Frontiers in Clinical Drug Research - Hematology [1 ed.] 9781681083674, 9781681083681

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Frontiers in Clinical Drug Research - Hematology (Volume 3)

Edited by Atta-ur-Rahman, FRS Honorary Life Fellow, Kings College, University of Cambridge, Cambridge, UK

 

Frontiers in Clinical Drug Research - Hematology Volume # 3 Editor: Atta-ur-Rahman ISSN (Online): 2352-3239 ISSN (Print): 2467-9585 ISBN (Online): 978-1-68108-367-4 ISBN (Print): 978-1-68108-368-1 ©2018, Bentham eBooks imprint. Published by Bentham Science Publishers – Sharjah, UAE. All Rights Reserved. First published in 2018.

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CONTENTS PREFACE ................................................................................................................................................ i LIST OF CONTRIBUTORS .................................................................................................................. ii CHAPTER 1 ADVANCES IN THE UNDERSTANDING AND TREATMENT OF IMMUNE THROMBOCYTOPENIA ...................................................................................................................... José Perdomo INTRODUCTION .......................................................................................................................... Platelet Biogenesis .................................................................................................................. Platelet Function ..................................................................................................................... Epidemiology, Aetiology and Disease Progression ................................................................ Platelet Destruction in ITP ........................................................................................... Recent Developments .................................................................................................... Impairment of Platelet Production in ITP .................................................................... Disease Characteristics and Therapies .................................................................................... Need for Novel Therapies ....................................................................................................... CONCLUDING REMARKS ......................................................................................................... CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ............................................................................................................................... CHAPTER 2 RECENT DEVELOPMENTS IN CHRONIC MYELOID LEUKEMIA BIOLOGY AND TREATMENT ................................................................................................................................ Massimiliano Bonifacio and Claudio Sorio INTRODUCTION .......................................................................................................................... PART I: BIOLOGY ....................................................................................................................... Signaling Mechanisms Altered in CML ................................................................................. Oncosuppressors Genes in CML: A Role for Cytosolic and Receptor-like PTPs .................. Oncogenic PTPs ...................................................................................................................... Non-coding RNAs in CML ..................................................................................................... Other Tumor Promoting Pathways ......................................................................................... Identifying Cancer Stem Cell in CML .................................................................................... Targeting LSCs ....................................................................................................................... PART II: CLINICS ........................................................................................................................ OPTIONS OF FRONT-LINE MANAGEMENT OF CML ........................................................ Imatinib ................................................................................................................................... Nilotinib .................................................................................................................................. Dasatinib ................................................................................................................................. Bosutinib and Ponatinib .......................................................................................................... THE SEQUENTIAL USE OF TKI IN SECOND OR FURTHER LINES OF TREATMENT BCR-ABL Mutations .............................................................................................................. After Imatinib Failure ............................................................................................................. After Failure of Nilotinib or Dasatinib ................................................................................... Indications for HSCT .............................................................................................................. THE PROGNOSTIC SIGNIFICANCE OF EARLY MOLECULAR RESPONSE ................ Imatinib ................................................................................................................................... Nilotinib .................................................................................................................................. Dasatinib ................................................................................................................................. Bosutinib ................................................................................................................................. Absolute vs Relative Reduction of BCR-ABL1 Levels .........................................................

1 1 2 5 6 7 8 10 12 15 17 17 17 17 17 29 30 31 31 32 34 34 34 36 37 38 38 38 40 41 42 43 43 44 44 45 45 47 48 48 48 49

Therapeutic Strategies Based on EMR ................................................................................... DEEP MOLECULAR RESPONSE AND DISCONTINUATION OF TREATMENT ........... Discontinuation Studies .......................................................................................................... Predictive Factors of DMR ..................................................................................................... The Impact of DMR on Survival ............................................................................................ PREDICTING RESPONSE TO TKI ............................................................................................ Risk Score Systems ................................................................................................................. Age .......................................................................................................................................... Comorbidities .......................................................................................................................... Gender ..................................................................................................................................... Cytogenetic at Diagnosis ........................................................................................................ Transcript Type ....................................................................................................................... Mutations ................................................................................................................................ Predicting Response to 2G TKI After Imatinib Failure .......................................................... New and Alternative Biomarkers of Response to Treatment ................................................. CONCLUDING REMARKS ......................................................................................................... ABBREVIATIONS ......................................................................................................................... CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ............................................................................................................................... CHAPTER 3 ROLE OF IMMUNOMODULATORY DRUGS IN THE TREATMENT OF LYMPHOID AND MYELOID MALIGNANCIES ............................................................................. Ota Fuchs INTRODUCTION .......................................................................................................................... CRL4CRBN as a Part of Ubiquitin-Proteasome System ........................................................ Regulation of CRBN-Associated Proteins in Multiple Myeloma by Lenalidomide .............. Biomarker of Response or Resistance to Lenalidomide and Pomalidomide .......................... Therapy for Multiple Myeloma Patients with Low CRBN Expression .................................. Importance of CRBN for Immunomodulatory Drugs Activity in Lymphoid Malignancies CLINICAL STUDIES IN LYMPHOID MALIGNANCIES ...................................................... Relapsed/ Refractory CLL ...................................................................................................... B-Cell NHL ............................................................................................................................. Multiple Myeloma .................................................................................................................. CLINICAL STUDIES IN MYELOID MALIGNANCIES ......................................................... Myelodysplastic Syndrome ..................................................................................................... Randomized Phase III Placebo-Controlled Study of Lenalidomide in del(5q) Patients ......... Further Clinical Studies of Lower Risk MDS Patients with del(5q) Treated with Lenalidomide .......................................................................................................................... Treatment of del(5q) Patients with Relapse during Lenalidomide Exposure ......................... Therapy with Lenalidomide in Combination with Another Drug in MDS ............................. Mechanisms of Action of Lenalidomide ................................................................................. Role of Cereblon in the Efficacy of Lenalidomide in del(5q) MDS and non-del(5q) MDS Further Prognostic Factors for the Efficacy of Lenalidomide in del(5q) MDS and nondel(5q) MDS ........................................................................................................................... High-Risk MDS and AML ...................................................................................................... CONCLUSION AND PERSPECTIVES ...................................................................................... CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ...............................................................................................................................

49 50 50 51 52 52 52 53 53 54 54 54 55 56 56 57 57 58 58 58 58 73 74 76 78 80 81 81 83 83 83 85 87 87 93 94 95 95 98 101 104 106 108 109 109 109 109

CHAPTER 4 PEDIATRIC HEMATOLOGICAL MALIGNANCIES – CLINICAL MANIFESTATION, TREATMENT AND FOLLOW-UP .................................................................. Naga Ramya Lanka and Krishna Kanth Pulicherla INTRODUCTION .......................................................................................................................... Pediatric Hematological Malignancies ................................................................................... Non-Hodgkin’s Lymphoma .................................................................................................... Etiology of Hematological Malignancies in Children ............................................................ Signs and Symptoms ............................................................................................................... Occular Symptoms .................................................................................................................. Genitourinary Symptoms ........................................................................................................ Staging of Leukemia and Lymphomas ................................................................................... Staging of Leukemia ...................................................................................................... Staging of Lymphoma ............................................................................................................. Treatment Strategies ............................................................................................................... Side Effects of Treatment ....................................................................................................... Advanced Therapies to Treat Hematological Malignancies ................................................... Immunotherapy ....................................................................................................................... Immunotherapy Using Gene Transfer: CAR and TCR ........................................................... Targeted Therapy .................................................................................................................... After Treatment - Follow-up ......................................................................................... Paediatric Cancer Emergencies ............................................................................................... CONCLUSION ............................................................................................................................... ABBREVATIONS .......................................................................................................................... CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ............................................................................................................................... CHAPTER 5 NOVEL THERAPIES AND IMMUNOTHERAPEUTIC APPROACHES TO TREAT CHILDHOOD LEUKEMIA .................................................................................................... Natasha Ali INTRODUCTION .......................................................................................................................... Biology .................................................................................................................................... Literature Search ..................................................................................................................... ACUTE LYMPHOBLASTIC LEUKAEMIA .............................................................................. Nucleoside Analogues ............................................................................................................ Monoclonal Antibodies ........................................................................................................... Bispecific Antibodies and Diabody ........................................................................................ Advantages/Disadvantages of Monoclonal Antibodies ................................................. Proteasome Inhibitor ............................................................................................................... Tyrosine Kinase Inhibitors ...................................................................................................... CAR T Cells ............................................................................................................................ Toxicities of CAR T Cells .............................................................................................. ACUTE MYELOID LEUKAEMIA .............................................................................................. Nucleoside Antimetabolites .................................................................................................... Tyrosine Kinase Inhibitors ...................................................................................................... CAR T Cells ............................................................................................................................ Epigenetic Agents ................................................................................................................... Gemtuzumab Ozogamicin [GO] ............................................................................................. GO as Single Agent ................................................................................................................

138 138 140 142 143 145 147 147 151 151 152 153 158 163 163 164 164 166 168 170 170 172 172 172 172 184 185 186 189 190 191 191 192 192 193 193 194 195 197 197 197 198 199 200 201

GO Combined With Chemotherapy ........................................................................................ GO as Part of Conditioning OR Post Allogeneic HSCT ........................................................ Proteasome Inhibitors ............................................................................................................. STEM CELL TRANSPLANT IN CHILDHOOD ACUTE LEUKAEMIAS ............................ CONCLUSION ............................................................................................................................... ABBREVIATIONS ......................................................................................................................... CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ............................................................................................................................... CHAPTER 6 ERYTHROCYTE TURNOVER AND ERYTHROPOIETIC PATTERNS IN TWO DIFFERENT EXPERIMENTAL MOUSE MODELS OF ANEMIA ................................................ Sreoshi Chatterjee and Rajiv K. Saxena INTRODUCTION .......................................................................................................................... Erythroid Differentiation in Bone Marrow (BM) and Spleen ................................................ Erythrocyte Turnover in Circulation: Double In Vivo Biotinylation (DIB) ........................... INDUCTION OF ANEMIA ........................................................................................................... Cadmium Induced Anemia ..................................................................................................... Mouse Model of Cadmium Induced Anemia ................................................................. Autoimmune Hemolytic Anemia (AIHA) .............................................................................. Induction of Autoimmune Hemolytic Anemia in Mice .................................................. TURNOVER OF CIRCULATING ERYTHROCYTES IN ANEMIC MICE ......................... Double in vivo Biotinylation (DIB) of Erythrocytes in Anemic Mice ................................... Turnover Kinetics of Circulating Erythrocytes ....................................................................... Young vs. Aged Erythrocytes ......................................................................................... Reactive Oxygen Species (ROS) Generation in Circulating Erythrocytes ............................. Phosphatidylserine (PS) Externalization in Circulating Erythrocytes .................................... CD47 Expression in Circulating Erythrocytes ........................................................................ ERYTHROPOIETIC ACTIVITY IN CADMIUM EXPOSED MICE ..................................... Plasma Epo Concentration ...................................................................................................... Erythroid Precursors ............................................................................................................... Membrane bound Autoantibodies on Erythroid Cells in AIHA Mice .................................... ROS Generation in Erythroid Cells in Anemic Mice ............................................................. Apoptotic Activity in Erythroid Cells in Anemic Mice .......................................................... CONCLUDING REMARKS ......................................................................................................... CONCLUSION ............................................................................................................................... CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... DISCLOSURE ................................................................................................................................ ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ...............................................................................................................................

201 201 201 202 204 204 205 205 205 205 220 221 221 223 225 225 225 226 227 228 228 228 228 231 234 236 236 236 237 239 239 240 241 243 245 245 245 245 245

SUBJECT INDEX ................................................................................................................................ 252

i

PREFACE The present third volume of this eBook series: Frontiers in Clinical Drug Research – Hematology comprises six comprehensive chapters covering immune thrombocytopenia, chronic myeloid leukemia, lymphoid and myeloid malignancies, pediatric hematological malignancies, childhood leukemia and erythrocyte turnover and erythropoietic patterns in experimental mouse models of anemia. In chapter 1, Jose Perdomo presents the current understanding of etiology of immune thrombocytopenia for the enhancement and development of existing and new treatments. In chapter 2, Bonifacio and Sorio discuss the recent developments in the treatment of chronic myeloid leukemia treatment based on understanding that BCR-ABL1 oncogene is alone not responsible for the occurrence, progression and maintenance of disease. The emerging role of phosphatases in the pathogenesis of hematologic malignancies as new therapeutic approaches has been discussed. Ota Fuchs in chapter 3, review the role of various immunomodulatory drugs in the treatment of lymphoid and myeloid malignancies. Hematological malignancies arise in blood forming tissue such as in the bone marrow. Diagnosis at the initial stages could lead to the successful treatment and eventually to a decline in mortality rate. It requires a detailed understanding of etiology of disease. In chapters 4 and 5, various aspects of pediatric leukemia has been discussed. Lanka and Pulicherla in chapter 4, focus on the clinical strategies and treatment approaches to combat pediatric hematological malignancies. Natasha Ali in chapter 5 reviews the new therapeutic agents for treating pediatric leukemia including nucleoside analogues, monoclonal antibodies, CAR T cells, tyrosine kinase inhibitors, epigenetic agents and proteasome inhibitors. In the last chapter of this book, Chatterjee and Saxena have compared the erythrocyte turnover and erythropoietic patterns in two different experimental mouse models of anemia. I hope that the pharmaceutical scientists and postgraduate students will find these reviews valuable in order to seek updated and important content for the development of clinical trials and formulating research plans in these fields. I am grateful for the timely efforts made by the editorial personnel of Bentham Science Publishers, especially Mr. Mahmood Alam (Director Publications), Mr. Shehzad Naqvi (Senior Manager Publications) and Dr. Faryal Sami (Assistant Manager).

Prof. Atta-ur-Rahman, FRS Honorary  Life Fellow Kings College University of Cambridge Cambridge UK

ii

List of Contributors Claudio Sorio

Section of General Pathology, Department of Medicine, University of Verona, Italy

José Perdomo

Haematology Research Unit, St George and Sutherland Clinical School, University of New South Wales, Australia

Krishna Kanth Pulicherla

Department of Science and Technology, Ministry of Science and Technology, Govt. of India, Technology Bhavan, New Mehrauli Road, New Delhi-110016, India

Massimiliano Bonifacio

Section of Hematology, Department of Medicine, University of Verona, Italy

Naga Ramya Lanka

Acharya Nagarjuna University, Nagarjuna Nagar, Guntur-522510, India

Natasha Ali

Department of Pathology & Laboratory Medicine/Oncology, Aga Khan University, Karachi, Pakistan

Ota Fuchs

Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 20 Prague 2, Czech Republic

Rajiv K. Saxena

Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India

Sreoshi Chatterjee

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

Frontiers in Clinical Drug Research-Hematology, 2018, Vol. 3, 1-28

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CHAPTER 1

Advances in the Understanding and Treatment of Immune Thrombocytopenia José Perdomo* Haematology Research Unit, St George and Sutherland Clinical School, University of New South Wales, Australia. Abstract: Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by a platelet count of less than 100 x 109 platelets/L. ITP results from two distinct processes: accelerated platelet destruction and reduced platelet production. A distinction of the relative contribution of these pathologies should help guide more targeted treatment decisions. Mechanistically, decreased platelet production is caused by autoantibody-mediated damage to megakaryocytes, while increased clearance of antibody opsonized platelets has traditionally been attributed to the activity of splenic and hepatic macrophages. T cell mediated toxicity has also been described as a contributor to ITP pathogenesis. Recent observations of increased platelet apoptosis and glycoprotein desialylation associated with platelet clearance by hepatocytes provide new avenues for therapeutic intervention. The aim of ITP therapy is to attain sufficient platelet levels to achieve haemostasis. Significant improvements have been obtained with first line therapies such as corticosteroids and intravenous immunoglobulins. For unresponsive patients, second line therapies (splenectomy, rituximab, TPO receptor agonists) have proved beneficial. Nevertheless, the heterogeneous nature of ITP demands further understanding of the causal biological processes to provide personalized and more effective therapies. This chapter presents an account of the current understanding of the biology of ITP and discusses the existing and potential new treatments.

Keywords: Apoptosis, Autoantibodies, Autoimmunity, Desialylation, Immune thrombocytopenia, Immune thrombocytopenic purpura, ITP, IVIg, Megakaryocyte, Platelets, Splenectomy, Thrombopoiesis, TPO receptor agonists. INTRODUCTION Thrombocytopenia (low platelet count) is caused by several conditions of both immune or non-immune nature. The term immune thrombocytopenia (ITP) (previously known as idiopathic thrombocytopenic purpura) refers to an acquired Corresponding author José Perdomo: Haematology Research Unit, St George and Sutherland Clinical School, University of New South Wales, Australia; Tel: +61 2 9113 2004; Fax: +61 2 9113 2058; E-mail: [email protected] *

Atta-ur-Rahman (Ed.) All rights reserved-© 2018 Bentham Science Publishers

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condition of autoimmune origin. Thrombocytopenia due to other causes - such as viral infections, myelosuppressive therapy, bone marrow disorders, Helicobacter pylori infection and drugs - is termed secondary ITP. Unlike secondary ITP, primary ITP is of unknown aetiology. This Chapter will concentrate on primary ITP, will discuss current treatment strategies and will highlight the latest advances in the understanding of ITP biology and the potential for diagnosis and novel therapies. A condition consistent with symptoms of ITP was termed Werlhof’s disease and was described in the 18th century [1]. After the discovery of platelets and their role in haemostasis it was understood that the disorder then known as purpura was due to reduced platelet numbers. The incidence of ITP is up to 3.3 cases per 100, 000 people per annum [2]. Prevalence ranges from 9.5 to 23.6 cases per 100, 000 people [3, 4]. The higher prevalence figure is due to the chronic nature of ITP in many patients. Platelet Biogenesis Platelets are enucleated, discoid cells, 2-3 µm in diameter with a normal range in healthy adults of 150 – 400 x 109 per L of peripheral blood. Platelets are derived from megakaryocytes (MK), the platelet progenitor cells, which in turn are derived from haematopoietic stem cells. A number of specific transcription factors and cytokines drive megakaryocytic differentiation. Absence of the leucine zipper factor NF-E2 results in severe thrombocytopenia [5] while forced expression of NF-E2 boosts MK differentiation and platelet release [6]. NF-E2 enhances the expression of 3β-hydroxysteroid dehydrogenase, which leads to augmented estradiol production within MK. This autocrine hormonal signaling then triggers proplatelet formation (proplatelets are cytoplasmic branched projections from which platelets are released) [7]. Estrogen treatment increases MK population in the human bone marrow [8] and high estrogen doses induce MK differentiation and a corresponding increase in platelet formation in mice [9]. The role of the female hormone in thrombopoiesis is likely to be behind the observation of higher platelet counts in female adolescents [10], which correlates with increased estrogen production levels to maintain sexual characteristics. NF-E2 appears to be critical for the final stages of MK maturation and for proplatelet formation. This is exemplified by the capacity of NF-E2-/- cells to become MK which then fail to produce proplatelets [11]. The zinc finger proteins GATA-1 and its transcriptional co-regulator FOG-1 are essential haematopoietic factors and their absence causes embryonic lethality. Specific disruption of GATA-1 in the megakaryocytic lineage results in accumulation of immature MK and thrombocytopenia, while severe MK defects

Treatment of Immune Thrombocytopenia

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are observed in mice lacking FOG-1. Cells lacking GATA-1 fail to differentiate in vitro and in the presence of thrombopoietin, proliferate indefinitely in an immature state [12]. Importantly, restoration of GATA-1 expression results in terminal maturation into megakaryocytic and erythroid lineages [12]. Embryonic stem cells where GATA-1 was silenced by an inducible shRNA construct also proliferate in vitro in an undifferentiated stage. Upon resumption of endogenous GATA-1 expression these cells undergo differentiation and are capable of producing functional platelets in vivo [13]. The importance of the GATA-1/FOG-1 axis in platelet biology is exemplified by a point mutation in the glycoprotein Ib beta promoter that gives rise to a type of the Bernard-Soulier syndrome [14]. In addition, Ets domain transcription factors, specifically Fli-1, are thought to act together with GATA-1/FOG-1 in MK development. Absence of Fli-1 disrupts MK development and hemizygous expression of Fli-1 has been founds in patients with the Jacobsen or ParisTrousseau Syndrome [15]. During differentiation from a myeloid progenitor, megakaryocytic cells undergo sequential changes that include an initial stage of proliferation followed by differentiation and endomytosis without cell division. This gives rise to large, polyploid (4N, 8N, 16N, 32N, 64N) MK that maintain expression of the amplified genes. This magnified gene expression is likely to contribute to additional protein accumulation within the enlarged cell [16] and to a buildup of platelet-specific proteins. Apart from the transcriptional regulators mentioned above, MK differentiation and platelet release depend on the activity of cytokines in the microenvironment, in particular thrombopoietin (TPO), stem cell factor (SCF), interleukin (IL) 3 and IL-11. TPO is the ligand for the c-Mpl receptor (CD110) and is considered the principal cytokine that stimulates megakaryopoiesis [17]. TPO is required throughout MK development. Binding of TPO to c-Mpl causes receptor dimerization, activation of Jak2 followed by Jak2 self-phosphorylation and phosphorylation of c-Mpl. These phosphorylation steps are the triggers for intracellular signaling through the STAT and PI3K pathways. The consequences of changes in c-Mpl function are illustrated by observation of thrombocytopenia in patients with mutations causing c-Mpl loss or reduced function and thrombocytosis in those with constitutively activating mutations (reviewed by [18]). In mice, absence of TPO or c-Mpl causes a marked decrease (around 90%) in circulating platelet numbers. Of note, mice lacking both TPO and c-Mpl do no show further drop in platelet numbers. Moreover, the remaining platelets in these animals are functional in several assays and mice do no suffer

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from haemorrhagic complications [19]. These observations indicate that to some extent other factors can drive MK differentiation and production of normal platelets. In this sense, the primary role of TPO appears directed towards control of platelet numbers. It is important to note that TPO and c-Mpl are also centrally involved in the maintenance and expansion [20] and in the repair of DNA breaks of adult haematopoietic stem cells [21]. Evidence that other cytokines such as IL-3, IL-6 and IL-11 possessed thrombopoietic function emerged in the 1990s. It was shown that IL-11 alone or together with IL-3 [22] or IL-11 in combination with IL-6 promoted MK differentiation in vitro [23]. However, the hypothesis that these cytokines compensate for the absence of TPO or are responsible for certain level of MK differentiation or platelet production was not supported by in vivo observations. For example, mice deficient in c-Mpl and IL-3 receptor alpha did not show further reduction in platelet or MK numbers [24], indicating that in vivo IL-3 activity did not contribute to MK differentiation. Moreover, mice lacking both c-Mpl and either IL-6 or IL-11 receptor alpha did not show further reduction in platelet counts [25]. Indeed, platelet numbers in mice lacking either IL-6 or IL-11 receptors were no different from wild type [25]. It was later shown that the stimulatory effect of IL-6 observed in inflammatory thrombocytosis is actually mediated by TPO [26]. In vitro, IL-11 has been observed to stimulate MK maturation but, unlike TPO, does not promote proliferation [27]. In cases of cancer patients undergoing myeloablative therapy an inverse correlation between IL-11 levels and platelet counts has been observed [28]. In addition, administration of recombinant IL-11 increases MK and platelet counts in chemotherapy-related thrombocytopenia [29], indicating that in these conditions IL-11 is involved in thrombopoiesis. Altogether, these findings indicate that in normal physiology IL-3, IL-6 and IL-11 do not contribute in a significant manner to MK differentiation and platelet production. Stromal cell-derived factor-1 (SDF1, also known as CXCL12) is involved in MK localization within the bone marrow. Exogenous SDF-1 causes MK re-distribution and increases platelet production. Even though SDF-1 does not affect platelet production in vitro, its activity in the bone marrow indicates that it is important for thrombopoiesis [30]. An unexpected finding is that TPO appears to be dispensable for the final stages of proplatelet formation and platelet release [31]. In fact, there are indications that TPO has an inhibitory effect on proplatelet formation in vitro [32, 33]; however this inhibitory effect may in fact reflect additional maturation (e.g. endomytosis) of MK in the presence of TPO which causes a delay in pro-platelet formation. How this may play out in vivo is yet to be determined.

Treatment of Immune Thrombocytopenia

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Platelet Function The function of platelets as the principal agents of haemostasis has been recognised for a long time. The pioneering work of Bizzozero in the 1880s reached the conclusion ‘La conclusione delle mie ricerche non possa essere altro che questa: che la parte principale nella coagulazione del sangue spetta non ai globuli bianchi ma alle piastrine’ [34] ‘the conclusion of my investigations could not be other than this: that the principal part for blood coagulation resides not in the white cells but in the platelets’ (author’s translation). These observations, together with later findings regarding the role of platelets in the regulation of vascular permeability, cemented the role of platelets as the keepers of vascular integrity. Despite their well-established function in haemostasis, platelets have also critical roles in both innate and acquired immunity, bacterial and viral infections and inflammatory disease. That platelets have additional roles seems clear in retrospect, given that the normal levels of circulating platelets are more than sufficient for what is required for haemostasis. For instance, platelet counts of 100 x 109/L are sufficient to perform surgery [35]. Platelets are involved in acute phase immune response to infection [36]. In this case, their main role may be platelet activation and clotting to ensnare the invading organism and restrict the infection. Platelets express Toll-like receptors (TLR) [37] which are well known for their function in infection. TLRs interact with molecules such as lipopolysaccharide (LPS) from Gram negative bacteria and double stranded RNA. Interactions with LPS cause platelet activation and microparticle (vesicles of 0.1 to 1 µm) release which leads to thrombocytopenia and platelet sequestration in the lungs. Roles for platelets in acquired immunity, regulation of gene expression via micro RNA and regulation of vascular inflammation have also been documented [38]. One relatively recent finding is the active secretion of DNA and other cellular proteins such as histones, myeloperoxidase and elastase by activated neutrophils. In this process known as NETosis, neutrophil extracellular traps (NETs) are formed to entangle invading organisms and constrain their capacity to spread [39]. Leukocyte-platelet interactions have been known for many years and are mediated by P-selectin and its receptor on leukocytes, P-selectin glycoprotein ligand-1 (PSGL-1) [40]. There are also interactions between GPIb on platelets and leukocyte CD11b/CD18 [41]. Neutrophils can also interact with platelets via fibrinogen bound to GPIIb/IIIa [42]. In sepsis, activated platelets interact with neutrophils and induce neutrophil activation and induction of NETs [43]. Since a key role of NETosis is fighting infection, and platelets are central components of

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both NETosis induction and of NETs structure, platelets can also be considered innate immune cells [38]. Epidemiology, Aetiology and Disease Progression Primary ITP is defined as an isolated platelet counts of < 100 x 109/L (reference count 150 – 400 x 109/L) in the absence of other causes or conditions that may cause thrombocytopenia [44]. The presence of bleeding is not a diagnostic criterion for ITP. ITP is a relatively uncommon disease and its annual incidence has been estimated at 3.3 per 100,000 [2] (this assessment is based on European studies and there could be variations elsewhere). The prevalence of ITP has been calculated at 9.5 per 100,000 [4]. ITP is a condition of both children and adults, but paediatric ITP has some unique characteristics and tends to resolve spontaneously [45]. The discussion here refers to adult ITP only. Population studies conducted after 2000 have questioned earlier ideas that ITP affected predominantly young women. The median age for ITP patients is 56, but the frequency of ITP increases with age, with an incidence of more than double for those over 60 [46]. This might be due to changes in the immune system and susceptibility to infections in older patients. For younger patients (those under 60) there is a somewhat higher prevalence of ITP in women (ratio 1.7), but in the older cohort the male/female ratio approaches 1 [46]. There is diversity in presentation, with some patients showing signs of bleeding (petechiae, mucosal bleeding), while other patients have less bleeding tendencies even when presenting with very low platelet numbers [47]. The aetiology of ITP is yet to be fully described, nevertheless the basis for low platelet counts falls within the general categories of increased or accelerated platelet destruction and reduced platelet production. Due to its causative diversity and presentation it is now clear that ITP is not a discreet disease and this heterogeneity has led to some authors proposing the term ITP syndrome [47]. A plethora of triggers has been described for secondary ITP. These include Helicobacter pylori infection, viral infections (HIV, hepatitis C and cytomegalovirus), anti phospholipid syndrome, systemic lupus erythematosus, Evans syndrome, malignancy, some vaccines and transplantation. Several drugs such as quinine, quinidine, rifampicin and vancomycin also cause ITP (for a recent review see Chong et al. [48]). Heparin, which can cause heparininduced thrombocytopenia, is considered a distinct type of reaction and is not included in ITP-like conditions. In addition, there are several non-immune conditions that cause thrombocytopenia that should also be considered, for instance disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, bone marrow disorders, liver disease, congenital thrombocytopenia,

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chemo- and radio- therapy and portal hypertension. Primary ITP, on the other hand, is a diagnosis of exclusion and by definition there is no particular causative agent or specific laboratory tests. It is highly probable that many cases diagnosed as primary ITP are in fact secondary ITP with the cause yet to be identified. Currently there are diverse mechanisms implicated in the development and progression of primary ITP. Platelet Destruction in ITP The demonstration in the 1950s by the well-documented Harrington-Hollingworth experiment [49] demonstrated that a factor in blood was responsible for platelet destruction. In this setting no obvious changes were found in the MK of test subjects after examination of bone marrow aspirates, indicating that the effect was due to platelet destruction in the peripheral blood. These investigators also examined one patient in more detail and showed that the responsible factor was present in the globulin fraction of plasma [49]. Subsequently, it was revealed that these activities were IgG autoantibodies that recognised platelet antigens [50 52]. The nature of these antiplatelet autoantibodies is consistent with expansion of antibody expressing B cell clones, suggesting that the development of plateletspecific antibodies in ITP is driven by persistent stimuli by platelet antigens [53]. Platelet reactive IgA and IgM antibodies are less common but they are also found in ITP patients [54, 55]. An important consideration is the fact that autoantibodies are not detected in all ITP patients. Different proportions (ranging from 20% to almost 80%) have been reported [50 - 52, 55, 56]. The variations observed are likely due to the heterogeneous nature of ITP, the number of patients analysed in different studies and the methodologies used for autoantibody detection. Overall 30-40% of patients have no detectable autoantibodies [57]. Autoantibodies recognise abundant platelet glycoproteins, mainly the fibrinogen receptor, GPIIb/IIIa or the von Willebrand factor receptor, GPIb/IX [58, 59]. Antibodies against other platelet antigens such as GPIV and GPIa/IIa have also been described [54, 55, 60] but appear to be rare. It is likely that only autoantibodies against abundant platelet glycoproteins manifest clinical ITP signs and are therefore found more often in laboratory analyses. Opsonized platelets are destroyed by splenic and hepatic Fcγ baring macrophages. This view is supported by observations of decreased platelet survival when using Indium-111 tropolonate labelled autologous platelets transfused into ITP patients [61]. Since virtually all patients had reduced platelet survival, this would indicate that autoantibodies were present in all patients even if in many cases they remained undetectable. Platelet uptake by macrophages is unlikely to be the only mechanism by which

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autoantibodies trigger platelet consumption. There is increased complement fixation on platelets in the presence of autoantibodies [62]. Accumulation of a membrane attack complex contributes to phagocytosis but it can also lyse platelets directly. The activity of autoantibodies could be determined by the target antigens. Autoantibodies against the GPIb/IX complex may have specific pathogenic actions and are less responsive to some therapeutic interventions [63, 64]. A multicentre study found significantly less response to intravenous immunoglobulin treatment in patients with anti GPIb/IX autoantibodies [65]. There is some evidence that an anti GPIbα autoantibody induced P-selectin and phosphatidylserine externalization on platelets, clustering of GPIbα on lipid rafts and activation of FcγRIIa [66]. It is known that localization of GPIb/IX to lipid rafts is required for interactions with FcγRIIa [67]. On the other hand, platelet activation determined as PAC-1 antibody binding to GPIIb/IIIa or by P-selectin expression was not detected in ITP platelets [68], therefore the role of platelet activation in ITP is yet to be fully established. The involvement of FcγRIIa in anti GPIIb/IIIa autoantibody activity is still to be clarified. Li et al. found that the activity of anti GPIIb/IIIa monoclonal antibodies was decreased by inhibition of FcγRIIa [69]. In these experiments, anti GPIbα antibodies were not affected by FcγRIIa neutralization [69]. Since these antibodies were generated in mice they may not emulate the behaviour of human ITP autoantibodies. It has been proposed that there is a failing of peripheral tolerance that gives rise to ITP [47]. There is increasing evidence that dysregulation of T cells is part of ITP pathogenesis. Several studies have shown decreased number of CD4+ T cells in ITP [70, 71]. Impaired T cell activity can be temporarily restored by immune suppressive treatments such as dexamethasone [72] or with TPO mimetics [73]. Others found no differences in the frequency of T cells in ITP patients relative to controls [74, 75]. Similar frequency of T cells, however, may not reflect their activity and T cells from chronic ITP patients demonstrated reduced immunosupresive activity in vitro [74]. The functional role of Tregs was also shown in a murine model of ITP [76]. Overall, current evidence indicates that compromised Treg activity is part of a pathogenic progression that allows ITP to occur. Recent Developments Anti GPIb/IX antibodies have been shown to induce platelet desialylation (removal of sialic acid moieties from platelet glycoproteins, mainly GPIbα). Desialylated platelets are consumed by hepatocytes through interactions with the Ashwell-Morell receptor (AMR) (also known as asialoglycoprotein receptor or

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ASGPR). There are indications that desialylated platelets may also be targeted by cytotoxic T cells [77]. Platelet desialylation was detected in mice injected with anti mouse GPIbα antibodies and platelet clearance was reduced by inhibition of the AMR receptor or by prevention of desialylation using an enzymatic inhibitor [78]. A strong anti GPIb/IX autoantibody from an ITP patient was found to induce platelet activation and desialylation in vitro [79]. A study of 61 ITP patients found indications of desialylation, especially in those with anti GPIb/IX antibodies [80]. The differences, however, were minor and this has to be corroborated in additional studies, especially in view of reports that there is desialylation in platelets from patients with anti GPIIb/IIIa autoantibodies [77]. In an animal model of ITP using an anti GPIIb antibody, Leytin et al. showed that there was induction of platelet apoptosis as measured by mitochondrial membrane depolarization, PS exposure and caspase 3 activation [81]. Indeed, it had already been demonstrated that ITP platelets possessed apoptotic characteristics [82]. Soon after, seminal work by Mason et al. established that platelet life-span is controlled by apoptosis with critical involvement of pro-apoptotic factors Bak and Bax [83]. This implicates platelet apoptosis as a mechanism that may affect platelet survival in ITP. Recent work found markers of apoptosis in ITP platelets, in particular PS exposure and mitochondrial membrane depolarization [68]. The authors also showed that ITP serum or purified IgG induced mitochondrial membrane depolarization in healthy platelets. Caspase 3 activation could not be demonstrated in in vitro treated platelets [68], and this is consistent with other reports in which no caspase 3 activation was observed in treated MK [55]. Taken together, these studies indicate that platelet life-span reduction in ITP is in part due to increased platelet apoptosis. The role of infection in the pathogenesis of ITP is becoming important and merits consideration. ITP patients are at higher risk of infection, but this may be due to the toxic effects of some therapies such as immunosuppressant drugs or corticosteroids or to decreased platelet numbers. The causative role of H. pylori in many cases of ITP has already been established. In a systematic review of 25 studies, Stasi et al. evaluated the platelet counts of 696 ITP patients after H. pylori eradication and found a complete response of 42.7% [84]. Eradication therapy appears to be more effective in patients with higher platelet counts. For those with