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 Hoffbrand’s Essential Haematology This title is also available as an e-book. For more details, please see www.wiley.com/buy/9781118408674 or scan this QR code:  Hoffbrand’s Essential Haematology A. Victor Hoffbrand MA DM FRCP FRCPath FRCP(Edin) DSc FMe...

 Hoffbrand’s Essential Haematology This title is also available as an e-book. For more details, please see www.wiley.com/buy/9781118408674 or scan this QR code:  Hoffbrand’s Essential Haematology A. Victor Hoffbrand MA DM FRCP FRCPath FRCP(Edin) DSc FMedSci Emeritus Professor of Haematology University College London London, UK Paul A. H. Moss PhD MRCP FRCPath Professor of Haematology University of Birmingham Birmingham, UK Seventh Edition This edition first published 2016 © 2016 by John Wiley & Sons Ltd Previous editions: 1980, 1984, 1993, 2001, 2006, 2011 Registered office: John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell The right of the author to be identified as the author of this work has been asserted in accordance with the UK Copyright, Designs and Patents Act 1988. All rights reserved. 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No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. Library of Congress Cataloging-in-Publication Data Hoffbrand, A. V., author. [Essential haematology] Hoffbrand’s essential haematology / A. Victor Hoffbrand, Paul A. H. Moss. — Seventh edition. p. ; cm. — (Essentials) Includes index. ISBN 978-1-118-40867-4 (pbk.) I. Moss, P. A. H., author. II. Title. III. Series: Essentials (Wiley-Blackwell (Firm)). [DNLM: 1. Hematologic Diseases. WH 120] RC633 616.1′5—dc23 2015019097 A catalogue record for this book is available from the British Library. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Cover image: P242/0207. Blood cells, SEM. NATIONAL CANCER INSTITUTE/SCIENCE PHOTO LIBRARY. Blood cells and platelets. Coloured Scanning Elec-tron micrograph (SEM) of human blood showing red and white cells and platelets. Red blood cells (erythrocytes) have a characteristic biconcave- disc shape and are numerous. These large cells contain haemoglobin, a red pigment by which oxygen is transported around the body. They are more numerous than white blood cells (yellow). White blood cells (leucocytes) are rounded cells with microvilli projections from the cell surface. Leucocytes play an important role in the immune response of the body. Platelets are smaller cells (pink) that play a major role in blood clotting. Set in 10/12 Adobe Garamond Pro by Aptara Inc., New Delhi, India 1 2016 Contents Preface to the Seventh Edition vi 16 Myelodysplasia 177 Preface to the First Edition vii How to use your textbook viii 17 Acute lymphoblastic leukaemia 186 About the companion website x 18 The chronic lymphoid leukaemias 197 1 Haemopoiesis 1 19 Hodgkin lymphoma 205 2 Erythropoiesis and general aspects of 20 Non-Hodgkin lymphoma 213 anaemia 11 21 Multiple myeloma and related 3 Hypochromic anaemias 27 disorders 228 4 Iron overload 41 22 Aplastic anaemia and bone 5 Megaloblastic anaemias and other marrow failure 242 macrocytic anaemias 48 23 Stem cell transplantation 250 6 Haemolytic anaemias 60 24 Platelets, blood coagulation and 7 Genetic disorders of haemoglobin 72 haemostasis 264 8 The white cells 1: granulocytes, 25 Bleeding disorders caused by monocytes and their benign disorders 87 vascular and platelet abnormalities 278 9 The white cells 2: lymphocytes and 26 Coagulation disorders 290 their benign disorders 102 27 Thrombosis 1: pathogenesis and 10 The spleen 116 diagnosis 302 11 The aetiology and genetics of 28 Thombosis 2: treatment 311 haematological malignancies 122 29 Haematological changes in systemic 12 Management of haematological disease 321 malignancy 135 30 Blood transfusion 333 13 Acute myeloid leukaemia 145 31 Pregnancy and neonatal haematology 346 14 Chronic myeloid leukaemia 156 Appendix: World Health Organization classification of 15 Myeloproliferative disease 165 tumours of the haematopoietic and lymphoid tissues 352 Index 357 Preface to the Seventh Edition There have been remarkable advances in the understanding of the pathogenesis of diseases of the blood and lymphatic system and in the treatment of these diseases, since the 6th Edition of Essential Haematology was published in 2011. This new knowledge is due largely to the application of next generation sequencing of DNA which has enabled the detection of the genetic mutations, inherited or acquired, that underlie these diseases. As examples, sequencing has revealed the CALR mutation underlying a substantial propor- tion of patients with myeloproliferative diseases and the MYD88 mutation present in almost all cases of Waldenström’s macrogobulinaemia. Multiple ‘driver’ gene mutations affecting signalling pathways and epi- genetic reactions involved in cell proliferation and survival have been discovered which underlie myelodys- plasia, acute myeloid and lymphoblastic leukaemias, chronic lymphocytic leukaemia and the lymphomas. The complexity of the molecular changes underlying the malignant diseases and the relevance of this to their sensitivity or resistance to therapy is becoming apparent. This new knowledge has been accompanied by spectacular improvements in therapy. Inhibition of the B cell receptor signalling pathway has transformed the life expectancy in many patients with resist- ant chronic lymphocytic leukaemia and some of the B cell lymphomas resistant to other therapy. JAK2 inhibitors are improving the quality of life and survival in primary myelofibrosis. Survival in myeloma is improving remarkably with new proteasome inhibitory and immunomodulatory drugs. Life expectancy has also improved for patients with diseases such as thalassaemia major receiving multiple transfusions with the worldwide introduction of orally active iron chelating agents. New anticoagulants which directly inhibit at a single point in the coagulation cascade and rarely need monitoring are now used commonly in preference to warfarin for the treatment and prevention of arterial and venous thrombosis. These advances in knowledge have been incorporated as new text, diagrams and tables for this seventh edition. New multiple choice questions have been added to the website and short summary boxes are included at the end of each chapter. We thank Dr Trevor Baglin for his helpful suggestions for the coagulation section of the book. We wish to thank our publishers Wiley‐Blackwell and the staff who have helped us with the production of this 7th Edition. We also thank Jane Fallows for once more producing clear, expertly drawn scientific diagrams. We hope it will be widely used both by undergraduates and by postgraduates in medicine and related sciences wishing to gain a grounding in one of the most exciting and advanced fields of medicine. Victor Hoffbrand Paul Moss Preface to the First Edition The major changes that have occurred in all fields of medicine over the last decade have been accompanied by an increased understanding of the biochemical, physiological and immunological processes involved in normal blood cell formation and function and the disturbances that may occur in different diseases. At the same time, the range of treatment available for patients with diseases of the blood and blood‐forming organs has widened and improved substantially as understanding of the disease processes has increased and new drugs and means of support care have been introduced. We hope the present book will enable the medical student of the 1980s to grasp the essential features of modern clinical and laboratory haematology and to achieve an understanding of how many of the manifes- tations of blood diseases can be explained with this new knowledge of the disease processes. We would like to thank many colleagues and assistants who have helped with the preparation of the book. In particular, Dr H.G. Prentice cared for the patients whose haematological responses are illustrated in Figs 5.3 and 7.8 and Dr J. McLaughlin supplied Fig. 8.6. Dr S. Knowles reviewed critically the final manuscript and made many helpful suggestions. Any remaining errors are, however, our own. We also thank Mr J.B. Irwin and R.W. McPhee who drew many excellent diagrams, Mr Cedric Gilson for expert photomicrography, Mrs T. Charalambos, Mrs B. Elliot, Mrs M. Evans and Miss J. Allaway for typing the manuscript, and Mr Tony Russell of Blackwell Scientific Publications for his invaluable help and patience. AVH, JEP 1980 viii / How to use your textbook How to use your textbook Features contained within your textbook Every chapter begins CHAPTER 1 with a list of Key topics Haemopoiesis of the chapter. Key topics Site of haemopoiesis 2 Haemopoietic stem and progenitor cells 2 Bone marrow stroma 4 The regulation of haemopoiesis 4 Haemopoietic growth factors 4 Growth factor receptors and signal transduction 6 Adhesion molecules 8 The cell cycle 8 Transcription factors 8 Epigenetics 8 Apoptosis 9 Hoffbrand’s Essential Haematology, Seventh Edition. By A. Victor Hoffbrand and Paul A. H. Moss. Published 2016 by John Wiley & Sons Ltd. 10 / Chapter 1: Haemopoiesis proteins that are involved in mediating apoptosis following DNA membrane loses integrity. There is usually an inflammatory damage, such as p53 and ATM, are also frequently mutated and infiltrate in response to spillage of cell contents. Autophagy is therefore inactivated in haemopoietic malignancies. the digestion of cell organelles by lysosomes. It may be involved Necrosis is death of cells and adjacent cells due to ischaemia, in cell death but in some situations also in maintaining cell chemical trauma or hyperthermia. The cells swell, the plasma survival by recycling nutrients. Summary Haemopoiesis (blood cell formation) arises from Adhesion molecules are a large family of glycoproteins pluripotent stem cells in the bone marrow. Stem cells that mediate attachment of marrow precursors and give rise to progenitor cells which, after cell divisions mature leucocytes and platelets to extracellular matrix, and differentiation, form red cells, granulocytes endothelium and to each other. (neutrophils, eosinophils and basophils), monocytes, Epigenetics refers to changes in DNA and chromatin platelets and B and T lymphocytes. that affect gene expression other than those that Every chapter ends Haemopoetic tissue occupies about 50% of the marrow space in normal adult marrow. Haemopoiesis affect DNA sequence. Histone modification and DNA methylation are two important examples relevant to with a Summary that can in adults is confined to the central skeleton but in infants and young children haemopoietic tissue haemopoiesis and haematological malignancies. Transcription factors are molecules that bind to DNA be used for study and extends down the long bones of the arms and legs. Stem cells reside in the bone marrow in niches formed and control the transcription of specific genes or gene families. revision purposes. by stromal cells and circulate in the blood. Growth factors attach to specific cell receptors and Apoptosis is a physiological process of cell death resulting from activation of caspases. The intracellular produce a cascade of phosphorylation events to the ratio of pro‐apoptotic proteins (e.g. BAX) to anti‐ cell nucleus. Transcription factors carry the message apoptotic proteins (e.g. BCL‐2) determines the cell to those genes that are to be ‘switched on’, to stimulate susceptibility to apoptosis. cell division, differentiation, functional activity or suppress apoptosis. Now visit www.wileyessential.com/haematology to test yourself on this chapter.  How to use your textbook / ix 110 / Chapter 9: White cells: lymphocytes Chapter 9: White cells: lymphocytes / 111 ‐DR) molecules, whereas the CD8 molecule recognizes class Antigen‐specific immune responses are generated in sec- I (HLA‐A, ‐B and ‐C) molecules (see Fig. 23.5). The antigen ondary lymphoid organs and commence when antigen is Germinal follicle Follicular dendritic cells recognition site of the TCR is joined to several other subunits carried into a lymph node (Fig. 9.10) on dendritic cells. B cells Mantle zone in the CD3 complex which together mediate signal transduc- recognize antigen through their surface immunoglobulin and tion. Depending on their cytokine production, CD4+ T cells although most antibody responses require help from antigen‐ Marginal zone can be broadly subdivided into T helper type 1 (Th1) and Th2 specific T cells, some antigens such as polysaccharides can lead cells. Th1 cells produce mainly IL‐2, TNF‐b and g‐interferon to T‐cell independent B cell antibody production. In the fol- Plasma cells (IFN‐g), and are important in boosting cell‐mediated immu- licle, germinal centres arise as a result of continuing response nity (and granuloma formation), whereas Th2 cells produce to antigenic stimulation (Fig. 9.11). These consist of follicular Bone IL‐4 and IL‐10 and are mainly responsible for providing help dendritic cells (FDCs), which are loaded with antigen, B cells marrow for antibody production. and activated T cells which have migrated up from the T zone. Apoptosis Memory B cells Naive B cells Lymph from extravascular tissue space Afferent lymphatics B cell proliferation IgG class switching and Subcapsular sinus and somatic hypermutation generation of memory Your textbook is Primary follicle Marginal zone Positive selection of B cells B cell or plasma cell full of photographs, Germinal Follicles (B cells) by binding to follicular dendritic cells or apoptosis of B cells illustrations and tables. Secondary centre T zone follicle Mantle zone Medullary cord Figure 9.11 Generation of a germinal centre. B cells activated by antigen migrate from the T zone to the follicle where they undergo massive proliferation. Cells enter the dark zone as centroblasts and accumulate mutations in their immunoglobulin V genes. Cells then pass back into the light zone (Fig. 9.10) as centrocytes. Only those cells that can interact with antigen on follicular dendritic cells and receive Efferent lymphatics signals from antigen‐specific T cells (Fig. 9.9) are selected and migrate out as plasma cells and memory cells. Cells not selected die by apoptosis. Lymph returned to venous blood (a) Proliferating B cells move to the dark zone of the germinal Table 9.3 Causes of lymphocytosis. centre as centroblasts where they undergo somatic mutation of their immunoglobulin variable‐region genes (Fig. 9.11). Their Infections progeny are known as centrocytes and these must be selected acute: infectious mononucleosis, rubella, pertussis, for survival by antigen on FDCs, otherwise they undergo apop- mumps, acute infectious lymphocytosis, infectious hepatitis, tosis. If selected they become memory B cells or plasma cells cytomegalovirus, HIV, herpes simplex or zoster chronic: tuberculosis, toxoplasmosis, brucellosis, syphilis (Fig. 9.11). Plasma cells migrate to the bone marrow and other sites in the RE system and produce high‐affinity antibody. Chronic lymphoid leukaemias (see Chapter 18) Acute lymphoblastic leukaemia (Chapter 17) Lymphocytosis Non‐Hodgkin lymphoma (some) (Chapter 20) Lymphocytosis often occurs in infants and young children in Thyrotoxicosis response to infections that produce a neutrophil reaction in HIV, human immunodeficiency virus. adults. Conditions particularly associated with lymphocytosis are listed in Table 9.3. Glandular fever is a general term for a disease character- Infectious mononucleosis ized by fever, sore throat, lymphadenopathy and atypical lymphocytes in the blood. It may be caused by primary infec- This is caused by primary infection with EBV and occurs only (b) tion with Epstein–Barr virus (EBV), cytomegalovirus, human in a minority of infected individuals – in most cases infection immunodeficiency virus (HIV) or Toxoplasma. EBV infection, is subclinical. The disease is characterized by a lymphocytosis Figure 9.10 (a) Structure of a lymph node. (b) Lymph node showing germinal follicles surrounded by a darker mantle zone rim and otherwise known as infectious mononucleosis, is the most caused by clonal expansions of T cells reacting against B lym- lighter, more diffuse marginal and T‐zone areas. common cause. phocytes infected with EBV. The disease is associated with a Chapter 5: Macrocytic anaemias / 59 Differential diagnosis of macrocytic anaemias The laboratory features of particular importance are the shape of macrocytes (oval in megaloblastic anaemia), the pres- The clinical history and physical examination may suggest B12 ence of hypersegmented neutrophils, of leucopenia and throm- or folate deficiency as the cause. Diet, drugs, alcohol intake, bocytopenia in megaloblastic anaemia, and the bone marrow family history, history suggestive of malabsorption, presence appearance. Assay of serum B12 and folate is essential. Exclusion of autoimmune diseases or other associations with pernicious of alcoholism (particularly if the patient is not anaemic), liver anaemia (Table 5.4), previous gastrointestinal disease or opera- and thyroid function tests, and bone marrow examination for tions are all important. The presence of jaundice, glossitis or myelodysplasia, aplasia or myeloma are important in the inves- neuropathy are also important indications of megaloblastic tigation of macrocytosis not caused by B12 or folate deficiency. anaemia. Summary Macrocytic anaemias show an increased size of is autoimmune gastritis, resulting in severe deficiency circulating red cells (MCV >98 fL). Causes include of intrinsic factor, a glycoprotein made in the stomach vitamin B12 (B12, cobalamin) or folate deficiency, which facilitates B12 absorption by the ileum. The website icon alcohol, liver disease, hypothyroidism, myelodysplasia, Other gastrointestinal diseases as well as a vegan diet paraproteinaemia, cytotoxic drugs, aplastic anaemia, may cause B12 deficiency. pregnancy and the neonatal period. B12 or folate deficiency cause megaloblastic anaemia, Folate deficiency may be caused by a poor diet, malabsorption (e.g. gluten‐induced enteropathy) indicates that you can find in which the bone marrow erythroblasts have a typical abnormal appearance. or excess cell turnover (e.g. pregnancy, haemolytic anaemias, malignancy). accompanying multiple choice Folates take part in biochemical reactions in DNA Treatment of B12 deficiency is usually with injections questions and answers on the synthesis. B12 has an indirect role by its involvement in of hydroxocobalamin and of folate deficiency with oral folate metabolism. folic (pteroylglutamic) acid. book’s companion website. B12 deficiency may also cause a neuropathy due to Rare causes of megaloblastic anaemia include inborn damage to the spinal cord and peripheral nerves. errors of B12 or folate transport or metabolism, and B12 deficiency is usually caused by B12 malabsorption defects of DNA synthesis not related to B12 or folate. brought about by pernicious anaemia in which there Now visit www.wileyessential.com/haematology to test yourself on this chapter. About the companion website Don’t forget to visit the companion website for this book: www.wileyessential.com/haematology There you will find valuable material designed to enhance your learning, including: Interactive multiple choice questions Figures and tables from the book Scan this QR code to visit the companion website CHAPTER 1 Haemopoiesis Key topics Site of haemopoiesis 2 Haemopoietic stem and progenitor cells 2 Bone marrow stroma 4 The regulation of haemopoiesis 4 Haemopoietic growth factors 4 Growth factor receptors and signal transduction 6 Adhesion molecules 8 The cell cycle 8 Transcription factors 8 Epigenetics 8 Apoptosis 9 Hoffbrand’s Essential Haematology, Seventh Edition. By A. Victor Hoffbrand and Paul A. H. Moss. Published 2016 by John Wiley & Sons Ltd. 2 / Chapter 1: Haemopoiesis This first chapter is concerned with the general aspects of blood cell formation (haemopoiesis). The processes that regulate ­haemopoiesis and the early stages of formation of red cells (erythropoiesis), granulocytes and monocytes (myelopoiesis) and platelets (thrombopoiesis) are also discussed. Site of haemopoiesis In the first few weeks of gestation the yolk sac is a transient site of haemopoiesis. However, definitive haemopoiesis derives from a population of stem cells first observed on the AGM (aorta‐gonads‐mesonephros) region. These common precur- sors of endothelial and haemopoietic cells (haemangioblasts) are believed to seed the liver, spleen and bone marrow. From 6 weeks until 6–7 months of fetal life, the liver and spleen are the major haemopoietic organs and continue to produce Figure 1.1 Normal bone marrow trephine biopsy (posterior iliac blood cells until about 2 weeks after birth (Table 1.1; see crest). Haematoxylin and eosin stain; approximately 50% of the Fig. 7.1b). The placenta also contributes to fetal haemopoi- intertrabecular tissue is haemopoietic tissue and 50% is fat. esis. The bone marrow is the most important site from 6–7 months of fetal life. During normal childhood and adult life the marrow is the only source of new blood cells. The develop- in bone marrow. Many of the cells are dormant and in mice ing cells are situated outside the bone marrow sinuses; mature it has been estimated that they enter cell cycle approximately cells are released into the sinus spaces, the marrow microcircu- every 20 weeks. Although its exact phenotype is unknown, on lation and so into the general circulation. immunological testing the HSC is CD34+ CD38− and negative In infancy all the bone marrow is haemopoietic but during for lineage markers (Lin−) and has the appearance of a small or childhood there is progressive fatty replacement of marrow medium‐sized lymphocyte (see Fig. 23.3). The cells reside in throughout the long bones so that in adult life haemopoietic specialized osteoblastic or vascular ‘niches’. marrow is confined to the central skeleton and proximal ends Cell differentiation occurs from the stem cell via committed of the femurs and humeri (Table 1.1). Even in these haemo- haemopoietic progenitors which are restricted in their devel- poietic areas, approximately 50% of the marrow consists of fat opmental potential (Fig. 1.2). The existence of the separate (Fig. 1.1). The remaining fatty marrow is capable of reversion progenitor cells can be demonstrated by in vitro culture tech- to haemopoiesis and in many diseases there is also expansion niques. Very early progenitors are assayed by culture on bone of haemopoiesis down the long bones. Moreover, the liver and marrow stroma as long‐term culture initiating cells, whereas spleen can resume their fetal haemopoietic role (‘extramedul- late progenitors are generally assayed in semi‐solid media. An lary haemopoiesis’). example is the earliest detectable mixed myeloid precursor which gives rise to granulocytes, erythrocytes, monocytes and Haemopoietic stem and progenitor cells megakaryocytes and is termed CFU (colony‐forming unit)‐ Haemopoiesis starts with a pluripotential stem cell that can GEMM (Fig. 1.2). The bone marrow is also the primary site by asymmetric cell division self‐renew but also give rise to of origin of lymphocytes, which differentiate from a common the separate cell lineages. These cells are able to repopulate a lymphoid precursor. The spleen, lymph nodes and thymus are bone marrow from which all stem cells have been eliminated secondary sites of lymphocyte production (see Chapter 9). by lethal irradiation or chemotherapy. This haemopoietic stem The stem cell has the capability for self‐renewal (Fig. 1.3) cell (HSC) is rare, perhaps 1 in every 20 million nucleated cells so that marrow cellularity remains constant in a normal healthy steady state. There is considerable amplification in the system: one stem cell is capable of producing about 106 mature blood Table 1.1 Sites of haemopoiesis. cells after 20 cell divisions (Fig. 1.3). In humans HSCs are capable of about 50 cell divisions, telomere shortening affect- Fetus 0–2 months (yolk sac) ing viability. Under normal conditions most are dormant. With 2–7 months (liver, spleen) aging, the number of stem cells falls and the relative proportion giving rise to lymphoid rather than myeloid progenitors also 5–9 months (bone marrow) falls. Stem cells also accumulate genetic mutations with age, Infants Bone marrow (practically all bones) an average of 8 at age 60, and these, either passenger or driver, may be present in tumours arising from these stem cells (see Adults Vertebrae, ribs, sternum, skull, sacrum and Chapter 11). The precursor cells are capable of responding to pelvis, proximal ends of femur haemopoietic growth factors with increased production of one  Chapter 1: Haemopoiesis / 3 Pluripotent stem cell CFUGEMM Common myeloid progenitor cell Common lymphoid progenitor cell CFUbaso BFUE CFUGMEo Erythroid CFUEo progenitors Eosinophil progenitor CFUGM CFUMeg Granulocyte CFUE Megakary- monocyte Thymus ocyte progenitor progenitor CFU-M CFU-G B T NK Red Platelets Monocytes Neutrophils Eosinophils Basophils Lymphocytes NK cell cells Figure 1.2 Diagrammatic representation of the bone marrow pluripotent stem cell and the cell lines that arise from it. Various progenitor cells can be identified by culture in semi‐solid medium by the type of colony they form. It is possible that an erythroid/megakaryocytic progenitor may be formed before the common lymphoid progenitor diverges from the mixed granulocytic/monocyte/eosinophil myeloid progenitor. Baso, basophil; BFU, burst‐forming unit; CFU, colony‐forming unit; E, erythroid; Eo, eosinophil; GEMM, granulocyte, erythroid, monocyte and megakaryocyte; GM, granulocyte, monocyte; Meg, megakaryocyte; NK, natural killer. Multiplication Self Differentiation renewal (a) Mature cells Stem Multipotent Recognizable cells progenitor cells committed marrow (b) precursors Figure 1.3 (a) Bone marrow cells are increasingly differentiated and lose the capacity for self‐renewal as they mature. (b) A single stem cell gives rise, after multiple cell divisions (shown by vertical lines), to >106 mature cells. 4 / Chapter 1: Haemopoiesis or other cell line when the need arises. The development of the growth factors such as granulocyte colony‐stimulating factor mature cells (red cells, granulocytes, monocytes, megakaryo- (G‐CSF) (see p. 91). The reverse process of stem cell homing cytes and lymphocytes) is considered further in other sections appears to depend on a chemokine gradient in which the of this book. stromal‐derived factor 1 (SDF‐1) which binds to its receptor CXCR4 on HSC is critical. Several critical interactions main- Bone marrow stroma tain stem cell viability and production in the stroma including stem cell factor (SCF) and jagged proteins expressed on stroma The bone marrow forms a suitable environment for stem cell and their respective receptors KIT and NOTCH expressed on survival, self‐renewal and formation of differentiated progeni- stem cells. tor cells. It is composed of stromal cells and a microvascular network (Fig. 1.4). The stromal cells include mesenchymal The regulation of haemopoiesis stem cells, adipocytes, fibroblasts, osteoblasts, endothelial cells and macrophages and they secrete extracellular molecules such Haemopoiesis starts with stem cell division in which one as collagen, glycoproteins (fibronectin and thrombospondin) cell replaces the stem cell (self‐renewal ) and the other is and glycosaminoglycans (hyaluronic acid and chondroi- committed to differentiation. These early committed pro- tin derivatives) to form an extracellular matrix. In addition, genitors express low levels of transcription factors that may stromal cells secrete several growth factors necessary for stem commit them to discrete cell lineages. Which cell lineage is cell survival. selected for differentiation may depend both on chance and Mesenchymal stem cells are critical in stromal cell forma- on the external signals received by progenitor cells. Several tion. Together with osteoblasts or endothelial cells they form transcription factors (see p. 8) regulate survival of stem cells niches and provide the growth factors, adhesion molecules (e.g. SCL, GATA‐2, NOTCH‐1), whereas others are involved and cytokines which support stem cells, e.g. the protein in differentiation along the major cell lineages. For instance, jagged, on stromal cells, binds to a receptor NOTCH1 on PU.1 and the CEBP family commit cells to the myeloid stem cells which then becomes a transcription factor involved lineage, whereas GATA‐2 and then GATA‐1 and FOG‐1 have in the cell cycle. essential roles in erythropoietic and megakaryocytic differen- Stem cells are able to traffic around the body and are found tiation. These transcription factors interact so that reinforce- in peripheral blood in low numbers. In order to exit the bone ment of one transcription programme may suppress that of marrow, cells must cross the blood vessel endothelium and another lineage. The transcription factors induce synthesis of this process of mobilization is enhanced by administration of proteins specific to a cell lineage. For example, the erythroid‐ specific genes for globin and haem synthesis have binding motifs for GATA‐1. Stem cell Extracellular Haemopoietic growth factors matrix The haemopoietic growth factors are glycoprotein hormones that regulate the proliferation and differentiation of haemo- poietic progenitor cells and the function of mature blood cells. They may act locally at the site where they are produced by cell–cell contact or circulate in plasma. They also bind to the extracellular matrix to form niches to which stem and progenitor cells adhere. The growth factors may cause cell proliferation but can also stimulate differentiation, matura- Macrophage Mesenchymal tion, prevent apoptosis and affect the function of mature cells stem cell (Fig. 1.5). Endothelial cell Fibroblast or osteoblast They share a number of common properties (Table 1.2) and act at different stages of haemopoiesis (Table 1.3; Fig. 1.6). Adhesion molecule Ligand Stromal cells are the major source of growth factors except for Growth factor Growth factor receptor erythropoietin, 90% of which is synthesized in the kidney, and thrombopoietin, made largely in the liver. An important feature of growth factor action is that two or more factors Figure 1.4 Haemopoiesis occurs in a suitable microenvironment may synergize in stimulating a particular cell to proliferate or (‘niche’) provided by a stromal matrix on which stem cells grow differentiate. Moreover, the action of one growth factor on and divide. The niche may be vascular (lined by endothelium) or a cell may stimulate production of another growth factor or endosteal (lined by osteoblasts). There are specific recognition growth factor receptor. SCF and FLT3 ligand (FLT3‐L) act and adhesion sites (see p. 8); extracellular glycoproteins and other locally on the pluripotential stem cells and on early myeloid compounds are involved in the binding. and lymphoid progenitors (Fig. 1.6). Interleukin‐3 (IL‐3) and  Chapter 1: Haemopoiesis / 5 Early cell G-CSF Proliferation Monocyte Differentiation G-CSF Neutrophil G-CSF Maturation Suppression of apoptosis G-CSF Late cell G-CSF Activation of Functional phagocytosis, activation killing, secretion Figure 1.5 Growth factors may stimulate proliferation of early bone marrow cells, direct differentiation to one or other cell type, stimulate cell maturation, suppress apoptosis or affect the function of mature non‐dividing cells, as illustrated here for granulocyte colony‐stimulating factor (G‐CSF) for an early myeloid progenitor and a neutrophil. Table 1.2 General characteristics of myeloid and lymphoid granuloctye–macrophage colony‐stimulating factor (GM‐ growth factors. CSF) are multipotential growth factors with overlapping activi- Glycoproteins that act at very low concentrations ties. G‐CSF and thrombopoietin enhance the effects of SCF, FLT‐L, IL‐3 and GM‐CSF on survival and differentiation of Act hierarchically the early haemopoietic cells. Usually produced by many cell types These factors maintain a pool of haemopoietic stem and progenitor cells on which later‐acting factors, erythropoietin, Usually affect more than one lineage G‐CSF, macrophage colony‐stimulating factor (M‐CSF), IL‐5 Usually active on stem/progenitor cells and on differentiated and thrombopoietin, act to increase production of one or cells other cell lineage in response to the body’s need. Granulocyte and monocyte formation, for example, can be stimulated by Usually show synergistic or additive interactions with other infection or inflammation through release of IL‐1 and tumour growth factors necrosis factor (TNF) which then stimulate stromal cells to Often act on the neoplastic equivalent of a normal cell produce growth factors in an interacting network (see Fig. 8.4). In contrast, cytokines, such as transforming growth factor‐β Multiple actions: proliferation, differentiation, maturation, (TGF‐β) and γ‐interferon (IFN‐γ), can exert a negative effect functional activation, prevention of apoptosis of progenitor on haemopoiesis and may have a role in the development of cells aplastic anaemia (see p. 244). 6 / Chapter 1: Haemopoiesis Table 1.3 Haemopoietic growth factors. Growth factor receptors and signal transduction Act on stromal cells IL‐1 The biological effects of growth factors are mediated through TNF specific receptors on target cells. Many receptors (e.g. erythro- Act on pluripotential stem cells poietin (epo) receptor (R), GMCSF‐R) are from the haemat- SCF opoietin receptor superfamily which dimerize after binding FLT3‐L their ligand. VEGF Dimerization of the receptor leads to activation of a complex Act on multipotential progenitor cells series of intracellular signal transduction pathways, of which IL‐3 the three major ones are the JAK/STAT, the mitogen‐activated GM‐CSF protein (MAP) kinase and the phosphatidylinositol 3 (PI3) IL‐6 kinase pathways (Fig. 1.7; see Fig. 15.2). The Janus‐associated G‐CSF kinase (JAK) proteins are a family of four tyrosine‐specific Thrombopoietin protein kinases that associate with the intracellular domains of Act on committed progenitor cells the growth factor receptors (Fig. 1.7). A growth factor mol- G‐CSF* ecule binds simultaneously to the extracellular domains of M‐CSF two or three receptor molecules, resulting in their aggregation. IL‐5 (eosinophil‐CSF) Receptor aggregation induces activation of the JAKs which Erythropoietin now phosphorylate members of the signal transducer and acti- Thrombopoietin* vator of transcription (STAT) family of transcription factors. CSF, colony‐stimulating factor; FLT3‐L, FLT3 ligand; G‐CSF, granulocyte colony‐ stimulating factor; GM‐CSF, granulocyte–macrophage colony‐stimulating factor; This results in their dimerization and translocation from the IL, interleukin; M‐CSF, macrophage colony‐stimulating factor; SCF, stem cell cell cytoplasm across the nuclear membrane to the cell nucleus. factor; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor. Within the nucleus STAT dimers activate transcription of spe- * These also act synergistically with early acting factors on pluripotential progenitors. cific genes. A model for control of gene expression by a tran- scription factor is shown in Fig. 1.8. The clinical importance of SCF FLT3-L PSC IL-3 IL-3 TPO CFU-GEMM GM-CSF GM-CSF BFUEMeg CFU-GMEo BFUE EPO CFUMeg CFUGM CFUEo M-CSF G-CSF IL-5 CFUE CFUM CFUG Red cells Platelets Monocytes Neutrophils Eosinophils Figure 1.6 A diagram of the role of growth factors in normal haemopoiesis. Multiple growth factors act on the earlier marrow stem and progenitor cells. EPO, erythropoietin; PSC, pluripotential stem cell; SCF, stem cell factor; TPO, thrombopoietin; FLT3‐L, FLT3 ligand. For other abbreviations see Fig. 1.2.  Chapter 1: Haemopoiesis / 7 Growth factor Plasma membrane PI3Kinase JAK AKT JAK Blocked STATs RAS apoptosis RAF Nucleus Active STAT dimers MAP kinase MYC, FOS M Gene expression Activation of gene expression G2 G1 E2F S Rb p53 DNA damage Figure 1.7 Control of haemopoiesis by growth factors. The factors act on cells expressing the corresponding receptors. Binding of a growth factor to its receptor activates the JAK/STAT, MAPK and phosphatidyl‐inositol 3‐kinase (PI3K) pathways (see Fig. 15.2) which leads to transcriptional activation of specific genes. E2F is a transcription factor needed for cell transition from G1 to S phase. E2F is inhibited by the tumour suppressor gene Rb (retinoblastoma) which can be indirectly activated by p53. The synthesis and degradation of different cyclins stimulates the cell to pass through the different phases of the cell cycle. The growth factors may also suppress apoptosis by activating AKT (protein kinase B). Transactivation domain RNA polymerase + Transcription DNA-binding accessory factors domain Enhancer TATA box Structural DNA sequence sequence gene (promotor) Figure 1.8 Model for control of gene expression by a transcription factor. The DNA‐binding domain of a transcription factor binds a specific enhancer sequence adjacent to a structural gene. The transactivation domain then binds a molecule of RNA polymerase, thus augmenting its binding to the TATA box. The RNA polymerase now initiates transcription of the structural gene to form mRNA. Translation of the mRNA by the ribosomes generates the protein encoded by the gene. 8 / Chapter 1: Haemopoiesis this pathway is revealed by the finding of an activating muta- is further partitioned into classical mitosis, in which nuclear tion of the JAK2 gene as the cause of polycythaemia rubra vera division is accomplished, and cytokinesis, in which cell fission (see p. 166). occurs. JAK can also activate the MAPK pathway, which is regu- Interphase is divided into three main stages: a G1 phase, lated by RAS and controls proliferation. PI3 kinases phopho- in which the cell begins to commit to replication, an S phase, rylate inositol lipids which have a wide range of downstream during which DNA content doubles and the chromosomes effects including activation of AKT leading to block of apopto- replicate, and the G2 phase, in which the cell organelles are sis and other actions (Fig. 1.7; see Fig. 15.2). Different domains copied and cytoplasmic volume is increased. If cells rest prior of the intracellular receptor protein may signal for the different to division they enter a G0 state where they can remain for processes (e.g. proliferation or suppression of apoptosis) medi- long periods of time. The number of cells at each stage of the ated by growth factors. cell cycle can be assessed by exposing cells to a chemical or A second smaller group of growth factors, including SCF, ­radiolabel that gets incorporated into newly generated DNA or FLT‐3L and M‐CSF (Table 1.3), bind to receptors that have by flow cytometry. an extracellular immunoglobulin‐like domain linked via The cell cycle is controlled by two checkpoints which act as a transmembrane bridge to a cytoplasmic tyrosine kinase brakes to coordinate the division process at the end of the G1 domain. Growth factor binding results in dimerization of and G2 phases. Two major classes of molecules control these these receptors and consequent activation of the tyrosine checkpoints, cyclin‐dependent protein kinases (Cdk), which kinase domain. Phosphorylation of tyrosine residues in the phosophorylate downstream protein targets, and cyclins, receptor itself generates binding sites for signalling proteins which bind to Cdks and regulate their activity. An example which initiate complex cascades of biochemical events result- of the importance of these systems is demonstrated by mantle ing in changes in gene expression, cell proliferation and pre- cell lymphoma which results from the constitutive activation vention of apoptosis. of cyclin D1 as a result of a chromosomal translocation (see p. 223). Adhesion molecules Transcription factors A large family of glycoprotein molecules termed adhesion mol- ecules mediate the attachment of marrow precursors, leuco- Transcription factors regulate gene expression by controlling cytes and platelets to various components of the extracellular the transcription of specific genes or gene families (Fig. 1.8). matrix, to endothelium, to other surfaces and to each other. Typically, they contain at least two domains: a DNA‐binding The adhesion molecules on the surface of leucocytes are termed domain, such as a leucine zipper or helix–loop–helix motif receptors and these interact with proteins termed ligands on which binds to a specific DNA sequence, and an activation the surface of target cells, e.g. endothelium. The adhesion mol- domain, which contributes to assembly of the transcription ecules are important in the development and maintenance of complex at a gene promoter. Mutation, deletion or transloca- inflammatory and immune responses, and in platelet–vessel tion of transcription factors underlie many cases of haemato- wall and leucocyte–vessel wall interactions. logical neoplasms (see Chapter 11). The pattern of expression of adhesion molecules on tumour cells may determine their mode of spread and tissue localiza- Epigenetics tion (e.g. the pattern of metastasis of carcinoma cells or non‐ Hodgkin lymphoma cells into a follicular or diffuse pattern). This refers to changes in DNA and chromatin that affect gene The adhesion molecules may also determine whether or not expression other than those that affect DNA sequence. cells circulate in the bloodstream or remain fixed in tissues. Cellular DNA is packaged by wrapping it around histones, They may also partly determine whether or not tumour cells a group of specialized nuclear proteins. The complex is tightly are susceptible to the body’s immune defences. compacted as chromatin. In order for the DNA code to be read, transcription factors and other proteins need to physically The cell cycle attach to DNA. Histones act as custodians for this access and so for gene expression. Histones may be modified by meth- The cell division cycle, generally known simply as the cell ylation, acetylation and phosphorylation which can result in cycle, is a complex process that lies at the heart of haemopoi- increased or decreased gene expression and so changes in cell esis. Dysregulation of cell proliferation is also the key to the phenotype. Epigenetics also includes changes to DNA itself, development of malignant disease. The duration of the cell such as methylation which regulates gene expression in normal cycle is variable between different tissues but the basic prin- and tumour tissues. The methylation of cytosine residues to ciples remain constant. The cycle is divided into the mitotic methyl cytosine results in inhibition of gene transcription. The phase (M phase), during which the cell physically divides, and genes DNMT 3A and B are involved in the methylation, and interphase, during which the chromosomes are duplicated and TET 1,2,3 and IDH1 and 2 in the hydroxylation and there- cell growth occurs prior to division (Fig. 1.7). The M phase fore breakdown of methylcytosine and restoration of gene  Chapter 1: Haemopoiesis / 9 expression (see Fig. 16.1). These genes are frequently mutated role in sensing DNA damage. It activates apoptosis by raising in the myeloid malignancies (see Chapters 13, 15 and 16). the cell level of BAX which then increases cytochrome c release (Fig. 1.9). P53 also shuts down the cell cycle to stop the damaged cell from dividing (Fig. 1.7). The cellular level of p53 Apoptosis is rigidly controlled by a second protein, MDM2. Following Apoptosis (programmed cell death) is a regulated process of death, apoptotic cells display molecules that lead to their inges- physiological cell death in which individual cells are triggered tion by macrophages. to activate intracellular proteins that lead to the death of the As well as molecules that mediate apoptosis there are several cell. Morphologically it is characterized by cell shrinkage, intracellular proteins that protect cells from apoptosis. The best condensation of the nuclear chromatin, fragmentation of the characterized example is BCL‐2. BCL‐2 is the prototype of a nucleus and cleavage of DNA at internucleosomal sites. It is an family of related proteins, some of which are anti‐apoptotic important process for maintaining tissue homeostasis in hae- and some, like BAX, pro‐apoptotic. The intracellular ratio of mopoiesis and lymphocyte development. BAX and BCL‐2 determines the relative susceptibility of cells to Apoptosis results from the action of intracellular cysteine apoptosis (e.g. determines the lifespan of platelets) and may act proteases called caspases which are activated following cleavage through regulation of cytochrome c release from mitochondria. and lead to endonuclease digestion of DNA and disintegration Many of the genetic changes associated with malignant of the cell skeleton (Fig. 1.9). There are two major pathways disease lead to a reduced rate of apoptosis and hence prolonged by which caspases can be activated. The first is by signalling cell survival. The clearest example is the translocation of the through membrane proteins such as Fas or TNF receptor via BCL‐2 gene to the immunoglobulin heavy chain locus in the their intracellular death domain. An example of this mecha- t(14;18) translocation in follicular lymphoma (see p. 222). Over- nism is shown by activated cytotoxic T cells expressing Fas expression of the BCL‐2 protein makes the malignant B cells less ligand which induces apoptosis in target cells. The second susceptible to apoptosis. Apoptosis is the normal fate for most pathway is via the release of cytochrome c from mitochondria. B cells undergoing selection in the lymphoid germinal centres. Cytochrome c binds to APAF‐1 which then activates caspases. Several translocations leading to the generation of fusion pro- DNA damage induced by irradiation or chemotherapy may teins, such as t(9;22), t(1;14) and t(15;17), also result in inhibi- act through this pathway. The protein p53 has an important tion of apoptosis (see Chapter 11). In addition, genes encoding Death factor e.g. Fas ligand APOPTOSIS Caspases Death Release of domain cytochrome c Procaspases Increased Inhibits BAX protein p53 BCL-2 BAX gene expression Increased DNA BCL-2 damage Cytotoxic Survival factor drugs e.g. growth factor Radiation Figure 1.9 Representation of apoptosis. Apoptosis is initiated via two main stimuli: (i) signalling through cell membrane receptors such as FAS or tumour necrosis factor (TNF) receptor; or (ii) release of cytochrome c from mitochondria. Membrane receptors signal apoptosis through an intracellular death domain leading to activation of caspases which digest DNA. Cytochrome c binds to the cytoplasmic protein Apaf‐1 leading to activation of caspases. The intracellular ratio of pro‐apoptotic (e.g. BAX) or anti‐apoptotic (e.g. BCL‐2) members of the BCL‐2 family may influence mitochondrial cytochrome c release. Growth factors raise the level of BCL‐2 inhibiting cytochrome c release, whereas DNA damage, by activating p53, raises the level of BAX which enhances cytochrome c release.  10 / Chapter 1: Haemopoiesis proteins that are involved in mediating apoptosis following DNA membrane loses integrity. There is usually an inflammatory damage, such as p53 and ATM, are also frequently mutated and infiltrate in response to spillage of cell contents. Autophagy is therefore inactivated in haemopoietic malignancies. the digestion of cell organelles by lysosomes. It may be involved Necrosis is death of cells and adjacent cells due to ischaemia, in cell death but in some situations also in maintaining cell chemical trauma or hyperthermia. The cells swell, the plasma survival by recycling nutrients. Summary Haemopoiesis (blood cell formation) arises from Adhesion molecules are a large family of glycoproteins pluripotent stem cells in the bone marrow. Stem cells that mediate attachment of marrow precursors and give rise to progenitor cells which, after cell divisions mature leucocytes and platelets to extracellular matrix, and differentiation, form red cells, granulocytes endothelium and to each other. (neutrophils, eosinophils and basophils), monocytes, Epigenetics refers to changes in DNA and chromatin platelets and B and T lymphocytes. that affect gene expression other than those that Haemopoetic tissue occupies about 50% of the affect DNA sequence. Histone modification and DNA marrow space in normal adult marrow. Haemopoiesis methylation are two important examples relevant to in adults is confined to the central skeleton but in haemopoiesis and haematological malignancies. infants and young children haemopoietic tissue Transcription factors are molecules that bind to DNA extends down the long bones of the arms and legs. and control the transcription of specific genes or gene Stem cells reside in the bone marrow in niches formed families. by stromal cells and circulate in the blood. Apoptosis is a physiological process of cell death Growth factors attach to specific cell receptors and resulting from activation of caspases. The intracellular produce a cascade of phosphorylation events to the ratio of pro‐apoptotic proteins (e.g. BAX) to anti‐ cell nucleus. Transcription factors carry the message