seropositivity, 245 coeruloplasmin insufficiency, 176 Conditioning regimen, 164, 241, 242, 245, 246, 263 conventional cytogenetics, 69, 75, 87, 88, 92–94, 96, 108 copper insufficiency, 176 copper supplementation, 182 cord blood, 246, 249, 270 CpG islands, 87, 90 cryptic deletions, 87 cytogenetic aberrations, 92, 107, 257, 259 Cytokines, 89, 148, 150, 151, 205, 206, 209, 210

D deferasirox, 191, 193–196, 198 deferiprone, 193–195, 198 deferoxamine, 193–195, 197–199 Detoxification, 5, 6, 8, 11, 17–20, 90 diagnostic criteria, 32, 40, 43–45, 51, 260, 268 DNA methylation, 87, 90, 91, 109, 227 DNA repair, 5, 6, 11–13, 16–18, 20, 90, 91, 258 Donor, 153, 240, 243–246, 249, 257, 258, 263, 270 Donor and recipient, 245 E Epigenetics, 109 Erythroid stimulating agents (ESAs), 205 Erythropoietin, 46, 47, 89, 140, 175, 205 F flow cytometric scoring system, 136, 138 flow cytometry, 45, 110, 121–124, 126–129, 131–133, 136–140, 259 Fluorescence in situ hybridization (FISH), 46, 67, 108 G Gene expression, 16, 63, 89, 93, 95, 96, 109, 168, 176, 206, 270 Gene mutation, 66, 169, 178, 179, 181, 182, 257, 269 gene silencing, 87, 91, 231 genomic changes, 87, 94, 96 Granulocyte-macrophage colony stimulating factors (GM-CSF), 3, 151, 209, 210, 268

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286 H Hematopoietic stem cell transplantation (HSCT), 257 Hemochromatosis gene mutation, 176 HLA mismatching, 245 I ICUS, 36, 45–48, 52 IDUS, 36, 45–48, 52, 268 immunohistochemistry, 25, 35, 48 ineffective hematopoiesis, 87 IPSS, 2, 4, 10, 44, 50, 51, 69, 72, 74, 91, 94, 107, 108, 110–115, 138–140, 147, 149, 152, 197, 198, 206, 208, 219, 220, 239–241, 245, 249, 261 iron chelation, 177, 182, 183, 190, 191, 194–196, 198–200, 222, 223 iron chelation therapy, 178, 182, 183, 190, 191, 194, 196, 199, 200 iron overload, 12, 107, 110, 175–178, 180–182, 187, 188, 192, 196–200, 205, 208, 223 J Juvenile myelomonocytic leukemia (JMML), 253, 266 K Karyotype evolution, 60, 63, 64, 68, 75 L leukemogenesis, 58, 87, 90 loss of heterozygosity, 66, 75, 87, 92 M matched siblings, 244 matched unrelated donors (MUD), 245 Microarray, 91–93, 95, 96, 163 Molecular genetics, 268 Myelodysplastic syndromes (MDS), 1, 5, 25, 43, 55, 87, 121, 145, 159, 205 Myeloid leukemia of Down syndrome, 263, 264 N Next generation sequencing, 96 non-transferin bound iron (NTBI), 178 Noonan syndrome, 253, 266

Index P peripheral blood stem cell (PBSC), 243 polymorphisms, 6–8, 10, 13, 14, 16–18, 133 prognosis, 39, 55, 57–61, 65–67, 69–72, 74, 88, 90, 94, 103, 104, 106–109, 111, 114, 121, 138, 139, 164, 167–169, 190, 192, 197, 200, 208, 210, 220, 221, 224, 226, 230, 239, 261, 270 prognostic score, 103, 107, 111, 112, 115, 219 R RAEB, 8, 27, 28, 35, 38, 39, 114, 133, 136, 138, 139, 165, 179, 210, 246, 254, 261 RAMCD, 179 RARS, 8, 28, 29, 34, 37–39, 89, 106, 160, 165, 168, 169, 177, 179, 182, 206, 211, 260 Refractory anemia, 25, 27, 28, 34, 37, 38, 94, 106 ring sideroblasts, 28, 29, 32, 37, 38, 44, 111, 131, 160, 168, 284 S SNP-Analysis, 92 susceptibility, 5, 7, 8, 17–20 systemic mastocytosis, 25, 28, 32, 34, 35, 39, 48, 162 T thrombocytopenia, 2, 28, 36, 106, 160, 162, 165, 177, 210, 211, 224, 225, 257, 261, 264, 265, 267 Thrombopoietic agents, 210 Thrombopoietin, 168, 211 Transcription factor, 88, 162, 264 transfusional siderosis, 194 U Uniparental Disomy, 87, 90, 92, 109, 163 unrelated donor, 245, 263, 270 W WPSS, 44, 50, 51, 108, 110, 114, 115, 138