Myeloid Malignancies: An Atlas of Investigation and Diagnosis

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An Atlas of Investigation and Diagnosis

Related titles: Lymphoid Malignancies: an Atlas of Investigation and Diagnosis E Matutes, B Bain, A Wotherspoon ISBN 978 1 904392 67 5 Problem Solving in Haematology G Smith ISBN 978 1 84692 005 9 Problem Solving in Oncology D O’Donnell, M Leahy, M Marples, A Protheroe, P Selby ISBN 978 1 904392 84 2

Website: www.clinicalpublishing.co.uk

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MYELOID MALIGNANCIES

A companion volume in the series comprises an illustrated guide to lymphoid malignancies.

MYELOID MALIGNANCIES Barbara J Bain • Estella Matutes

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This Atlas provides a visual presentation of myeloproliferative neoplasms and myeloid leukaemia, meeting the needs of haematologists and clinical chemists. Highly illustrated throughout with colour photographs and diagrams, it covers clinical presentation, haematological and pathological features, immunophenotyping and cytogenetic and genetic abnormalities. This volume is a unique addition to the literature and an essential reference for haematologists.

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MYELOID MALIGNANCIES

Bain • Matutes

An Atlas of Investigation and Diagnosis

ISBN: 978 1 84692 055 4

CLINICAL PUBLISHING

CLINICAL PUBLISHING

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An Atlas of Investigation and Diagnosis

MYELOID MALIGNANCIES Barbara J Bain

MB BS, FRACP, FRCPath Professor of Diagnostic Haematology Faculty of Medicine, Imperial College, London, UK and Honorary Consultant Haematologist St Mary's Hospital NHS Trust, London, UK

Estella Matutes

MD, PhD, FRCPath Reader in Haemato-Oncology Institute of Cancer Research, London, UK and Consultant Haematologist The Royal Marsden NHS Foundation Trust, London, UK

CLINICAL PUBLISHING OXFORD

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Clinical Publishing an imprint of Atlas Medical Publishing Ltd Oxford Centre for Innovation Mill Street, Oxford OX2 0JX, UK Tel: +44 1865 811116 Fax: +44 1865 251550 Email: [email protected] Web: www.clinicalpublishing.co.uk Distributed in USA and Canada by: Clinical Publishing 30 Amberwood Parkway Ashland OH 44805 USA Tel: 800-247-6553 (toll free within US and Canada) Fax: 419-281-6883 Email: [email protected] Distributed in UK and Rest of W orld by: Marston Book Services Ltd PO Box 269 Abingdon Oxon OX14 4YN, UK Tel: +44 1235 465500 Fax: +44 1235 465555 Email: [email protected]

© Atlas Medical Publishing Ltd 2010 First published 2010 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Clinical Publishing or Atlas Medical Publishing Ltd. Although every effort has been made to ensure that all owners of copyright material have been acknowledged in this publication, we would be glad to acknowledge in subsequent reprints or editions any omissions brought to our attention. A catalogue record of this book is available from the British Library ISBN print 978 1 84692 055 4 ISBN e-book 978 1 84692 614 3 The publisher makes no representation, express or implied, that the dosages in this book are correct. Readers must therefore always check the product information and clinical procedures with the most up-to-date published product information and data sheets provided by the manufacturers and the most recent codes of conduct and safety regulations. The authors and the publisher do not accept any liability for any errors in the text or for the misuse or misapplication of material in this work. Colour reproduction by RDC Publishing Group, Kuala Lumpur, Malaysia Printed by Henry Ling Ltd, The Dorset Press, Dorchester, Dorset, UK

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Contents Acknowledgements

vi

Abbreviations

vi

1 Molecular basis and classification of myeloid neoplasms

1

2 Acute myeloid leukaemia

7

3 Myeloproliferative neoplasms

57

4 Chronic myeloid leukaemia

61

5 Chronic eosinophilic leukaemia

71

6 Polycythaemia vera

75

7 Essential thrombocythaemia

81

8 Idiopathic or primary myelofibrosis

85

9 Systemic mastocytosis

93

10 Myelodysplastic syndromes

99

11 Myelodysplastic/myeloproliferative neoplasms

121

12 Chronic myelomonocytic leukaemia

125

13 Atypical chronic myeloid leukaemia

129

14 Juvenile myelomonocytic leukaemia

133

Index

136

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Acknowledgements We should like to thank Mr Ricardo Morilla and Dr John Swansbury, both from the Royal Marsden Hospital, who have contributed illustrations of cytogenetic analysis and flow cytometry. They are individually acknowledged in the legends to the relevant figures. We also wish to acknowledge, with gratitude, the leadership of Professor Daniel Catovsky and the late Professor David Galton, together with other members of the

FAB group, and that of Professor John Goldman, in the field of haematological malignancy, over the last 40 years. They and other colleagues at St Mary’s Hospital, Hammersmith Hospital, and the Royal Marsden Hospital have generously shared their knowledge with us. Barbara J Bain Estella Matutes

Abbreviations aCML atypical chronic myeloid leukaemia ALIP abnormal localization of immature precursors ALL acute lymphoblastic leukaemia AML acute myeloid leukaemia ATRA all-trans-retinoic acid BM bone marrow c cytoplasmic CAE chloroacetate esterase CD cluster of differentiation CEL chronic eosinophilic leukaemia CML chronic myeloid leukaemia CMML chronic myelomonocytic leukaemia CNS central nervous system DIC disseminated intravascular coagulation FAB French–American–British FISH fluorescence in situ hybridization FSC forward light scatter G-CSF granulocyte colony-stimulating factor H&E haematoxylin and eosin Hb haemoglobin concentration Hct haematocrit HIV human immunodeficiency virus ICUS idiopathic cytopenia of undetermined significance IPSS international prognostic scoring system ITD internal tandem duplication JMD juxtamembrane domain JMML juvenile myelomonocytic leukaemia LDC lymphoid dendritic cell

MDS myelodysplastic syndrome/syndromes MGG May–Grünwald–Giemsa MPD myeloproliferative disorder/disorders MPN myeloproliferative neoplasm/neoplasms MPO myeloperoxidase NK natural killer NSE non-specific esterase PAS periodic acid-Schiff PCR polymerase chain reaction Ph Philadelphia PTD partial tandem duplication RAEB refractory anaemia with excess blasts RARS-T refractory anaemia with ring sideroblasts and thrombocytosis RBC red cell count RCMD refractory cytopenia with multilineage dysplasia RCMD-RS refractory cytopenia with multilineage dysplasia and ringed sideroblasts RCUD refractory cytopenia with unilineage dysplasia RT-PCR reverse transcriptase polymerase chain reaction RQ-PCR real time quantitative polymerase chain reaction SBB Sudan black B SSC sideways light scatter TdT terminal deoxynucleotidyl transferase TKD tyrosine kinase domain WBC white cell count WHO World Health Organization wt wild type

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Chapter 1

1

Molecular basis and classification of myeloid neoplasms Normal haemopoiesis In the adult, normal haemopoiesis occurs predominantly in the bone marrow, although haemopoietic stem cells circulate in the blood stream and the potential for haemopoiesis in liver, spleen or other tissues is retained. All blood cells are derived ultimately from a pluripotent haemopoietic stem cell, able to give rise to lymphoid and myeloid lineages [1]. The pluripotent stem cells are capable not only of self renewal but also of generating multipotent myeloid stem cells and the common lymphoid stem cells

(Figure 1.1). The multipotent stem cell gives rise in turn to committed progenitor cells from which cells of the major myeloid lineages are derived. Differentiation and maturation are controlled by a variety of cytokines which are to some extent specific for particular cell lines. In addition, the microenvironment and accessory cells such as fibroblasts and fat cells have a role in the differentiation and maturation of stem cells. Cells of haemopoietic origin include mast cells and osteoclasts.

Pluripotent haemopoietic stem cell

Common lymphoid stem cell

Multipotent myeloid stem cell

Bi-, tri- and multipotent progenitor cells Dendritic cell precursor

Erythroid lineage Basophil lineage

Neutrophil lineage

Megakaryocyte lineage Monocyte lineage

Mast cell lineage

T lineage

B NK lineage lineage

Eosinophil lineage

Figure 1.1 A diagram of the stem cell hierarchy and myeloid and lymphoid differentiation pathways. Abbreviation: NK, natural killer.

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Molecular basis and classification of myeloid neoplasms

Pluripotent haemopoietic stem cell

Multipotent myeloid stem cell

Ph-positive CML, PDGFRA- and FGFR1- neoplasms, mixed lineage acute leukaemia

Most AML, MDS, most MPD, plasmacytoid dendritic cell precursor neoplasm Possibly some AML

Bi-, tri- and multipotent progenitor cells

Erythroid lineage

Neutrophil lineage

Basophil lineage

‘Lymphoid’ and ‘myeloid’ dendritic cells

Megakaryocyte lineage

Monocyte lineage

Mast cell lineage

Eosinophil lineage

Figure 1.2 A diagram of the stem cell hierarchy and myeloid differentiation pathways showing the cell in which the causative mutation appears to occur in various haematological neoplasms. Abbreviations: AML acute myeloid leukaemia; CML chronic myeloid leukaemia; MDS myelodysplastic syndrome(s); MPD myeloproliferative disorder(s).

Myeloid neoplasms arise from mutation in a haemopoietic stem cell or progenitor cell (Figure 1.2). Many neoplasms, including most types of acute myeloid leukaemia (AML) and the myelodysplastic syndromes (MDS) arise from a mutated multipotent stem cell. Some chronic myeloid leukaemias arise from mutation in a pluripotent stem cell so that at one stage of the disease the leukaemia may manifest itself as a lymphoid leukaemia or lymphoma. This is true of Philadelphia (Ph)-positive chronic myeloid leukaemia associated with a BCR-ABL1 fusion gene (in which B-lineage and less often T-lineage blast transformation can occur) and of FGFR1-related neoplasms, which at various stages of the disease may be manifest as chronic eosinophilic leukaemia, T-lineage lymphoblastic leukaemia/lymphoma, B-lineage lymphoblastic leukaemia/lymphoma or AML. It is possible that some subtypes of AML arise in a mutated committed progenitor cell without the capacity to differentiate into cells of erythroid or megakaryocyte lineages.

The molecular basis of haematological neoplasms In common with other neoplasms, haematological neoplasms can be viewed as acquired genetic diseases in the sense that they result from genetic alteration in a stem cell that gives rise to an abnormal clone of cells, the behaviour of which is responsible for the disease phenotype. The host immune response also has a role in disease development since the body’s immune response includes some ability to recognize tumour cells and destroy them.

Classification of haematological neoplasms Classification of haematological neoplasms is moving from a period when classification was largely based on clinicopathological features, including morphology and, to a

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Molecular basis and classification of myeloid neoplasms 3

lesser extent, immunophenotype, to a period when definitions are based to some extent on identified molecular abnormalities. Although certain syndromes are defined mainly on the basis of the genetic abnormality these must be interpreted in the light of the clinicopathological features. Thus t(9;22)(q34;q11) and BCR-ABL1 fusion are the hallmark of chronic myelogenous leukaemia (CML) but they can also be observed in acute lymphoblastic leukaemia (ALL) and, uncommonly, AML. Similarly t(15;17)(q22;q12) is the hallmark of acute promyelocytic leukaemia, including its variant form but can be observed, albeit rarely, in transformation of a chronic myeloproliferative neoplasm (MPN). The conditions that are defined largely on a molecular basis are CML, the FIP1L1-PDGFRA syndrome and MPD associated with rearrangement of PDGFRB and FGFR1 genes. A second group of disorders are currently defined on the basis of clinicopathological/morphological features supplemented by cytogenetic/molecular genetic information. This applies to AML, MDS, polycythaemia vera, essential thrombocythaemia, primary myelofibrosis, systemic mastocytosis and juvenile myelomonocytic leukaemia (JMML). There remains a third group of disorders where the disease definition is essentially based on clinicopathological/morphological features, even though relevant cytogenetic/molecular genetic abnormalities are sometimes found. At present chronic myelomonocytic leukaemia (CMML) and atypical chronic myeloid leukaemia (aCML) fall into this group. Although MDS has been placed in the second group, there is only a single cytogenetically defined entity and otherwise its definition remains largely clinicopathological and morphological; it has long been suspected that specific genetic abnormalities should be identifiable in subgroups of MDS but these have been slow to reveal themselves. Myeloid neoplasms have been classified by various expert groups under the aegis of the World Health Organization (WHO) as shown, in simplified form, in Tab le 1.1 (overleaf).

Oncogenic mechanisms Oncogenic mechanisms differ between the chronic MPN and AML. The essential difference between the genetic events in the two groups of disorders is that in MPN they result in an expanded clone of proliferating cells able to

differentiate into end cells of one or more myeloid lineages, whereas in AML cells continue to proliferate but are mainly unable to differentiate to end cells. Mutations in myeloid malignancies include novel fusion genes and mutated genes. Fusion genes can result from a translocation, inversion, insertion or cryptic deletion. Mutated genes may harbour a point mutation, a partial duplication or a small insertion or deletion that alters the reading frame. Genes can be triplicated as the result of trisomy. Genes can be amplified (multiple copies) in double minute chromosomes or in homogeneously staining regions within chromosomes. There can also be epigenetic effects, such as an altered methylation status that alters gene expression. All these changes are related to the formation or activation of oncogenes. In addition, deletion or inactivation of tumour suppressor genes can contribute to oncogenesis. In MPN there is often a mutation in a gene encoding a protein on a signalling pathway between the surface membrane and the nucleus; often this protein is a tyrosine kinase that becomes constitutively activated as a result of the mutation. The neoplastic cells are thus able to proliferate and differentiate without being dependent on growth factors. Examples of such constitutively activated tyrosine kinases include the product of the BCR-ABL1 fusion gene in CML, and the product of a mutated JAK2 gene (JAK2 V617F) in almost all cases of polycythaemia vera and in some cases of essential thrombocythaemia, primary myelofibrosis and refractory anaemia with ring sideroblasts and thrombocytosis (RARS-T). In AML there appears to be a need for at least two mutations to convey the leukaemic phenotype to the neoplastic cells and in some types of AML there are multiple mutations. Particularly in AML with multilineage dysplasia, secondary AML, therapy-related AML and AML in the elderly there are likely to have been multiple mutational events (which can include those leading to loss of activity of tumour suppressor genes). The first genetic subtypes of AML recognized were those associated with recurrent cytogenetic abnormalities that gave rise to fusion genes. Specifically these were: t(15;17)(q22;q12) associated with a PML-RARA fusion gene; t(8;21)(q22;q22) associated with RUNX1-CBFA2T 1; and either inv(16)(p13q22) or t(16;16)(p13;q22) associated with CBFB-MYH11. Each of these subtypes was found to have characteristic cytological features. More recently, genetic subtypes of AML have been recognized, mainly among patients with normal cytogenetic analysis, that are characterized by gene mutation without

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Molecular basis and classification of myeloid neoplasms

Table 1.1 An overview of the classification of myeloid neoplasms

Category

Important subcategories

Acute myeloid leukaemia (AML)

Therapy-related myeloid neoplasms AML with recurrent cytogenetic/genetic abnormalities AML with myelodysplasia-related changes AML not otherwise categorized

The myelodysplastic syndromes (MDS)

Refractory cytopenia, including refractory anaemia, with unilineage dysplasia Refractory anaemia with ring sideroblasts Refractory cytopenia with multilineage dysplasia (with or without ring sideroblasts) Refractory anaemia with excess blasts 5q– syndrome Myelodysplastic syndrome, unclassifiable Childhood myelodysplastic syndrome

Myeloproliferative neoplasm (MPN)

Chronic myelogenous leukaemia (with BCR-ABL1 fusion gene) Chronic neutrophilic leukaemia (occasionally associated with JAK2 V617F mutation) Chronic eosinophilic leukaemias and other chronic myeloid leukaemias (including those associated with rearrangement of the PDGFRA, PDGFRB and FGFR1 genes)* Polycythaemia vera (usually has JAK2 V617F mutation) Essential thrombocythaemia (often has JAK2 V617F mutation) Myelofibrosis (often has JAK2 V617F mutation) Mast cell disease Cutaneous mastocytosis including urticaria pigmentosa Systemic mastocytosis (usually associated with KITD816V mutation) Mast cell leukaemia

The myelodysplastic/ myeloproliferative neoplasms (MDS/MPN)

Chronic myelomonocytic leukaemia Atypical chronic myeloid leukaemia Juvenile myelomonocytic leukaemia (often associated with either PTPN11 or NF1 or RAS mutation)

* In the WHO 2008 classification, myeloid and lymphoid neoplasms associated with rearrangement of neoplasms PDGFRA, PDGFRB and FGFR1 are assigned to a separate category.

chromosomal rearrangement. Specifically these are associated with mutations in either NPM1 [2] or CEBPA [3]. Neither is associated with distinctive cytological features. It has been postulated that for any case of AML

there is a need for two different types of mutation, one designated type I to indicate a mutation that conveys a proliferation or survival advantage to the cells and another, designated type II, which interferes with differentiation [4].

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Molecular basis and classification of myeloid neoplasms 5

Table 1.2 Type I and type II mutations that can interact in the pathogenesis of AML

Class II mutation (interferes with differentiation)

Class I mutation (conveys proliferation or survival advantage) (reported incidence in subtype shown in brackets)

RUNX1-CBFA2T, usually resulting from t(8;21)(q22;q22)

KIT mutation (12–47% of cases) NRAS (c 10%) FLT3-ITD (c 4%)

CBFB-MYH11, usually resulting from inv(16)(p13q22) or t(16;16)(p13;q22)

NRAS (c 30–40%) FLT3-ITD (c 7%) KIT mutation (22–47% of cases)

PML-RARA, usually resulting from t(15;17)(q22;q12)

FLT3-ITD (c 30%) NRAS (c 2%)

CEBPA mutated

FLT3-ITD

NPM1 mutated

FLT3-ITD

ITD, internal tandem duplication

Type I and type II mutations are associated with each other in a non-random manner. It is the type II mutation that can be related to the clinical and haematological phenotype of the disease but the type I mutation is also likely to be essential for leukaemogenesis and often affects prognosis (Table 1.2). In MDS, multiple genetic events occur, which can include changes in oncogenes and tumour suppressor genes. These processes are generally poorly understood. The net result is continuing cell proliferation but with ineffective haemopoiesis, i.e. with an increased rate of apoptotic death of haemopoietic cells in the bone marrow and a resultant failure of production of adequate numbers of end cells. The only subtype of MDS so far linked to a specific cytogenetic abnormality is the 5q– syndrome, in which there is an interstitial deletion of part of the long arm of chromosome 5; several candidate genes that are often deleted have been identified of which RPS14 appears the most likely to be relevant [5]. A deletion of the tumour suppressor gene TP53 at 17p13.1 occurs in some patients with MDS.

Aetiology The aetiology of most instances of myeloid neoplasms is unknown. AML, MDS and MDS/MPN can result from exposure to radiation, anticancer chemotherapy and chemical carcinogens such as benzene. Cigarette smoking also increases the incidence of AML. CML can follow exposure to irradiation or topoisomerase-II-interactive drugs. Genetic predisposition also has an aetiological role. Down’s syndrome predisposes to transient leukaemia in the neonatal period and to acute megakaryoblastic leukaemia in infants. Inherited defects in proto-oncogenes can predispose to leukaemia, e.g. germline mutation in RUNX1 and in CEBPA predispose to AML. Germline mutation of NF1 in neurofibromatosis type 1 and of PTPN11 in Noonan syndrome predispose to JMML. Inherited defects in tumour suppressor genes likewise predispose to various types of leukaemia. Germline mutation of TP53 in the Li Fraumeni syndrome, of RB1 in familial retinoblastoma families and of WT1 in familial Wilms’ tumour families predispose to AML.

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References 1 Nissen-Druey C, Tichelli A, Meyer-Monard S (2005). Human hematopoietic colonies in health and disease. Acta Haematol, 113, 5–96. 2 Falini B, Nicoletti I, Martelli MF, Mecucci C (2007). Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features. Blood, 109, 874–885. 3 Preudhomme C, Sagot C, Boissel N, Cayuela J-M, Tigaud I, de Botton S, et al., for the ALFA Group (2002). Favourable prognostic significance of CEBPA mutations with de novo acute myeloid leukemia: a study from the acute Leukemia French Association (ALFA). Blood, 100, 2717–2723.

4 Gilliland DG (2001). Hematologic malignancies. Curr Opinions Hematol, 8, 189–191. 5 Ebert BL, Pretz J, Bosco J, Chang CY, Tamayo P, Galili N, et al. (2008). Identification of RPS14 as a 5qsyndrome gene by RNA interference screen. Nature, 451, 335–339.

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Chapter 2

7

Acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a malignant disease, usually resulting from mutation in a multipotent haemopoietic stem cell. Normal polyclonal haemopoietic cells in the bone marrow are replaced by a clone of neoplastic cell with a growth advantage over normal cells and with a pronounced defect in differentiation. There is usually neutropenia, anaemia and thrombocytopenia as a result both of the differentiation defect of the neoplastic cells and of the crowding out of normal cells. Leukaemic cells may also suppress the growth of normal cells. Occasionally AML results from a mutation in a pluripotent haemopoietic stem cell able to give rise to both lymphoid and myeloid cells. In other categories of acute leukaemia the mutated cell that gives rise to the leukaemic clone may be a cell already committed to the granulocyte–monocyte lineages. The point in the stem cell hierarchy where mutations occur in cases of apparently pure erythroid leukaemia and megakaryoblastic leukaemia has not been defined. Acute myeloid leukaemia usually arises de no vo . However, a significant minority of cases represent evolution of a preceding haematological disorder, which may have been a myeloproliferative or myelodysplastic disorder, aplastic anaemia or paroxysmal nocturnal haemoglobinuria; these cases are referred to as secondary AML. Others are therapy-related, following prior administration of cytotoxic drugs or exposure to radiation. Further aetiological factors include benzene and cigarette smoking. AML is more common in men than women. The prevalence rises exponentially with age to about 18/100 000/year above the age of 65 years [1]. The median age of onset is about 65 years.

Clinical features Clinical manifestations result either from the proliferation of leukaemic cells or from bone marrow failure that leads to a lack of normal cells. Leukaemic cells can infiltrate tissues, leading to hepatomegaly, splenomegaly, skin infiltrates and swollen gums. Tissue infiltration is particularly a feature when there is monocytic differentiation. As an indirect effect of the leukaemic proliferation there may be hyperuricaemia and occasionally renal failure. The lack of normal cells leads to clinical features of anaemia, neutropenia and thrombocytopenia. Thus there may be pallor, fatigue, breathlessness, fever due to opportunistic infections, purpura and visual impairment (due to retinal haemorrhage). In several subtypes of acute leukaemia, particularly but not only acute promyelocytic leukaemia, there is a profound coagulation defect as a result of both disseminated intravascular coagulation (DIC) and increased fibrinolysis. In such patients purpura and haemorrhagic manifestations are much more pronounced and can be lifethreatening. Occasionally patients with AML present with a tumour at an extramedullary site, e.g. soft tissues such as orbit, lymph nodes, or central nervous system (CNS), while the blood and bone marrow are still apparently normal. A tumour of this type is known as a granulocytic sarcoma or a myeloid sarcoma and it is more frequent in AML with differentiation, either granulocytic or monocytic (French–American–British (FAB) M2, M4 and M5 subtypes).

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Acute myeloid leukaemia

Haematological and pathological features Typically patients with AML have an increased white cell count (WBC) as the result of the presence in the blood of a large number of blast cells. These may be myeloblasts, monoblasts or megakaryoblasts. Because of the lack of differentiation, there are usually few maturing cells so that the neutrophil count is reduced, but occasionally differentiation occurs with the production of dysplastic neutrophils. Sometimes monocytes are increased and occasionally eosinophils. There is a normocytic normochromic anaemia with an inappropriately low reticulocyte count. In patients with dysplastic features or a preceding myelodysplastic syndrome (MDS), the anaemia may be macrocytic. The platelet count is often reduced. A bone marrow aspirate shows hypercellularity with an increase in blast cells. These may comprise almost all the bone marrow cells or there may also be maturing cells of neutrophil, eosinophil or monocyte lineage. As incorporated into the World Health Organization (WHO) classification, a blast cell count of 20% or more is now considered sufficient

for a diagnosis of AML [2]. In some specific instances with recurring cytogenetic abnormalities, the blast cell percentage can be even lower (see below) and in patients with myeloid sarcoma, the blast count is not considered in the diagnosis of AML. Erythropoiesis is usually greatly reduced but may be increased. Megakaryocytes are usually reduced but occasionally increased. There may be dysplasia of one, two or three lineages. Trephine biopsy sections show the features that would be expected from the aspirate; if a particulate, cellular aspirate is obtained trephine biopsy is not essential but if the marrow is hypocellular or fibrotic it becomes important. In rare cases, the bone marrow (aspirate and trephine biopsy specimen) is hypocellular with an increase in fat spaces but with blasts constituting a high percentage of the cells present. This group has been designated hypocellular or hypoplastic AML and does not correspond to a specific morphological or genetic category (Figures 2.1–2.4). Other relevant tests include a coagulation screen,

Table 2.1 A summary of the FAB classification of AML FAB category

Characteristics

M0

AML with minimal evidence of differentiation: MPO, SBB and NSE stains positive in fewer than 3% of blast cells

M1

AML without maturation: MPO and SBB stains positive in at least 3% of blast cells but fewer than 10% of maturing cells of granulocyte or monocyte lineage

M2

AML with maturation: more than 10% of maturing cells of granulocyte lineage and fewer than 20% of monocyte lineage

M3

Acute promyelocytic leukaemia: the dominant cell is either a hypergranular promyelocyte or a dysplastic hypogranular promyelocyte with a lobulated nucleus

M4

Acute myelomonocytic leukaemia: more than 20% of cells are of granulocyte lineage and more than 20% are of monocyte lineage

M5

Acute monocytic/monoblastic leukaemia: NSE positive, fewer than 20% of cells are of granulocyte lineage

M6

Erythroleukaemia: more than 50% of cells are erythroid with blasts constituting at least 30% of non-erythroid cells

M7

Acute megakaryoblastic leukaemia: the dominant cell is a megakaryoblast with megakaryocytes sometimes being increased in number and dysplastic

AML, acute myeloid leukaemia; MPO, myeloperoxidase; NSE, non-specific esterase; SBB, Sudan black B

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Acute myeloid leukaemia 9

including fibrinogen assay and assay of D-dimer, to identify DIC. Uric acid may be elevated and renal and liver function may be impaired. The FAB group classified AML, mainly on the basis of morphological features, into seven subtypes, which are summarized in Tab le 2.1 [3–7]. Diagnosis of AML according to the FAB classification, requires a minimum of

30% bone marrow blast cells (whereas in the WHO classification it has been modified to 20% blast cells). Assigning cases to the M0 and M7 categories requires immunophenotyping as well as morphology and cytochemistry. Haematological features differ between the FAB categories.

Figure 2.1 Trephine biopsy section from a patient with hypoplastic AML showing a markedly hypocellular bone marrow with blast cells. H&E, × 20 objective.

Figure 2.2 Trephine biopsy section from a patient with hypoplastic AML (same patient as Figure 2.1) showing that most cells present are blast cells. H&E, × 100 objective.

Figure 2.3 Trephine biopsy section from a patient with hypoplastic AML (same patient as Figure 2.1) showing that the majority of cells express the stem cell marker, CD34. Immunoperoxidase, CD34, × 50 objective.

Figure 2.4 Trephine biopsy section from a patient with hypoplastic AML (same patient as Figure 2.1) showing that the majority of cells express the stem cell marker, CD34. Immunoperoxidase, CD34, × 100 objective.

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Acute myeloid leukaemia

The FAB classification

M0 AML

The FAB categories are still of relevance since the initial assessment of any case is morphological; in FAB M3 AML the rapid and accurate morphological diagnosis is of considerable clinical importance. The features of the FAB categories will therefore be summarized and illustrated.

In FAB M0 AML (Figures 2.5–2.7) the dominant cell in the bone marrow is a blast cell which has no morphological features that identify it as myeloid. There are no Auer rods and no Sudan black B (SBB)- or myeloperoxidase (MPO)positive granules. The leukaemia is identified as myeloid by immunophenotyping or by ultrastructural cytochemistry (Figure 2.8).

Figure 2.5 Peripheral blood film of a patient with FAB M0 AML showing agranular blast cells with basophilic cytoplasm and a high nucleocytoplasmic ratio; some have a hand-mirror configuration. MGG, low power.

Figure 2.6 Bone marrow aspirate film from a patient with FAB M0 AML showing agranular blast cells with basophilic cytoplasm, which is forming blebs; cytoplasmic blebs are more characteristic of FAB M7 AML. MGG, high power.

Figure 2.7 Bone marrow aspirate film from a patient with FAB M0 AML showing agranular blast cells with a high nucleocytoplasmic ratio and basophilic cytoplasm; the blast cells show considerable variation in cell size. MGG, high power.

Figure 2.8 Ultrastructural cytochemistry showing MPO activity in blast cells from a patient with M0 AML. Unstained section, MPO reaction.

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Acute myeloid leukaemia 11

M1 AML In FAB M1 AML (Figures 2.9–2.14) the dominant cell in the bone marrow is a myeloblast, identified as such by the presence of Auer rods and by SBB or MPO positivity in at least 3% of blast cells; positivity in a lower percentage of cells is not significant since a low number of cytochemically-

Figure 2.9 Bone marrow aspirate film from a patient with FAB M1 AML showing two Auer rods adjacent to the nucleus of a crushed blast cell. Sometimes Auer rods are more easily discernible when a cell is crushed. MGG, low power.

Figure 2.11 Peripheral blood film of a patient with FAB M1 AML (same patient as Figure 2.10) showing strong myeloperoxidase activity that identifies the blast cells as myeloid. MPO, high power.

positive blast cells may represent residual normal cells. Auer rods are also positive for SBB and MPO. Blast cells also give positive reactions for naphthol AS-D chloroacetate esterase (CAE). This subtype of AML is distinguished from M2 and M4 AML by the lower number of maturing cells.

Figure 2.10 Peripheral blood film from a patient with FAB M1 AML showing pleomorphic medium sized to large blast cells with no obvious granules or Auer rods. MGG, low power.

Figure 2.12 Bone marrow aspirate film from a patient with FAB M1 AML showing medium sized blast cells with no granules or Auer rods. MGG, high power.

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Figure 2.13 Bone marrow aspirate film from a patient with FAB M1 AML (same patient as Figure 2.12) showing Sudan black B activity that identifies the blast cells as myeloid. MGG, high power.

Figure 2.14 Bone marrow trephine biopsy section from a patient with hypoplastic AML of FAB M1 type showing a markedly hypocellular marrow in which most of the recognizable cells are blast cells. H&E, low power.

M2 AML In FAB M2 AML (Figures 2.15–2.19) the bone marrow has at least 30% myeloblasts but more than 10% of cells are maturing cells of granulocyte lineage. These maturing cells are often dysplastic. Residual erythroid precursors and megakaryocytes may also be dysplastic. Usually myeloid

Figure 2.15 Peripheral blood film of a patient with FAB M2 AML showing very dysplastic neutrophils (hypogranular and often binucleated). MGG, high power.

cells are of neutrophil lineage, but sometimes they are of eosinophil lineage (Figure 2.20). Blast cells may contain Auer rods. Blasts and maturing cells are positive for SBB, MPO and CAE.

Figure 2.16 Bone marrow aspirate film from a patient with FAB M2 AML showing small to medium sized blast cells, a promyelocyte and a neutrophil. MGG, high power.

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Figure 2.17 Bone marrow aspirate film from a patient with FAB M2 AML (same patient as Figure 2.16) showing strong peroxidase activity in blast cells. However, note that the mature neutrophils are peroxidase deficient. MPO, high power.

Figure 2.18 Bone marrow aspirate film from a patient with FAB M2 AML (same patient as Figure 2.16) showing three blast cells, one of which contains an Auer rod. MPO, high power.

Figure 2.19 Bone marrow aspirate film from a patient with FAB M2 AML (same patient as Figure 2.16) showing an immature cell of eosinophil lineage with large peroxidasepositive granules. MPO, high power.

Figure 2.20 Bone marrow aspirate film from a patient with FAB M2 AML with eosinophilic differentiation. There are Charcot-Leyden crystals (blue) which reflect the increased death of cells of eosinophil lineage. MGG, low power.

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M3 AML FAB M3 AML is cytologically distinctive, the dominant cell being an abnormal promyelocyte. Peripheral blood blast cells may not be greatly elevated. The neoplastic cells vary between cases. In classical acute promyelocytic leukaemia they are hypergranular promyelocytes, packed with large

brightly staining granules and usually containing Auer rods, often in bundles (Figures 2.21–2.24). Cells with bundles of Auer rods are often referred to as faggot cells. There may also be giant granules. In the microgranular or hypogranular variant, granules are very fine and often not visible by light microscopy (Figures 2.25 and 2.26). The distinctive feature

Figure 2.21 Bone marrow aspirate film from a patient with FAB M3 AML showing leukaemic cells packed with fine granules. There are two cells in the centre with giant granules. MGG, medium power.

Figure 2.22 Bone marrow aspirate film from a patient with FAB M3 AML showing a cell with multiple Auer rods and leukaemic cell packed with granules, which is in mitosis. Cytological features in this case are intermediate between the classical and variant forms of the disease. MGG, high power.

Figure 2.23 Bone marrow trephine biopsy section from a patient with FAB M3 AML showing cells with plentiful granular cytoplasm and a central nucleus that has an immature chromatin pattern. H&E, low power.

Figure 2.24 Bone marrow trephine biopsy section from a patient with FAB M3 AML (same patient as Figure 2.23). There are some cells with a nucleus that is lobulated but has an immature chromatin pattern. H&E, low power.

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is the bilobed nucleus, the observation of which should lead to a search for occasional hypergranular cells. The bone marrow aspirate often shows a higher percentage of hypergranular cells than is present in the blood. In a third cytological variant the abnormal promyelocytes have cytoplasmic basophilia and blebs. The promyelocytes, both

Figure 2.25 Bone marrow aspirate film from a patient with FAB M3 AML, variant form, showing leukaemic promyelocytes with lobulated nuclei. A minority of cells have discernible granules and one cell is hypergranular. MGG, intermediate power.

Perox

Baso

in the classical and the variant form, show strong activity for MPO, SBB and CAE. This can be detected not only by cytochemical stains of bone marrow films but also from the printout of automated blood counters that employ peroxidase cytochemistry to perform a differential count (Figure 2.27).

Figure 2.26 Bone marrow aspirate film from a patient with FAB M3 AML, variant form, showing leukaemic promyelocytes with lobulated and irregular nuclei. There is one faggot cell. MGG, high power.

RVC H/VC

PLT scatter

Figure 2.27 Print-out from an Advia automated blood cell counter showing very strong peroxidase activity in a patient with M3 AML.

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M4 AML In FAB M4 AML (Figures 2.28 and 2.29) the bone marrow has at least 30% myeloblasts plus monoblasts and in addition has at least 20% of cells of granulocyte lineage (myeloblasts to polymorphonuclear cells) and at least 20% of cells of monocyte lineage (monoblasts to monocytes). There is often maturation. Granulocytic differentiation is usually neutrophilic but sometimes eosinophilic and in the

latter instance there will also be an increase in eosinophil myelocytes, sometimes with aberrantly staining purple granules (pro-eosinophilic granules). Blast cells may contain Auer rods. Depending on their lineage, leukaemic cells are positive for SBB, MPO and CAE (if granulocytic) or for non-specific esterase (NSE) (if monocytic). A double esterase stain for CAE and NSE is useful for identifying M4 AML (Figure 2.29).

Figure 2.28 Peripheral blood film of a patient with FAB M4 AML showing blasts and maturing cells of both granulocytic and monocytic lineages. MGG, high power.

Figure 2.29 Peripheral blood film of a patient with FAB M4 AML (same patient as Figure 2.28) showing that some cells are positive for chloroacetate esterase (orange-red, indicating granulocyte lineage) and others for non-specific esterase (brownish-black, indicating monocyte lineage). Double esterase, high power.

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M5 AML In FAB M5 AML the bone marrow has at least 30% blast cells (mainly monoblasts) and has fewer than 20% of cells of granulocyte lineage. The dominant cell may be a monoblast (M5a AML) (Figures 2.30–2.37) or there may be maturation to promonocytes and monocytes (M5b) (Figures 2.38–2.42). The promonocyte is a very primitive cell that resembles a monoblast except that its nucleus is

irregular or lobulated. In the WHO classification (see below) promonocytes are given the same significance as blast cells in making a diagnosis of AML. A NSE stain is positive, although the reaction may be negative in the most immature monoblasts. Some monoblasts have fine SBB- and MPO-positive granules. An interesting cytochemical stain that is no longer used in diagnosis is that for lysozyme (Figure 2.43).

Figure 2.30 Bone marrow aspirate film from a patient with FAB M5a AML showing mainly monoblasts. MGG, high power.

Figure 2.31 Bone marrow aspirate film from a patient with FAB M5a AML (same patient as Figure 2.30) showing a variety of cell types with two cells being in mitosis. MGG, high power.

Figure 2.32 Bone marrow aspirate film from a patient with FAB M5a AML (same patient as Figure 2.30) showing two monoblasts and a promonocyte (centre) with a cleft nucleus. MGG, high power.

Figure 2.33 Bone marrow aspirate film from a patient with FAB M5a AML (same patient as Figure 2.30) showing mainly monoblasts. MGG, high power.

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Figure 2.34 Bone marrow trephine biopsy section from a patient with M5a AML showing large blast cells with large nuclei, some of which are lobulated or contain a prominent nucleolus; there is also a prominent macrophage (top right). H&E, high power.

Figure 2.36 Ultrastructural examination of a monoblast from a patient with M5a AML showing a nucleus with euchromatin and a large nucleolus; the cytoplasm contains mitochondria and a ribosomal-lamellar complex (arrow). Lead nitrate and uranyl acetate stain.

Figure 2.35 Ultrastructural examination of a monoblast from a patient with M5a AML showing a nucleus with euchromatin and a large nucleolus; the cytoplasm contains mitochondria, endoplasmic reticulum, small bull’s eye granules and debris-laden vacuoles. Lead nitrate and uranyl acetate stain.

Figure 2.37 Ultrastructural examination of part of a monoblast from a patient with M5a AML showing mitochondria and a ribosomal-lamellar complex in cross-section. Lead nitrate and uranyl acetate stain.

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Figure 2.38 Peripheral blood film of a patient with FAB M5b AML showing a monoblast and two promonocytes. MGG, high power.

Figure 2.39 Peripheral blood film of a patient with FAB M5b AML showing a blast cell of uncertain lineage and four monoblasts/promonocytes. MGG, high power.

Figure 2.40 Peripheral blood film of a patient with FAB M5b AML showing four promonocytes. MGG, high power.

Figure 2.41 Bone marrow aspirate film from a patient with FAB M5b AML monoblasts and promonocytes. MGG, high power.

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Figure 2.42 Bone marrow trephine biopsy section from a patient with FAB M5b AML showing an intensely cellular marrow with numerous macrophages producing a ‘starry sky’ appearance (indicative of a high rate of cell death). The other cells are monoblasts and promonocytes. H&E, low power.

Figure 2.43 Lysozyme preparation. The clear spaces around the blast cells result from lysis of the organism Micrococcus lysodeikticus by lysozyme secreted by the leukaemic cells. Micrococcus lysodeikticus with MGG counterstain.

M6 AML In FAB M6 AML (Figures 2.44–2.55) the bone marrow has at least 50% erythroid cells and at least 30% of nonerythroid cells are blast cells (usually myeloblasts but could include monoblasts or megakaryoblasts). The erythroid cells

are often very dysplastic, e.g. giant forms or megaloblasts. Sometimes erythropoiesis is sideroblastic. Erythroid cells are often positive with a periodic acid-Schiff (PAS) stain (Figure 2.53). Dysplasia of other lineages is common.

Figure 2.44 Peripheral blood film of a patient with FAB M6 AML showing anisocytosis, poikilocytosis and an agranular platelet. MGG, high power.

Figure 2.45 Peripheral blood film of a patient with FAB M6 AML (same patient as Figure 2.44) showing a dysplastic erythroblast with a bilobed nucleus. MGG, high power.

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Figure 2.46 Peripheral blood film of a patient with FAB M6 AML (same patient as Figure 2.44) showing a proerythroblast and a late erythroblast with basophilic stippling. MGG, high power.

Figure 2.47 Peripheral blood film of a patient with FAB M6 AML (same patient as Figure 2.44) showing anisocytosis, poikilocytosis and a myeloblast. MGG, high power.

Figure 2.48 Peripheral blood film of a patient with FAB M6 AML (same patient as Figure 2.44) showing an unidentifiable cell and a dysplastic erythroblast with a lobulated nucleus. MGG, high power.

Figure 2.49 Peripheral blood film of a patient with FAB M6 AML (same patient as Figure 2.44) showing a megaloblast. MGG, high power.

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Figure 2.50 Bone marrow aspirate film from a patient with M6 AML showing dysplastic erythroblasts (e.g. lobulated nuclei and binucleate forms). MGG, high power.

Figure 2.51 Bone marrow aspirate film from a patient with M6 AML showing erythroblasts with cytoplasmic blebs. Such blebs can also be a feature of FAB M7 AML. MGG, high power.

Figure 2.52 Bone marrow aspirate film from a patient with M6 AML showing a dysplastic (binucleated) megakaryocyte. MGG, high power.

Figure 2.53 Bone marrow aspirate film from a patient with M6 AML showing PAS positivity of erythroblasts. PAS, high power.

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Figure 2.54 Ultrastructural examination of an immature cell from a patient with M6 AML. It has the morphological features of a blast cell but expression of glycophorin A identifies it as a primitive erythroid cell. Lead nitrate and uranyl acetate stain, immunogold technique.

Figure 2.55 Ultrastructural examination of a primitive cell from a patient with M6 AML. The nuclear outline is irregular and the cytoplasm contains vacuoles (arrow). The lineage has been identified with an immunogold technique using an antibody to Gerbich antigen. Lead nitrate and uranyl acetate stain, immunogold technique.

M7 AML In FAB M7 AML (Figures 2.56–2.66) the dominant bone marrow cell is a megakaryoblast, which often appears poorly differentiated; sometimes there are basophilic cytoplasmic protrusions. There may be differentiation to dysplastic megakaryocytes, sometimes including micromegakaryocytes. The blood film may show giant and dysplastic platelets,

Figure 2.56 Bone marrow aspirate film from a patient with FAB M7 AML showing megakaryoblasts. Note that one blast has cytoplasmic blebs. MGG, high power.

megakaryocyte fragments and circulating megakaryoblasts and micromegakaryocytes. Reactive fibrosis is common; this may lead to difficulty in aspiration so that trephine biopsy can be important in making the diagnosis. These cases with marked reactive fibrosis and pancytopenia have been referred to as ‘acute myelofibrosis’ [8].

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Figure 2.57 Ultrastructural examination of a megakaryoblast from a patient with FAB M7 AML. The lineage has been identified with an immunogold technique using an antibody to CD61. The arrows indicate deposits of gold where the antibody has bound to CD61 on the surface membrane of the blast cell. Lead nitrate and uranyl acetate stain, immunogold technique.

Figure 2.58 Ultrastructural examination of a megakaryoblast from a patient with FAB M7 AML. The lineage has been identified with a platelet peroxidase technique which shows a reaction product on the nuclear membrane and on the endoplasmic reticulum. Unstained section, platelet peroxidase reaction.

Figure 2.59 Bone marrow trephine biopsy section from a patient with FAB M7 AML showing a mixture of megakaryoblasts and mature megakaryocytes. H&E, low power.

Figure 2.60 Bone marrow trephine biopsy section from a patient with FAB M7 AML showing a mixture of granulocytes, megakaryoblasts and megakaryocytes. H&E, high power.

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Figure 2.61 Bone marrow trephine biopsy section from a patient with FAB M7 AML showing a mixture of megakaryoblasts and megakaryocytes; the megakaryocytes are present in increased numbers and many are hypolobated. H&E, low power.

Figure 2.62 Bone marrow trephine biopsy section from a patient with FAB M7 AML (same patient as Figure 2.61) showing a mixture of megakaryoblasts and megakaryocytes; many of the latter are micromegakaryocytes or are hypolobated. H&E, high power.

Figure 2.63 Bone marrow trephine biopsy section from a patient with FAB M7 AML (same patient as Figure 2.61) showing a mixture of megakaryoblasts and megakaryocytes; many of the megakaryocytes are micromegakaryocytes or are hypolobated. Giemsa, low power.

Figure 2.64 Bone marrow trephine biopsy section from a patient with FAB M7 AML (same patient as Figure 2.61) showing a mixture of micromegakaryocytes and megakaryoblasts. Giemsa, high power.

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Figure 2.65 Bone marrow trephine biopsy section from a patient with FAB M7 AML (same patient as Figure 2.61) showing increased reticulin deposition (grade 3/4). Reticulin, low power.

Figure 2.66 Bone marrow trephine biopsy section from a patient with FAB M7 AML (same patient as Figure 2.61) showing increased reticulin deposition (grade 3/4). Reticulin, high power.

Table 2.2 Monoclonal antibodies useful in the diagnosis of acute leukaemia

Antibody CD19 CD10 cCD79a cμ cCD3 CD2 CD1a CD4 CD7 CD13 CD14 CD15 CD33

Spectrum of a ctivity Pan-B cell (and some cases of FAB M2 AML) Common ALL (weaker in T-lineage ALL) Pan-B (but less specific than CD19, weaker in T-lineage ALL) Late B-lineage lymphoblasts (immunoglobulin M heavy chain) Pan-T cell Pan-T (and a minority of cases of AML) Some T ALL (and some thymocytes) T-lymphoid cells and cells of monocyte lineage Pan-T (and a minority of cases of AML) Pan-myeloid Maturing cells, particularly of monocyte lineage Maturing cells, particularly of monocyte lineage Pan-myeloid

Antibody CD64 CD117 cMPO cLysozyme CD41a CD62P CD235 CD34 HLA-DR CD45 Nuclear TdT

Spectrum of a ctivity Monocyte lineage Immature myeloid (and mast cells) Myeloid Myeloid (including monocytic) Megakaryocyte lineage (platelet glycoprotein IIb/IIIa) Platelets and megakaryocytes Erythroid (detects glycophorin) Stem cells Immature cells, activated cells and B-lineage cells Common leucocyte antigen B- and T-lineage blasts cells, blast cells of a significant minority of cases of AML

ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; c, cytoplasmic; CD, cluster of differentiation; MPO, myeloperoxidase; TdT, terminal deoxynucleotidyl transferase

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Immunophenotype Flow cytometry immunophenotyping is widely applied in the diagnosis and further investigation of AML (Figures 2.67–2.77) [9, 10]. A useful panel of antibodies for diagnosis of acute leukaemia is shown in Tab le 2.2. Immunophenotyping is essential for the diagnosis of M0, pure erythroid leukaemia and M7 AML, and should be

Normal BM

080186

routinely applied in all cases of acute leukaemia that are not obviously myeloid. In other patients with AML it can be useful for the recognition of an aberrant leukaemiaassociated phenotype that can subsequently be used for monitoring of minimal residual disease. If immunophenotyping is to be used for this purpose, a wide panel of

Normal BM

080186

Figure 2.67 Dot plots of flow cytometric immunophenotyping of normal bone marrow cells provided for comparison with leukaemic samples. The top left scatter plot shows how sideways light scatter (SSC) and CD45 (common leucocyte antigen) can be used to distinguish and gate on clusters of normal and abnormal haemopoietic and lymphoid cells; a gate can also be placed on the area where blast cells are usually found. Antigen expression by cells within each window (identified by a distinguishing colour on the basis of the SSC/CD45 expression) can then be identified in the other scatter plots. With thanks to Mr Ricardo Morilla.

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Figure 2.68 (Right) Dot plots of flow cytometric immunophenotyping of bone marrow cells from a patient with FAB M0 AML. The cells in the CD45-weak blast window (green) express the myeloid antigens CD13, CD33 and CD117. They also express the stem cell marker, CD34, but not terminal deoxynucleotidyl transferase (TdT). The majority also express HLA-DR. There are other myeloid markers that are not expressed (CD14, CD15, CD64, myeloperoxidase (MPO) and lysozyme). They show aberrant expression of CD7 but do not express other T-cell markers (CD2, cytoplasmic CD3) or B-cell markers (CD10, CD19, CD20, CD22, CD79a). With thanks to Mr Ricardo Morilla.

Figure 2.69 (Opposite, left) Dot plots of flow cytometric immunophenotyping of bone marrow cells from a patient with FAB M1 AML. The cells in the CD45-weak blast window (orange) express myeloid markers, CD13, CD33 and CD117, but only a minor population express myeloperoxidase (MPO), lysozyme or CD64. There is expression of HLA-DR and the stem cell marker, CD34 and some express TdT. There is no expression of CD14 or CD15, T-cell markers (CD2, cytoplasmic (cyt) CD3 and CD7) or of B-cell markers (CD10, CD19, CD79a). With thanks to Mr Ricardo Morilla.

Figure 2.70 (Opposite, right) Dot plots of flow cytometric immunophenotyping of bone marrow cells from a patient with FAB M2 AML. The cells in the CD45-positive blast window (khaki) express the myeloid markers, CD13, CD33 and CD117. The majority of cells express myeloperoxidase and a minority lysozyme; CD15 is negative. There is expression of HLA-DR and the stem cell marker, CD34, but not of TdT. There is no expression of T-cell markers (CD2, CD3 and CD7) or B-cell markers (CD10, CD19 and CD79a). With thanks to Mr Ricardo Morilla.

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Acute myeloid leukaemia 29 AML M1

AML M2

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Figure 2.71 Dot plots of flow cytometric immunophenotyping of bone marrow cells from a patient with FAB M3 AML. The cells in the CD45-positive blast window (blue-lilac) express the myeloid markers, CD13, CD33 and MPO (strong) and show partial expression of CD64 and CD117; they do not express CD11b, CD14, CD15 or TdT. The majority of cells do not express the stem cell marker, CD34, or HLA-DR; lack of expression of these two markers is characteristic of M3 AML. There is weak expression of CD2 but not of CD7 or cytoplasmic CD3; expression of CD2 can be a feature of this subtype of AML. There is no expression of B-cell markers (CD10, CD19, cytoplasmic CD22 and CD79a). The control dot plot displays a high level of background fluorescence, which is characteristic of M3 AML. With thanks to Mr Ricardo Morilla.

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Acute myeloid leukaemia 31 AML M4

Figure 2.72 Dot plots of flow cytometric immunophenotyping of bone marrow cells from a patient with FAB M4 AML. Gates have been placed on two cell populations apparent in the SSC/CD45 plot, one in the usual position of blast cells (yellow, R1) and one in a position where monoblasts may be found (grey, R2). The two populations differ in their light-scattering properties and CD45 expression and show subtle differences in expression of myeloid antigens. The granulocyte precursor population (yellow) shows weak CD45 expression with strong CD13, and is positive for CD33, CD117, HLA-DR and MPO (partial). There is lack of expression of other myeloid markers (CD14, CD64 [mainly negative] and lysozyme). The monocytic precursor population (grey) shows stronger CD45 expression and slightly higher sideways scatter (SSC). This population is positive for HLA-DR, CD14, CD64 and CD33 (strong). This pattern is characteristic of different stages of monocytic lineage maturation. There is very weak expression of lysozyme and no expression of CD13, MPO or CD117. Expression of CD117 is variable in the monocyte lineage; in this case the granulocytic population is positive and the monocytic negative. The differential expression of CD14 is consistent with the specificity of this marker for monocyte differentiation and maturation. Neither population expresses the stem cell marker, CD34, the mature granulocyte marker, CD15, T-cell markers (CD2, CD3 and CD7) or B-cell markers (CD10, CD22, CD79a and cytoplasmic μ chain). With thanks to Mr Ricardo Morilla.

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AML M5a BM

Figure 2.73 Dot plots of flow cytometric immunophenotyping of bone marrow cells from a patient with FAB M5a AML. The cells in the CD45-positive blast window (blue) express the myeloid markers, CD33 (strong) and CD64 (strong), heterogeneous CD15 and weak and heterogeneous CD13. They also express weak CD4, a marker which is not specific for the T- lineage, being often also expressed by cells of monocyte lineage. Other T-cell markers are negative as are B-cell markers. There is partial weak expression of lysozyme but not of MPO. The lack of expression of CD14 is consistent with immature rather than mature cells of monocyte lineage. With thanks to Mr Ricardo Morilla.

Figure 2.74 (Top, opposite) Dot plots of flow cytometric immunophenotyping of bone marrow cells from a patient with FAB M5b AML. The cells in the CD45-positive blast window (green) express the myeloid markers CD33 (strong) and CD64 and show weak expression of CD4 (a T-cell/monocyte marker). In comparison with the case of FAB M5a AML (Figure 2.73), some of the CD64-positive cells are also positive for CD14, indicating maturation. Some express MPO (weak) and lysozyme (strong), again indicating different stages of monocyte maturation. There is no expression of cytoplasmic CD3, B-cell markers (CD19, CD79a), CD15 or the stem cell marker, CD34. With thanks to Mr Ricardo Morilla.

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Acute myeloid leukaemia 33 AML M5b

AML M6

Figure 2.75 Dot plots of flow cytometric immunophenotyping of bone marrow cells from a patient with FAB M6 AML. Cells in the CD45-weak blast window (red, R1) express the myeloid markers CD13 and CD33; they are positive for CD34, the majority are positive for HLA-DR and a minority express CD64 (weak) and CD117. They do not express CD7, CD14 or CD19. There is a minority of cells expressing CD15 but these cells do not express CD34 (an observation that is expected since in myeloid cells CD15 is a marker of maturation, particularly of monocyte lineage). The most specific erythroid marker used, glycophorin, identifies a population of erythroid cells that also express CD71, the transferrin receptor. With thanks to Mr Ricardo Morilla.

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AML M7

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antibodies and multicolour flow cytometry is needed. Immunophenotyping with CD33 is recommended in patients in whom treatment with gemtuzumab ozogamicin is being considered. The immunophenotype can aid in the recognition of acute promyelocytic leukaemia, in which HLA-DR and CD34 are characteristically negative. In addition, some markers such as lysozyme, CD14 and CD64 may be useful indicators of monocytic differentiation. Immunofluorescence can be used to show the distribution of PML protein. In normal myeloid cells and in blast cells of most types of AML the distribution within the nucleus is in a small number of ‘nuclear bodies’, whereas in acute promyelocytic leukaemia there is a microparticulate distribution or diffuse cytoplasmic staining. This test can confirm the presence of PML-RARA fusion in cases in which a polymerase chain reaction (PCR) is negative because of atypical breakpoints [11].

Cytogenetic and molecular genetic abnormalities Many patients with AML have a clonal cytogenetic abnormality (Figures 2.78 and 2.79) demonstrable on classical cytogenetic analysis and often also by fluorescence in situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR). The underlying cytogenetic abnormality determines the clinical and haematological features of the disease, including the prognosis, and in some cases it determines the approach to treatment. It is thus of critical importance that it be applied in all cases of acute leukaemia. Of particular importance is the speedy diagnosis of acute promyelocytic leukaemia since this has specific therapeutic implications; conventional cytogenetic analysis is too slow for this purpose so that FISH or demonstration of abnormal distribution of PML protein is preferred.

Figure 2.76 (Far left) Dot plots of flow cytometric immunophenotyping of bone marrow cells from a patient with pure erythroid acute leukaemia. The gate (pink, R1) encompasses the CD45-negative blast-erythroid area of the scatter plot. The small cluster of ungated cells with strong CD45 expression and low sideways scatter is likely to represent lymphocytes and illustrates the use of gating to exclude irrelevant populations. Gated cells are negative for all tested myeloid, B-lymphocyte and T-lymphocyte markers. As expected of erythroid cells, they express glycophorin and the transferrin receptor, CD71. With thanks to Mr Ricardo Morilla.

Figure 2.77 (Left) Dot plots of flow cytometric immunophenotyping of bone marrow cells from a patient with FAB M7 AML. The CD45-weak gated cells (blue) show weak expression of CD13 and CD34, and strong express two platelet specific markers, CD41a (identifying the platelet glycoprotein IIb/IIIa complex) and CD62P (identifying P selectin, a platelet alpha granule constituent). The cells are mainly negative for CD33 and do not express other myeloid markers (CD15, CD117, MPO and lysozyme). Nor do they express B-lineage (CD19, CD79a) or T-lineage (CD3, CD7) markers or HLADR. With thanks to Mr Ricardo Morilla.

Figure 2.78 Metaphase showing trisomy 21 in a 3-yearold girl with acute megakaryoblastic leukaemia. All the metaphases analysed at diagnosis had the extra chromosome, raising the possibility that it was constitutional. However, there were no physical signs of Down’s syndrome. A fluorescence in situ hybridization (FISH) study of a buccal smear found only two RUNX1 signals, indicating that the gain was likely to be clonal, as the result of a somatic mutation. This was confirmed by another FISH study of bone marrow performed in remission a month later, which found only the normal two signals. With thanks to Dr John Swansbury.

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Figure 2.79 Partial karyogram showing t(8;16)(p11;p13). In this translocation, almost all of the short arm of a chromosome 8 has been moved to the short arm of one chromosome 16. This is an uncommon abnormality (<1% of AML) but it is well-known for being closely associated with a subtype of acute monocytic leukaemia with characteristic erythrophagocytosis. Disseminated intravascular coagulation is frequent and this may initially suggest a diagnosis of FAB M3 AML. The genes involved are MYST3 (previously known as MOZ) at 8p11.2 and CREBBP (previously known as CBP) at 16p13.3. With thanks to Dr John Swansbury.

Some of the recurrent cytogenetic abnormalities recognized in AML are shown in Table 2.3. Some of these karyotypic abnormalities were incorporated into the 2001 WHO classification of AML [2], which is summarized in Table 2.4. In addition, the WHO classification recognizes transient abnormal myelopoiesis in neonates with Down’s syndrome as transient AML (Figure 2.80); both transient abnormal myelopoiesis and acute megakaryoblastic leukaemia in infants with Down’s syndrome are associated with a GATA1 mutation, in addition to the constitutional trisomy 21 or equivalent abnormality. In the 2008 WHO classification AML associated with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) is considered a separate category. In addition to the genetic abnormalities that result from chromosomal rearrangement, there are recurrent genetic abnormalities in AML that have no cytogenetic correlate. They include point mutations, partial gene duplications and frame shift mutations. The more important of these are summarized in Table 2.5. Other point mutations, at least some of which relate to disease evolution, include mutations in TP53, NRAS, KRAS, KIT, PTPN11, CSF1R, NF1 and BRAF. Some of these mutations (e.g. FLT3-ITD) occur as a secondary abnormality in various cytogenetic categories of AML. Others (e.g. NPM1 and CEBPA mutations) occur mainly in patients with a normal karyotype and may

Table 2.3 Recurrent cytogenetic abnormalities in AML

Cytogenetic abnormality

Molecular abnormality

Percentage of cases

t(8;21)(q22;q22)

RUNX1-CBFA2T1

5% (higher in children)

t(15;17)(q22;q12)

PML-RARA

5%

inv(16)(p13.1q22) or t(16;16)(p13.1;q22)

CBFB-MYH11

5%

t(9;11)(p21;q23)

MLLT3-MLL

3% (higher in children)

inv(3)(q21q26.2) or t(3;3)(q21;q26.2)

RPN1-EVI1

1%

t(11;17)(q23;q21)

ZNF145-RARA

<1%

t(9;22)(q34;q11.2)

BCR-ABL1

<1%

t(6;9)(p23;q34.3)

DEK-NUP214

<1%

t(8;16)(p11;p13)

MYST3-CREBBP

<1%

t(1;22)(p13;q13)

RBM15-MKL1

<1%

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Figure 2.80 Peripheral blood film in transient abnormal myelopoiesis of Down’s syndrome showing poikilocytosis, a blast cell, an erythroblast and a micromegakaryocyte. MGG, high power.

Table 2.4 The 2008 WHO classification of acute myeloid leukaemia Therapy-related myeloid neoplasms AML with recurrent cytogenetic abnormalities* AML with t(8;21)(q22;q22); RUNX1-RUNX1T1 (AML1-ETO) AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 Acute promyelocytic leukaemia with t(15;17)(q22;q12); PML-RARA AML with t(9;11)(p22;q23); MLLT3-MLL AML with t(6;9)(p23;q34); DEK-NUP214 AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 AML (megakaryoblastic) with t(1;22)(p13;q13): RBM15-MKL1 AML with mutated NPM1 AML with mutated CEBPA AML with myelodysplasia-related changes (meeting one or more of the following three requirements) Following MDS or MDS/MPN With multilineage dysplasia† With myelodysplastic syndrome-related cytogenetic abnormality

AML not othe rwise categorized Minimally differentiated AML (similar to FAB M0) AML without maturation (similar to FAB M1) AML with maturation (similar to FAB M2) Acute myelomonocytic leukaemia (similar to FAB M4) Acute monoblastic and monocytic leukaemia (similar to FAB M5) Acute erythroid leukaemias (erythroid and myeloid or pure erythroleukaemia) (similar to FAB M6) Acute megakaryoblastic leukaemia (similar to FAB M7) Acute basophilic leukaemia Acute panmyelosis with myelofibrosis Myeloid sarcoma Myeloid proliferations related to D own’s syndrome Transient abnormal myelopoiesis Myeloid leukaemia associated with Down’s syndrome

*If therapy-related cases are found to have these recurrent cytogenetic abnormalities this should be noted, but they are categorized as therapy-related AML or MDS not as AML with recurrent cytogenetic abnormalities † Defined as having at least 50% of dysplastic cells in at least 2 lineages AML, acute myeloid leukaemia; FAB, French–American–British; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm

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Table 2.5 Recurrent molecular genetic abnormalities in AML

Molecular genetic abnormality

Approximate frequency

Prognostic significance

CEBPA mutated

10%

Favourable if not accompanied by FLT3-ITD

NPM1 mutated

30%

Favourable if not accompanied by FLT3-ITD

FLT3-ITD and high ratio of FLT3-ITD:wt FLT3

25–30%

Adverse

FLT3-TKD mutation

5%

Not known

FLT3-JMD mutation

1%

Not known

MLL-PTD

5–10%

Adverse

GATA1 mutated

Not applicable

Specifically associated with AML in Down’s syndrome; good prognosis

JAK2 V617F point mutation

4%

Adverse (higher rate of relapse)

AML, acute myeloid leukaemia; ITD, internal tandem duplication; JMD, juxtamembrane domain; PTD, partial tandem duplication; TKD, tyrosine kinase domain; wt, wild type

represent a primary abnormality. However, a single case may have both FLT3-ITD and an NPM1 mutation. NPM1 mutations can be detected by immunohistochemistry, which shows accumulation of the protein in the cytoplasm of the blasts when the gene is mutated [12].

The WHO classification The WHO classification requires knowledge of the patient’s medical history, morphological assessment and cytogenetic or molecular genetic analysis. Immunophenotyping is occasionally essential for diagnosis; it is also often applied even when the case is obviously myeloid in order both to exclude mixed phenotype acute leukaemia and to provide information on any aberrant phenotype. However, other than proving that a case is myeloid, immunophenotyping is not essential for the application of the WHO classification. The WHO classification is hierarchical. Cases are assigned to categories in the order shown in the following paragraphs.

Therapy-related AML The 2001 WHO classification recognized two broad groups of therapy-related AML although some overlap occurs

[13–15]. In the 2008 classification the two categories have been amalgamated. AML following alkylating agents (e.g. melphalan and chlorambucil) and the nitrosoureas (e.g. lomustine), platinum-based agents (carboplatin, cisplatin) and exposure to radiation is characterized by a latent phase of 5–10 years, preceding MDS, trilineage myelodysplasia and an association with abnormalities of chromosomes 5 and 7 (–5, 5q–, –7, 7q–) and with complex chromosomal abnormalities. There are also some recurring balanced translocations and other rearrangements that can occur following exposure to alkylating agents, e.g. t(6;9)(p23;q34), t(3;3)(q21;q26.2), inv(3)(q21q26.2) and t(8;16)(p11;p13). Therapy-related AML is more likely than de novo disease to have a hypocellular bone marrow, bone marrow fibrosis or both. AML following topoisomerase-II-interactive drugs (e.g. etoposide, anthracyclines, mitoxantrone) has a shorter latent phase of 1–3 years, a much weaker association with MDS and myelodysplasia and an association with recurrent balanced chromosomal translocations with specific breakpoints: 11q23 (MLL gene ), 11p15 (NUP98 gene) and 21q22 (RUNX1 gene) [16, 17]. Translocations observed include t(9;11)(p21;q23), t(8;21)(q22;q22),

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t(3;21)(q26;q22) and t(1;11)(q23;p15). Acute promyelocytic leukaemias with t(15;17)(q22;q12) and acute myelomonocytic leukaemia with eosinophilia and inv(16)(p13q22) also occur [18]. These leukaemias have similar cytological features to de novo cases with the same chromosomal rearrangement (see below) but a somewhat worse prognosis. A third category of ‘other’ includes patients who have been exposed to either to radiation or to drugs that do not fall into these two groups, such as antimetabolites (fludarabine, thiopurines) and antitubulin agents.

Figure 2.81 Bone marrow aspirate film in acute promyelocytic leukaemia showing hypergranular cells and Auer rods. MGG, high power.

Figure 2.83 Karyogram showing t(15;17)(q22;q21) associated with acute promyelocytic leukaemia. These abnormal chromosomes can be difficult to identify in the poor-quality metaphases that are often obtained in this type of leukaemia. Rapid confirmation by FISH may therefore be needed to permit optimal management of the patient. The genes involved are PML and RARA (encoding the retinoic acid receptor alpha). With thanks to Dr John Swansbury.

AML with recurrent cytogenetic/genetic abnormalities AML with t(15;17)(q22;q12) and P ML-RARA fusion has been described above (FAB M3 category) and further features are illustrated in Figures 2.81–2.83. AML associated with t(11;17)(q23;q21) and ZNF145-RARA fusion has some features similar to those of acute promyelocytic leukaemia but cytological features can be intermediate between the FAB M2 and M3 categories (Figure 2.84) and the same responsiveness to therapy with all-trans-retinoic acid (ATRA) is not seen.

Figure 2.82 Peripheral blood film in the variant form of acute promyelocytic leukaemia showing a variant promyelocyte with a crater-like indentation in the nucleus and a crystalline inclusion. MGG, high power.

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Figure 2.84 Bone marrow aspirate film from a patient with acute leukaemia associated with t(11;17)(q23;q21).

Figure 2.86 Peripheral blood film from a patient with AML (FAB M2 type) associated with t(8;21)(q22;q22) (same patient as Figure 2.85) showing peroxidase positivity. In two blast cells the peroxidase activity is restricted to the Golgi zone. MPO reaction, high power.

Figure 2.85 Peripheral blood film from a patient with AML (FAB M2 type) associated with t(8;21)(q22;q22) showing three blast cells with a typical clear Golgi zone in the nuclear hof. MGG, high power.

Figure 2.87 Peripheral blood film from a patient with AML (FAB M2 type) associated with t(8;21)(q22;q22) (same patient as Figure 2.85) showing SBB reactivity confined to the nuclear hof. SBB, high power.

Figures 2.88 Bone marrow aspirate film from a patient with AML associated with t(8;21)(q22;q22) showing the cytological features of AML of FAB M2 type. MGG, high power.

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AML with t(8;21)(q22;q22) and RUNX1-C B FA2T1 fusion is usually associated with the cytological features of FAB M2 AML [19] (Figures 2.85–2.90). The blast cells often have basophilic cytoplasm, a prominent Golgi zone

and a single long thin Auer rod. A variable degree of dysplasia may be present in granulocytic cells. Cells of eosinophil and basophil lineage may be increased while the monocytic component is scanty.

Figure 2.89 Karyogram of a patient with t(8;21)(q22;q22). Arrows indicate the two chromosomes that have been involved in the translocation, an abnormal chromosome 13, which has a small insertion, and a missing Y chromosome. The translocation t(8;21)(q22;q22) frequently shows an associated loss of a sex chromosome (the Y in males or an X in females) as a secondary abnormality. Sex chromosome loss is otherwise rare as a clonal event and its biological and clinical significance in association with t(8;21) is unknown. Non-clonal loss, especially of the Y, is common with increasing age and of no apparent clinical significance. With thanks to Dr John Swansbury.

Figure 2.90A, B FISH in a patient with a three-way translocation between chromosomes 8, 15 and 21, leading to RUNX1-CBFA2T1 (previously known as AML1-ETO) fusion. The study used the Vysis LSI AML1/ETO dual colour, dual fusion probe. A: In the metaphase shown, the FISH signals from top left to bottom right are (1) ETO (red) on the normal chromosome 8; (2) part of ETO on the abnormal 8; (3) ETO-AML1 fusion on an abnormal 21; (4) AML1 (green) on the normal 21; (5) part of AML1 on the abnormal 15; B: Two interphase nuclei from the same case are shown: the nucleus on the left has the same signal pattern as shown in the metaphase (two red signals, two green and one fusion). A small proportion of the nuclei had the pattern shown on the right. This is the pattern that is seen with a typical t(8;21)(q22;q22), having two fusion signals. From the FISH study it could be deduced that the original clone in this patient had the standard translocation between the ETO gene at 8q22 and the AML1 gene at 21q22. This had been masked by a subsequent translocation between the abnormal 8 and a chromosome 15, and this evolved clone had become more abundant. With thanks to Dr John Swansbury.

A

B

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AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22) and CBFB-MYH11 fusion is most often associated with the cytological features of FAB M4 AML (Figures 2.91–2.99). In addition, there is an increase of eosinophils and

Figure 2.91 Peripheral blood film from a patient with inv(16)(p13q22) showing blast cells and an abnormal partially degranulated eosinophil. MGG, low power.

Figure 2.93 Bone marrow aspirate film from a patient with inv(16)(p13q22) showing granulocyte and monocyte precursors, including an eosinophil precursor with very large pro-eosinophilic granules that have basophilic staining characteristics. MGG, high power.

eosinophil precursors; the latter have some deep purple proeosinophilic granules. The designation ‘M4Eo’ is often used.

Figure 2.92 Peripheral blood film from a patient with inv(16)(p13q22) showing promonocytes and an abnormal, vacuolated and partially degranulated, eosinophil. MGG, high power.

Figure 2.94 Bone marrow aspirate film from a patient with inv(16)(p13q22) showing blast cells and an eosinophil precursor with large pro-eosinophilic granules with basophilic staining characteristics. MGG, high power.

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Figure 2.95 Bone marrow aspirate film from a patient with t(16;16)(p13.1;q22) showing that the cytological features are the same as in cases associated with inv(16)(p13.1q22). MGG, high power.

Figure 2.97 Bone marrow trephine biopsy section from a patient with AML associated with inv(16)(p13q22) showing cells of monocyte lineage (irregular nuclei) and eosinophils and their precursors. H&E, high power.

Figure 2.98 Karyogram from a patient with inv(16)(p13q22). There is also trisomy for chromosome 22 and duplication of part of the long arm of a chromosome 2. Trisomy 22 is rare except in the presence of inv(16). Its clinical and biological significance in this context are unknown. This inversion arises when there is a break in both the short arm and the long arm of the chromosome. The inset shows the 16s with the centromeres in the same alignment and the telomeres moved. The 16s in the full karyotype are shown with the telomeres in the correct position and the centromere inverted. This karyotype also illustrates the variation that can occur in the size of the heterochromatic (dark) band near the centromere of the 16s. This is a normal, inherited feature but to the inexperienced it can sometimes lead to uncertainty as to whether or not an inversion is present. With thanks to Dr John Swansbury.

Figure 2.96 Ultrastructural examination of an abnormal basophil from a patient with inv(16)(p13q22) showing mature (m) and immature (i) granules in a cell that has a mature nucleus. Lead nitrate and uranyl acetate stain.

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Figure 2.100 Peripheral blood film of a patient with FAB M5a AML associated with t(9;11)(p21;q23) showing three monoblasts. MGG, high power. Figure 2.99 FISH in inv(16)(p13q22). There is a normal chromosome 16 at the left, with the paired signals of the Vysis LSI CBFB break-apart probe that locates to 16q22. On the right is the inverted 16, with the distal (green) part of CBFB translocated onto the short arm of the chromosome. With thanks to Dr John Swansbury.

Figure 2.101 Peripheral blood film of a patient with FAB M5a AML associated with t(9;11)(p21;q23) showing five monoblasts and a promonocyte. MGG, high power.

Figure 2.102 Bone marrow aspirate film of a patient with FAB M5a AML associated with t(9;11)(p21;q23) showing monoblasts. MGG, high power.

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AML with t(9;11)(p21;q23) and MLL- MLLT3 is typically associated with acute monocytic/monoblastic AML (Figures 2.100–2.103). Other cases of AML are associated with other translocations with a chromosome 11q23 breakpoint with rearrangement of the MLL gene. These cases can be regarded as cytogenetic variants within this category. Among these other 11q23 translocations is AML associated with t(11;19)(q23;p13) (Figures 2.104 and 2.105).

AML with t(1;22)(p13;q13) and RBM15-MKL1 fusion. This subtype of AML has been added to the 2008 WHO classification. It is typically of megakaryocytic lineage and occurs in infants (Figure 2.106).

Figure 2.103 Bone marrow aspirate film of a patient with FAB M4 AML associated with t(9;11)(p21;q23) showing cells of both neutrophil and monocyte lineages. MGG, high power.

Figure 2.104 Bone marrow aspirate of a patient with AML associated with t(11;19)(q23;p13). MGG, high power.

Figure 2.105 Partial karyogram showing t(11;19)(q23;p13). With thanks to Dr John Swansbury.

Figure 2.106 Peripheral blood film of an infant with acute megakaryoblastic leukaemia associated with t(1;22)(p13;q13) showing a megakaryoblast and a micromegakaryocyte. MGG, high power.

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AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) and RP N1-EVI1 fusion. This subtype of AML has become a specific category in the 2008 WHO classification. It often shows megakaryocyte dysplasia and has a poor prognosis (Figures 2.107 and 2.108). AML with t(6;9)(p23;q34.3) and DEK-NUP 214 fusion. This subtype of AML has been added to the 2008 WHO classification. It can occur de novo or be therapy related (in which case it is categorized as therapy-related AML). It is associated with neutrophilic and often basophilic differentiation. It has a poor prognosis (Figures 2.109–2.111).

AML with multilineage dysplasia AML with multilineage dysplasia is diagnosed if more than 50% of cells in at least 2 myeloid lineages show dysplastic features (Figures 2.112–2.117). In the 2001 WHO classification, cases are divided into two groups depending on whether they follow MDS or a myelodysplastic syndrome /myeloproliferative disorder (MDS/MPD) or occur de novo. In the 2008 classification the two groups have been amalgamated together with cases of MDS-related cytogenetic abnormalities.

Figure 2.107 Bone marrow aspirate film from a patient with inv(3)(q21q26) showing dysplastic megakaryocytes. MGG, high power.

Figure 2.108 Bone marrow aspirate film from a patient with inv(3)(q21q26) showing a dysplastic megakaryocyte. MGG, high power.

Figure 2.109 Bone marrow aspirate film from a patient with t(6;9)(p23;q34) showing neutrophilic and basophilic differentiation. The basophils have scanty granules and are vacuolated. MGG, high power.

Figure 2.110 Bone marrow aspirate film from a patient with t(6;9)(p23;q34). Basophilic differentiation is confirmed by metachromatic staining with toluidine blue. Toluidine blue high power.

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Figure 2.111 Karyogram showing (6;9)(p23;q34).The arrows indicate the two chromosomes involved in the translocation. The shortening of the short arm of chromosome 6 is subtle and can be overlooked, while the extra material on the long arm of chromosome 9 can misleadingly resemble that seen in the Philadelphia translocation. With thanks to Dr John Swansbury.

Figure 2.112 Peripheral blood film of a patient with AML with multilineage dysplasia showing blast cells, a hypogranular neutrophil and a giant platelet. MGG, high power.

Figure 2.113 Bone marrow aspirate film from a patient with AML with multilineage dysplasia (same patient as Figure 2.112), showing blast cells and a hypogranular neutrophil band form. MGG, high power.

Figure 2.114 Bone marrow aspirate film from a patient with AML with multilineage dysplasia (same patient as Figure 2.112), showing hypogranular band forms and a giant multinucleated neutrophil precursor. MGG, high power.

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Figure 2.115 Bone marrow aspirate film from a patient with AML with multilineage dysplasia (same patient as Figure 2.112), showing a dysplastic megakaryocyte. MGG, high power.

Figure 2.116 Bone marrow aspirate film from a patient with AML with multilineage dysplasia (same patient as Figure 2.112), showing a dysplastic megakaryocyte, which has the normal positive reaction for non-specific esterase. Double esterase, high power.

Figure 2.117 Bone marrow aspirate film from a patient with AML with multilineage dysplasia showing expression of platelet glycoprotein Ib by platelets and by a dysplastic micromegakaryocyte. Immunoperoxidase for CD42b, high power.

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AML not othe rwise categorized Once cases have been assigned to the above three categories a considerable number of cases are left. They can be further classified according to cytological features into categories that resemble the FAB categories except that all the subtypes discussed above have been removed and that only 20% of blast cells in blood or bone marrow are needed to make the diagnosis. It will be seen from Table 2.4 that the first seven categories resemble FAB categories. FAB M3 does not appear because all cases with that morphology fall into the category of AML with recurrent genetic/cytogenetic

abnormalities (or t-AML). Acute erythroleukaemia has been expanded to include also pure erythroid cases, in which there is erythroid predominance (more than 80% of bone marrow cells) and no significant myeloblastic component (Figures 2.118–2.120). In these cases, the leukaemic erythroblasts may be poorly differentiated or, in other patients, may show differentiation readily recognized by microscopic examination. Three categories are new, not specifically related to the FAB classification.

Figure 2.118 Bone marrow aspirate film from a patient with pure erythroid leukaemia showing dysplastic erythroblasts at all stages of development, from proerythroblasts to mature erythroblasts. However, early forms predominate. MGG, high power.

Figure 2.119 Bone marrow aspirate film from a patient with pure erythroid leukaemia (same patient as Figure 2.118) showing dysplastic erythroblasts at all stages of development, from proerythroblasts to mature erythroblasts. However, early forms predominate. MGG, high power.

Figure 2.120 Ultrastructural examination of an early and a late erythroblast from a patient with erythroleukaemia. The lineage has been identified with an immunogold technique using an antibody to Gerbich antigen. ‘e’ indicates an erythroblast nucleus. Lead nitrate and uranyl acetate stain, immunogold technique.

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Acute basophilic leukaemia This is an acute myeloid leukaemia with differentiation being predominantly basophilic (Figures 2.121–2.123). Some patients have symptoms related to histamine excess. The leukaemic cells are mainly basophil precursors with few

Figure 2.121 Peripheral blood film of a patient with acute basophilic leukaemia showing blast cells with vacuoles or basophilic granules. MGG, high power.

mature basophils. The blast cells have vacuolated cytoplasm or contain a small number of basophilic granules. Ultrastructural examination may reveal the presence of granules typical of mast cells as well as those typical of basophils (Figures 2.124–2.128).

Figure 2.122 Peripheral blood film of a patient with acute basophilic leukaemia (same patient as Figure 2.121) showing confirmation of the lineage by demonstration of metachromatic staining with toluidine blue. Toluidine blue, high power.

Figure 2.123 Peripheral blood film of a patient with acute basophilic leukaemia showing a blast cell with a bilobed nucleus and basophilic granules. MGG, high power. Figure 2.124 Ultrastructural cytochemistry of a blast cell in acute basophilic leukaemia showing peroxidasepositive granules (arrow). MPO activity was not detected by light microscopy. Unstained section, MPO reaction.

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Figure 2.125 Ultrastructural examination of a blast cell in acute basophilic leukaemia showing mitochondria, endoplasmic reticulum, granules and a cytoplasmic vacuole. Lead nitrate and uranyl acetate stain.

Figure 2.126 Ultrastructural examination showing part of a blast cell in acute basophilic leukaemia. The finely particulate granule contents are typical of early basophil granules. Lead nitrate and uranyl acetate stain.

Figure 2.127 Ultrastructural examination of part of a blast cell in acute basophilic leukaemia showing theta granules (arrow) typical of mast cells. A mixture of basophil-type and mast cell-type granules can also be seen in acute transformation of chronic myeloid leukaemia but this case was Philadelphia negative. Lead nitrate and uranyl acetate stain.

Figure 2.128 Ultrastructural examination of part of a blast cell in acute basophilic leukaemia showing whorled granule contents (arrow) typical of mast cell granules. Lead nitrate and uranyl acetate stain.

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Figure 2.129 Peripheral blood film of a patient with acute panmyelosis showing red cell crenation (probably a storage artefact) and a hypogranular neutrophil. MGG, high power.

Figure 2.130 Bone marrow trephine biopsy section from a patient with acute panmyelosis (same patient as Figure 2.112) showing a mixture of immature and mature cells in a fibrous stroma. H&E, low power.

Figure 2.131 Bone marrow trephine biopsy section from a patient with acute panmyelosis (same patient as Figure 2.112) showing myeloblasts identified by peroxidase activity. Immunohistochemistry, anti-MPO, high power.

Figure 2.132 Bone marrow trephine biopsy section from a patient with acute panmyelosis (same patient as Figure 2.112) showing immature erythroid cells that are reactive with the Ulex europaeus lectin. Immunohistochemistry, Ulex europaeus lectin, high power.

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Figure 2.133 Bone marrow trephine biopsy section from a patient with acute panmyelosis (same patient as Figure 2.112) showing micromegakaryocytes identified by reactivity for CD61. Immunohistochemistry, CD61, high power.

Figure 2.134 Bone marrow trephine biopsy section from a patient with acute panmyelosis (same patient as Figure 2.112) showing increased small blood vessels demonstrated by endothelial cell expression of von Willebrand factor. Immunohistochemistry, anti-von Willebrand factor, high power.

Acute panmyelosis with myelofibrosis This is a rare type of AML characterized by the presence of blast cells and differentiating cells of various myeloid lineages with reactive deposition of reticulin (Figures 2.129–2.135). The peripheral blood often shows pancytopenia with few blast cells and bone marrow may be inaspirable, so that diagnosis rests on trephine biopsy histology and immunohistochemistry.

Plasmacytoid dendritic cell leukaemias Most of these cases were included in the 2001 WHO classification within the blastoid NK lymphoma category.

Figure 2.135 Bone marrow trephine biopsy section from a patient with acute panmyelosis (same patient as Figure 2.112) showing a megakaryoblast expressing von Willebrand antigen. Immunohistochemistry, anti-von Willebrand factor, high power.

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Diagnosis and differential diagnosis The main differential diagnosis is high-grade MDS. Making the distinction requires consideration of the blast cell percentage and any cytogenetic abnormalities that are present. The FAB classifications used a cut-off point of 30% bone marrow blast cells. In the WHO classification this has been replaced by a cut-off point of 20% blast cells in either blood or bone marrow. There are certain cytogenetic abnormalities that indicate a diagnosis of AML rather than MDS, regardless of the blast percentage. The WHO has identified t(8;21), t(15;17) and inv(16). To these should probably be added t(1;22)(p13;q13). In the case of Ph-positive BCR-ABL1positive AML, the differential diagnosis is blast crisis of chronic myelogenous leukaemia. Very rarely, non-neoplastic conditions can be confused with AML. The bone marrow picture of ‘maturation arrest’ in patients with agranulocytosis has been confused with acute promyelocytic leukaemia. A similar picture can be seen in patients who have received granulocyte colony-stimulating factor (G-CSF) for neutropenia of various aetiologies. Megaloblastic anaemia due to deficiency of vitamin B12 or folic acid has been confused with acute erythroleukaemia. Careful attention to cytological details will avoid these errors. The promyelocytes of agranulocytosis have very prominent granules but they do not have Auer rods or giant granules; they also have a very prominent Golgi zone, which is not usually seen in the promyelocytes of acute promyelocytic leukaemia. Patients with severe megaloblastic anaemia can have gross dysplasia and a great excess of proerythroblasts that can give the impression of acute leukaemia; usually giant metamyelocytes are easily detected. If there is the slightest doubt as to the diagnosis a trial of B12 and folate therapy should be given, even if assays are normal. Hypoplastic AML can be confused with aplastic anaemia and aplastic MDS but good quality histological sections permit the distinction.

Prognosis Prognosis is related both to the nature of the leukaemia and biological factors in the patient. Prognosis worsens with age, in part because there is a greater prevalence of adverse cytogenetic abnormalities in older patients and is worse if there is co-morbidity (increased serum bilirubin or creatinine). Prognosis can be related to disease bulk, being

worse with a higher white cell count or higher lactate dehydrogenase. Prognosis has been reported to be worse in AML with trilineage myelodysplasia and is worse for therapyrelated AML than for de novo cases, with those related to alkylating agents having a significantly worse prognosis that those related to topoisomerase-II-interactive drugs. The best prognosis is seen in patients with t(15;17) and promyelocytic leukaemia. A relatively good prognosis is associated with t(8;21) and inv(16). Poor prognostic cytogenetic abnormalities include t(9;22), t(6;9), inv(3), t(3;3), monosomy 7 and complex karyotypic abnormalities. An intermediate prognosis is associated with t(9;11), normal cytogenetic analysis and –Y (the latter likely to be a nonclonal age-related abnormality). In patients with a normal karyotype, certain molecular genetic abnormalities are of prognostic significance, particularly CEBPA and NPM1 mutations which, in the absence of FLT 3-ITD, are prognostically favourable (Table 2.5). Conversely, FLT3ITD worsens the prognosis when associated with either normal cytogenetics or various recurrent cytogenetic abnormalities [20–22]. Gene expression profiling has identified, within the group of patients with a normal karyotype, two subsets which differ in outcome. Although the poor prognostic group included higher number of cases with FLT3-ITD, there were also patients in this subset without this abnormality [23]. Overexpression of BAALC, ERG and NM1 have also been related to adverse prognosis [24].

Treatment Most subtypes of AML are treated with intensive combination chemotherapy with the aim of achieving and then maintaining a complete remission (less than 5% bone marrow blast cells and recovery of blood counts). In patients with an adverse prognosis, allogeneic transplantation should be considered, when possible. In patients with a relatively good prognosis, transplantation should be reserved for those who relapse and enter a second remission. The standard treatment of acute promyelocytic leukaemia is with chemotherapy plus all-trans-retinoic acid (ATRA). In elderly patients chemotherapy can be avoided and ATRA can be combined with arsenic trioxide [25]. It is possible that, when more data are available, this combination of drugs will be extended to younger patients. Gemtuzumab ozogamicin can be added for patients with adverse prognostic factors.

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References 1 Estey E, Garcia-Manero G, Ferrajoli A, Faderl S, Verstovsek S, Jones D and Kantarjian H (2006). Use of all-trans retinoic acid plus arsenic trioxide as an alternative to chemotherapy in untreated acute promyelocytic leukemia. Blood, 107, 3469-3473. 2 Jaffe ES, Harris NL, Stein H, Vardiman JW (2001). World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon. 3 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR and Sultan C (1976). Proposals for the classification of the acute leukaemias (FAB cooperative group). Br J Haematol, 33, 451–458. 4 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR and Sultan C (1980). A variant form of acute hypergranular promyelocytic leukaemia (M3). Br J Haematol, 44, 169–170. 5 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR and Sultan C (1985). Criteria for the diagnosis of acute leukemia of megakaryocytic lineage (M7): a report of the French–American–British cooperative group. Ann Intern Med, 103, 460–462. 6 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR and Sultan C (1985). Proposed revised criteria for the classification of acute myeloid leukemia. Ann Intern Med, 103, 626–629. 7 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR and Sultan C (1991). Proposal for the recognition of minimally differentiated acute myeloid leukaemia (AML M0). Br J Haematol, 78, 325–329. 8 Bain BJ, Catovsky D, O’Brien M, Prentice HG, Lawlor E, Kumaran TO e t al. (1981). Megakaryoblastic leukemia presenting as acute myelofibrosis – a study of four cases with the platelet-peroxidase reaction. Blood, 58, 206–213. 9 Bain BJ, Barnett D, Linch D, Matutes E, Reilly JT; General Haematology Task Force of the British Committee for Standards in Haematology (BCSH), British Society of Haematology (2002). Revised guideline on immunophenotyping in acute leukaemias and chronic lymphoproliferative disorders. Clin Lab Haematol, 24, 1–13.

10 Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A and van’t Veer MB (1995). Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL). Leukemia, 9, 1783–1786. 11 Mistry AR, Pedersen EW, Solomon E, Grimwade D (2003). The molecular pathogenesis of acute promyelocytic leukaemia: implications for the clinical management of the disease. Blood Rev, 17, 71–97. 12 Falini B, Nicoletti I, Martelli MF, Mecucci C (2007). Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features. Blood, 109, 874–885. 13 Mauritzson N, Albin M, Rylander L, Billstrom R, Ahlgren T, Mikoczy Z et al. (2002). Pooled analysis of clinical and cytogenetic features in treatment-related and de no vo 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. 14 Smith SM, Le Beau MM, Huo D, Karrison T, Sobecks RM, Anastasi J e t al. (2003). Clinical-cytogenetic associations in 306 patients with therapy-related myelodysplasia and myeloid leukemia: the University of Chicago series. Blood, 102, 43–52. 15 Rund D, Krichevsky S, Bar-Cohen S, Goldschmidt N, Kedmi M, Malik E et al. (2005). Therapy-related leukemia: clinical characteristics and analysis of new molecular risk factors in 96 adult patients. Leukemia, 19, 1919–1928. 16 Bloomfield CD, Archer KJ, Mrozek K, Lillington DM, Kaneko Y, Head DR et al. (2002). 11q23 balanced chromosome aberrations in treatment-related myelodysplastic syndromes and acute leukemia: report from an international workshop. Genes, Chromosomes Cancer, 33, 331–345. 17 Slovak ML, Bedell V, Popplewell L, Arber DA, Schoch C, Slater R et al. (2002). 21q22 balanced chromosome aberrations in therapy related haemopoietic disorders: report from an international workshop. Ge ne s, Chromosomes Cancer, 33, 379–344.

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18 Andersen MK, Larson RA, Muritzson N, Schnittger S, Jhanwar SC, Pedersen-Bjergaard J (2002). Balanced chromosome abnormalities inv(16) and t(15;17) in therapy related myelodysplastic syndromes and acute leukemia: report from an international workshop. Genes Chromosomes Cancer, 33, 395–400. 19 Applebaum FR, Kopecky KJ, Tallman MS, Slovak ML, Gundacker HM, Kim HT et al. (2006). The clinical spectrum of adult acute myeloid leukaemia associated with core binding factor translocations. Br J Haematol, 135, 165–173. 20 Thiede C, Steudel C, Mohr B, Schaich H, Schakel U, Platzbecker U et al. (2002). Analysis of FLT3–activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood, 99, 4326–4335. 21 Schnittger S, Schoch C, Dugas M, Kern W, Staib P, Wuchter C et al. (2002). Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood, 100, 59–66.

22 Thiede C, Koch S, Creutzig E, Steudel C, Illmer T, Schaich M et al. (2006). Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukaemia (AML). Blo o d, 107, 4011–1020. 23 Radmacher MD, Marcucci G, Ruppert AS, Mrozek K, Whitman SP, Vardiman JW et al. (2006). Independent confirmation of a prognostic gene-expression signature in adult acute myeloid leukemia with a normal karyotype: a Cancer and Leukemia Group B study. Blood, 108, 1677–1683. 24 Baldus CD, Mrózek K, Marcucci G, Bloomfield CD (2007). Clinical outcome of de novo acute myeloid leukaemia patients with normal cytogenetics is affected by molecular genetic alterations: a concise review. Br J Haematol, 137, 387–400. 25 Estey E, Dohner H (2006). Acute myeloid leukaemia. Lancet, 368, 1894–1907.

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Myeloproliferative neoplasms

The concept of the myeloproliferative disorders (MPD) or myeloproliferative neoplasms (MPN) as a group of closely related disorders was formulated by William Dameshek in 1951 [1]. His editorial on this subject commenced with the words “With accumulating experience, it becomes more and more evident that the bone marrow cells – erythroblasts, granulocytes, megakaryocytes – often proliferate en masse or as a unit rather than as individual elements.” The six disorders he included in this group were chronic granulocytic leukaemia, polycythaemia vera, thrombocythaemia, megakaryocytic leukaemia, idiopathic or agnogenic myeloid metaplasia of the spleen (and liver) and erythroleukaemia (di Guglielmo’s syndrome). The concept of a group of related disorders was based on shared features and on the observation of transitional forms and the evolution of one condition into another. There was at this stage no conception of the neoplastic nature of these conditions. Dameshek suspected that the cause was a myelostimulatory factor, perhaps adrenocorticotropic hormone. He also viewed the myelofibrosis as an intrinsic part of the disease with reticulum cells and fibroblasts participating in the process. With the advances in knowledge in the last 50 years it is now possible to refine Dameshek’s ideas. The MPN are now known to be haematological neoplasms characterized, at least in their early stages, by effective proliferation of myeloid cells of at least one lineage. The World Health Organization (WHO) classification now suggests use of the term ‘myeloproliferative neoplasm’. Evolution to myelofibrosis is possible in all. The myeloid metaplasia in the spleen and liver is, as Dameshek suspected, an intrinsic part of these disorders, not a compensatory phenomenon, but the fibrosis is a reactive process. All of these conditions have the potential to evolve into acute myeloid leukaemia

(AML) but with a variable frequency. Transformation is common in chronic granulocytic leukaemia (see below) and less common in other MPN. With the discovery 35 years ago of a recurrent cytogenetic abnormality, t(9;22)(q34;q11) [2, 3] and the subsequent discovery of the BCR-ABL1 fusion gene [4] in chronic granulocytic leukaemia, it was recognized as a quite distinct entity within the MPN. Recent scientific advances have confirmed the close relationship of polycythaemia vera, essential thrombocythaemia and idiopathic myelofibrosis. In 2005 four groups almost simultaneously described a point mutation in the JAK2 gene, JAK2 V617F, which is present in virtually all cases of polycythaemia vera and in about half of cases of essential thrombocythaemia and idiopathic myelofibrosis [5–9]. A point mutation in the MPL gene, MPL W515L, is found in a lower percentage of cases of essential thrombocythaemia and idiopathic myelofibrosis (Figure 3.1). Cases of essential thrombocythaemia with a JAK2 mutation have disease features closer to polycythaemia vera than do other cases. As further conditions were recognized that shared features with the classical MPN, this category of haematological neoplasm was expanded to include other chronic myeloid leukaemias – atypical (Philadelphianegative) chronic myeloid leukaemia (aCML), neutrophilic leukaemia (Figure 3.2) and chronic eosinophilic leukaemia. Systemic mastocytosis could also reasonably be assigned to the MPN group since the mast cell is of myeloid origin and in the 2008 WHO classification this has been done. With the development of the French–American–British (FAB) classification of myelodysplastic syndromes (MDS) [10, 11] there was a recognition of two broad groups of haematological neoplasms, one characterized by effective proliferation (MPN) and one by ineffective (MDS). Di

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Polycythaemia vera

Essential thrombocythaemia

Idiopathic myelofibrosis

Figure 3.1 Diagram showing the frequency of JAK2 V617F (blue) and MPL W515L (red) mutations in patients with polycythaemia vera, essential thrombocythaemia and chronic idiopathic myelofibrosis.

Figure 3.2 Peripheral blood film in chronic neutrophilic leukaemia. MGG stain, high power.

Guglielmo’s syndrome has often been ignored in later classifications. It falls more naturally into the MDS group (refractory anaemia) or AML rather than into MPN, cases with increased blast cells being assigned to the FAB M6 category of AML. The FAB group regarded the condition known as chronic myelomonocytic leukaemia (CMML) as MDS since there could be dysplastic features in various myeloid lineages and anaemia sometimes occurred. This caused some problems since dysplastic features were, if anything, more common in aCML than in CMML. The problem with CMML was resolved by the 2001 WHO classification, which divided the chronic myeloid neoplasms into three rather than two groups [12]. The classification recognized the existence of overlap syndromes (MDS/MPN) in which there were features of both MPN (effective haemopoiesis and overproduction of end cells of at least one lineage) and MDS (ineffective haemopoiesis and underproduction of cells of at least one myeloid lineage, usually accompanied by dysplasia) [13]. CMML and aCML, together with juvenile myelomonocytic leukaemia (JMML), were assigned to this new group. The overlap

syndromes also include refractory anaemia with ring sideroblasts and thrombocytosis (RARS-T); these cases often have a JAK2 mutation and are thus closely related to the classical MPN. In the WHO classification, there is an ‘unclassifiable’ category for cases that do not meet the criteria for any more specific category. Mast cell disorders are a group by themselves rather than being recognized as a MPN in the 2001 classification but in the 2008 classification are grouped with other MPN. With advancing knowledge of the molecular mechanisms underlying chronic haematological neoplasms, the three broad groups of MPN, MDS and MDS/MPN may prove constricting. Cases with the same molecular abnormality will sometimes fall into the MPN category and sometimes into the MDS/MPN group. The next decade is likely to see a reappraisal of the basis of our classifications. Until recently myeloproliferative disorders have generally been categorized as shown in Table 3.1, but it is likely that following publication of the 2008 modifications of the WHO classification the classification shown in Table 3.2 will be used increasingly.

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Table 3.1 The myeloproliferative disorders (2001 WHO classification)

Chronic myelogenous leukaemia (chronic granulocytic leukaemia) Chronic neutrophilic leukaemia Chronic eosinophilic leukaemia Polycythaemia vera Essential thrombocythaemia Chronic idiopathic myelofibrosis Chronic myeloproliferative disorder, unclassifiable

Table 3.2 The myeloproliferative neoplasms and related conditions (2008 WHO classification)

Chronic myelogenous leukaemia (chronic granulocytic leukaemia), BCR-ABL1+ Chronic neutrophilic leukaemia Chronic eosinophilic leukaemia, not otherwise specified Polycythaemia vera Essential thrombocythaemia Primary myelofibrosis Mastocytosis Myeloproliferative neoplasm, unclassifiable Myeloid and lymphoid ne oplasms associated with e osinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1 Myeloid and lymphoid neoplasms associated with eosinophilia and abnormalities of PDGFRA Myeloid neoplasms associated with eosinophilia and abnormalities of PDGFRB Myeloid and lymphoid neoplasms associated with eosinophilia and abnormalities of FGFR1

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References 1 Dameshek W (1951). Some speculations on the myeloproliferative syndromes. Blood, 6, 372–375. 2 Nowell PC and Hungerford DA (1960). Chromosome studies on normal and leukemic human leukocytes. J Natl Cancer Inst, 5, 85–109. 3 Rowley JD (1973). A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine staining and Giemsa staining. Nature, 243, 290–293. 4 Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR and Grosveld G (1984). Philadelphia chromosome breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell, 36, 93–99. 5 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. (2005). A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med, 352, 1779–1790. 6 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S et al. (2005). Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet, 365, 1054–1061. 7 Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L et al. (2005). Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood, 106, 2162–2168. 8 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Staerk J et al. (2005). A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature, 434, 1144–1148.

9 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ et al. (2005). Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell, 7, 387–397. 10 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR and Sultan C (1982). Proposals for the classification of the myelodysplastic syndromes. Br J Haematol, 51, 189–199. 11 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR and Sultan C (1985). Proposed revised criteria for the classification of acute myeloid leukemia. Ann Intern Med, 103, 626–629. 12 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, IARC Press, Lyon. 13 Vardiman JW (2001). Myelodysplastic/myeloproliferative disease: introduction. In: Jaffe ES, Harris NL, Stein H and Vardiman JW (eds), World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 47–48.

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Chronic myeloid leukaemia

Chronic myeloid leukaemia (CML), also known as chronic granulocytic leukaemia and chronic myelogenous leukaemia, is a chronic myeloproliferative neoplasm that results from a t(9;22)(q34;q11.2) translocation occurring in a pluripotent lymphoid-myeloid haemopoietic stem cell. The translocation gives rise to an abbreviated chromosome 22 (22q–) referred to as the Philadelphia (Ph) chromosome. It also leads to formation of a BCR-ABL1 fusion gene that encodes a constitutively activated tyrosine kinase. CML is a biphasic or triphasic disease. Following a chronic phase of variable length, there is transformation to acute leukaemia (‘blast transformation’) that is usually myeloid but sometimes lymphoblastic or mixed lineage. An accelerated phase can precede acute transformation. Some patients present in acute transformation without a clinically apparent chronic phase. Aetiological factors include irradiation [1] and probably exposure to topoisomerase-IIinteractive drugs [2].

Clinical features Some patients are diagnosed as a result of an incidental blood count. Others have symptoms such as fatigue, lethargy, weight loss, abdominal discomfort and early satiety. Physical examination may show splenomegaly and less often hepatomegaly. During the accelerated phase there may be refractory splenomegaly. Acute transformation is characterized by variable combinations of pallor, bruising and bleeding, lymphadenopathy, soft tissue or bone infiltration and increasing splenomegaly.

Haematological and pathological features The blood count shows leucocytosis and anaemia. The majority of patients have a normal platelet count or thrombocytosis but a minority have thrombocytopenia. The leucocytosis is the result of a generalized increased in myeloid cells [3] (Figures 4.1 and 4.2). There is neutrophilia, basophilia and often eosinophilia. Granulocyte precursors are also present in the blood but orderly differentiation is preserved so that blast cells are less numerous than promyelocytes, which in turn are less numerous than myelocytes. The two most numerous cells in the differential count are myelocytes and mature neutrophils. Prominent monocytosis is rarely present and is associated with a BCR-ABL1 fusion gene encoding a p190 protein rather than the p210 protein more usually seen in CML. The anaemia is mild to moderate with normocytic normochromic red cells. Patients who present with thrombocytosis with little or no elevation of the white cell count and who are found to have a BCR-ABL1 fusion gene should be regarded as having a variant form of CML. During the accelerated phase there may be worsening anaemia (with anisocytosis and poikilocytosis), thrombocytopenia, increasing blast cells and increasing numbers of basophils. When acute transformation occurs, blast cells usually appear in the blood in increasing numbers and anaemia and thrombocytopenia are very common (Figures 4.3–4.7). There may or may not be neutropenia. In those patients in whom blast transformation occurs at an extramedullary site, there may initially be no new abnormality present in the blood or bone marrow. Bone marrow aspiration shows a markedly hypercellular marrow with an increase of all myeloid cells (Figure 4.8).

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Figure 4.1 Peripheral blood film of a patient with CML showing neutrophils and their precursors, one basophil and one eosinophil precursor. MGG, high power.

Figure 4.3 Peripheral blood film of a patient with CML in myeloid transformation showing three blast cells. MGG, high power.

Figure 4.2 Peripheral blood film of a patient with CML showing granulocytes (neutrophils, eosinophils and a basophil) and their precursors. MGG, high power.

Figure 4.4 Peripheral blood film of a patient with CML in myeloid transformation showing three blast cells and dysplastic maturing cells. MGG, high power.

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Figure 4.5 Peripheral blood film of a patient with CML in myeloid transformation showing a micromegakaryocyte. MGG, high power.

Figure 4.7 Peripheral blood film of a patient with CML in mixed myeloid/lymphoblastic transformation (same patient as Figure 4.6), showing small and medium sized blast cells. MGG, low power.

Figure 4.6 Peripheral blood film of a patient with CML in mixed myeloid/lymphoblastic transformation showing blast cells and dysplastic myeloid cells. MGG, low power.

Figure 4.8 Bone marrow aspirate film from a patient with CML showing hypercellularity. MGG, low power.

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The myeloid:erythroid ratio is greatly increased (Figure 4.9). Megakaryocytes are usually increased and, on average, are smaller than normal and relatively hypolobated. There may be a mild to moderate increase in reticulin deposition. Sea-blue histiocytes and pseudo-Gaucher cells can be increased and reflect increased cell turnover (Figure 4.10).

Figure 4.9 Bone marrow aspirate film from a patient with CML showing a hypolobated megakaryocyte and increased cells of all granulocyte lineages. MGG, low power.

Figure 4.11 Bone marrow aspirate in megakaryoblastic transformation of CML showing a blast cell of undetermined lineage (left) and a megakaryoblast (right). MGG, high power.

When blast transformation occurs the bone marrow aspirate shows infiltration by blast cells that can be identified on immunophenotyping as myeloblasts, megakaryoblasts, lymphoblasts or a combination of these. When there is megakaryoblastic transformation, micromegakaryocytes may be prominent (Figures 4.11–1.14).

Figure 4.10 Bone marrow aspirate film from a patient receiving imatinib for CML showing a sea-blue histiocyte containing several erythrocytes. MGG, high power.

Figure 4.12 Bone marrow aspirate in megakaryoblastic transformation of CML showing a micromegakaryocyte to the right of an eosinophil myelocyte (same patient as Figure 4.11). MGG, high power.

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Trephine biopsy sections show orderly granulopoiesis with expansion of the paratrabecular and periarteriolar cuff of immature myeloid cells (Figure 4.15). Sometimes storage cells are markedly increased (Figure 4.16). In the accelerated phase and blast transformation trephine biopsy is particularly useful for showing dysplastic megakaryocytes

in a fibrotic marrow; osteosclerosis may also be revealed (Figures 4.17 and 4.18). In the accelerated phase, dysplastic features and increased blast cells and basophils can be seen. Morphological and clinical criteria for accelerated phase suggested in the World Health Organization (WHO) 2001

Figure 4.13 Bone marrow aspirate in megakaryoblastic transformation of CML showing a micromegakaryocyte budding off sheets of platelets (same patient as Figure 4.11). MGG, high power.

Figure 4.14 Bone marrow aspirate in megakaryoblastic transformation of CML showing a binucleate micromegakaryocyte (same patient as Figure 4.11). MGG, high power.

Figure 4.15 Bone marrow trephine biopsy section from a patient with CML showing an increase of megakaryocytes and of granulocytes and their precursors. H&E, low power.

Figure 4.16 Bone marrow trephine biopsy section from a patient with CML showing increased granulocytes and granulocyte precursors and large numbers of pseudoGaucher cells. H&E, low power.

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Figure 4.17 Bone marrow trephine biopsy section from a patient with CML in transformation showing increased numbers of dysplastic megakaryocytes and osteomyelosclerosis. H&E, low power.

Figure 4.18 Bone marrow trephine biopsy section from a patient with CML in transformation showing increased numbers of dysplastic megakaryocytes and deposition of fine collagen fibres (same patient as Figure 4.17). H&E, high power.

and 2008 classifications are: 1) persistent or increasing leucocytosis WBC (>10 × 109/l) and/or persistent or increasing splenomegaly unresponsive to therapy, 2) persistent thrombocytosis (platelet count >1000 × 109/l) uncontrolled by therapy, 3) persistent thrombocytopenia (platelet count <100 × 109/l) unrelated to therapy, 4) 20% or more basophils in the peripheral blood and 5) 10–19% myeloblasts in the blood or bone marrow. Blast transformation is characterized by increasing replacement of chronic phase maturing cells by blast cells. Once the blast percentage in the blood of bone marrow reaches 20% this diagnosis is made. Depending on the specific genetic events that lead to the transformation, these may be myeloblasts, megakaryoblasts, lymphoblasts or a mixture of blast cells belonging to different lineages. Blast cells may also co-express antigens typical of different lineages. In some patients blast crisis involves mainly cells of megakaryocyte lineage – megakaryoblasts and megakaryocytes, often including micromegakaryocytes; in these patients the platelet count may be normal or high. Sometimes there is maturation largely to eosinophils. Basophil precursors are often prominent and occasionally a basophilic blast crisis occurs. Trephine biopsy sections may show, in addition to increasing numbers of blast cells and sometimes megakaryocytes, increased reticulin deposition, collagen formation and osteosclerosis. Initially the

accumulation of blast cells may be focal and this is better detected by trephine biopsy than by an aspirate. Blast crisis can also occur at extramedullary sites such as bone or lymph nodes without evidence of transformation in the blood or bone marrow. A range of other pathological features can occur following stem cell transplantation. Figure 4.19 shows one such posttransplant complication.

Immunophenotype The immunophenotype is not relevant during chronic phase disease. During accelerated phase or acute transformation, it can be used to characterize the blast cells (Figure 4.20). Myeloblasts are often quite primitive cells with little cytochemical evidence of their lineage so that immunophenotyping is needed for their recognition. Lymphoblastic transformation is usually B-lineage; uncommonly it is T-lineage. Immunohistochemistry to detect CD34 (or terminal deoxynucleotidyl transferase) is useful for highlighting blast cells in accelerated phase and blast transformation. Use of monoclonal antibodies directed at platelet glycoproteins highlights small dysplastic megakaryocytes in trephine biopsy sections; these often occur in sheets in advanced phase disease.

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Figure 4.19 Peripheral blood film of a patient with CML following allogeneic stem cell transplantation showing erythrocyte fragments resulting from micro-angiopathic haemolytic anaemia. MGG, high power.

073222

Figure 4.20 Dot plot showing flow cytometry immunophenotyping in CML. Sideways (SSC) and forward (FSC) light scatter have been used to gate on cells of granulocyte lineage (green, P2) and monocyte lineage (red, P1); ungated data are black. There are expanded populations of cells of granulocyte lineage and of monocyte lineage. The granulocyte population shows a mature myeloid phenotype with expression of CD11b, CD13, CD14, CD45, HLA-DR and CD10 (normally expressed with moderate intensity on neutrophils), and strong expression of MPO. In this sample CD11b is strongly expressed on the cells of granulocyte lineage and more weakly on those of monocyte lineage, in both lineages indicating maturation. As expected, the monocyte population show strong expression of CD14 and CD64. Co-expression of CD13 and CD34 by a small proportion of cells of granulocyte lineage reflects the presence of a low proportion of blast cells With thanks to Mr Ricardo Morilla.

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Cytogenetic and molecular genetic abnormalities The characteristic cytogenetic abnormality, t(9;22)(q34;q11.2) (Figure 4.21), can be demonstrated by conventional cytogenetic analysis or by fluorescence in situ hybridization (FISH) analysis using probes for BCR and ABL1. Simple and complex translocations also occur but all lead to formation of a BCR-ABL1 fusion gene. This can also be demonstrated by the polymerase chain reaction (PCR). Different breakpoints lead to BCR-ABL1 fusion genes encoding proteins of various lengths. Most typical is p210 with p190 (typical of Ph-positive acute lymphoblastic leukaemia) and p230 occurring less frequently. Accelerated phase and blast crisis are associated with clonal evolution, often shown by additional cytogenetic and molecular abnormalities, e.g. +8, +19, +22q– (i.e. an extra copy of the Ph chromosome) or i(17q) and mutations affecting RAS genes or TP53. The acquisition of an extra cytogenetic abnormality not present at diagnosis has been suggested as a criterion for accelerated phase disease in the WHO classification.

Molecular genetic analysis is also used to monitor response to treatment, real time quantitative PCR (RQPCR) being most useful for this purpose. Molecular analysis can also be used to demonstrate the mechanism of resistance to the primary treatment, imatinib (see below), and predict the likelihood of response to alternative therapies. A common mechanism of resistance is further mutation in the BCR-ABL1 gene, with some mutations causing resistance also to alternative targeted therapy.

Diagnosis and differential diagnosis The differential count and blood film of CML are so characteristic that there is hardly ever any difficulty distinguishing this condition from reactive leucocytosis. The diagnosis is confirmed by either cytogenetic or molecular genetic analysis. One or other of these should be carried out in all cases since sometimes Ph-negative myeloid leukaemias can resemble the Ph-positive condition. Rarely it is necessary to recognize cases of apparent essential thrombocythaemia as a forme fruste of CML in which there

Figure 4.21 A karyogram of a patient with CML showing t(9;22)(q34;q11.2). The derivative chromosome, 22q–, is known as the Philadelphia (Ph) chromosome. There has also been duplication of part of the long arm of a chromosome 17, with the duplicated segment being attached to the short arm of a chromosome 19. Some secondary abnormalities, such as gain of the abnormal 22 or presence of an isochromosome of the long arm of a chromosome 17, are common in CML. The secondary abnormality shown here is rare. With thanks to Dr John Swansbury.

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is thrombocytosis with little leucocytosis; the presence of t(9;22) or BCR-ABL1 fusion means that the diagnosis is not essential thrombocythaemia. A precise diagnosis is essential for directing treatment. CML needs to be distinguished from atypical chronic myeloid leukaemia (a Ph-negative condition) and from chronic myelomonocytic leukaemia. Morphology is usually sufficient; difficult cases may need molecular genetics.

Prognosis Prognosis is greatly affected by the phase of the disease with accelerated phase being prognostically worse than chronic phase and blast transformation being prognostically very adverse. For patients in chronic phase, several prognostic scoring systems have been developed but their applicability with current treatment is uncertain. The system proposed by Sokal et al. [4] remained relevant in imatinib-treated patients in one study [5] but not in another [6]. In the second of these studies the Hasford score [7] was not an independent risk factor but age, peripheral blood blast percentage and increased bone marrow basophils remained prognostically adverse [6]. In imatinib-treated patients (see below), response to therapy is of considerable prognostic importance. In several studies complete cytogenetic response at 1 year was found to be prognostically favourable [5, 6] as was a good molecular response (e.g. greater than 3 logs reduction in leukaemic cells) [5, 8].

Treatment Historically CML was treated with busulphan then with hydroxycarbamide (previously known as hydroxyurea) and later with interferon or, when possible, stem cell transplantation. The development of imatinib, a tyrosine kinase inhibitor that is active against the BCR-ABL1 gene product, has revolutionized the treatment of this disease. It is now first line treatment, being used in a starting dose of 400 mg daily, increasing in a stepwise manner to 800 mg daily if the cytogenetic response is inadequate [9]. Imatinib therapy was associated with an 83% event-free survival and 89% overall survival at 5 years in one study [5] and an 88% overall survival at 5 years in another [6]. Allogeneic transplantation and to a lesser extent interferon therapy

remain options for patients who are refractory to imatinib. The necessary duration of imatinib therapy is not yet established. If therapy is stopped after a minimum of 2 years of continuous molecular remission, around 50% of patients relapse [10]; a second remission may be achieved with readministration of imatinib. It is important to monitor the cytogenetic and molecular response during imatinib therapy so that an inadequate response is detected and alternative methods of treatment can be considered [11]. Alternative treatments include other tyrosine kinase inhibitors, e.g. dasatinib and nilotinib, interferon and homoharringtonine. At present stem cell transplantation is still sometimes used in children but in adults its use is confined to patients with a suboptimal response to the tyrosine kinase inhibitors.

References 1 Little MP, Weiss HA, Boice JD, Draby SC, Day NE and Muirhead CR (1999). Risks of leukemia in Japanese atomic bomb survivors, in women treated for cervical cancer, and in patients treated for ankylosing spondylitis. Radiat Res, 152, 280–292. 2 Pedersen-Bjergaard J, Brøndum-Nielsen K, Karle H and Johansson B (1997). Chemotherapy-related lateoccurring Philadelphia chromosome in AML, ALL and CML. Similar events related to treatment with DNA topoisomerase II inhibitors? Leukemia, 11, 1571–1574. 3 Spiers AS, Bain BJ and Turner JE (1977). The peripheral blood in chronic granulocytic leukaemia. Study of 50 untreated Philadelphia-positive cases. Scand J Haematol, 18, 25–38. 4 Sokal JE, Cox EB, Baccarani M, Tura S, Gomez GA, Robertson JE et al. (1984). Prognostic discrimination in ‘good-risk’ chronic granulocytic leukemia. Blood, 63, 789–799. 5 Druker BJ, Guilhot F, O’Brien SG, Gathmann I, Kantarjian H, Gattermann N et al., IRIS investigators (2006). Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med, 355, 2408–2417. 6 Kantarjian HM, Talpaz M, O’Brien S, Jones D, Giles F, Garcia-Manero G et al. (2006). Survival benefit with imatinib mesylate versus interferon-alpha-based regimens in newly diagnosed chronic-phase chronic myelogenous leukemia. Blood, 108, 1835–1840.

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7 Hasford J, Pfirrmann M, Hehlmann R, Allan NC, Baccarani M, Kluin-Nelemans JC et al. (1998). A new prognostic score for survival of patients with chronic myeloid leukemia treated with interferon alfa. Writing Committee for the Collaborative CML Prognostic Factors Project Group. J Natl Cancer Inst, 90, 850–858. 8 Press RD, Love Z, Tronnes AA, Yang R, Tran T, Mongoue-Tchokote S et al. (2006). BCR-ABL mRNA levels at and after the time of a complete cytogenetic response (CCR) predict the duration of CCR in imatinib mesylate-treated patients with CML. Blo o d, 107, 4250–4256. 9 Goldman JM (2007). How I treat chronic myeloid leukaemia in the imatinib era. Blood, 110, 2828–2837.

10 Rousselot P, Huguet F, Rea D, Legros L, Cayuela JM, Maarek O e t al. (2007). Imatinib mesylate discontinuation in patients with chronic myelogenous leukemia in complete molecular remission for more than 2 years. Blood, 109, 58–60. 11 Baccarani M, Saglio G. Goldman J, Hochhaus A, Simonsson B, Appelbaum F et al. (2006). Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. Blood, 108, 1809–1820.

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Chronic eosinophilic leukaemia

Chronic eosinophilic leukaemia (CEL) designates a type of Philadelphia (Ph)-negative BCR-ABL1-negative chronic myeloid leukaemia in which eosinophils and their precursors constitute the dominant neoplastic cell. However, the causative mutation occurs in a multipotent myeloid stem cell (or often a pluripotent lymphoid-myeloid stem cell) so that other lineages are also part of the neoplastic clone. The distinction from chronic myelomonocytic leukaemia with eosinophilia and atypical chronic myeloid leukaemia with eosinophilia is somewhat artificial. It is very important to recognize cytogenetic/molecular genetic sub-groups of CEL since their clinicopathological features and their prognoses differ. CEL was arbitrarily defined in the 2001 World Health Organization (WHO) classification [1], as a BCR-ABL1negative haematological neoplasm in which the eosinophil count is at least 1.5 × 109/l, other causes of eosinophilia are excluded and there is some positive evidence that the process is neoplastic (e.g. clonal eosinophils or increased blast cells in blood or bone marrow). In the 2008 WHO classification, specific categories for CEL associated with rearrangement of PDGFRA, PDGFRB or FGFR1 were recognized with remaining cases being designated ‘CEL, not otherwise specified’. In the case of PDGFRA and FGFR1 rearrangement, neoplasms may be lymphoid as well as myeloid.

Clinical features Clinical features may include those expected in a chronic myeloid leukaemia, such as hepatomegaly, splenomegaly and symptoms of anaemia. Other features, which are attributable more specifically to the eosinophil proliferation and release of eosinophil granule contents, include cardiac

damage, pulmonary infiltration with cough and dyspnoea, gastrointestinal symptoms, neuropathy and vasculitis.

Haematological and pathological features The blood film shows increased eosinophils and may or may not show an increase of neutrophils and monocytes or the presence of blast cells [2, 3]. Eosinophils may be cytologically normal or abnormal (Figures 5.1 and 5.2). A bone marrow aspirate shows an increase of eosinophils and their precursors. Charcot–Leyden crystals may be present in macrophages. In some patients there is an increase of blast cells (but these are, by definition, less than 20% of bone marrow and peripheral blood cells) or an increase of cells of other myeloid lineages, sometimes including mast cells (seen in association with both PDGFRA and PDGFRB rearrangement). Transformation to acute myeloid leukaemia can occur. In cases associated with FIP1L1-PDGFRA fusion there can also be transformation to T- lineage acute lymphoblastic leukaemia/lymphoblastic lymphoma [4] and in cases associated with FGFR1 to both T- and B-lineage acute lymphoblastic leukaemia/lymphoblastic lymphoma [5]. One patient with FIP1L1-PDGFRA fusion who presented with simultaneous acute eosinophilic leukaemia and Tlymphoblastic lymphoma has been reported [4].

Immunophenotype The immunophenotype is not relevant to diagnosis or management except in those patients who, at presentation, have acute leukaemia or lymphoblastic lymphoma or in whom acute transformation occurs.

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Figure 5.1 Peripheral blood film of a patient with CEL associated with FIP1L1-PDGFRA showing degranulated eosinophils. MGG, × 100 objective.

Cytogenetic and molecular genetic abnormalities Certain specific cytogenetic/molecular genetic entities can present as eosinophilic leukaemia (Table 5.1) [6–11]. In other patients there are less specific abnormalities such as trisomy 8 or 20q–. Patients with suspected eosinophilic leukaemia should be specifically investigated for FIP1L1PDGFRA fusion and translocations with a 5q31-q33 breakpoint (suggesting PDGFRB rearrangement). Since these subgroups of eosinophilic leukaemia are responsive to imatinib, cytogenetic/molecular genetic investigation is crucial for their management. Cytogenetic analysis will also lead to detection of patients with translocations that result in FGFR1 rearrangement (Figures 5.3 and 5.4). The JAK2 V617F mutation has been reported in three patients but may not represent the primary genetic change [12].

Diagnosis and differential diagnosis The differential diagnosis is reactive eosinophilia and the idiopathic hypereosinophilic syndrome [13]. Appropriate investigations include those for allergies, parasitic infection and lymphoid neoplasms, as well as those directed

Figure 5.2 Peripheral blood film of a patient with CEL associated with FIP1L1-PDGFRA showing degranulated and hypolobated eosinophils (same patient as Figure 5.1). MGG, × 100 objective.

specifically at confirming a diagnosis of eosinophilic leukaemia. The diagnosis of idiopathic hypereosinophilic syndrome cannot be made unless patients have been thoroughly investigated for reactive eosinophilia, eosinophilic leukaemia and aberrant cytokine-secreting T lymphocytes. If a clonal abnormality of myeloid cells is found, the diagnosis is eosinophilic leukaemia not the idiopathic hypereosinophilic syndrome. It is important that observation of an increase of mast cells does not lead to a misdiagnosis of systemic mastocytosis.

Prognosis Since the introduction of imatinib therapy, the prognosis of CEL associated with rearrangement of the PDGFRA gene or the PDGFRB gene is relatively good, with the median survival being considerably in excess of 2 years (median follow-up of 25 months) [14] and increasing with longer follow-up of cohorts of patients. In contrast, the prognosis of cases associated with rearrangement of the FGFR1 gene is poor, as a result of the lack of any specific effective treatment and the frequency of early transformation to acute myeloid or acute lymphoblastic leukaemia.

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Table 5.1 Cytogenetic/molecular genetic entities that can present as eosinophilic leukaemia or related conditions

Category

Cytogenetics

Molecular genetics

PDGFRA rearranged

Usually cryptic interstitial deletion at 4q12

FIP1L1-PDGFRA

Rarely t(4;22)(q12;q11) or other translocations with a 4q12 breakpoint

BCR-PDGFRA or other fusion genes incorporating part of PDGFRA

PDGFRB rearranged

Usually t(5;12)(q31-q33;p13)

ETV6-PDGFRB

FGFR1 rearranged

Most often t(8;13)(p11;q12)

ZNF198-FGFR1

Sometimes t(6;8)(q27;p11)

FGFR1OP1-FGFR1

Sometimes t(8;9)(p11;q33)

CEP1-FGFR1

Rarely other translocations with an 8p11 breakpoint

Other fusion genes incorporating part of FGFR1

Usually t(5;12)(q31-q35;p13)

ETV6-PDGFRB (see above)

Rarely t(5;12)(q31;p13)

ETV6-ASC2

Rarely t(9;12)(q34;p13)

ETV6-ABL1

Rarely t(9;12)(q22;p12)

ETV6-SYK

Rarely t(8;9)(p22;p24)

PCM1-JAK2

ETV6 rearranged

JAK2 rearranged

Figure 5.3 Peripheral blood film of a patient with chronic myelomonocytic leukaemia with eosinophilia with 8p11 (FGFR1) rearrangement. With thanks to Dr Donald MacDonald, London. MGG, × 100 objective.

Figure 5.4 Bone marrow aspirate film of a patient with chronic myelomonocytic leukaemia with eosinophilia with 8p11 (FGFR1) rearrangement, same patient as Figure 5.3. With thanks to Dr Donald MacDonald. MGG, × 100 objective.

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Treatment Cases of CEL associated with rearrangement of the PDGFRA gene or the PDGFRB gene are treated with the tyrosine kinase inhibitor, imatinib. They are more sensitive to this drug than are cases of Ph-positive chronic myeloid leukaemia. There is not yet any specific treatment for cases associated with rearrangement of the FGFR1 gene. For these patients, and for those with no demonstrated relevant molecular lesion, treatment with hydroxycarbamide, interferon or corticosteroids can lower the eosinophil count. Because of the poor prognosis, haemopoietic stem cell transplantation should be considered for suitable patients with rearrangement of the FGFR1 gene.

References 1 Bain B, Pierre R, Imbert M, Vardiman JW, Brunning RD and Flandrin G (2001). Chronic eosinophilic leukaemia and the hypereosinophilic syndrome. In: Jaffe ES, Harris NL, Stein H and Vardiman JW (eds). World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 29–31. 2 Bain BJ (2003). Cytogenetic and molecular genetic aspects of eosinophilic leukaemia. Br J Haematol, 122, 173–179. 3 Fletcher S and Bain B (2007). Eosinophilic leukaemia. Brit Med Bull, 81–82, 115–127. 4 Metzgeroth G, Walz C, Score J, Siebert R, Schnittger S, Haferlach C et al. (2007). Recurrent finding of the FIP1L1-PDGFRA fusion gene in eosinophilia-associated acute myeloid leukemia and lymphoblastic T-cell lymphoma. Leukemia, 21, 1183–1188. 5 Macdonald D, Reiter A and Cross NC (2002). The 8p11 myeloproliferative syndrome: a distinct clinical entity caused by constitutive activation of FGFR1. Ac ta Haematol, 107, 101–107. 6 Bain BJ and Fletcher S (2007). Chronic eosinophilic leukemias and the myeloproliferative variant of the hypereosinophilic syndrome. Im m uno l Alle rgy Clin North Am, 27, 377–388. 7 Bain BJ, Gilliland DG, Vardiman JW and Horny H-P (2008). Chronic eosinophilic leukaemia, not otherwise specified. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J and Vardiman JW (eds), World Health Organization Classification of Tumours:

Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 51–53. 8 Bain B, Gilliland DG, Vardiman J and Horny H-P (2008). Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J and Vardiman JW (eds), World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 68–73. 9 Bain B, Gilliland DG, Horny H-P and Vardiman J (2008). Myeloid and lymphoid neoplasms associated with PDGFRA rearrangement. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J and Vardiman JW (eds), Wo rld He alth Organizatio n Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 68–73. 10 Bain B, Gilliland DG, Horny H-P and Vardiman J (2008). Myeloid neoplasms associated with PDGFRB rearrangement. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J and Vardiman JW (eds), Wo rld He alth Organizatio n Classific atio n o f Tum o urs: Patho lo gy and Ge ne tic s o f Tum o urs o f Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 71–72. 11 Bain B, Gilliland DG, Horny H-P and Vardiman J (2008). Myeloid and lymphoid neoplasms associated with FGFR1 rearrangement. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J and Vardiman JW (eds), Wo rld He alth Organizatio n Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 72–73. 12 Helbig G, Stella-Holowiecka B, Majewski M, Lewandowska M and Holowiecki J (2007). Interferon induces a good molecular response in a patient with chronic eosinophilic leukemia (CEL) carrying the JAK2V617F point mutation. Haematologica, 92, e118–e119. 13 Tefferi A, Patnaik MM and Pardanani A (2006). Eosinophilia: secondary, clonal and idiopathic. Br J Haematol, 133, 468–492. 14 Baccarani M, Cilloni D, Rondoni M, Ottaviani F, Messa F, Merante S et al. (2007). The efficacy of imatinib mesylate in patients with FIP1L1-PDGFR-positive hypereosinophilic syndrome. Results of a multicenter study. Haematologica, 92, 1173–1179.

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Polycythaemia vera

Polycythaemia vera or polycythaemia rubra vera is a myeloproliferative neoplasm (MPN) in which the total volume of red cells in the circulation is increased as a result of mutation of a multipotent haemopoietic stem cell. This is a disease mainly of middle and old age with an incidence of 1–3/100,000 per year. A familial predisposition occurs [1, 2].

Clinical features Presentation may be with vascular complications such as cerebrovascular accident, myocardial infarction, splanchnic (mesenteric, hepatic or portal) vein thrombosis or peripheral ischaemia or gangrene. The incidence of venous thromboembolism is also increased. Patients may complain of itch, often exacerbated by a hot bath, and there is an increased incidence of peptic ulcer; both these disease features are related to histamine excess. Sometime the diagnosis is made incidentally when a blood count is performed for an unrelated reason. On examination, there may be a plethoric complexion, conjunctival injection or palpable splenomegaly, in addition to physical signs as a result of vascular complications. The disease can progress to post-polycythaemic myelofibrosis. Acute transformation can also occur.

Haematological and pathological features The blood count shows an increase in the red cell count (RBC), haemoglobin concentration (Hb) and haematocrit (Hct), except in those patients who have complicating iron deficiency. The white cell count (WBC) and the platelet count are sometimes also increased. The increased WBC is the result of neutrophilia; the absolute count of basophils is also often increased. The blood film appears ‘packed’ as a result of the increased viscosity of the blood (Figure 6.1). The bone marrow aspirate is hypercellular as a result of erythroid and granulocytic hyperplasia (Figures 6.2 and 6.3). Megakaryocyte numbers are often increased and megakaryocytes are pleomorphic with both large and small

Figure 6.1 Peripheral blood film of a patient with polycythaemia vera showing a ‘packed’ film and one basophil. MGG, high power.

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forms. Trephine biopsy confirms the features seen on the aspirate (Figures 6.4 and 6.5). The megakaryocytes may be clustered. Reticulin may be increased but characteristically this increase is slight (Figure 6.6). Serum vitamin B12 levels are increased as a result of an increase in leucocyte-derived B12 binding protein (haptocorrin). The neutrophil alkaline phosphatase score is

increased but this test is now rarely used in diagnosis. Serum erythropoietin concentration is decreased. Spontaneous growth of erythroid colonies in the absence of exogenous erythropoietin is characteristic. Radio-isotopic studies show that the total red cell volume is increased and the plasma volume is also often increased.

Figure 6.2 Bone marrow aspirate film of a patient with polycythaemia vera showing a hypercellular fragment. MGG, low power.

Figure 6.3 Bone marrow aspirate film of a patient with polycythaemia vera showing erythroid hyperplasia. MGG, high power.

Figure 6.4 Bone marrow trephine biopsy section from a patient with polycythaemia vera showing increased erythropoiesis and a cluster of megakaryocytes. H&E, low power.

Figure 6.5 Bone marrow trephine biopsy section from a patient with polycythaemia vera showing sea-blue histiocytes resulting from increased cell turnover. H&E, high power.

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del(20)(q11.2q13.3)

del(20)(q11.2q13.1)

Figure 6.7 Partial karyogram showing two examples of 20q–. Deletion of part of the long arms of chromosome 20 occurs in two forms, as illustrated. The normal 20 is shown on the left of each pair. There is a larger deletion in which both dark bands are lost and a smaller deletion in which dark band q13.2 is retained. Figure 6.6 Bone marrow trephine biopsy section from a patient with polycythaemia vera showing normal reticulin. Reticulin, high power.

Immunophenotype The immunophenotype is not relevant unless transformation occurs.

Cytogenetic and molecular genetic abnormalities A clonal cytogenetic abnormality may be present but such abnormalities are not specific to the disease. In addition, they do not represent the primary event since they may be present in only a proportion of clonal cells. Those most often observed are 20q– (Figure 6.7), trisomy 8, trisomy 9 and trisomy 1. A characteristic molecular abnormality, a V617F mutation in exon 14 of the JAK2 gene, was reported almost simultaneously by a number of research groups and is observed in about 95% of patients if sensitive techniques are used [3–9]. The other 5% of patients may have other JAK2 mutations, specifically in exon 12 [10]. The mutation occurs in a pluripotent lymphoid-myeloid stem cell [11] and thus both T and B lymphocytes can carry the mutation. It may be homozygous as a result of mitotic recombination of 9p leading to uniparental disomy. Somatic JAK2 mutations occur also in patients with a familial predisposition to polycythaemia [2]. The JAK2 V617F mutation may not be the primary event in the pathogenesis of this disease since it

is sometimes absent when acute transformation occurs. Although the JAK2 V617F mutation is becoming increasingly important in the diagnosis of polycythaemia vera it is not specific for this condition. It is also seen not infrequently (around 50% of cases) in essential thrombocythaemia, primary myelofibrosis and refractory anaemia with ring sideroblasts and thrombocytosis (RARS-T). It occurs less often in other myelo proliferative neoplasms. JAK2 mutation may be the marker of an occult MPN presenting as splanchnic vein thrombosis [12].

Diagnosis and differential diagnosis The main differential diagnoses of polycythaemia vera are relative polycythaemia (increased Hb as a result of reduced plasma volume) and secondary (true) polycythaemia (as a result of hypoxic cardiac or lung disease or inappropriate erythropoietin synthesis in renal disease, e.g. renal carcinoma). Occasionally other MPN need to be distinguished from polycythaemia vera, e.g. essential thrombocythaemia. The discovery of JAK2 mutations in virtually all patients with polycythaemia vera has led to proposals for new diagnostic criteria (Table 6.1) [13]. Patients who present with mesenteric, hepatic or portal vein

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Table 6.1 Proposed criteria for diagnosis of polycythaemia vera* [13]

Major criteria Haemoglobin concentration >18.5 g/dl (men) or >16.5 g/dl (women) or other evidence of increased red cell volume (appropriate elevation of haematocrit or red cell mass) (altitude to be taken into account) Presence of JAK2 V617F or other functionally similar mutation Minor criteria Bone marrow biopsy showing hypercellularity for age with prominent erythroid, granulocytic and megakaryocytic proliferation Serum erythropoietin below the reference range for normal Endogenous erythroid colony growth in vitro * Diagnosis requires both major criteria and one minor criterion or the presence of the first major criterion and two minor criteria

thrombosis should be screened for JAK2 mutations, even if there are no other features suggestive of a MPN. The diagnosis of polycythaemia vera may be missed when there is co-existing iron deficiency. However, in these patients the red cell indices may be suggestive in that the microcytosis is striking for the degree of anaemia and the RBC is either increased or is not as low as expected. The WBC and basophil count will not be affected by iron deficiency but it should be noted that the platelet count may be increased in uncomplicated iron deficiency anaemia.

Prognosis If the Hb/Hct are controlled and if low-dose aspirin is prescribed, life expectancy is improved compared with untreated patients but the death rate is nevertheless increased about twofold in comparison with the general (age- and gender-matched) population [14]. Most deaths are vascular in nature. A WBC greater than 15 × 109/l [15], higher age [14] and a previous history of thrombosis [14] have been associated with a higher rate of vascular complications. The ratio of mutated JAK2 to wild type

JAK2 correlates with major thrombotic events [14]. Although it is customary to attempt to lower the platelet count (see below), two large studies have not found an elevated platelet count indicative of thrombosis [14]. When transformation to acute leukaemia occurs, prognosis is poor.

Treatment The aim of treatment is to reduce the Hb and Hct to normal, reduce the platelet count to normal and control the hyperaggregability of the platelets. This is achieved by venesection and aspirin if there is isolated polycythaemia or, if the platelet count is also increased, by hydroxycarbamide in combination with low-dose aspirin (75–100 mg daily). In one large prospective study hydroxycarbamide was not associated with an increased risk of acute leukaemia in comparison with phlebotomy, whereas an increased risk was seen with busulfan, pipobroman and 32P [14]. Use of 32P is, however, appropriate in older patients. The Hct should be reduced to less than 0.55 l/l; whether it is necessary to reduce it to less than 0.50 l/l is not established [16]. The platelet count is customarily reduced to less than 600 × 109/l [16].

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References 1 Kralovics R, Stockton DW and Prchal JT (2003). Clonal hematopoiesis in familial polycythemia vera suggests the involvement of multiple mutational events in the early pathogenesis of the disease. Blood, 102, 3793–3797. 2 Cario H, Goerttler PS, Steimle C, Levine RL and Pahl HL (2005). The JAK2V617F mutation is acquired secondary to the predisposing alteration in familial polycythaemia vera. Br J Haematol, 130, 800–801. 3 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. (2005). A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med, 352, 1779–1790. 4 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S et al. (2005). Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet, 365, 1054–1061. 5 Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L et al. (2005). Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood, 106, 2162–2168. 6 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Staerk J et al. (2005). A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature, 434, 1144–1148. 7 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ et al. (2005). Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell, 7, 387–397. 8 Zhao R, Xing S, Li Z, Fu X, Li Q, Krantz SB et al. (2005). Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem, 280, 22788–22792. 9 Campbell PJ and Green A (2006). The myeloproliferative disorders. N Engl J Med, 355, 2452–2466. 10 Cazzola M (2007). Somatic mutations of JAK2 exon 12 as a molecular basis of erythrocytosis. Haematologica, 92, 1585–1589. 11 Delhommeau F, Dupont S, Tonetti C, Masse A, Godin I, Le Couedic JP et al. (2007). Evidence that the JAK2 G1849T (V617F) mutation occurs in a lymphomyeloid progenitor in polycythemia vera and idiopathic myelofibrosis. Blood, 109, 71–77.

12 Boissinot M, Lippert E, Girodon F, Dobo I, Fouassier M, Masliah C et al. (2007). Latent myeloproliferative disorder revealed by the JAK2-V617F mutation and endogenous megakaryocytic colonies in patients with splanchnic vein thrombosis. Blood, 108, 3223–3224. 13 Tefferi A, Thiele J, Orazi A, Kvasnicka HM, Barbui T, Hanson CA et al. (2007). Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood, 110, 1092–1097. 14 Finazzi G and Barbui T (2007). How I treat patients with polycythemia vera. Blood, 109, 5104–5111. 15 Landolfi R, Di Gennaro L, Barbui T, De Stefano V, Finazzi G, Marfisi R et al. European Collaboration on Low-dose Aspirin in Polycythaemia vera (ECLAP) (2007). Leukocytosis as a major thrombotic risk factor in patients with polycythemia vera. Blood, 109, 2446–2452. 16 DiNisio M, Barbui T, Di Gennaro L, Borrekki G, Finazzi G, Landolfi R et al., European Collaboration on Low-dose Aspirin in Polycythaemia Vera (ECLAP) Investigators (2007). The haematocrit and platelet target in polycythemia vera. Br J Haematol, 136, 249–259.

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Essential thrombocythaemia

Essential thrombocythaemia is a myeloproliferative neoplasm (MPN) characterized by sustained overproduction of platelets and resultant thrombocytosis. The incidence is 1–3/100,000/year. The disease arises as a result of mutation in a multipotent or pluripotent haemopoietic stem cell. A familial predisposition exists [1].

Clinical features An incidental diagnosis when a blood count is performed for some unrelated condition is the most frequent presentation. Those patients who are symptomatic usually present with cerebrovascular symptoms (e.g. transient ischaemic attacks, transient hearing or visual loss, recurrent headaches), peripheral ischaemia, erythromelalgia, arterial or venous thrombosis or haemorrhage. The spleen may be palpable but usually it is not. The liver is less often enlarged. Haemorrhage is sometimes due to acquired von Willebrand disease in patients with a very high platelet count, as a result of removal of large multimers of von Willebrand factor from the circulation by platelets. Evolution to myelofibrosis can occur. Development of myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML) occurs in a small minority of patients; cytogenetic evolution may be present at this time.

Haematological and pathological features The blood count and film show thrombocytosis. The white cell count (WBC) is sometimes increased. The absolute basophil count is occasionally increased and there may be small numbers of circulating granulocyte precursors.

Platelets typically include large forms and sometimes hypogranular forms (Figure 7.1). The haemoglobin concentration (Hb) is normal unless there has been haemorrhage. The neutrophil alkaline phosphatase score is often increased, particularly in patients with a JAK2 mutation [2]. Bone marrow aspiration (Figure 7.2) and trephine biopsy (Figures 7.3 and 7.4) show normal or mildly increased cellularity. Megakaryocytes are increased and on average are larger than normal with large well lobulated nuclei. Emperipolesis is increased. There may be granulocytic hyperplasia. Erythropoiesis is usually normal. On trephine biopsy sections, the megakaryocytes are often clustered. Reticulin may be mildly increased. The World Health Organization (WHO) classification [3] makes a distinction between essential thrombocythaemia and pre-fibrotic myelofibrosis. In essential thrombocythaemia the megakaryocytes are large to giant, in loose clusters or dispersed, with hyperlobated nuclei. In prefibrotic myelofibrosis the megakaryocytes are markedly abnormal with mainly large but also some small forms, they are clustered and sometimes abnormally situated near to the bone trabeculae; they have abnormal nuclei, including some with abnormal chromatin clumping and some that are cloud-like. Any increase in reticulin is minor at this stage.

Immunophenotype The immunophenotype is not relevant unless acute transformation occurs.

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Figure 7.1 Peripheral blood film of a patient with essential thrombocythaemia showing thrombocytosis and platelet anisocytosis. MGG, high power.

Figure 7.2 Bone marrow aspirate film of a patient with essential thrombocythaemia showing increased numbers of large megakaryocytes. MGG, low power.

Figure 7.3 Bone marrow trephine biopsy section from a patient with essential thrombocythaemia showing normal cellularity and increased numbers of mainly large megakaryocytes present in loose clusters. H&E, low power.

Figure 7.4 Bone marrow trephine biopsy section from a patient with essential thrombocythaemia showing a hypercellular marrow with increased numbers of mainly large megakaryocytes. H&E, low power.

Cytogenetic and molecular genetic abnormalities Clonal cytogenetic abnormalities may be present, although there is no specific disease-associated abnormality. They are present in only a small minority of patients with trisomy 8 and 9 being most often seen. The Philadelphia (Ph)

chromosome and BCR-ABL1 fusion gene are not found. DNA analysis shows a significant proportion of patients, around 50–60%, to have an activating mutation in the JAK2 V617F gene [4]. This may not, however, be the initiating

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event in development of the disease since it is sometimes absent when acute transformation occurs. The mutation is also sometimes found in T lymphocytes [5]. In comparison with patients without the mutation, these patients have some disease characteristics that are closer to those of polycythaemia, e.g. a higher Hb, a higher WBC, a more cellular bone marrow and an increased risk of venous thrombosis. For pregnant women, there is also a worse fetal outcome. Evolution into polycythaemia can occur during the course of the disease. A small minority (about 1%) of patients with essential thrombocythaemia have a mutation in the MPL gene, which encodes the thrombopoietin receptor [6]. Mutations reported include MPL W515L and MPL W515K. Patients who present with thrombocytosis and are found to have t(9;22) and a BCR-ABL1 fusion gene should be classified as a variant of chronic myeloid leukaemia (CML) rather than as essential thrombocythaemia, even if the WBC is normal.

Diagnosis and differential diagnosis A platelet count of 600 × 109/l is often taken as one of the diagnostic criteria for a diagnosis of essential thrombocythaemia. However, a JAK2 mutation has been found in patients with platelet counts ranging from 450 to 600 × 109/l, so that a lower cut-off point of 450 × 109/l appears appropriate and has been adopted in the 2008 WHO classification. Familial thrombocytosis should be considered in the differential diagnosis. In most ethnic groups this is rare but a common African polymorphism of the MPL gene, MPLBaltimore (found in 7% of African Americans), can cause significant thrombocytosis in heterozygotes, mean count 424 (observed range 320–505) × 109/l [7] and more marked thrombocytosis in homozygotes. The differential diagnosis includes reactive thrombocytosis, polycythaemia vera with coexisting iron deficiency, pre-fibrotic or early myelofibrosis, CML and refractory sideroblastic anaemia with thrombocytosis (RARS-T), a myelodysplastic/myeloproliferative disorder identified in the WHO classification [8]. In patients who are iron deficient, it may not be possible to make a distinction between essential thrombocythaemia and polycythaemia vera; serum erythropoietin is generally reduced in polycythaemia but it is sometimes reduced in essential thrombocythaemia and thus

cannot be used to make a distinction. Since marked thrombocytosis without leucocytosis can represent a forme fruste of CML, it is important to exclude the presence of a BCR-ABL1 fusion gene, at least in patients who do not have a JAK2 mutation; making the correct diagnosis has therapeutic implications because of the imatinib sensitivity of BCR-ABL1-positive cases. The discovery that the JAK2 V617F mutation is present in a significant proportion of patients with essential thrombocythaemia has led to proposals for new diagnostic criteria (Table 7.1) [9].

Prognosis Survival in the first decade after diagnosis is near normal but thereafter is clearly reduced [10]. An increased WBC, either greater than 8.7 (the median count in the study) [11] or greater than 15 × 109/l [10], has been associated with a

Table 7.1 Proposed criteria for a diagnosis of essential thrombocythaemia* [9]

Sustained increase of platelet count to at least 450 × 109/l Bone marrow biopsy specimen showing proliferation mainly of the megakaryocyte lineage with increased numbers of enlarged, mature megakaryocytes; no significant increase or left shift of neutrophil granulopoiesis or erythropoiesis Does not meet WHO criteria for a diagnosis of polycythaemia vera†, primary myelofibrosis, chronic myeloid leukaemia or myelodysplastic syndrome Demonstration of JAK2 V617F or other clonal marker or, in the absence of a clonal marker, no evidence of a reactive thrombocytosis *At least the first three criteria must be met; usually all four will be met † Iron-deficient polycythaemia should be excluded

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greater thrombotic risk. Age greater than 60 years, WBC greater than 15 × 109/l, coexisting diabetes mellitus and cigarette smoking are independent adverse prognostic factors [10]. The presence of a JAK2 mutation correlates with a greater risk of thrombosis [12]. Splenectomy should be avoided since it leads to a marked increase in platelet count and may shorten survival.

Treatment All patients should be advised to stop smoking. Patients with a moderate increase of the platelet count (e.g. <1000 × 109/l) under the age of 50–60 years and without cardiovascular risk factors require no treatment other than low-dose aspirin (100 mg daily) to correct platelet hyperaggregability. In those under the age of 40 years, even a platelet count of greater than 1000 × 109/l may not require treatment [13]. When more specific treatment is indicated, hydroxycarbamide (previously hydroxyurea) is most often used with anagrelide being an alternative. Aspirin should probably not be co-administered with anagrelide since the combination has been associated with an increased risk of haemorrhage. Interferon may be efficacious and may be useful during pregnancy. In elderly patients 32P is appropriate.

References 1 Randi ML, Fabris F, Vio C and Girolami A (1987). Familial thrombocythemia and/or thrombocytosis – apparently a rare disorder. Acta Haematol, 78, 63. 2 Basquiera AL, Fassetta F, Soria N, Barral JM, Ricchi B and García JJ (2007). Accuracy of leukocyte alkaline phosphatase score to predict JAK2 V617F mutation. Haematologica, 92, 704–705. 3 Imbert M, Pierre R, Thiele J, Vardiman JW, Brunning RD and Flandrin G (2001). Essential thrombocythaemia. In: Jaffe ES, Harris NL, Stein H and Vardiman JW (eds), T he Wo rld He alth Organizatio n Classific atio n o f Tum o urs: Patho lo gy and Ge ne tic s o f Tum o urs o f Haem o po ietic and Lym pho id Tissues. IARC Press, Lyon, pp. 39–41. 4 Campbell PJ and Green A (2006). The myeloproliferative disorders. N Engl J Med, 355, 2452–2466.

5 Larsen TS, Christensen JH, Hasselbalch HC and Pallisgaard N (2007). The JAK2 V617F mutation involves B- and T-lymphocyte lineages in a subgroup of patients with Philadelphia chromosome-negative chronic myeloproliferative disorders. Br J Haematol, 136, 745–751. 6 Pardanani AD, Levine RL, Lasho T, Pikman Y, Mesa RA, Wadleigh M et al. (2006). MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood, 108, 3472–3476. 7 Moliterno AR, Williams DM, Gutierrez-Alamillo LI, Salvatori R, Ingersoll RG and Spivak JL (2004). MPLBaltimore: a thrombopoietin receptor polymorphism associated with thrombocytosis. Proc Natl Acad Sci U S A, 101, 11444–11447. 8 Bain B, Vardiman JW, Imbert M and Pierre R (2001). Myelodysplastic/myeloproliferative disease, unclassifiable. In: Jaffe ES, Harris NL, Stein H and Vardiman JW (eds), Wo rld He alth Organizatio n Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 58–59. 9 Tefferi A, Thiele J, Orazi A, Kvasnicka HM, Barbui T, Hanson CA et al. (2007). Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood, 110, 1092–1097. 10 Wolanskyj AP, Schwager SM, McClure RF, Larson DR and Tefferi A (2006). Essential thrombocythemia beyond the first decade: life expectancy, long-term complication rates and prognostic factors. Mayo Clin Proc, 81, 159–166. 11 Carobbio A, Finazzi G, Guerini V, Spinelli O, Delaini F, Marchiolo R et al. (2007). Leukocytosis is a risk factor for thrombosis in essential thrombocythemia: interaction with treatment, standard risk factors, and JAK2 mutation status. Blood, 109, 2310–2313. 12 Finazzi G, Rambaldi A, Guerini V, Carobbo A and Barbui T (2007). Risk of thrombosis in patients with essential thrombocythemia and polycythemia vera according to JAK2 V617F mutation status. Haematologica, 92, 135–136. 13 Tefferi A, Gangat N and Wolanskyj AP (2006). Management of extreme thrombocytosis in otherwise low-risk essential thrombocythemia; does number matter? Blood, 108, 2493–2494.

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Idiopathic or primary myelofibrosis

Idiopathic or primary myelofibrosis indicates a myeloproliferative neoplasm (MPN) characterized by reactive bone marrow fibrosis. The causative mutation is in a multipotent or, more likely, a pluripotent haemopoietic stem cell. A multitude of names have been used for this condition, which indicates that none is wholly satisfactory. The International Working Group for Myelofibrosis Research and Treatment recommended use of the term ‘primary myelofibrosis’ [1] and this was accepted for the 2008 WHO classification. However, the condition is not idiopathic nor is it primary. The myelofibrosis is reactive, secondary to a myeloid neoplasm. Myelofibrosis with myeloid metaplasia has also been used but ‘myeloid metaplasia’, a term used in this context to indicate extramedullary haemopoiesis, can also occur with other forms of bone marrow disease with myelofibrosis, e.g. in the inherited condition, osteopetrosis. Since the condition is not of unknown origin (agnogenic), agnogenic myeloid metaplasia is similarly an unsatisfactory term. Chronic granulocytic–megakaryocytic myelosis is not inaccurate but does not convey the information that myelofibrosis is a characteristic disease feature. Chronic megakaryocytic leukaemia has also been suggested. ‘Myeloproliferative myelofibrosis’ could be suggested as a more appropriate name than those that are customarily used. The incidence is 0.3–1.5/100 000/year with a median age of onset of 65 to 75 years. A similar condition can occur following polycythaemia vera or essential thrombocythaemia, in which case the terms ‘post-polycythaemia myelofibrosis’ and ‘post-essential thrombocythaemia myelofibrosis’ are used. The disease may evolve into an accelerated phase with an increasing white cell count (WBC) and increasing blast cells and can transform into acute myeloid leukaemia (AML).

Clinical features Clinical features may be absent in the early stages of the disease. With disease progression, the spleen becomes palpable and later there is hepatomegaly. In late stage disease there may be massive splenomegaly and hepatomegaly. Other disease features relate to cytopenia and include the clinical features of anaemia and thrombocytopenia. Some patients have gout or renal calculi as a result of hyperuricaemia. With advanced disease there is fatigue, weight loss, low-grade fever, night sweats, generalized wasting and haemorrhage as a result of thrombocytopenia. Cardiac failure and portal hypertension can occur.

Haematological and pathological features In early stage disease there may be neutrophilia and thrombocytosis. As the disease advances, there is progressive pancytopenia. The blood film is leucoerythroblastic and shows poikilocytosis. Teardrop poikilocytes are particularly characteristic (Figures 8.1 and 8.2). Platelets show abnormal variation in size, including giant forms, and they may also be hypogranular. Ultrastructural examination shows morphological abnormalities of platelets (Figure 8.3) In addition to circulating erythroblasts, myelocytes and promyelocytes, there may also be blast cells (Figure 8.4) and occasional bare megakaryocyte nuclei or micromegakaryocytes (Figures 8.5 and 8.6). With disease progression, there may be increased myeloid proliferation. This can be characterized by differentiation to a specific lineage, e.g. megakaryocytic (Figures 8.7 and 8.8)

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Figure 8.1 Peripheral blood film of a patient with primary myelofibrosis showing anisocytosis, poikilocytosis, teardrop poikilocytes and a myelocyte. MGG, high power.

Figure 8.2 Peripheral blood film of a patient with primary myelofibrosis showing teardrop poikilocytes and an erythroblast (same patient as Figure 8.1). MGG, high power.

Figure 8.3 Ultrastructural examination of platelets in primary myelofibrosis. Lead nitrate and uranyl acetate stain.

Figure 8.4 Peripheral blood film of a patient with primary myelofibrosis showing anisocytosis, poikilocytosis and a blast cell. MGG, high power.

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Figure 8.5 Peripheral blood film of a patient with primary myelofibrosis showing a ‘bare’ megakaryocyte nucleus; on ultrastructural examination such nuclei show a thin rim of cytoplasm. MGG, high power.

Figure 8.6 Peripheral blood film of a patient with primary myelofibrosis showing a micromegakaryocyte and large, hypogranular platelets. MGG, high power.

Figure 8.7 Peripheral blood film of a patient with primary myelofibrosis in megakaryocytic/megakaryoblastic transformation showing a blast cell, two erythroblasts and circulating micromegakaryocytes. MGG, high power.

Figure 8.8 Bone marrow aspirate of a patient with primary myelofibrosis in megakaryocytic/megakaryoblastic transformation showing numerous micromegakaryocytes. MGG, high power.

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or eosinophilic (Figures 8.9–8.12). Megakaryocyte involvement is often prominent. Dysplasia can be striking. In early stage disease there is a cellular bone marrow with increased reticulin deposition. The likelihood of increased reticulin can often be anticipated from the H&E stained sections, which show wide open sinusoids, ‘streaming’ and sometimes a twisting artefact. Haemopoietic precursors may be present within sinusoids. Megakaryocytes (Figure 8.13) are dysplastic, clustered and sometimes abnormally sited in a paratrabecular position. The WHO classification recognizes a pre-fibrotic phase of myelofibrosis in which there is usually thrombocytosis and the marrow is hypercellular with little or no increase in reticulin deposition [1, 2]; megakaryocytes are clustered and dysplastic (typically there are hyperchromatic or large, cloud-like nuclei) and there is an increased frequency of bare megakaryocyte nuclei. Lymphoid nodules are increased.

In late stage disease the number of haemopoietic precursors diminishes and the marrow is increasingly occupied by fibroblasts and collagen (Figures 8.14 and 8.15). Megakaryocytes can remain more prominent than other haemopoietic cells. Angiogenesis is increased. CD34-positive cells are increased in the bone marrow earlier in the disease but decreased in advanced disease, numbers correlating inversely with the number in the peripheral blood [3]. The stromal changes of myelofibrosis are likely to be secondary to the action of cytokines such as transforming growth-factor β produced by megakaryocytes, monocytes and other myeloid cells, possibly aggravated by autoimmune responses to the altered stroma [4]. Splenic histology shows haemopoietic cells in the red pulp. In the liver they are located within sinusoids.

Figure 8.9 Peripheral blood film of a patient with primary myelofibrosis with evolution to predominantly eosinophil differentiation showing two blast cells and a hypogranular eosinophil. MGG, high power.

Figure 8.10 Peripheral blood film of a patient with primary myelofibrosis with evolution to predominantly eosinophil differentiation showing a granulocyte of uncertain lineage and an eosinophil precursor. Note that the eosinophil precursor has both eosinophilic and pro-eosinophilic (purple) granules (same patient as Figure 8.9). MGG, high power.

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Figure 8.11 Peripheral blood film of a patient with primary myelofibrosis with evolution to predominantly eosinophil differentiation showing a mature eosinophil with both eosinophilic and pro-eosinophilic granules and another granulocyte of uncertain lineage but possibly an abnormal eosinophil (same patient as Figure 8.9). MGG, high power.

Figure 8.13 Bone marrow trephine biopsy section in primary myelofibrosis showing clusters of dysplastic megakaryocytes. H&E, low power.

Figure 8.12 Peripheral blood film of a patient with primary myelofibrosis with evolution to predominantly eosinophil differentiation showing a highly abnormal eosinophil precursor (same patient as Figure 8.9). MGG, high power.

Figure 8.14 Bone marrow trephine biopsy section showing extensive fibrosis. H&E, low power.

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Figure 8.15 Bone marrow trephine biopsy section showing a marked increase in reticulin. Reticulin, low power.

Immunophenotype The immunophenotype of neoplastic cells is not relevant, except in characterizing blast transformation. Immunohistochemistry on trephine biopsy sections can be useful for highlighting vessels, CD34-positive haemopoietic progenitors and dysplastic megakaryocytes.

progression to massive splenomegaly than occurs in patients without the mutation [10]. An MPL gain-of function mutation, either MPL W515L or MPL W515K, is found in about 5% of patients and may coexist with a JAK2 V617F mutation [11].

Cytogenetic and molecular genetic abnormalities

Diagnosis and differential diagnosis

Cytogenetic analysis is not always successful because of the difficulty in aspirating bone marrow. A variety of clonal cytogenetic abnormalities have been observed, including trisomy 8, trisomy 9, trisomy 1q, del(13q) and del(20q) [5, 6]. Two of the more specific abnormalities are del(13)(q11~13q14~22) [5] and der(6)t(1;6)(q21~23;p21.3) [7]. The Philadelphia (Ph) chromosome and BCR-ABL1 fusion gene are not found. An activating mutation in the JAK2 gene, JAK2 V617F, is found in 50–60% of patients with primary myelofibrosis and is almost invariably found in patients with preceding polycythaemia vera [8]. Recombination can lead to transition from heterozygosity to homozygosity during the course of the illness. The mutation occurs in a pluripotent lymphoid–myeloid stem cell [9] and can be found in both T and B lymphocytes. Patients with this mutation have been found to have more likelihood of pruritis and thrombosis, a higher WBC, higher haemoglobin (Hb), lower transfusion requirement and, for those with homozygosity, more

The differential diagnosis of early stage disease includes essential thrombocythaemia. In contrast to the megakaryocytes of essential thrombocythaemia, which are large but cytologically fairly normal, those of pre-fibrotic myelofibrosis are markedly abnormal – mainly large but some small, with some nuclei showing abnormal chromatin clumping or being large, pale and ‘cloud-like’. In later stage disease the differential diagnosis includes other causes of bone marrow fibrosis. Because of the frequent occurrence of dysplasia of myeloid cells, there is usually no difficulty in making a distinction from bone marrow fibrosis secondary to non-haemopoietic malignancy or bone disease, but if difficulty does occur the problem can be resolved by immunohistochemistry. It is important to be aware of the wide range of conditions that can cause increased reticulin deposition and even collagen fibrosis [12] in order to avoid misdiagnosis of primary myelofibrosis. The discovery that the JAK2 V617F mutation is present in a significant proportion of patients with primary myelofibrosis has led to proposals for adoption of new

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diagnostic criteria (Table 8.1) [1]. It should be noted that ‘acute myelofibrosis’ is a different disease. It represents AML, often acute megakaryoblastic leukaemia, with a marked stromal response. Acute panmyelosis is also a different disease, this term indicating an acute myeloid neoplasm with trilineage involvement and with reactive fibrosis. In these two conditions there is marked reticulin fibrosis but collagen deposition is not prominent.

Prognosis Myelofibrosis is a chronic condition but is associated with a very significant shortening of life. Median survival is about 5 years. Survival is longer when the diagnosis is pre-fibrotic myelofibrosis. A worse prognosis is associated with age above 70 years, a Hb concentration less than 10 g/dl, a platelet count less than 100 × 109/l, an abnormal karyotype and a higher number of circulating CD34-positive cells [3,

13]. The JAK2 V617F mutation has been associated with a higher risk of leukaemic transformation [10] and, in two of three studies, with a worse survival [8, 10].

Treatment Treatment is essentially palliative. It includes erythropoietin, danazol and blood transfusion. Thalidomide was not found efficacious in a randomized trial [14]. Lenalidomide is indicated in the minority of patients with del(5q). Splenectomy is sometimes performed in order to reduce physical discomfort, improve the thrombocytopenia and lessen the transfusion requirement, although the operative mortality is high and splenectomy can lead to thrombotic complications. The only curative treatment, allogeneic stem cell transplantation, is not often feasible since patients are usually elderly.

Table 8.1 Proposed revised criteria* for diagnosis of primary myelofibrosis [1]

Major criteria 1. Presence of megakaryocyte proliferation and atypia,† usually accompanied by either reticulin and/or collagen fibrosis; in the absence of significant reticulin fibrosis, the megakaryocyte changes must be accompanied by an increased bone marrow cellularity characterized by granulocytic proliferation and often decreased erythropoiesis (i.e. pre-fibrotic cellular-phase disease) 2. Not meeting WHO criteria for polycythaemia vera, chronic myeloid leukaemia, myelodysplastic syndrome or other myeloid neoplasms 3. Demonstration of JAK2 V617F or other clonal marker (e.g. MPL W515L/K) or, in the absence of a clonal marker, no evidence of bone marrow fibrosis due to underlying inflammatory or other neoplastic diseases Minor criteria 1. Leukoerythroblastosis 2. Increase in serum lactate dehydrogenase level 3. Anaemia 4. Palpable splenomegaly * Diagnosis requires meeting all 3 major criteria and 2 minor criteria. Small to large megakaryocytes with an aberrant nuclear/cytoplasmic ratio and hyperchromatic, bulbous, or irregularly folded nuclei and dense clustering †

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Idiopathic or primary myelofibrosis

References 1 Tefferi A, Thiele J, Orazi A , Kvasnicka HM, Barbui T, Hanson CA et al. (2007). Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood, 110, 1092–1097. 2 Thiele J, Pierre R, Imbert M, Vardiman JW, Brunning RD and Flandrin G (2001). Chronic idiopathic myelofibrosis. In: Jaffe ES, Harris NL, Stein H and Vardiman JW (eds), Wo rld He alth Organizatio n Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 35–38. 3 Ni H, Barosi G, Rondelli D and Hoffman R (2005). Studies of the site and distribution of CD34+ cells in idiopathic myelofibrosis. Am J Clin Patho l, 123, 833–839. 4 Tefferi A (2007). Primary myelofibrosis and its paraneoplastic stromal effects. Hae m ato lo gic a, 92, 577–579. 5 Tefferi A, Mesa RA, Schroeder G, Hanson CA, Li CY and Dewald GW (2001). Cytogenetic findings and their clinical relevance in myelofibrosis with myeloid metaplasia. Br J Haematol, 113, 763–771. 6 Reilly JT (2002). Cytogenetic and molecular genetic aspects of idiopathic myelofibrosis. Acta Haematol, 108, 113–119. 7 Dingli D, Grand FH, Mahaffey V, Spurbeck J, Ross FM, Watmore AE et al. (2005). Der(6)t(1;6)(q21-23;p21.3): a specific cytogenetic abnormality in myelofibrosis with myeloid metaplasia. Br J Haematol, 130, 229–232. 8 Campbell PJ and Green A (2006). The myeloproliferative disorders. N Engl J Med, 355, 2452–2466.

9 Delhommeau F, Dupont S, Tonetti C, Masse A, Godin I, Le Couedic JP et al. (2007). Evidence that the JAK2 G1849T (V617F) mutation occurs in a lymphomyeloid progenitor in polycythemia vera and idiopathic myelofibrosis. Blood, 109, 71–77. 10 Barosi G, Bergamaschi G, Marchetti M, Vannucchi AM, Guglielmelli P, Antoniolo E et al. (2007). JAK2 V617F mutational status predicts progression to large splenomegaly and leukemic transformation in primary myelofibrosis. Blood. 110, 4030–4036. 11 Pardanani AD, Levine RL, Lasho T, Pikman Y, Mesa RA, Wadleigh M et al. (2006). MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood, 108, 3472–3476. 12 Kuter DJ, Bain B, Mufti G, Bagg A and Hasserjian RP (2007). Bone marrow fibrosis: pathophysiology and clinical significance of increased bone marrow stromal fibres. Br J Haematol, 139, 351–362. 13 Strasser-Weippl K, Steurer M, Kees M, Augustin F, Tzankov A, Dirnhofer S e t al. (2006). Age and hemoglobin level emerge as most important clinical prognostic parameters in patients with osteomyelofibrosis: introduction of a simplified prognostic score. Leuk Lymphoma, 47, 441–450. 14 Abgrall JF, Guibaud I, Bastie JN, Flesch M, Rossi JF, Lacotte-Thierry L et al., Groupe Ouest-Est Leucemies et Maladies du Sang (GOELAMS) (2006). Thalidomide versus placebo in myeloid metaplasia with myelofibrosis: a prospective, randomized, double-blind, multicenter study. Hematologica, 91, 1027–1032.

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Systemic mastocytosis

Systemic mastocytosis is a myeloproliferative neoplasm (MPN) with prominent involvement of mast cells [1–4]. Some cases have an associated myelodysplastic syndrome or a specific myeloproliferative or myelodysplastic/myelo proliferative neoplasm involving other lineages; among these associated conditions chronic myelomonocytic leukaemia (CMML) is most often observed. The disease may terminate in acute myeloid leukaemia (AML), most often not acute mast cell leukaemia. There is an unexplained association of systemic mastocytosis with AML with a t(8;21)(q22;q22).

Clinical features Presentation is in adults, usually from the third decade onwards, with a slight male predominance. Patients may present with clinical symptoms related to release of mast cell products [5] or with features such as hepatomegaly and splenomegaly characteristic of MPD [4]. Organomegaly is not usually marked. Mast cell release of histamine and other inflammatory mediators can cause flushing, tachycardia and collapse, sometimes triggered by insect bites or exposure to certain drugs. There may be gastrointestinal symptoms (diarrhoea or peptic ulceration) as a result of infiltration or chronic histamine release. Some patients have bone pain. Some have urticaria pigmentosa, a skin lesion resulting from cutaneous mast cell infiltration; this is characterized by pigmentation due to melanin deposition and urticaria on stroking. Striking systemic symptoms due to release of inflammatory mediators can occur in patients whose mastocytosis is indolent.

Haematological and pathological features The blood count and film may be normal or there may be anaemia, leucocytosis, monocytosis or eosinophilia. With more advanced disease there may be leucopenia, neutropenia and thrombocytopenia. Sometimes there are small numbers of circulating mast cells (Figure 9.1). Eosinophils are sometimes degranulated (Figure 9.2). The bone marrow is infiltrated to a variable extent in the great majority of patients, with infiltration often being preferentially paratrabecular or periarteriolar (Figures 9.3–9.5). Infiltrates are multifocal and cohesive in comparison with the scattered mast cells present in reactive conditions. The mast cells are often morphologically abnormal, being spindle-shaped with elongated nuclei, hypogranularity or both. They can be readily detected on a Giemsa stain (Figure 9.6) and their nature can be confirmed by immunohistochemistry (see below). Granulocytic hyperplasia, neutrophilic and eosinophilic, is common. On trephine biopsy sections, eosinophils may be apparent around the periphery of the mast cell infiltrates. Lymphocytes are sometimes also increased and may similarly surround the mast cell infiltrates (Figure 9.7). There may be increased macrophages and fibroblasts. Bones may show osteoporosis, osteosclerosis or both (Figure 9.8). The bone marrow aspirate is considerable less useful in diagnosis than the trephine biopsy but may show dysplastic mast cells, often in low numbers and within or adjacent to fragments (Figure 9.9). These are often oval or spindleshaped, sometimes with cytoplasmic tails (Figure 9.10) with fewer granules than are seen in normal mast cells. They are positive for napthol AS-D chloroacetate esterase. Rare cases have well-granulated round mast cells; these cytological features have been associated with the imatinib-sensitive

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Figure 9.1 Peripheral blood film of a patient with systemic mastocytosis showing two abnormal mast cells. MGG, high power.

Figure 9.3 Bone marrow trephine biopsy sections from a patient with systemic mastocytosis showing paratrabecular infiltration. New bone formation is also apparent. H&E, low power.

Figure 9.2 Peripheral blood film of a patient with systemic mastocytosis showing two cytologically abnormal eosinophils. MGG, high power.

Figure 9.4 Bone marrow trephine biopsy sections from a patient with systemic mastocytosis (same patient as Figure 9.3) showing periarteriolar infiltration. The arteriole is in cross section and a capillary can be seen entering it (bottom right). Increased eosinophils are apparent in the adjacent marrow. H&E, low power.

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Figure 9.5 Bone marrow trephine biopsy sections from a patient with systemic mastocytosis (same patient as Figure 9.3) showing periarteriolar infiltration. The arteriole is seen in longitudinal section. Increased eosinophils are again apparent in the adjacent marrow. H&E, low power.

Figure 9.6 Bone marrow trephine biopsy sections from a patient with systemic mastocytosis (same patient as Figure 9.3) showing mast cells within fibrous tissue. Giemsa, low power.

Figure 9.7 Bone marrow trephine biopsy sections from a patient with systemic mastocytosis (same patient as Figure 9.3) showing a cohesive mast cell infiltrate surrounded by lymphocytes. H&E, low power.

Figure 9.8 Radiology of shoulder in a patient with systemic mastocytosis showing mixed osteolytic and osteosclerotic lesions.

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Figure 9.9 Bone marrow trephine biopsy sections from a patient with systemic mastocytosis (same patient as Figure 9.3) showing round and spindle-shaped mast cells. Mast cell tryptase, immunoperoxidase, low power.

Figure 9.11 Bone marrow aspirate from a patient with systemic mastocytosis showing mast cell precursors and an abnormal spindle-shaped mast cell, which is hypogranular and has a tail. MGG, low power.

Figure 9.10 Bone marrow aspirate from a patient with systemic mastocytosis showing a fragment in which abnormal spindle-shaped mast cells can be identified. MGG, high power.

KIT F522C mutation so are an indication for molecular analysis [6]. There may also be infiltration of skin (in the dermis), liver (periportal) [7], spleen (red and white pulp) [8], lymph nodes (particularly paracortex) [9], gastrointestinal tract (mucosa) and many other organs. Liver infiltration can result in portal hypertension [10]. Bone infiltration can be associated with osteosclerosis, osteolysis, mixed osteosclerosis and osteolysis or osteoporosis. Serum mast cell tryptase is significantly elevated, a concentration of greater than 20 ng/l being useful in diagnosis. A significant elevation is an indication for further investigation of a patient with urticaria pigmentosa.

Immunophenotype Normal and neoplastic mast cells express CD9, CD33, CD44, CD45, CD68R, CD117, histidine decarboxylase and mast cell tryptase and may express mast cell chymase [4, 11–13]. Aberrant expression of CD2, CD25 or both is common for neoplastic mast cells. Flow cytometry with gating on CD117-positive cells is useful to demonstrate a low percentage of mast cells showing aberrant antigen expression. Immunohistochemical staining of trephine

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biopsy sections for mast cell tryptase (Figure 9.11), CD2 and CD25 is recommended. The mast cells also show aberrant expression of nuclear phosphorylated STAT5, a downstream target of mutated KIT [14]; similar activity was, however, seen in a patient with chronic eosinophilic leukaemia (CEL) resulting from a FIP1L1-PDGFRA fusion gene.

Cytogenetic and molecular genetic abnormalities

worse prognosis. Worse prognosis has also been related to presence of hepatosplenomegaly, absence of cutaneous lesions, anaemia, thrombocytopenia, elevated lactate dehydrogenase, elevated alkaline phosphatase and increased numbers and abnormal morphology of bone marrow mast cells. Systemic symptoms are not necessarily prognostically adverse if they are the result of release of mast cell inflammatory mediators rather than infiltration.

Treatment

Clonal cytogenetic abnormalities are sometimes present but there is no specific abnormality associated with systemic mastocytosis. Trisomy 8, trisomy 9 and miscellaneous translocations and deletions have been observed [15]. If sensitive techniques, such as microdissection, are used, almost all cases have an activating mutation of the KIT gene, most often D816V [3, 16].

There is as yet no specific treatment. Antihistamines can generally prevent the effects of histamine release. Other patients may benefit from corticosteroids or interferon. Imatinib is of benefit in some of the uncommon patients who lack the KIT D816V mutation, e.g. those who instead have KIT F522C.

Diagnosis and differential diagnosis

References

An important differential diagnosis is CEL associated with a FIP1L1-PDGFRA fusion gene [17, 18]. These patients usually have increased bone marrow mast cells and sometimes there are cohesive infiltrates so that the condition simulates systemic mastocytosis. A mast cell infiltrate has also been observed in patients with a MPN associated with ETV6-PDGFRB fusion [19]. Distinguishing these two MPN from systemic mastocytosis is clinically important since the former are sensitive to imatinib, whereas the great majority of cases of systemic mastocytosis are not. Because of the very elongated mast cells, the bone marrow trephine biopsy sections may simulate primary myelofibrosis. Occasionally the spaced nuclei with plentiful cytoplasm give a resemblance to hairy cell leukaemia. As long as the possibility of systemic mastocytosis is considered, and appropriate immunohistochemical stains are used, the diagnosis is easily made.

1 Bain BJ (1999). Systemic mastocytosis and other mast cell neoplasms. Br J Haematol, 106, 9–17. 2 Valent P, Akin C, Escribano L, Födinger M, Hartmann K, Brockow K e t al. (2007). Standards and standardization in mastocytosis: consensus statements on diagnostics, treatment recommendations and response criteria. Eur J Clin Inve st, 37, 435–453. PMID: 17537151. 3 Orfao A, Garcia-Montero AC, Sanchez L and Escribano L for the Spanish Network on Mastocytosis (REMA) (2007). Recent advances in the understanding of mastocytosis: the role of KIT mutations. Br J Haematol, 138, 12–30. PMID: 17555444. 4 Horny H-P, Valent P, Metcalf DD, Bennett JM, Bain B, Akin C and Escribano L (2008). Mast cell diseaseMastocytosis. . In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J and Vardiman JW (eds), Wo rld He alth Organizatio n Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 57–63In press. 5 Valhabhji J, Robinson S, Johnston D, Bellamy M, Davies W and Bain BJ (2000). Unexplained loss of consciousness: systemic mastocytosis. J Roy Soc Med, 93, 141–142.

Prognosis Prognosis is very variable. Some patients who present with urticaria pigmentosa and have only minor bone marrow infiltration have a long survival. Those who present with an overt MPN or a myelodysplastic syndrome have a much

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6 Akin C, Fumo G, Yavuz AS, Lipsky PE, Neckers L and Metcalfe DD (2004). A novel form of mastocytosis associated with a transmembrane c-kit mutation and response to imatinib. Blood, 103, 3222–3225. 7 Horny H-P, Kaiserling E, Campbell M, Parwaresch MR and Lennert K (1989). Liver findings in generalized mastocytosis. A clinicopathologic study. Cancer, 63, 532–538. 8 Horny H-P, Ruck MT and Kaiserling E (1992). Spleen findings in generalized mastocytosis. A clinicopathologic study. Cancer, 70, 459–468. 9 Horny H-P, Kaiserling E, Parwaresch MR and Lennert K (1992). Lymph node findings in generalized mastocytosis. Histopathology, 21, 439–446. 10 Addada J, Lloyd J and Bain B (2004). Teaching Cases from the Royal Marsden and St Mary’s Hospitals; Case 27: Ascites and oedema in a patient with systemic mastocytosis. Leuk Lymphoma, 45, 1713–1715. 11 Escribano L, Orfao A, Villarrubia J, Diaz-Agustin B, Cervero C, Rios A et al. (1998). Immunophenotypic characterization of human bone marrow mast cells. A flow cytometric study of normal and pathological bone marrow samples. Analy Cell Pathol, 16, 151–159. 12 Sotlar K, Horny H-P, Simonitsch I, Krokowski M, Aichberger KJ, Mayerhofer M et al. (2004). CD25 indicates the neoplastic phenotype of mast cells: a novel immunohistochemical marker for the diagnosis of systemic mastocytosis (SM) in routinely processed bone marrow biopsy specimens. Am J Surg Patho l, 28, 1319–1325. 13 Krauth MT, Födinger Rebuzzi ML, Greul R, Chott A and Valent P (2007). Aggressive systemic mastocytosis with sarcoma-like growth in the skeleton, leukemic progression, and partial loss of mast cell differentiation antigens. Haematologica, 92, e126–e129.

14 Toro TZ, Hsieh FH, Bodo J, Dong HY and His ED (2007). Detection of phospho-STAT5 in mast cells: a reliable phenotypic marker of systemic mast cell disease that reflects constitutive tyrosine kinase activation. Br J Haematol, 139, 31–40. PMID: 17662084. 15 Gupta R, Bain BJ and Knight CL (2002). Cytogenetic and molecular genetic abnormalities in systemic mastocytosis. Acta Haematologica, 107, 123–128. 16 Sotlar K, Fridrich C, Mall A, Jaussi R, Bultmann B, Valent P and Horny H-P (2002). Detection of c-kit point mutation Asp-816 —> Val in microdissected pooled single mast cells and leukemic cells in a patient with systemic mastocytosis and concomitant chronic myelomonocytic leukemia. Leuk Res, 26, 979–984. 17 Bain BJ (2004). Relationship between idiopathic hypereosinophilic syndrome, eosinophilic leukemia, and systemic mastocytosis. Am J Hematol, 77, 82–85. 18 Bain BJ, Gilliland DG, Vardiman JW and Horny H-P (2008). Myeloproliferative Myeloid and lymphoid neoplasms associated with eosinophilia and rearrangement abnormalities of PDGFRA, PDGFRB or FGFR1. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J and Vardiman JW (eds), World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 67-73. In press. 19 Walz C, Metzgeroth G, Haferlach C, Schmitt-Graeff A, Fabarius A, Hagen V et al. (2007). Characterization of three new imatinib-responsive fusion genes in chronic myeloproliferative disorders generated by disruption of the platelet-derived growth factor receptor beta gene. Hematologica, 92,163–169.

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Myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a heterogeneous group of haematological neoplasms characterized by ineffective and dysplastic haemopoiesis. The ineffectiveness of haemopoiesis is apparent from the usual coexistence of a hypercellular bone marrow and peripheral cytopenia. The responsible mutation occurs in a multipotent haemopoietic stem cell, although the defect in maturation may be apparent in only one or two lineages. The incidence in adults is 3–4/100,000/year with the condition being about twice as common in men as in women. The incidence rises exponentially with age. Aetiological factors include anti-cancer chemotherapy and irradiation. There are also predisposing conditions, such as aplastic anaemia (congenital or acquired). MDS can evolve into acute myeloid leukaemia (AML), but in some patients death from the consequences of bone marrow failure occurs without transformation. A minority of patients who are transfusion-dependent die from iron overload.

Figure 10.1 Peripheral blood film of a patient with refractory anaemia (FAB and WHO classifications) showing anisocytosis. MGG, high power.

Clinical features In some patients the diagnosis is made incidentally, e.g. when a blood count done for another reason shows anaemia. Other patients present with features of bone marrow failure, such as infection, bleeding or symptoms of anaemia.

Haematological and pathological features The blood film and bone marrow aspirate are critical in diagnosis. The peripheral blood may show cytopenia (anaemia, neutropenia, thrombocytopenia) and various dysplastic features (Figures 10.1–10.9). Monocytes are sometimes increased and less often neutrophils or platelets. The dysplastic features that may be present include hypogranular neutrophils, hypolobated neutrophils and macrocytosis. A dimorphic blood film, sometimes with

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Figure 10.2 Peripheral blood film of a patient with refractory anaemia (FAB and WHO classifications) showing an oval macrocyte. The neutrophil is cytologically normal. MGG, high power.

Figure 10.3 Peripheral blood film of a patient with refractory anaemia with ring sideroblasts (FAB and WHO classifications) showing dimorphic red cells. MGG, high power.

Figure 10.4 Peripheral blood film of a patient with refractory anaemia with ring sideroblasts (FAB and WHO classifications) (same patient as Figure 10.3) showing dimorphic red cells and an erythrocyte with prominent Pappenheimer bodies. MGG, high power.

Figure 10.5 Peripheral blood film of a patient with refractory anaemia (FAB classification)/refractory anaemia with multilineage dysplasia (WHO classification) showing anisocytosis and an acquired Pelger–Huët anomaly. MGG, high power.

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Figure 10.6 Peripheral blood film of a patient with refractory anaemia with ring sideroblasts (FAB classification)/refractory anaemia with multilineage dysplasia and ring sideroblasts (WHO classification) showing anisocytosis, poikilocytosis and an erythrocyte with prominent basophilic stippling. In this patient there is anisochromasia rather than a distinctly dimorphic film. MGG, high power.

Figure 10.7 Peripheral blood film of a patient with refractory anaemia with excess of blasts (FAB classification)/refractory anaemia with excess of blasts 1 (WHO classification) showing anisocytosis, poikilocytosis and a cell with prominent basophilic stippling. MGG, high power.

Figure 10.8 Peripheral blood film of a patient with refractory anaemia with excess of blasts (FAB classification)/refractory anaemia with excess of blasts 2 (WHO classification) showing anisocytosis and a cell with prominent Pappenheimer bodies. MGG, high power.

Figure 10.9 Peripheral blood film of a patient with refractory anaemia with excess of blasts (FAB classification)/refractory anaemia with excess of blasts 2 (WHO classification) (same patient as Figure 10.8) confirming the nature of the Pappenheimer bodies. Perls’ stain, high power.

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Pappenheimer bodies, is seen in sideroblastic anaemia; usually the two populations are: 1) hypochromic microcytes and 2) normochromic macrocytes. There may be platelets that are large, hypogranular or both. There may be circulating

erythroblasts, myeloblasts, granulocyte precursors, monoblasts or promonocytes and immature monocytes. The bone marrow aspirate is usually hypercellular and shows dysplasia of one, two or three lineages and there may

Figure 10.10 Bone marrow aspirate film from a patient with refractory anaemia (FAB and WHO classifications) (same patient as Figure 10.1) showing a macronormoblast and cytologically normal granulocyte precursors. MGG, high power.

Figure 10.11 Bone marrow aspirate film from a patient with refractory anaemia (FAB and WHO classifications) (same patient as Figure 10.2) showing a late erythroblast with an irregular nucleus (top left) and a binucleate early erythroblast (bottom right). MGG, high power.

Figure 10.12 Bone marrow aspirate film from a patient with refractory anaemia (FAB and WHO classifications) (same patient as Figures 10.2 and 10.11) showing left shifted erythropoiesis (increased early erythroblasts) and dysplastic late erythroblasts. MGG, high power.

Figure 10.13 Bone marrow aspirate film from a patient with refractory anaemia with ring sideroblasts (FAB and WHO classifications) showing a late erythroblast with fine cytoplasmic granules and virtually absent haemoglobin; the granules may represent basophilic stippling. MGG, high power.

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also be an increase in blast cells (Figures 10.10–10.33). Blast cells sometimes contain Auer rods or, much less often, giant granules. Erythroblasts may show nuclear lobulation or fragmentation, binuclearity or multinuclearity, defective

haemoglobinization or megaloblastosis. Granulopoiesis may show hypolobation and a defect of primary or secondary granulation or both. Megakaryocytes may be very small (micromegakaryocytes), normal size but hypolobated, or

Figure 10.14 Bone marrow aspirate film from a patient with refractory anaemia with ring sideroblasts (FAB and WHO classifications) (same patient as Figure 10.13) showing a late erythroblast with Pappenheimer bodies and very little haemoglobin. MGG, high power.

Figure 10.15 Bone marrow aspirate film from a patient with refractory anaemia with ring sideroblasts (FAB and WHO classifications) (same patient as Figure 10.13) showing a giant late erythroblast with large cytoplasmic vacuoles and Pappenheimer bodies. There is also a myeloblast (middle left). MGG, high power.

Figure 10.16 Bone marrow aspirate film from a patient with refractory anaemia with ring sideroblasts (FAB and WHO classifications) (same patient as Figure 10.13) showing two ring sideroblasts and another abnormal sideroblast. Perls’ stain, high power.

Figure 10.17 Bone marrow aspirate film from a patient with refractory anaemia with ring sideroblasts (FAB and WHO classifications) showing an erythrocyte and an erythroblast with Pappenheimer bodies. MGG, high power.

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Figure 10.18 Bone marrow aspirate film from a patient with refractory anaemia with ring sideroblasts (FAB and WHO classifications) (same patient as Figure 10.17) with at least three ring sideroblasts. Perls’ stain, high power.

Figure 10.19 Bone marrow aspirate film from a patient with refractory anaemia (FAB classification)/refractory anaemia with multilineage dysplasia (WHO classification) (same patient as Figure 10.5) showing two erythroblasts joined by a cytoplasmic bridge. MGG, high power.

Figure 10.20 Bone marrow aspirate film from a patient with refractory anaemia (FAB classification)/refractory anaemia with multilineage dysplasia (WHO classification) (same patient as Figures 10.5 and 10.19) showing a binucleate micromegakaryocyte. MGG, high power.

Figure 10.21 Bone marrow aspirate film from a patient with refractory anaemia (FAB classification)/refractory anaemia with multilineage dysplasia (WHO classification) (same patient as Figures 10.5 and 10.19) showing an abnormal sideroblast. Perls’ stain, high power.

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Figure 10.22 Bone marrow aspirate film from a patient with refractory anaemia (FAB classification)/refractory anaemia with multilineage dysplasia (WHO classification) (same patient as Figures 10.5 and 10.19) showing a macrocyte with two large siderotic granules. Perls’ stain, high power.

Figure 10.23 Bone marrow aspirate film from a patient with refractory anaemia with ring sideroblasts (FAB classification)/refractory anaemia with multilineage dysplasia and ring sideroblasts (WHO classification) (same patient as Figure 10.6) showing hypercellularity and a marked increase in megakaryocytes. MGG, low power.

Figure 10.24 Bone marrow aspirate film from a patient with refractory anaemia with ring sideroblasts (FAB classification)/refractory anaemia with multilineage dysplasia and ring sideroblasts (WHO classification) (same patient as Figure 10.6) showing gross erythroid dysplasia: an erythroblast with a lobulated nucleus (top left), an erythroblast with empty cytoplasm (centre left) and a macronormoblast (centre). MGG, high power.

Figure 10.25 Bone marrow aspirate film from a patient with refractory anaemia with ring sideroblasts (FAB classification)/refractory anaemia with multilineage dysplasia and ring sideroblasts (WHO classification) (same patient as Figure 10.6) showing gross erythroid dysplasia, e.g. a giant binucleate megaloblast (centre). There is also a defect of neutrophil segmentation. MGG, high power.

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Figure 10.26 Bone marrow aspirate film from a patient with refractory anaemia with ring sideroblasts (FAB classification)/refractory anaemia with multilineage dysplasia and ring sideroblasts (WHO classification) (same patient as Figure 10.6) showing increased erythropoiesis and gross erythroid dysplasia; features include cytoplasmic bridging, binuclearity and defective haemoglobinization. MGG, high power.

Figure 10.27 Bone marrow aspirate film from a patient with refractory anaemia with ring sideroblasts (FAB classification)/refractory anaemia with multilineage dysplasia and ring sideroblasts (WHO classification) (same patient as Figure 10.6) showing numerous ring sideroblasts. Perls’ stain, high power.

Figure 10.28 Bone marrow aspirate film from a patient with refractory anaemia with excess of blasts (FAB classification)/refractory anaemia with excess of blasts 1 (WHO classification) (same patient as Figure 10.7) showing two erythroblasts with heavy basophilic stippling. MGG, high power.

Figure 10.29 Bone marrow aspirate film from a patient with refractory anaemia with excess of blasts (FAB classification)/refractory anaemia with excess of blasts 1 (WHO classification) (same patient as Figure 10.7) showing a cytoplasmic bridge between two myelocytes. MGG, high power.

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Figure 10.30 Bone marrow aspirate film from a patient with refractory anaemia with excess of blasts (FAB classification)/refractory anaemia with excess of blasts 1 (WHO classification) (same patient as Figure 10.7) showing a neutrophil with two Döhle bodies. Döhle bodies are a feature of infection and inflammation but can also be seen in MDS. MGG, high power.

Figure 10.31 Bone marrow aspirate film from a patient with the 5q– syndrome (WHO classification) showing a hypolobated megakaryocyte, a blast cell and two other neutrophil precursors. MGG, high power.

Figure 10.32 Bone marrow aspirate film from a patient with the 5q– syndrome (WHO classification) showing hypolobated megakaryocytes. MGG, high power.

Figure 10.33 Bone marrow aspirate film from a patient with therapy-related MDS (WHO classification) showing a hypogranular neutrophil band form. MGG, high power.

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multinucleated. There is an increased rate of apoptosis and macrophages may be increased. A Perls’ stain for haemosiderin may show ring sideroblasts, other abnormal sideroblasts and increased iron stores (Figures 10.16, 10.18, 10.21, 10.22 and 10.27). Erythroblasts may be positive with a periodic acid–Schiff (PAS) stain. A trephine (core) biopsy (Figures 10.34-10.38) is essential whenever an inadequate aspirate is obtained, whether this is for technical reasons or because the bone marrow is fibrotic or hypoplastic. It may only be possible to make a reliable distinction between aplastic anaemia, hypoplastic MDS and hypoplastic AML by means of a core biopsy. Sections of a core biopsy also show the bone marrow architecture, permitting the recognition of larger than normal erythroid islands, erythroid islands with all erythroblasts at the same stage of development, abnormal localization of blasts and promyelocytes other than against the bone or in a periarteriolar position (known as abnormal localization of immature precursors, ALIP), megakaryocyte clustering and megakaryocytes abnormally located adjacent to bone. The use of immunohistochemistry with a CD34

antibody helps in the recognition of blast cells, and antibodies detecting CD41 or CD61 in the recognition of megakaryocytes, particularly micromegakaryocytes. Various non-specific abnormalities may be present, e.g. increased iron stores, increased small blood vessels, increased mast cells and increased reticulin deposition. Initial investigation of patients with suspected MDS should include history (including remote and recent drug history), physical examination, full blood count, differential count, reticulocyte count and blood film, bone marrow aspirate including iron stain and cytogenetic analysis, serum ferritin and serum erythropoietin. Consideration should be given to the need for testing for human immunodeficiency virus (HIV) infection and for assaying serum vitamin B12, red cell folate and serum copper. MDS has been further subcategorized on the basis of haematological features, by both the French–American– British (FAB) (Table 10.1) [1, 2] and the World Health Organization (WHO) (Tables 10.2–10.4) [3, 4] expert groups. These categorizations are of considerable prognostic significance.

Figure 10.34 Bone marrow trephine biopsy section from a patient with refractory anaemia (FAB and WHO classifications) (same patient as Figure 10.2) showing hypercellularity, increased erythropoiesis and increased macrophages. H&E, low power.

Figure 10.35 Bone marrow trephine biopsy section from a patient with refractory anaemia (FAB and WHO classifications) (same patient as Figure 10.2) showing hypercellularity, increased erythropoiesis and increased macrophages. The cells with halos (a shrinkage artefact) are late erythroblasts while the cells with large pale nuclei and linear nucleoli are proerythroblasts and early erythroblasts. H&E, intermediate power.

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Figure 10.36 Bone marrow trephine biopsy section from a patient with refractory anaemia (FAB and WHO classifications) (same patient as Figure 10.2) showing myeloblasts in a normal paratrabecular location. H&E, high power.

Figure 10.37 Bone marrow trephine biopsy section from a patient with refractory anaemia (FAB and WHO classifications) (same patient as Figure 10.1) showing hypercellularity and a large lymphoid aggregate. Reactive lymphoid aggregates can be seen in patients with myeloid neoplasms. H&E, low power.

Figure 10.38 Bone marrow trephine biopsy section from a patient with refractory anaemia (FAB and WHO classifications) (same patient as Figure 10.1) showing normal reticulin (grade 2/4). Reticulin, low power.

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Table 10.1 The French–American–British (FAB) classification of myelodysplastic syndromes [modified from 2] Category

Peripheral blood c riteria

Bone marrow criteria

Refractory anaemia (RA) or refractory cytopenia*

Anaemia,* Blasts <1% Monocytes <1 × 109/l

Blasts <5%, ringed sideroblasts <15% of erythroblasts

Refractory anaemia with ringed sideroblasts (RARS)

Anaemia Blasts <1% Monocytes <1 × 109/l

Blasts <5%, ringed sideroblasts >15% of erythroblasts

Refractory anaemia with excess of blasts (RAEB)

Anaemia Blasts >1% Monocytes <1 × 109/l

or

Blasts ≥5% but

blasts <5% Refractory anaemia with excess of blasts in transformation (RAEB-T)

Anaemia, blasts ≥5

and

blasts <20%

or

Blasts ≥20% or blasts <30%

or If blasts not increased, Auer rods Chronic myelomonocytic leukaemia (CMML)

Monocytes >1 × 109/l granulocytes often increased blasts <5%

*Or in the case of refractory cytopenia either neutropenia or thrombocytopenia

Blasts up to 20% Promonocytes often increased

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Table 10.2 The 2001 WHO classification of myelodysplastic syndromes [3] Disease

Peripheral blood findings

Bone marrow findings

Refractory anaemia (RA)

Anaemia Blasts rarely seen and always <1%

Dysplasia confined to erythroid lineage <5% blasts <15% ringed sideroblasts

Refractory anaemia with ringed sideroblasts (RARS)

Anaemia No blasts

Dysplasia confined to erythroid lineage <5% blasts ≥15% ringed sideroblasts

Refractory cytopenia with multilineage dysplasia (RCMD)*

Cytopenias (bicytopenia or pancytopenia) No or rare blasts No Auer rods <1 × 109/l monocytes

Dysplasia in ≥10% of the cells of two or more myeloid cell lineages <5% blasts <15% ringed sideroblasts No Auer rods

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

Cytopenias (bicytopenia or pancytopenia) No or rare blasts No Auer rods <1 × 109/l monocytes

Dysplasia in ≥10% of the cells of two or more myeloid cell lineages <5% blasts ≥15% ringed sideroblasts No Auer rods

Refractory anaemia with excess blasts-1 (RAEB-1)*

Cytopenias <5% blasts No Auer rods <1 × 109/l monocytes

Unilineage or multilineage dysplasia 5–9% blasts No Auer rods

Refractory anaemia with excess blasts-2 (RAEB-2)*

Cytopenias 5–19% blasts Auer rods sometimes present <1 × 109/l monocytes

Unilineage or multilineage dysplasia 10–19% blasts Auer rods sometimes present

Myelodysplastic syndromeunclassified (MDS-U)*

Cytopenias No or rare blasts No Auer rods

Unilineage dysplasia <5% blasts No Auer rods

MDS associated with isolated del(5q)

Anaemia, platelet count usual normal or elevated <5% blasts

Megakaryocytes in normal or increased numbers but with hypolobated nuclei <5% blasts No Auer rods 5q– as sole cytogenetic abnormality

* If cases are therapy-related, this should be specified and it should be further specified whether cases are alkylating agent-related (the majority) or topoisomerase II-interactive-drug-related (a small minority); therapy-related cases are categorized with therapy-related AML

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Table 10.3 Outline of the 2008 W orld Health Organization classification of myelodysplastic syndromes [4]

Disease

Peripheral blood

Bone marrow

Refractory cytopenia with unilineage dysplasia (RCUD) (includes refractory anaemia, refractory neutropenia and refractory thrombocytopenia)

Cytopenia* No or rare blasts† No Auer rods

Unilineage dysplasia ≥10% <15% ring sideroblasts <5% blasts† No Auer rods

Refractory anaemia with ring sideroblasts (RARS)

Anaemia* No blasts No Auer rods

≥15% ring sideroblasts Erythroid dysplasia only (No Auer rods) <5% blasts†

Refractory cytopenia with multilineage dysplasia with or without ring sideroblasts (RCMD±RS)

Cytopenia(s) No or rare blasts† No Auer rods <1 × 109/l monocytes

Dysplasia in ≥10% in ≥ two myeloid lineages <5% blasts† No Auer rods

Refractory anaemia with excess blasts-1 (RAEB-1)

Cytopenia(s) < 5% blasts‡ No Auer rods <1 × 109/l monocytes

Unilineage or multilineage dysplasia 5–9% blasts‡ No Auer rods

Refractory anaemia with excess blasts-2 (RAEB-2)

Cytopenia(s) 5–19% blasts** Auer rods ±** <1 × 109/l monocytes

Unilineage or multilineage dysplasia 10–19% blasts** Auer rods ± **

Myelodysplastic syndrome, unclassified (MDS-U)

Cytopenias <5% blasts Does not fit any of the other categories

Unequivocal dysplasia but in <10% of cells or for any reason does not fit one of the other categories

Myelodysplastic syndrome associated with isolated 5q–

Anaemia No or rare blasts

Normal or increased megakaryocytes Hypolobated nuclei <5% blasts No Auer rods Isolated 5q–

* Bicytopenia may occur but if there is pancytopenia that leads to the designation of MDS, unclassified. If the marrow myeloblast percentage is less than 5% but there are 2–4% myeloblasts in the blood, the diagnostic classification is RAEB-1. If the marrow myeloblast percentage is <5% and there are 1% myeloblasts in the blood, the case should be classified as MDS-U. ‡ Either 2–4% peripheral blood blasts or 5–9% bone marrow blasts qualifies a case for this designation. ** Either 5–19% peripheral blood blasts or 10–19% bone marrow blasts or Auer rods qualifies a case for this designation. Cases with Auer rods and <5% myeloblasts in the blood and <10% in the marrow should be classified as RAEB-2. †

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Table 10.4 Outline of the 2008 W orld Health Organisation criteria for designation as myelodysplastic syndrome, unclassified [4]

Otherwise meets criteria for refractory cytopenia with unilineage dysplasia (RCUD) or refractory cytopenia with multilineage dysplasia (RCMD) but with 1% blasts in the peripheral blood Otherwise meets criteria for refractory cytopenia with unilineage dysplasia (RCUD) but there is pancytopenia Persistent cytopenias with <10% dysplasia in any lineage, but with cytogenetic abnormalities considered as presumptive evidence of MDS*† * Complex karyotypic abnormality, −7 or del(7q), −5 or del(5q), i(17q) or t(17p), −13 or del(13q), del(11q), del(12p) or t(12p), del(9q), idic(X)(q13), t(11;16)(q23;p13.3), t(3;21)(q26.2;q22.1), t(1;3)(p36.3;q21.2), t(2;11)(p21;q23), inv(3)(q21q26.2), t(6;9)(p23;q34). † The presence of −Y (which does not necessarily indicate a clonal abnormality), +8 or del(20q) is not considered sufficient evidence for assignment to this category.

Immunophenotype The immunophenotype is not widely used in diagnosis, although characteristic abnormalities are often present and could contribute to diagnosis when the diagnosis is not certain from other disease features. Immunophenotyping may show abnormal maturation of myeloid lineages, e.g. abnormal light scatter, abnormal antigen expression (e.g. stronger or weaker than normal or asynchronous expression of different antigens) or there may be aberrant expression of non-myeloid antigens (CD5, CD7, CD56) [5]. Immunophenotype may also reveal abnormal maturation in a lineage that is not cytologically dysplastic. However, it should be noted that considerable experience in the interpretation is needed. In addition, it must be noted that immunophenotypic features give evidence of dysplasia, rather than specifically evidence of MDS, so that a diagnosis of MDS cannot be based solely on immunophenotyping. For example, CD56 can be expressed aberrantly on regenerating cells and CD64 can be expressed on the neutrophils in sepsis [5]. Abnormalities have also been reported in patients with autoimmune disease [6]. Patients with an increased blast count may have increased numbers of CD34-positive cells but it should be noted that not all blast cells are CD34 positive (Figure 10.39). In these circumstances CD117 and terminal deoxynucleotidyl transferase (TdT) expression may also be increased, helping to identify a population of early progenitor/stem cells.

Cytogenetic and molecular genetic abnormalities Cytogenetic abnormalities are present in 40–60% of patients with MDS. They are more frequent in higher grade MDS (i.e. MDS with an increase of blast cells and a worse prognosis) and more frequent in therapy-related than de no vo disease. Cytogenetic abnormalities are often unbalanced, e.g. trisomy 8, 19 or 21, monosomy 5 or 7, 1q+, 5q–, 7q–, 12p+, 17q– or 20q–. Certain balanced translocations and other chromosomal rearrangements also occur, e.g. inv(3)(q21q26.2) or t(3;3)(q21;q26.2). The most frequent abnormalities in de novo MDS are +8 (10%), abnormalities of chromosome 5 (10%), abnormalities of chromosome 7 (10%), 20q– (5–8%) and abnormality of chromosome 17 (3–5%). The most frequent abnormalities in therapy-related MDS are abnormalities of chromosome 7 (50% of cases) and abnormalities of chromosome 5 (40% of cases). The 5q– abnormality (Figures 10.40 and 10.41) correlates with normal sized but hypolobated megakaryocytes (see Figures 10.31 and 10.32). Loss of 17p correlates with granulocyte dysplasia (hypolobated and vacuolated neutrophils) [7]. Cytogenetic analysis can be of value in supporting a diagnosis of MDS in patients with cytopenia in whom the cytological evidence of dysplasia is minimal [4, 8]. Cytogenetic analysis gives valuable information as to prognosis (see below).

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MDS

Figure 10.39 Dot plot of flow cytometric immunophenotyping in a patient with MDS. Sideways (SSC) and forward (FSC) light scattering have been used to gate on cells of granulocytic lineage (green, P2) and monocytic lineage (red, P1); ungated data are black. Among the granulocytic population fewer than 5% of cells are expressing markers of immaturity: CD34, terminal deoxynucleotidyl transferase and CD117. The majority of mature myeloid cells show expression of the common leucocyte antigen (CD45) and typical myeloid markers (CD13, CD33 and CD11b), with down regulation of CD10 and HLA-DR. The monocytic population shows stronger expression of CD4, CD14, CD64 and HLA-DR than is shown by the cells of granulocyte lineage. CD71 (transferrin receptor) is expressed by a proportion of cells of both lineages but this may represent non-specific binding of the antigen to the surface membrane. There is little expression of CD56 by either lineage. Flow cytometric signs of MDS include reduced SSC by mature neutrophils, down regulation of neutrophil expression of CD10 and HLA-DR and aberrant expression of granulocytic, monocytic and erythroid markers. With thanks to Mr Ricardo Morilla, London.

Diagnosis and differential diagnosis The differential diagnosis includes aplastic anaemia, AML, the myelodysplastic/myeloproliferative disorders, reactive conditions with associated dysplasia and various inherited conditions that can cause unilineage or multilineage dysplasia. Inherited conditions such as congenital dyserythropoietic anaemia, congenital sideroblastic anaemia and mitochondrial cytopathies, such as Pearson’s syndrome, have to be considered in the differential diagnosis,

particularly in children. The differential diagnosis with aplastic anaemia arises in cases of hypocellular MDS. High quality trephine biopsy sections, supplemented by immunohistochemistry for CD34, CD117 and TdT, are important in making the distinction. The significance of clonal cytogenetic abnormalities in this setting is controversial since such clones in patients with aplastic anaemia do not necessarily

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Figure 10.40 Karyogram showing 5q– (arrow). The 5q– syndrome is characterized by macrocytic anaemia, normal numbers of blast cells and a relatively good prognosis. There is a wide range in the amount of chromosome material deleted, from a single band up to almost the whole long arm. Breakpoints anywhere between 5q11 and 5q35 have been described. However, band 5q31 is the most commonly deleted region. The example shown is of intermediate size. With thanks to Dr John Swansbury.

persist or indicate imminent disease progression. AML enters into the differential diagnosis in patients with borderline blast cell counts and in those with fibrotic marrows. It should be acknowledged that the choice of any particular blast count to separate MDS from AML is arbitrary. In addition, if the blast count is in the region of 15–25% it is important to count at least 500 cells in order to have a precise count. In the WHO classification, the blast cell count is irrelevant in those with t(8;21)(q22;q22), t(15;17)(q22;q12), inv(16)(p13.1q22) or t(16;16)(p13.1;q22) in whom a diagnosis of AML is made regardless of the blast count. MDS with fibrosis can be confused with the WHO AML category of acute panmyelosis; the distinction is made by the larger proportion of blasts cells, including proerythroblasts, in acute panmyelosis. The differential diagnosis of suspected MDS without an increase in the blast cell count includes exposure to toxic chemicals and drugs (e.g. arsenic, lead), deficiency states (deficiency of vitamin B12, folic acid or copper) and dysplasia secondary to infections (e.g. HIV infection, tuberculosis, leishmaniasis), acute severe illness or

Figure 10.41 Fluorescence in situ hybridization (FISH) from a patient with loss of at least part of the long arm of chromosome 5. Two small marker chromosomes were present. The FISH probes used are the Vysis LSI EGR1 (5q31) SpectrumOrange/ D5S23 and D5S721 SpectrumGreen Probe. On the right of the metaphase is the normal 5 with the green signal on the short arm of the chromosome and the red signal at q31 on the long arm. The green signals at the top and bottom of the metaphase identify the marker chromosomes as being derived from the missing part of chromosome 5, which has lost most of the long arm including the EGR1 gene. It should be noted that interphase FISH studies using probes to score loss of a chromosome or part of a chromosome are not reliable for low-level clones: there are technical reasons why a FISH signal may appear to be missing and so a low level of random or technical loss is normal. With thanks to Dr John Swansbury.

autoimmune disease. The clinical history and a constant awareness of the wide range of conditions able to cause dysplasia are essential to avoid serious mis-diagnosis. In order to have a designation for patients with cytopenia of unknown origin without defining features of MDS, the term ‘Idiopathic Cytopenia of Undetermined Significance (ICUS)’ was suggested by the International Working Group on Morphology of Myelodysplastic Syndromes (IWGMMDS) (Lisbon, May, 2005) and use of the term was subsequently recommended in the 2008 WHO classification. Such patients need close follow-up as in some such patients the condition does evolve into overt MDS or AML.

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Prognosis Prognosis is dependent on age, gender (worse overall survival in males), co-morbidity, the severity of any cytopenia, the number of blast cells and the presence and nature of any cytogenetic abnormalities. A high serum lactate dehydrogenase is prognostically adverse [9]. Both the FAB and WHO categories (Tab le 10.5) [10] are of

prognostic significance. Therapy-related MDS has a much worse prognosis than de no vo disease. Disease characteristics that contribute to prognosis have been combined in an international prognostic scoring system (IPSS) [11] (Table 10.6). More recent studies have given information on the significance of a greater range of

Table 10.5 Prognosis according to WHO category [10] Category

Number

Median survival (years)

Cumulative leukaemic transformation at 1 year (%)

Refractory anaemia

22

2.3

12.8

Refractory anaemia with ring sideroblasts

34

4.8

5.9

Refractory cytopenia

40

2.3

15.5

Refractory cytopenia with ring sideroblasts

28

2.7

7.7

Refractory anaemia with excess of blasts 1

55

0.9

38

Refractory anaemia with excess of blasts 2

34

0.6

45.2

MDS with isolated del(5q)

17

2.4

20.5

MDS, unclassified

6

5.2

17

Table 10.6 The International Prognostic Scoring System for MDS [1 1]

Score Prognostic variables % blasts Karyotype † Cytopenias‡

0

0.5

1.0

1.5

2

<5 Good 0–1

5–10 Intermediate 2–3

− Poor

11–20 −

20–30* −

* Cases with 20–30% blasts are classified as AML not MDS in the WHO classification. Good prognosis karyotype – normal, Y, del(5q), del(20q); Poor prognosis karyotype – complex (≥3 abnormalities) or chromosome 7 abnormalities; Intermediate prognosis karyotype – other abnormalities. ‡ Cytopenias – Hb <10 g/dl, neutrophil count <1.5 × 109/l, platelet count <100 × 109/l. †

Individual scores are summed and cases are then assigned to four risk groups, indicative of an increasingly bad prognosis. A score of 0 is indicative of low risk; a score of 1 is indicative of intermediate risk-1; a score of 1.5–2.0 is indicative of intermediate risk-2; a score of ≥2.5 is indicative of high risk.

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cytogenetic abnormalities (Table 10.7) [10, 12, 13]. There would appear to be a need for a meta-analysis to permit interpretation of all the data now available and to give new information on the prognostic significance of less common abnormalities. With the availability of new data it should be possible to refine the IPPS score. The presence of dysplasia in more than one lineage indicates a worse prognosis than if dysplasia is confined to a

single lineage [14]. The presence of peripheral blood blast cells in patients who would otherwise be regarded as having low-grade MDS correlates with a worse prognosis [15]. The presence of Auer rods in patients with a low blast count is associated with adverse prognosis [16] and leads to their being classified as RAEB2 in the WHO classification. Histological features that are prognostically adverse include the presence of fibrosis and an increased number of CD34-

Table 10.7 Comparison of prognostic significance of cytogenetic abnormalities in four studies [10–13] Greenberg et al. 1997 [11]

Sole et al. 2005 [10]

Bernasconi et al. 2007 [12]

Haase et al. 2007 [13]

Number studied

816

968

492

1237

Good

Normal del(5q) del(20q) −Y

Normal del(5q) del(11q) del(12p) del(20q) −Y

Normal add(1q) del(5q) trisomy 8 del(11)(q14q23) del(12p) 20q–

Normal trisomy 1 add(1q) t(1q) del(5q) t(7q) del(9p) del(12p) t(15q) t(17q) del(20q) trisomy 21 monosomy 21 −X −Y

Intermediate

All other

3q2126 abnormalities +8 +9 t(11q) del(17p)

Monosomy 7 del(7)(q31q35)

del(7q) monosomy 7 trisomy 8 t(11q23) anomalies of 19 3q rearrangement

Poor

Complex (≥3) chromosome 7 abnormalities

Complex (>2 abnormalities) monosomy 7 del(7q) i(17q)

Complex 3q abnormalities

Complex (≥3)* t(5q)

Unknown

All other

*Prognosis of 4–6 anomalies was worse than that of 3 anomalies.

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positive cells. With the advent of more effective treatments, their availability to the individual patient is also likely to influence prognosis.

Treatment Choice of treatment depends on age, symptoms and prognosis of the individual patient. Some patients require no treatment. Others require symptomatic treatment, particularly blood and sometimes platelet transfusion. In patients with 5q–, lenalidomide is indicated [17]. It can lead not only to haematological response and transfusion independence but also to complete cytogenetic response in approaching half of patients. Patients who do best are those with the 5q– syndrome (as defined in the WHO classification) but patients with increased blast cells or additional cytogenetic abnormalities can also respond to this drug. Patients with monosomy 7 have been found to benefit from azacytidine. In patients with neither 5q– nor monosomy 7 who have symptomatic anaemia, a combination of erythropoietin and granulocyte colonystimulating factor (G-CSF) may avoid the need for transfusion. Lenalidomide can also be of benefit but is less efficacious than in those with 5q– [18]; it is appropriate as second-line treatment in patients who do not respond to erythropoietin with or without G-CSF. Demethylating agents, azacytidine and decitabine, can be useful in patients with worse prognosis MDS and in those who have not responded to erythropoietin [19]. Patients with a hypocellular marrow may respond to antithymocyte globulin. In patients who are transfusiondependent but in whom the prognosis is otherwise relatively good, serum ferritin should be monitored and, if necessary, iron chelation therapy should be given.

References 1 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR and Sultan C (1982). Proposals for the classification of the myelodysplastic syndromes. Br J Haematol, 51, 189–199. 2 Bain BJ (2010). Leukaemia Diagnosis, 4rd Edition, Blackwell Publishing, Oxford, pp. 219–260.

3 Brunning RD, Bennett JM, Flandrin G, Matutes E, Head D, Vardiman JW and Harris NL (2001). Myelodysplastic syndromes: introduction. In: Jaffe ES, Harris NL, Stein H and Vardiman JW (eds), World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC Press, Lyon, pp. 63–67. 4 Brunning R, Orazi A, Germing U, LeBeau M, Porwit A, Baumann I, Vardiman J and Hellstrom-Lindberg E (2008). Myelodysplastic syndromes/neoplasms, overview. In press. 5 Kussick SJ (2007). Multiparametric flow cytometry in the diagnosis of myelodysplastic syndromes and related disorders. Lab Med, 38, 305–313. 6 Steinbach F, Henke F, Krause B, Thiele B, Burmester GR and Hiepe F (2000). Monocytes from SLE patients are severely altered in phenotype and lineage flexibility. Ann Rheum Dis, 59, 283–288. 7 Lai JL, Preudhomme C, Zandecki M, Flactif M, Vanrumbeke M, Lepelley P 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. 8 Steensma DP, Dewald GW, Hodnefield JM, Tefferi A and Hanson CA (2003). Clonal cytogenetic abnormalities on bone marrow specimens without clear morphological evidence of dysplasia: a forme fruste of myelodysplasia? Leuk Res, 27, 235–242. 9 Germing U, Hildebrandt B, Pfeilstöcker M, Nösslinger T, Valent P, Fonatsch C 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. 10 Sole F, Luno E, Sanzo C, Espinet B, Sanz GF, Cervera J 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. 11 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.

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12 Bernasconi P, Klersy C, Boni M, Cavigliano PM, Calatroni S, Giardini I et al. (2007). World Health Organization classification in combination with cytogenetic markers improves the prognostic stratification of patients with de novo primary myelodysplastic syndromes. Br J Haematol, 137, 193–205. 13 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. 14 Verburgh E, Achten R, Louw VJ, Brusselmans C, Delforge M, Boogaerts M et al. (2007). A new disease categorization of low-grade myelodysplastic syndromes based on the expression of cytopenia and dysplasia in one versus more than one lineage improves on the WHO classification. Leukemia, 21, 668–677. 15 Knipp S, Strupp C, Gattermann N, Hildebrandt B, Schapira M, Giagounidis A et al. (2007). Presence of peripheral blasts in refractory anemia and refractory cytopenia with multilineage dysplasia predicts an unfavourable outcome. Leuk Res, 32, 33–37.

16 Willis MS, McKenna RW, Peterson LC, Coad JE, and Kroft SH (2005). Low blast count myeloid disorders with Auer rods: a clinicopathologic analysis of 9 cases. Am J Clin Pathol, 124, 191–198. 17 List A, Dewald G, Bennett J, Giagounidis A, Raza A, Feldman E et al., Myelodysplastic syndrome-003 study Investigators (2006). Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion. N Engl J Med, 355, 1456–1465. 18 Raza A, Reeves JE, Feldman EJ, Dewald GW, Bennett JM, Deeg HJ et al. (2008). Phase 2 study of lenalidomide in transfusion-dependent, low-risk, and intermediate-risk-1 myelodysplastic syndromes with karyotypes other than deletion 5q. Blood, 111, 86–93. 19 Kantarjian H, Oki Y, Garcia-Manero G, Huang X, O’Brien S, Cortes J et al. (2007). Results of a randomized study of 3 schedules of low-dose decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic leukaemia. Blood, 109, 52–57.

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Myelodysplastic/ myeloproliferative neoplasms In the French–American–British (FAB) classification, chronic haematological malignancies were classified either as a myelodysplastic syndrome (MDS) or as a myeloproliferative disorder or disease (MPD). Chronic myelomonocytic leukaemia (CMML) was regarded as MDS, whereas atypical chronic myeloid leukaemia (aCML) was regarded as an MPD. This was never very satisfactory since, although CMML could have dysplastic features, this was equally true of aCML. This problem was resolved by the 2001 World Health Organization (WHO) classification which adopted the idea of a category for conditions with features overlapping between these two groups of neoplastic conditions, MDS/MPD [1] (Table 11.1). In the 2008 WHO classification the designation was changed to myelodysplastic/myeloproliferative neoplasms (MDS/MPN). This group includes only patients who have overlapping features at diagnosis. Patients with an MPN who develop dysplastic features as a result of disease evolution are not included.

Table 11.1 The myelodysplastic/myeloproliferative neoplasms

Chronic myelomonocytic leukaemia Atypical chronic myeloid leukaemia Juvenile myelomonocytic leukaemia Myelodysplastic/myeloproliferative neoplasm, unclassifiable

Similarly, patients with MDS who develop proliferative features as a result of disease acceleration or evolution are excluded. It is possible that some patients who have overlapping features at presentation may represent a disease in evolution from a previous MDS or MPN, but if this cannot be proven then they belong in this group. Other patients may have a condition with genuine overlapping features from disease initiation. By definition, the WHO classification excludes patients from this group who have BCR-ABL1 fusion or isolated 5q–. Juvenile myelomonocytic leukaemia (JMML) includes conditions that would previously have been categorized as juvenile myeloid leukaemia or as the monosomy 7 syndrome. The unclassifiable group include a number of patients with refractory anaemia with ring sideroblasts and thrombocytosis (RARS-T) (Figures 11.1–11.4), who often have a JAK2 V617F mutation [2]. Patients with a JAK2 mutation may form a discrete group. In around half of them the mutation is homozygous (due to mitotic recombination), differentiating this group of patients from those with essential thrombocythaemia [3]. Patients with a JAK2 mutation have a higher red cell count, higher white cell count (WBC) and better prognosis than those without a mutation [3]. Patients with MDS/MPN, unclassifiable, who lack a JAK2 mutation, form a more heterogeneous group than those with the mutation. Some of them have features typical of MDS but also have an elevated WBC.

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Figure 11.1 Peripheral blood (PB) film of a patient with refractory anaemia with ring sideroblasts and thrombocytosis (RARS-T) showing macrocytosis, hypogranular platelets and a neutrophil demonstrating the acquired Pelger-Hüet anomaly (evidence of myelodysplasia) plus thrombocytosis with large platelets (evidence of myeloproliferation). MGG, high power.

Figure 11.2 PB film of a patient with RARS-T showing macrocytes, a teardrop poikilocyte, a hypochromic microcyte and a platelet with a single giant granule (evidence of myelodysplasia) plus thrombocytosis (evidence of myeloproliferation). MGG, high power.

Figure 11.3 Bone marrow (BM) film of a patient with RARS-T showing increased numbers of large, hyperlobated megakaryocytes. The patient had a JAK2 V617F mutation. Low power, MGG.

Figure 11.4 BM film from a patient with RARS-T (same patient as Figure 11.3) showing two ring sideroblasts and another abnormal sideroblast. Perls’ stain, high power.

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References 1 Vardiman J (2001). Myelodysplastic/myeloproliferative diseases: introduction. In: Jaffe ES, Harris NL, Stein H and Vardiman JW (eds), World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, IARC, Lyon. 2 Wang SA, Hasserjian RP, Loew JM, Sechman EV, Jones D, Hao S et al. (2006). Refractory anemia with ringed sideroblasts associated with marked thrombocytosis harbors JAK2 mutation and shows overlapping myeloproliferative and myelodysplastic features. Leukaemia, 20, 1641–1644.

3 Schmitt-Graeff AH, Teo SS, Olschewski M, Schaub F, Haxelmans S, Kirn A et al. (2008). JAK2 V617F mutation status identifies subtypes of refractory anemia with ringed sideroblasts associated with marked thrombocytosis. Haematologica, 93, 34–40.

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Chronic myelomonocytic leukaemia

Chronic myelomonocytic leukaemia (CMML) is a haematological neoplasm characterized by both dysplastic features and proliferative features (monocytosis and sometimes neutrophilia). It is therefore classified in the World Health Organization (WHO) classification as one of the myelodysplastic/myeloproliferative neoplasms (MDS/MPN). In the French–American–British (FAB) classification it was included in the category of myelodysplastic syndrome (MDS). The clinical and haematological features cover a spectrum from predominantly myelodysplastic to predominantly myeloproliferative.

Clinical features CMML is mainly a disease of the elderly with a moderate male predominance. Some patients present with symptoms of anaemia, low-grade fever and night sweats, weight loss or bleeding. In others the diagnosis is incidental. Splenomegaly is sometimes detected. Hepatomegaly, lymphadenopathy and skin infiltration are less common. Organomegaly is more common in patients with a high white cell count (WBC). Serous effusions are occasionally seen.

Haematological and pathological features There is usually anaemia and sometimes there is macrocytosis. There may be leucopenia or leucocytosis. By definition, the monocyte count is greater than 1 × 109/l and monocytes are usually greater than 10% of leucocytes. In some patients the monocytes are all mature but in others there are immature or cytologically abnormal monocytes, e.g. with

cytoplasmic basophilia, heavy granulation or hypersegmented nuclei (Figures 12.1 and 12.2). Cells of other lineages may show dysplastic features (Figure 12.3). There may be small numbers of circulating blast cells and promonocytes but they do not exceed 20%. In some patients there is also neutrophilia. Neutropenia can also occur. Neutrophil precursors are generally less than 10% of leucocytes. Occasionally the eosinophil count is increased but, in the 2008 WHO classification, cases with rearrangement of PDGFRB are excluded from the category of CMML. The platelet count may be normal or reduced. The monocytes stain for non-specific esterases, e.g. alpha naphthyl acetate esterase and alpha naphthyl butyrate esterase. The bone marrow is hypercellular. Granulocytic hyperplasia is common. Sometimes abnormal monocytes and their precursors are prominent but this is not always so (Figure 12.4). Staining for non-specific esterase can help to identify the monocytic component. Monoblasts plus promonocytes are less than 20% of cells. There may be dysplastic features in one, two or three myeloid lineages but sometimes dysplasia is minimal. Dysplastic features can include dysplastic neutrophils, ring sideroblasts and micromegakaryocytes. Auer rods are rarely seen. The trephine biopsy usually shows hypercellularity and granulocytic hyperplasia, with or without obvious hyperplasia of the monocyte lineage. Immunohistochemistry using CD68R and CD163 monoclonal antibodies can help to identify the monocytic component [1]. In a significant minority of patients there are nodules of plasmacytoid monocytes, which have been shown to be part of the neoplastic clone [2]. Reticulin deposition may be increased. Splenic infiltration is mainly in the red pulp. Plasmacytoid monocytes may be present in the spleen and also in lymph nodes.

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Figure 12.1 Peripheral blood film of a patient with CMML showing a neutrophil and three abnormal monocytes. MGG, high power.

Figure 12.2 Peripheral blood film of a patient with CMML showing two abnormal monocytes. MGG, high power.

Figure 12.3 Peripheral blood film of a patient with CMML (same patient as Figure 12.1) showing a band form, a hypogranular neutrophil, a neutrophil with an abnormally shaped nucleus, a large hypogranular platelet and poikilocytes including acanthocytes. MGG, high power.

Figure 12.4 Bone marrow aspirate film of a patient with CMML showing an abnormal monocyte and two precursors. MGG, high power.

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Immunophenotype

Prognosis

The immunophenotype is not generally necessary for diagnosis. However, sometimes immunophenotyping identifies a monocytic component in the bone marrow that is not apparent morphologically and sometimes an aberrant immunophenotype is useful in supporting the diagnosis. On flow cytometry, leukaemic cells are shown to express CD13 and CD33 with variable expression of CD14, CD64 and CD68R. On immunohistochemistry they express lysozyme, CD68R and CD163.

The median survival in reported series is around 2–3 years. The prognosis can be related to the number of blasts plus promonocytes in blood and bone marrow. Thus cases can be divided into CMML-1 with blasts plus promonocytes being less than 5% in the blood and less than 10% in the marrow, and CMML-2 in which blasts are either 5–19% in the blood or 10–19% in the marrow or both. CMML-2 has both a shorter median survival (15 months cf. 20 months) and a significantly higher probability of transformation to AML (24% cf. 14%) [4]. A WBC greater than 13 × 109/l, low haemoglobin concentration (Hb), elevated lactate dehydrogenase, increased percentage of CD34-positive cells and the presence of splenomegaly have also been found to be prognostically adverse [4–7]. In multivariate analysis, Hb less than 12 g/dl, an absolute lymphocyte count of greater than 2.5 × 109/l, circulating immature myeloid cells and either elevated lactate dehydrogenase or increased bone marrow blast cells (<5%, 5–10% or >10%) were found to be independent risk factors [8]. In another multivariate analysis adverse prognosis was associated with male gender, Hb less than 10 g/dl, lymphocyte count greater than 2.5 × 109/l and CMML-2 [4].

Cytogenetic and molecular genetic abnormalities There is no specific cytogenetic or molecular genetic abnormality. Cytogenetic abnormalities are present in 40–50% of patients and include trisomy 8, monosomy 7, 7q–, abnormalities of 12p and i(17q). By definition, the BCR-ABL1 fusion gene and rearrangement of PDGFRA or PDGFRB are not detected. In cases with eosinophilia, the latter two rearrangements should be specifically sought by molecular and cytogenetic analysis. RAS mutations are often present. The JAK2 V617F mutation is uncommon [3].

Diagnosis and differential diagnosis When there is no cytogenetic or molecular genetic evidence of clonality, the possibility of reactive monocytosis must be considered. If there is minimal dysplasia it becomes important to exclude other potential causes of monocytosis and to observe the patient for a period, e.g. 3 months, to make sure that no cause emerges and that the monocytosis persists. Atypical chronic myeloid leukaemia (aCML) is important in the differential diagnosis. Dysplasia is a feature of both CMML and aCML. The distinction is made by the higher monocyte count and the lower count of granulocyte precursors in CMML. Eosinophilia and basophilia are more likely to be a feature of aCML. Sometimes AML with monocytic differentiation (FAB classification AML M5b) enters into the differential diagnosis. The distinction is made by the sum of blast cells and promonocytes being less than 20% in the blood and bone marrow in CMML.

Treatment There is no specific treatment. High counts can be reduced by hydroxycarbamide. Anaemia may respond to erythropoietin but otherwise blood transfusion may be necessary.

References 1 (2006). Chronic myelomonocytic leukemia: the role of bone marrow biopsy immunohistology. Mod Pathol, 19, 1536–1545. 2 Vermi W, Facchetti F, Rosati S, Vergoni F, Rossi E, Festa S et al. (2004). Nodal and extranodal tumor-forming accumulation of plasmacytoid monocytes/interferonproducing cells associated with myeloid disorders. Am J Surg Pathol, 28, 585–595.

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3 Johan MF, Goodeve AC, Bowen DT, Frew ME and Reilly JT (2005). JAK2 V617F mutation is uncommon in chronic myelomonocytic leukaemia. Br J Haematol, 130, 968. 4 Germing U, Strupp C, Knipp S, Kuendgen A, Giagounidis A, Hildebrandt B et al. (2007). Chronic myelomonocytic leukemia in the light of the WHO proposals. Haematologica, 92, 974–977. 5 Woodlock TJ, Seshi B, Sham RL, Cyran EM and Bennett JM (1994). Use of cell surface antigen phenotype in guiding therapeutic decisions in chronic myelomonocytic leukemia. Leuk Res, 18, 173–181.

6 Onida F, Kantarjian HM, Smith TL, Ball G, Keating MJ, Estey EH et al. (2002). Prognostic features and scoring systems in chronic myelomonocytic leukemia: a retrospective analysis of 213 patients. Blo o d, 99, 840–849. 7 Germing U, Strupp C, Alvado M and Gattermann N (2002). New prognostic parameters for chronic myelomonocytic leukemia? Blood, 100, 731–732. 8 Beran M, Wen S, Shen Y, Onida F, Jelinek J, Cortes J et al. (2007). Prognostic factors and risk assessment in chronic myelomonocytic leukemia: validation study of the M.D. Anderson Prognostic Scoring System. Le uk Lymphoma, 48, 1150–1160.

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Atypical chronic myeloid leukaemia

Atypical chronic myeloid leukaemia (aCML) is a Phnegative, BCR-ABL1-negative haematological neoplasm characterized by leucocytosis, an increase of granulocyte precursors in the peripheral blood and dysplastic features. In the World Health Organization (WHO) classification it is categorized as one of the myelodysplastic/myeloproliferative neoplasms (MDS/MPN).

Clinical features Patients may present with symptoms of anaemia or with infection or bruising. The spleen may be enlarged and less often the liver. aCML is mainly a disease of the elderly. It is a rare condition.

Haematological and pathological features The white cell count (WBC) is increased due to an increase in neutrophils and their precursors and often also eosinophils and basophils [1, 2] (Figures 13.1–13.3). Marked eosinophilia is not usual. Granulocyte precursors are 10–20% of leucocytes. Monocytes are increased but less so than in chronic myelomonocytic leukaemia (CMML), being usually less than 10%. There is usually anaemia and there may also be thrombocytopenia. Peripheral blood cells usually show dysplastic features such as macrocytosis, hypogranular neutrophils, acquired Pelger-Huët anomaly and platelet anisocytosis, but sometimes dysplastic features are very minor. A minority of patients have abnormal chromatin clumping in neutrophils, sometimes together with hypolobation or hypogranularity. A few blast cells may be present but they are usually less than 5% and, by definition, always less than 20%.

Bone marrow cellularity is increased due to an increase in cells of granulocyte lineage (Figures 13.4 and 13.5). All lineages may show dysplasia, which may include ring sideroblasts and dysplastic megakaryocytes including micromegakaryocytes. Blast cells are, by definition, less than 20%. The trephine biopsy shows similar features (Figure 13.6). Reticulin deposition may be increased (Figure 13.7). Sometimes it can be difficult to distinguish between CMML and aCML. This distinction can be aided by cytochemistry (non-specific esterase positivity) to identify the monocytic component in CMML.

Immunophenotype Immunophenotyping is not needed for diagnosis but with specific antibody panels can demonstrate aberrant expression of antigens, providing evidence of dysplastic maturation. In blast transformation immunophenotyping will identify the lineage. Immunophenotyping by flow cytometry (using CD14) or immunohistochemistry (using CD68R or CD163) can help to identify the monocytic component in CMML.

Cytogenetic and molecular genetic abnormalities Clonal cytogenetic abnormalities are present in the majority of patients but those found are not specific for this disease. The most frequently observed abnormalities are trisomy 8 and del(20q) [3]. Clonal molecular genetic abnormalities (e.g. RAS mutations or, occasionally, a JAK2 V617F mutation) may be present but these are likewise not specific.

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Figure 13.1 Peripheral blood film of a patient with aCML who had a very high white cell count and presented with features of hyperviscosity, showing mainly neutrophil precursors. MGG, high power.

Figure 13.3 Peripheral blood film of a patient with aCML (same patient as Figure 13.2), showing a basophil, a myelocyte and two monocytes, one of which is immature. There is also anisocytosis and a very bizarre platelet. MGG, high power.

Figure 13.2 Peripheral blood film of a patient with aCML, showing a dysplastic neutrophil, a myelocyte and a monocyte. There is also anisocytosis and stomatocytosis. MGG, high power.

Figure 13.4 Bone marrow aspirate film of a patient with aCML (same patient as Figure 13.2), showing hypercellularity. MGG, low power.

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Figure 13.5 Bone marrow aspirate film of a patient with aCML (same patient as Figure 13.2), showing that hypercellularity is due to an increase of neutrophils and eosinophils and their precursors. MGG, high power.

Figure 13.6 Bone marrow trephine biopsy section from a patient with aCML (same patient as Figure 13.2) showing disorganisation of the architecture and an increase of granulocytes and their precursors. H&E, low power.

By definition, in the WHO classification of 2008, cases with t(5;12)(q31-33;p12) and other cases with rearrangement of PDGFRB (who usually have eosinophilia) are regarded as a specific entity and are not included in aCML. Cases with BCR-ABL1 or PDGFRA rearrangement are also specifically excluded, but usually aCML is clinically and haematologically different from these conditions. In the 2008 WHO classification, some patients with iso(17q) (i.e. an isochromosome of 17q), who usually have prominent granulocytic dysplasia, are categorized as aCML but more often they are assigned to the CMML or MDS/MPN, unclassifiable categories.

Diagnosis and differential diagnosis The differential diagnosis includes BCR-ABL1-positive chronic myeloid leukaemia (CML) and CMML. Features that differentiate aCML from CML include more dysplasia, more monocytosis, less consistent eosinophilia and basophilia, less probability of thrombocytosis and more probability of thrombocytopenia in aCML. If CML presents in accelerated phase it can be indistinguishable from aCML without cytogenetic and molecular genetic analysis. Features that differentiate aCML from CMML include

Figure 13.7 Bone marrow trephine biopsy section from a patient with aCML (same patient as Figure 13.2) showing increased reticulin deposition (note that some of the reticulin relates to two blood vessels and is therefore normal). Reticulin, low power.

more numerous immature granulocytes in the peripheral blood in aCML, less prominent monocytosis and often more dysplasia. In cases with a higher blast count, acute

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myeloid leukaemia (AML) is included in the differential diagnosis. The distinction is based on the blast percentage, the presence of 20% or more blast cells in the blood or bone marrow leading to a diagnosis of AML.

Prognosis The prognosis is poor, worse than that of either CML or CMML. Reported median survivals have varied from 1 to 3 years. Poor prognostic features include advanced age, a higher WBC, a lower haemoglobin concentration and thrombocytopenia [4]. Death may be the result of bone marrow failure or evolution to AML.

Treatment Cytoreductive treatment with hydroxycarbamide and supportive treatment for anaemia may be needed, but treatment is not very effective and offers only palliation.

References 1 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick H 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, 746–754. 2 Hernandez JM, del Canizo MC, Cuneo A, Garcia JL, Gutierrez NC, Gonzalez M et al. (2000). Clinical, hematological and cytogenetic characteristics of atypical chronic myeloid leukemia. Ann Oncol, 11, 441–444. 3 Martiat P, Michaux JL and Rodhain J (1991). Philadelphia-negative (Ph-) chronic myeloid leukemia (CML): comparison with Ph+ CML and chronic myelomonocytic leukemia. The Groupe Francais de Cytogenetique Hematologique. Blood, 78, 205–211. 4 Breccia M, Biondo F, Latagliata R, Carmosino L, Mandelli F and Alimena G (2006). Identification of risk factors in atypical chronic myeloid leukemia. Haematologica, 91, 1566–1568.

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Juvenile myelomonocytic leukaemia

Juvenile myelomonocytic leukaemia (JMML) is a rare myelodysplastic/myeloproliferative haematological neoplasm that occurs predominantly in young children, particularly children with one of a number of predisposing inherited conditions [1–3]. Both neurofibromatosis type 1 (NF1 mutation, about 10% of cases of JMML) and Noonan’s syndrome (PTPN11 mutation) predispose to JMML.

Clinical features Most affected children are under the age of 3 years but age of presentation ranges from early infancy to adolescence. Boys are affected about twice as often as girls. Children present with systemic symptoms and clinical features resulting from anaemia, neutropenia and thrombocytopenia. Hepatomegaly and splenomegaly are usually marked and tonsillar enlargement is common. Lung infiltration leads to cough, tachypnoea and sometimes death from pulmonary insufficiency [4]. In addition, there may be eczema and lymphadenopathy. Children with neurofibromatosis type 1 may have café-au-lait spots and children with Noonan’s syndrome, abnormal facies and cardiac anomalies.

Haematological and pathological features There is anaemia and thrombocytopenia. The anaemia is normocytic (the majority), macrocytic or microcytic (a small minority). The white cell count (WBC) is moderately to markedly elevated (Figures 14.1–14.3). The neutrophil count is usually increased. The monocyte count is increased, a count of at least 1 × 109/l being one of the criteria for diagnosis of this condition. There are increased

numbers of neutrophil precursors including some blast cells (usually less than 5% and by definition always less than 20%). Eosinophilia and basophilia are less prominent than in Philadelphia (Ph)-positive chronic myeloid leukaemia. There are usually circulating nucleated red cells. The bone marrow is hypercellular, due to neutrophilic and to a lesser extent monocytic hyperplasia (Figure 14.4). Dyserythropoiesis and dysgranulopoiesis are common but Auer rods are not seen. Megakaryocytes are often decreased. Blast cells (plus promonocytes) are, by definition, less than 20%. Haemoglobin F is often increased for age and there may be other features also more typical of fetal erythropoiesis, such as a reduced erythrocyte concentration of haemoglobin A2 and carbonic anhydrase, and increased expression of the i antigen. These features of fetal erythropoiesis are seen in those children without monosomy 7 [4]. Haemopoietic stem cells show a marked in vitro hypersensitivity to granulocytemonocyte colony-stimulating factor (GM-CSF) [5]. Common non-specific findings include hyperglobulinaemia (due to an increase in polyclonal immunoglobulins) and an increased prevalence of autoantibodies. The direct antiglobulin test may be positive. Skin infiltration is in the dermis, spleen infiltration in the red pulp, and liver infiltration in the sinusoids and portal tracts. In the lungs, leukaemic cells infiltrate from capillaries into the alveolar septae and alveolae.

Immunophenotype Immunophenotyping is not usually useful in making the diagnosis although immunohistochemistry can be used to identify monocytic and granulocytic populations in the bone marrow and other tissues.

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Figure 14.1 Peripheral blood film from a child with JMML showing a neutrophil, a promyelocyte and abnormal monocytes. MGG, high power.

Figure 14.2 Peripheral blood film from a child with JMML showing a neutrophil and highly abnormal monocytes (same child as Figure 14.1). MGG, high power.

Figure 14.3 Peripheral blood film from a child with JMML showing a dysplastic neutrophil and several blast cells. MGG, high power.

Figure 14.4 Bone marrow aspirate film from a child with JMML showing increased granulocyte and monocyte precursors and a dysplastic (binucleated and hypogranular) neutrophil. MGG, high power.

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Cytogenetic and molecular genetic abnormalities Clonal cytogenetic abnormalities, e.g. trisomy 8 or monosomy 7, may be present but are seen in only a minority of patients. The Ph chromosome and the BCR-ABL1 fusion gene are absent. Molecular genetic changes involving both oncogenes and tumour suppressor genes are common. RAS mutations (NRAS or KRAS) are found in about a quarter of patients and PTPN11 mutations in about a third [4]. The normal NF1 allele is lost in children with neurofibromatosis as a result of recombination leading to uniparental disomy [6]. RAS, PTPN11 and NF1 involvement appear to be mutually exclusive; these are alternative mechanisms for dysregulation of the GM-CSF receptor–RAS–RAF– MAPK–ERK signal transduction pathway.

Diagnosis and differential diagnosis The differential diagnosis includes reactive bone marrow dysfunction, e.g. due to Epstein–Barr virus, cytomegalovirus and human herpesvirus 6 infection [7]. Children with Noonan’s syndrome suffer both an increased incidence of JMML and a morphologically similar transient haematological disorder (of uncertain nature) [8]; a period of observation is therefore needed before a firm diagnosis can be made.

Prognosis The prognosis is poor with the median survival being 1 year or less. Worse prognosis is associated with age above 2 years, thrombocytopenia and a high haemoglobin F percentage. Spontaneous improvement, despite persisting clonal cells, lasting a period of years has been observed in some patients with JMML associated with RAS mutation [9].

Treatment The prognosis with chemotherapy is poor so allogeneic haemopoietic stem cell transplantation should be considered [10].

References 1 Hasle H (1994). Myelodysplastic syndromes in childhood – classification, epidemiology, and treatment. Leuk Lymphoma, 13, 11–26. 2 Stiller CA, Chessells JM and Fitchett M (1994). Neurofibromatosis and childhood leukaemia/lymphoma: a population based UKCCSG study. Br J Cancer, 70, 969–972. 3 Passmore J, Chessells J, Kempski H, Hann IM, Brownbill PA and Stiller CA (2003). Paediatric MDS and JMML in the UK: a population based study of incidence and survival. Br J Haematol, 121, 758–767. 4 Flotho C, Kratz CP and Niemeyer CM (2007). How a rare pediatric neoplasia can give important insights into biological concepts: a perspective on juvenile myelomonocytic leukaemia. Haematologica, 92, 1441–1446. 5 Emanuel PD, Bates LJ, Castleberry RP, Gualtieri RJ and Zuckerman KS (1991). Selective hypersensitivity to granulocyte-macrophage colony-stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors. Blood, 77, 925–929. 6 Flotho C, Steinemann D, Mullighan CG, Neale G, Mayer K, Kratz CP et al. (2007). Genome-wide single nucleotide polymorphism analysis in juvenile myelomonocytic leukemia identifies uniparental disomy surrounding the NF1 locus in cases associated with neurofibromatosis, but not in cases with mutant RAS or PTPN11. Oncogene, 26, 5816–5821. 7 Pinkel D (1998). Differentiating juvenile myelomonocytic leukemia from infectious disease. Blood, 91, 365–367. 8 Ferraris S, Lanza C, Barisone E, Bertorello N, Farinasso D and Miniero R (2002). Transient abnormal myelopoiesis in Noonan syndrome. J Pediatr Hematol Oncol, 24, 763–764. 9 Matsuda K, Shimada A, Yoshida N, Ogawa A, Watanabe A, Yajima S et al. (2007). Spontaneous improvement of hematologic abnormalities in patients having juvenile myelomonocytic leukemia with specific RAS mutations. Blood, 109, 5477–5480. 10 Locatelli F, Nöllke P, Zecca M, Korthof E, Lanino E, Peters C et al., on behalf of the European Working Group of Childhood MDS (EWOG-MDS) and the European Blood and Marrow Transplantation (EBMT) Group (2005). Hematopoietic stem cell transplantation (HSCT) in children with juvenile myelomonocytic leukemia (JMML): results of the EWOG-MDS/EBMT trial. Blood, 105, 410–419.

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Index Note: Page numbers in italic refer to tables acanthocytes 126 aCML, see atypical chronic myeloid leukaemia acute lymphoblastic leukaemia (ALL) 3 acute myeloid leukaemia (AML) 4 aetiology 5, 7 basophilic 50–1 clinical features 7 cytogenetic and molecular genetic abnormalities 3–5, 35–7, 45–6 definition 7 diagnosis/differential diagnosis 54, 115, 132 FAB classification 8, 9, 10–26 haematological and pathological features 8–9 immunophenotyping 26, 27–35 with multilineage dysplasia 46–8 not otherwise categorized 49 prognosis and treatment 54 with recurrent cytogenetic/genetic abnormalities 39–46 therapy-related 38–9 WHO classification 37, 38–53 acute promyelocytic leukaemia (APL) 3, 39 aetiological factors 5, 7 alkylating agents 38 all-trans-retinoic acid 39, 54 AML, see acute myeloid leukaemia AML1-ETO fusion, see RUNX1CBFA2T1 fusion anaemia aplastic 114 iron deficiency 78 megaloblastic 54 micro-angiopathic haemolytic 67 normocytic/normochromic 8 see also refractory anaemia, refractory anaemia with excess of blasts in transformation (RAEB-T); refractory anaemia with excess blasts-1/2 (RAEB-1/2); refractory anaemia with ring sideroblasts (RARS); refractory

anaemia, see also (continued) anaemia with ring sideroblasts and thrombocytosis (RARS-T) anagrelide 84 anisocytosis 20, 21, 85, 86, 99, 100–1, 130 antihistamines 97 antithymocyte globulin 118 arsenic trioxide 54 aspirin 78, 84 atypical chronic myeloid leukaemia (aCML) 3, 127, 129–32 Auer rods 11, 13, 14, 41, 103, 117 azacytidine 118 basophilic leukaemia, acute 50–1 BCR-ABL1 fusion 2, 3, 57, 61, 68, 83, 131 blast cells aCML 129 acute basophilic leukaemia 50–1 AML 8, 9, 10, 11, 13, 40, 50–1 CD34 positive 113, 114 CML 61, 62, 66 MDS 102–7, 117 peroxidase positive 13, 15, 40, 50 primary myelofibrosis 85, 86 blast crisis 64, 68 bone marrow hypercellular 8, 61, 63, 75, 76, 105, 125, 126, 129, 130–1 hypocellular 8, 9, 12 ‘starry sky’ 20 CBFB-MYH11 fusion 3, 5, 42 CEBPA 4, 5 CEL, see chronic eosinophilic leukaemia cerebrovascular symptoms 81 Charcot–Leyden crystals 13, 71 chloroacetate esterase (CAE) 11 chronic eosinophilic leukaemia (CEL) 71–4, 97 chronic granulocytic leukaemia, see chronic myeloid leukaemia (CML) chronic myelogenous leukaemia, see chronic myeloid leukaemia chronic myeloid leukaemia (CML) aetiology 5

chronic myeloid leukaemia (CML) (continued) atypical (aCML) 3, 127, 129–32 clinical features 61 cytogenetic and molecular genetic abnormalities 2, 3, 68 diagnosis/differential diagnosis 68–9, 131 haematological and pathological features 61–6 immunophenotype 66–7 prognosis and treatment 69 chronic myelomonocytic leukaemia (CMML) 3, 58, 110, 121 clinical features 125 cytogenetic and molecular genetic abnormalities 127 diagnosis/differential diagnosis 127, 131 haematological and pathological features 110, 125–6 immunophenotype 127 prognosis and treatment 127 cigarette smoking 5, 7, 84 classification 2–3, 4 AML 8, 9, 37 MDS 111–12 MDS/MPN 4, 121 MPN 4, 57–8, 59 CML, see chronic myeloid leukaemia CMML, see chronic myelomonocytic leukaemia collagen formation 66, 88, 90 corticosteroids 74 CREBBP (CBP) 36 cytokines 88 cytopenia idiopathic of undetermined significance 115 refractory 116 refractory with ring sideroblasts 111, 116 Dameshek, William 57 decitabine 118 deficiency states 115 DEK-NUP214 fusion 46 Di Guglielmo’s syndrome 57–8 disseminated intravascular coagulation (DIC) 7

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Döhle bodies 107 Down’s syndrome 5, 36, 37 dyserythropoiesis 133 dysgranulopoiesis 133

gemtuzumab ozogamicin 54 granulocyte colony-stimulating factor (G-CSF) 54, 118, 133 granulocyte precursors 61, 62, 129, 130

EGR1 115 emperipolesis 81 eosinophilia, reactive 72 eosinophilic leukaemia, chronic 71–4, 97 eosinophils 42, 71, 72, 73 epigenetic effects 3 erythroblasts 20, 21, 86, 102 Pappenheimer bodies 103 PAS-positive 20, 22, 108 erythrocytes basophilic stippling 101 fragmentation 67 Pappenheimer bodies 100, 103 erythroid cells, primitive 23 erythroid dysplasia 105, 106 erythroid leukaemia, pure 49 erythroleukaemia, acute 49 erythropoiesis fetal 133 increased 76 shifted 102 erythropoietin, serum 76, 118 ETO 41 ETV6 73, 73 ETV6-PDGFRB fusion 97

haematocrit (Hct) 78, 78 haemoglobin concentration (Hb) 78, 78, 127 haemopoiesis, normal 1–2 Hasford score 69 hepatomegaly 85, 93, 97 histamine release, mast cells 93 histiocytes, sea-blue 64, 76 hydroxycarbamide 74, 78, 84, 127, 132 hypereosinophilic syndrome, idiopathic 72 hydroxyurea, see hydroxycarbamide hyperuricaemia 7, 85 hyperviscosity 130

imatinib 69, 72, 74, 97 immunophenotyping aCML 129 AML 26, 27–35 CML 66–7 CMML 127 MDS 113–14 normal bone marrow cells 27 systemic mastocytosis 96–7 interferon 74, 84 International Prognostic Scoring System (IPSS), MDS 116 faggot cells 14, 15 International Working Group on fetal erythropoiesis 133 Morphology of Myelodysplastic FGFR1 rearrangement 2, 3, 59, 72, 73, 74 Syndromes 115 fibrosis International Working Group for bone marrow 88, 89 Myelofibrosis Research and reactive 23 Treatment 85 FIP1L1-PDGFRA fusion 71, 72 inversions 5q- syndrome 4, 5, 107, 113, 115, 118 inv(3)(q21q26.2) 46 FLT3-ITD mutations 5, 38 inv(16)(p13q22) 42–4, 54, 115 fluorescence in situ hybridization (FISH) iron chelation therapy 118 AML 35 iron deficiency 78 CML 68 iron overload 99, 118 5q- syndrome 115 French–American–British (FAB) JAK2 mutations 3, 57, 72, 73, 77, 82–3, classification 90–1, 121–2 AML 8, 9, 10–26 juvenile myelomonocytic leukaemia MDS 57–8, 110, 121 (JMML) 3, 58, 121, 133–5 fusion genes 3–4 BCR-ABL1 2, 3, 57, 61, 68, 83, 131 karyograms CBFB-MYH11 42 20q- 77 DEK-NUP214 46 AML 36, 39, 41, 43 ETV6-PDGFRB 97 CML 68 FIP1L1-PDGFRA 71, 72 5q- syndrome 115 PML-RARA 3, 39 polycythaemia vera 77 RPN1-EVI1 46 t(8;21)(q22;q22) 41 RUNX1-CBFA2T1 3 trisomy 22 43 ZNF145-RARA 39

KIT mutations 5, 97 lactate dehydrogenase 116 lenalidomide 91, 118 Li Fraumeni syndrome 5 lung infiltration 133 lymphoid aggregates 109 lysozyme 20 macrocytosis 99, 100–1, 122 macronormoblast 102 macrophages, bone marrow 20 mast cell granules 51 mast cells dysplasia 93–6 histamine release 93 mastocytosis, systemic 93–7 MDS, see myelodysplastic syndromes megakaryoblasts 23, 24, 45 megakaryocytes 8, 22, 24, 25, 75–6, 88, 89 ‘bare’ 85, 87 clustering 76 dysplastic 46–8, 65, 66 hypolobated 107, 113 large 81, 82 megakaryocytic/megakaryoblastic transformation 85, 87, 88 megaloblasts 21, 24, 25, 105 giant binucleate 105 ultrastructure 24 Micrococcus lysodeikticus 20 micromegakaryocytes 23, 45, 63, 64, 85, 87, 103, 104 MLL 38 monoblasts 18–19, 44, 125 monocytosis 61, 125, 126, 129, 130 monosomy 5 113 monosomy 7 113, 118, 127 MPL mutations 57, 83, 90 MPL-Baltimore 83 MPN, see myeloproliferative neoplasms myeloblasts 11, 12, 103 myelocytes 106 myelodysplastic syndrome-unclassified (MDS-U) 111, 113 myelodysplastic syndromes (MDS) 99 associated with isolated del(5q) 111 classification 3, 4, 5, 108, 110–13 clinical features 99 cytogenetic and molecular genetic abnormalities 5, 113–14, 117 diagnosis/differential diagnosis 54, 114–15, 116 haematological and pathological features 99–109 immunophenotype 113 incidence 99

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thrombocythaemia, essential (continued) prognosis 83 thrombocytosis 81, 82, 83, 121, 122 thromboembolism 75, 77–8 topoisomerase-II-interactive drugs 38–9 toxic drugs/chemicals 38–9, 115 TP53 5, 68 transforming growth factor-β 88 translocations 3, 5 eosinophilic leukaemia 73 t(1;22)(p13;q13) 45 t(3;3)(q21;q26.2) 46 t(6;9)(p23;q34) 46, 47 radiation exposure 5, 38 t(8;16)(p11;p13) 36 RAS mutations 68, 127, 135 t(8;21)(q22;q22) 3, 5, 40, 41, 115 RB1 5 t(9;11)(p21;q23) 44, 45 refractory anaemia 108–9, 110, 111 t(9;22)(q34;q11.2) 57, 61, 68 refractory anaemia with excess of blasts t(11;17)(q23;q21) 39, 40 (RAEB) 1, 106–7, 110, 111, 116 t(11;19)(q23;p13) 45 refractory anaemia with excess of blasts in t(15;17)(q22;q12) 3, 5, 39, 115 transformation (RAEB-T) 110 neurofibromatosis type 1 (NF1) 5, 133, 135 t(16;16)(p13.1;q22) 43, 115 refractory anaemia with ring sideroblasts neutrophil alkaline phosphatase 81 trisomy 1q 90 (RARS) 102–6, 110, 111, 116 neutrophils trisomy 8 72, 90, 97, 113, 127, 129 refractory anaemia with ring sideroblasts chromatin clumping 129 trisomy 9 97 and thrombocytosis (RARS-T) 58, 77, dysplasia 134 trisomy 19 113 121, 122 increases 129, 130 trisomy 21 35, 113 refractory cytopenia with multilineage segmentation, defect 105 trisomy 22 43 dysplasia (RCMD) 111 nitrosureas 38 tryptase, mast cell 96 refractory cytopenia with multilineage non-specific esterase (NSE) 16, 17 tumour suppressor genes 5, 68 dysplasia and ringed sideroblasts Noonan’s syndrome 5, 133, 135 tumours, extramedullary 7 (RCMD-RS) 111, 116 NPM1 4, 5, 38, 38 20q- 72, 77, 129 reticulin 26, 66, 109, 125, 129, 131 NUP98 38 tyrosine kinase inhibitors 69, 74, 97 retinoblastoma, familial 5 ring sideroblasts 100–1, 103–6 oncogenic mechanisms 3–4 urticaria pigmentosa 93, 97 RPN1-EVI1 fusion 46 osteomyelosclerosis 65, 66 RPS14 5 osteoporosis 93, 95 vascular disease, polycythaemia vera 75, RUNX1 38 osteosclerosis 93, 95 77–8 RUNX1-CBFA2T1 fusion 3, 5, 41 32P 78, 84 vitamin B12 54, 76 sex chromosome loss 41 pancytopenia 85 von Willebrand factor 53, 81 sideroblasts 100–1, 103–6 panmyelosis spleen, infiltration 125 acute 91 white cell count (WBC) 8, 75, 78 splenectomy 91 acute with myelofibrosis 52–3 Wilms’ tumour 5 splenomegaly 61, 85, 93, 97 Pappenheimer bodies 100, 101, 103 World Health Organization (WHO) stem cell transplantation 54, 66, 67, 69 PDGFRA 59, 73 classification 3, 4 stem cells 1–2 PDGFRB rearrangement 3, 59, 73, 125, AML 37, 38–53 stomatocytosis 130 131 MDS 111–12 Sudan Black B (SBB) 11, 12, 16 Pelger–Huët anomaly 100, 122 MDS/MPN 121, 121 periodic acid-Schiff (PAS) stain 20, 22, 108 MPN 58, 59, 81 therapy-related disease 7 Philadelphia (Ph) chromosome 2, 61, 68 AML 37, 38–9 plasmacytoid dendritic cell leukaemia 53 ZNF145-RARA fusion 39 MDS 107, 113, 116 platelets 20, 83 theta granules 51 anisocytosis 82 thrombocythaemia, essential 57, 59, giant granule 122 68–9, 81–4 hypogranular 20, 85, 87, 102, 126 clinical features 81 morphological abnormalities 85, 86 diagnosis 83, 83 PML-RARA fusion 3, 5, 39 haematological and pathological poikilocytes 126 features 81, 82 teardrop 85, 86, 122 myelodysplastic syndromes (MDS) (continued) prognosis and treatment 116–18 therapy-related 107, 113, 116 myelodysplastic/myeloproliferative neoplasms (MDS/MPN) 4, 121–2 myelofibrosis ‘acute’ 23, 91 differential diagnosis 90 idiopathic (primary) 85–91, 97 pre-fibrotic phase 88 myeloid sarcoma 37 myeloperoxidase (MPO) 10, 11 myeloproliferative neoplasms (MPN) 57 classification 4, 57–8, 59 oncogenic mechanisms 3, 57, 58 see also individual disorders MYST3 36

poikilocytosis 20, 21, 85, 86, 101 polycythaemia relative 77 secondary (true) 77 polycythaemia vera 3, 75–8 proerythroblasts 21 promonocytes 17, 19, 42, 44, 125 promyelocytes 12, 14–15, 54 proto-oncogenes 5 pseudo-Gaucher cells 64, 65 PTPN11 mutations 135