Molecular Hematology

Cover
Title Page
Copyright Page
Dedication Page
Contents
List of contributors
Foreword
Preface
Chapter 1 Beginnings: the molecular pathology of hemoglobin
	Introduction
	Normal structure and function of hemoglobin and the globin genes
		The structure and function of hemoglobin
		The structure of the globin gene loci
		Transcription, processing, and translation of globin RNA
		The regulation of globin gene transcription
		The regulation of developmental globin switching
	Molecular pathology of the globin genes
		The  thalassemias
		Rare mutations causing  thalassemia
		β Thalassemia
		Rare forms of β thalassemia
		δβ Thalassemia and hereditary persistence of fetal hemoglobin (HPFH)
	The pathophysiology and clinical phenotypes of thalassemia
		The α thalassemias
		The β thalassemias
		Development of therapies
	Conclusion
	Further reading
		General background
		Hemoglobin genetics and structural variants
		Hemoglobin switching
		The β thalassemias
		The α thalassemias
		Evolutionary background of hemoglobin disorders
		Molecular basis of prevention and management of hemoglobin disorders
Chapter 2 Stem cells
	Introduction
	Stem cell definitions and distinctions
	Hematopoietic stem cell concepts and their origin
		The cellular compartment model
		Models of lineage commitment
	Molecular regulation of hematopoiesis
		Cell-intrinsic regulators of hematopoiesis
		Cell-extrinsic regulators
		Components of the hematopoietic microenvironmental niche
	Trafficking of primitive hematopoietic cells
		Hematopoietic ontogeny
		Homing and engraftment of HSPCs following infusion
		Egress of HSCs from bone marrow under physiological conditions
	Manipulating hematopoietic stem cells for clinical use
		Mobilization of HSCs
		Isolating stem cells for manipulation
		HSC from induced pluripotent stem cells
		Functional analysis of HSCs
	Summary
	Further reading
		Regulation of hematopoiesis
		Hematopoietic stem cell niche
		Hematopoietic stem cell quiescence
		Isolation of hematopoietic stem cells
		Hematopoietic stem/progenitor cell expansion
		Hematopoietic stem cell engraftment
Chapter 3 The genetics of acute myeloid leukemia
	Introduction
	AML with recurrent cytogenetic abnormalities
		Core-binding factor leukemias
		Acute promyelocytic leukemia
		KMT2A gene rearrangements (11q23)
		Other rare chromosomal translocations
		Numerical chromosomal abnormalities
	Molecular genetic aberrations not detectable by conventional cytogenetics
		Driver gene mutations
		NPM1
	Activated signaling
		FLT3
		c-KIT
	Myeloid transcription factors
		RUNX1
		CEBPA
	Tumor suppressors
		TP53
		WT1
		BCOR
	Spliceosome genes
	Cohesin complex gene
	DNA methylation
		DNMT3A
		IDH1 and IDH2
		EZH2
		TET2
	Chromatin modifiers
		ASXL1
		KTM2A (formerly MLL)
	Germline predisposition
	AML therapies and MRD monitoring targeted by genetics
	Summary
	Further reading
		Genetics of AML
		Core-binding factor leukemias
		NPM1
		Activated signaling
		Myeloid transcription factors (RUNX1, CEBPA)
		Tumor suppressors (BCOR, TP53)
		Spliceosome genes
		Cohesin complex genes
		DNA methylation (EHZ2, TET2, IDH1/IDH2, DNMT3A)
		Chromatin modifiers
		Germline predisposition
		AML therapies and MRD monitoring targeted by genetics
Chapter 4 Molecular diagnostics and risk assessment in myeloid malignancies
	Introduction
		A disease continuum
		General methodology of cytogenetic analysis in hematologic malignancies
	Methods of detection
		Genetic and clinical consequences of chromosomal translocation
	Karyotypic evolution
	Clinical implications
	Treatment selection
		Recurrent cytogenetic abnormalities in MDS
		Recurrent cytogenetic aberrations in AML
	Therapy-related chromosomal aberrations in AML post cytotoxic therapy
		Recurrently mutated genes in MDS
	Mutations in splicing factors
	Epigenetic regulators
	Histone modification
	DNA methylation
	Cohesin complex
	Transcription factors
	Cell signaling genes
		TP53
		Recurrently mutated genes in AML
		Differences and similarities of the WHO classification 2022 and the ICC 2022
	References
	Further reading
Chapter 5 Molecular basis of acute lymphoblastic leukemia
	Introduction
	B-cell precursor acute lymphoblastic leukemia
	High hyperdiploid ALL
	Hypodiploid ALL
	Intrachromosomal amplification of chromosome 21 (iAMP21)
	ETV6::RUNX1 and ETV6::RUNX1-like ALL
	TCF3-rearranged ALL (TCF3::PBX1 and TCF3::HLF)
	KMT2A-rearranged ALL
	BCR::ABL1 (Ph+) and Ph-like ALL
	DUX4-rearranged ALL
	MEF2D-rearranged ALL
	ZNF384-rearranged leukemia
	PAX5-driven ALL (PAX5alt and PAX5 P80R)
	CDX2/UBTF ALL
	NUTM1-rearranged ALL
	IKZF1 N159Y
	Other subtypes of B-ALL
	T-cell precursor acute lymphoblastic leukemia (T-ALL)
		Early T-cell precursor (ETP) ALL, immature T-ALL, and BCL11B-activated leukemia
	TLX1 and TLX3-driven T-ALL
	TAL1- and class II basic helix-loop-helix (bHLH) factor-deregulated T-ALL
	Other subtypes of T-ALL
	Summary
	Further reading
Chapter 6 Chronic myelogenous leukemia
	Introduction
	Epidemiology
	Clinical presentation and natural history
	Prognostic models
	Molecular biology
		BCR::ABL1 oncogene
		BCR::ABL1 kinase signaling pathways
	Therapy
		General
		Frontline CML therapy
		Selection of frontline TKI therapy
		Discontinuation of TKI therapy and TFR
		Which treatment endpoints/milestones are clinically relevant in CML?
		Monitoring response and resistance
		Distinguishing CML failure due to toxicities/intolerance versus true resistance
		Management of TKI “Failure” due to side effects/intolerance
		Management of CML post-TKI resistance (true CML failure)
		Other therapies
		Pregnancy and CML
	Allogeneic hematopoietic stem cell transplantation
	Therapy of CML-AP and CML-BP
	Philadelphia chromosome-negative CML
	Summary
	Further reading
		Molecular biology of CML
		Prognostic factors and models
		Interferon-α
		Allogeneic hematopoietic stem cell transplantation
		Imatinib and other tyrosine kinase inhibitors
		Mechanisms of resistance to tyrosine kinase inhibitors
Chapter 7 Myeloproliferative neoplasms
	Introduction
	JAK–STAT pathways with a focus on JAK2
	The JAK2 V617F mutation in Philadelphia-negative MPNs
	JAK2 V617F: One mutation yet multiple phenotypes
	Other JAK2 mutations
		JAK2 exon12 mutations
		JAK2 variants and eosinophilia
		Germline JAK2 mutations
		JAK2 germline haplotype
	MPL mutations in ET and MF
	CALR mutations in ET and MF
	Other somatic mutations in ET, MF, and PV
		Reticulin deposition in MPN
	Chronic neutrophilic leukemia
		Chronic eosinophilic leukemia and other eosinophilic disorders
	Integration of molecular information into diagnostic algorithms and prognostication
	Conclusions
	Further reading
		Introduction
		The JAK2 V617F mutation in Philadelphia- negative MPNs
		JAK2 exon12 mutations
		JAK2 variants and eosinophilia
		Germline JAK2 mutations
		JAK2 germline haplotype
		MPL mutations in ET and MF
		CALR mutations in ET and MF
		Other somatic mutations in ET, ET, and ET
		Chronic neutrophilic leukemia
		Chronic eosinophilic leukemia and other eosinophilic disorders
		Integration of molecular information into diagnostic algorithms and prognostication
Chapter 8 Lymphoma genetics
	Introduction
	Techniques
	Burkitt lymphoma
	Diffuse large B-cell lymphoma, not otherwise specified
	Mantle cell lymphoma
	Follicular lymphoma
	Lymphoplasmacytic lymphoma
	Marginal zone lymphoma
	Chronic lymphocytic leukemia/small lymphocytic lymphoma
	Anaplastic large cell lymphoma
	Nodal T-follicular helper (TFH) cell lymphoma
	Peripheral T-cell lymphoma, not otherwise specified
	Conclusions
	Further reading
		Introduction
		Techniques
		Burkitt lymphoma
		Diffuse large B-celllymphoma
		Mantle cell lymphoma
		Follicular lymphoma
		Lymphoplasmacytic lymphoma
		Marginal zone lymphomas
		CLL/SLL
		Anaplastic large cell lymphoma
		Nodal T-follicularhelper (TFH) cell lymphoma
		Peripheral T-celllymphoma, not otherwise specified
Chapter 9 Molecular basis of chronic lymphocytic leukemia
	Introduction and definition
	Epidemiology
	Genetic factors
	Environmental factors
	Molecular biology of CLL
		IGHV mutations status
		Chromosome abnormalities
		Somatic mutations
		MicroRNA changes
		Epigenetic factors
	B-cell receptor signaling pathway
	BCL2 family
	CLL microenvironment
	Impaired immunity in CLL
	Richter transformation
		Definition
		Morphology and immunophenotype
		Clonal relatedness between CLL and lymphoma
		Biology of Richter’s DLBCL
	Conclusion
	Further reading
Chapter 10 The molecular biology of multiple myeloma
	Introduction
	Normal plasma cell development
	Development of myeloma cell
		Hyperdiploidy/trisomies
		IgH translocations
		Cyclin D dysregulation
	Progression to multiple myeloma
		Del (13q)
		RAS abnormalities
		MYC signaling
		Aberrant NF-B signaling
		Deletion of 17p [del(17p)] including TP53
		Chromosome 1q gain, amplification
		Del (1p32)
		Bone marrow microenvironment
	Classification and risk stratification of myeloma
	Conclusion
	Further reading
Chapter 11 The molecular basis of bone marrow failure syndromes and red cell enzymopathies
	Introduction
	Conditions associated with bone marrow failure
	Acquired bone marrow failure syndromes
		Aplastic anemia (AA)
		Paroxysmal nocturnal hemoglobinuria
	Inherited GPI deficiency
	Inherited bone marrow failure syndromes
		Fanconi anemia
	Dyskeratosis congenita and the telomere biology disorders (TBD)
	Diamond–Blackfan anemia
	Red cell enzyme deficiencies
		Pyruvate kinase deficiency
		Glucose 6-phosphate dehydrogenase deficiency
		Pyrimidine 5-nucleotidase deficiency
	Concluding remarks
	Further reading
		Bone marrow failure syndromes
		Paroxysmal nocturnal hemoglobinuria
		Fanconi anemia
		Dyskeratosis congenita
		Diamond–Blackfan anaemia
		Red cell enzyme deficiencies
Chapter 12 Anemia of chronic disease
	Introduction and definition
	General principles
	Diagnosis
		Iron parameters
		Serum ferritin
		Bone marrow studies
	Pathogenesis
		Hypoferremia
	Principles of treatment
	Future treatments
	Further reading
		General
		Diagnosis
		Mechanisms of hypoferremia
		Hepcidin and anemia
		Clinical studies
Chapter 13 Molecular basis of iron metabolism
	Introduction
	Mechanisms of iron transport
		General principles
		Intestinal iron transport
		Other iron uptake mechanisms
	Sites of iron storage
	Regulation of iron homeostasis
	Iron disorders
		Iron-deficiency disorders
		Iron overload disorders
		HFE-related hemochromatosis
		Non-HFE-related hemochromatosis
		Other forms of hemochromatosis
		Ferroportin disease
		African siderosis
		Abnormal iron distribution
		Atransferrinemia
		Aceruloplasminemia
	Conclusions
	Further reading
		General
		Regulation of iron homeostasis
		Iron overload disorders
		Abnormal iron distribution
Chapter 14 Perspectives in genomics and sickle cell disease therapeutics
	Introduction
	Epidemiology of sickle cell disease
	Pathophysiology
	Mortality and life expectancy of SCD
	Management of SCD
		Current known genetic determinants of outcomes in sickle cell disease
	Exploring the missing heritability of fetal hemoglobin in Africa to uncover therapeutic targets
	Investigating the prospect of RNA therapy for SCD
	Developing genetic risk models for SCD complications
	Conclusion and perspectives
	Further reading
Chapter 15 Molecular coagulation and thrombophilia
	Introduction
	Blood coagulation
	Regulation of blood coagulation
		Protein C anticoagulant system
	Molecular genetics of venous thromboembolism
		Factor V gene mutations causing APC resistance
		Deficiency of antithrombin
		Protein C deficiency
		Protein S deficiency
		Prothrombin gain-of-function mutations
		Other genetic susceptibilities for VTE
	Severe thrombophilia is a multigenic disease
	Management of thrombophilia
	Conclusions
	Further reading
		Blood coagulation: introduction and regulation
		Molecular genetics of venous thromboembolism, APC resistance, and FV Leiden
		Antithrombin deficiency
		Protein C system and protein C deficiency
		Protein S and protein S deficiency
		Prothrombin gene mutations
		VTE and genome-wideassociation studies
		Management of thrombophilia
Chapter 16 The molecular basis of hemophilia
	Introduction: clinical features of hemophilia
	Inheritance of hemophilia
	Molecular basis of hemophilia A
	Molecular basis of hemophilia B
	Inhibitor formation: etiology and clinical implications
	Therapeutic applications of molecular biology to patient care
		Carrier testing
		Antenatal diagnosis of hemophilia
		Recombinant factor concentrates
		Non-factor therapies
		Gene therapy for hemophilia (see also Chapter 25)
	Conclusions
		Hemophilia resources on the internet
	Further reading
		Introduction
		Hemophilia A
		Hemophilia B
		Inhibitors
		Carrier testing and antenatal diagnosis
		Hemophilia therapeutics – Current and future
Chapter 17 The molecular basis of von Willebrand disease
	Introduction
	Function of von Willebrand factor in primary hemostasis
	Function of von Willebrand factor in intrinsic coagulation
	Gene organization, synthesis, and multimeric structure of von Willebrand factor
	von Willebrand disease and its classification
	Genetic defects in von Willebrand disease
		Type 1 VWD
		Type 2 VWD
		Type 3 VWD
	Treatment of von Willebrand disease
		Desmopressin
		Transfusional therapies
	New therapies for von Willebrand disease
		von Willebrand disease resources on the internet
	Further reading
		General
		von Willebrand factor
		von Willebrand disease and its classification
		Genetic defects in von Willebrand disease
		Treatment of von Willebrand disease
Chapter 18 Platelet disorders
	Normal platelet function
	Diagnosis of platelet defects in bleeding disorders
	Platelet adhesion disorders
		Bernard–Soulier syndrome
		GPV
		GPIb and  monoallelic mutations leading to thrombocytopenia and large platelets
		Platelet-type von Willebrand disease
	Collagen receptor defects
		α2β1 integrin
		GPVI
	Platelet aggregation defects
		Glanzmann thrombasthenia
	Agonist receptor defects
		ADP and ATP receptor defects
		Other primary agonist receptor defects
	Defects in intracellular signaling pathways
	Platelet secretion defects (storage pool disease)
		Defects of α-granules
		Gray platelet syndrome
		Quebec platelet disorder
	Dense (δ) granule defects
		Hermansky–Pudlak syndrome
		Chédiak–Higashi syndrome
		Griscelli syndrome
		Wiskott–Aldrich syndrome
	Procoagulant regulation defects
		Scott syndrome
		Stormorken syndrome
	Giant platelet syndromes and cytoskeletal defects
		Medich giant platelet syndrome
	Transcription factor defects
		Glycosylation defects
	Platelet protein polymorphisms and tendency to thrombosis
	Further reading
		Overview of platelet function
		Laboratory assessment of platelet disorders
		Overview of inherited platelet disorders
		“Next-generation”sequencing methods for molecular diagnosis of bleeding disorders of platelet origin
		Bernard–Soulier syndrome overview
		New GPIbα mutations
		New GPIbβ mutations
		Velocardiofacial syndrome
		New GPIX mutations
		Variant forms
		Platelet-typevon Willebrand disease
		Collagen receptor defects α2β1 integrin
		GPVI
		Glanzmann thrombasthenia overview
		αIIb defects
		β3 defects
		Kindlins and leukocyte adhesion defect
		ADP and ATP receptor defects
		Other primary agonist receptor defects
		Intracellular signaling pathway defects
		Platelet secretion defects (storage pool disease) Gray platelet syndrome
		Quebec platelet disorder
		Hermansky–Pudlak syndrome
		Chédiak–Higashi syndrome
		Griscelli syndrome
		Wiskott–Aldrich syndrome
		Procoagulant regulation defects
		Scott syndrome
		Stormorken syndrome
		Giant platelet syndromes and cytoskeletal defects
		Glycosylation defects
		Transcription factor defects
		Platelet protein polymorphisms and tendency to thrombosis
Chapter 19 The molecular basis of blood cell alloantigens
	Introduction
	The major histocompatibility complex
		HLA antigens
		HLA class I genes
		HLA class II genes
		Function
		Identification of HLA gene polymorphism
		Molecular typing techniques
		PCR-SSP
		Reverse PCR-SSOP
		Sequencing-based typing
		Next generation sequencing
		Long-read sequencing/third-generation sequencing
		DNA microarray genotyping
		The molecular basis of platelet-specific antigens or HPA
		HPA allele frequencies
		Molecular detection methods
		HPA alloimmunization and clinical implications
	Post-transfusion purpura
	Platelet refractoriness
	Neonatal alloimmune thrombocytopenia
		Incidence of and HPA immunogenicity
		Characteristics
		Treatment
		Counseling
		HNA antigen systems
		HNA allele frequency
		HNA detection methods
		Clinical significance of HNA
	Neonatal alloimmune neutropenia
	Transfusion-related acute lung injury
		HNA and allograft rejection
	Red blood cell antigens and molecular typing
	Hemolytic disease of the fetus and newborn
		Rh system
		RH genes
		Immunogenicity of RhD and prevention of immunization
		Kell
		Immunogenicity of K and the mechanism of fetal anemia
		Monitoring, treatment, and prevention of HDFN
	Conclusions
	Further reading
Chapter 20 Functions of blood group antigens
	Introduction
	Anion-exchanger protein 1 (band 3 protein)
		Transmembrane domain
		C-terminal cytoplasmic domain
		N-terminal cytoplasmic domain
		Nitric oxide export
		Deficiencies and mutations of AE1
	Rh proteins
	Glycophorins C and D
	Blood group antigen proteins that function as adhesion molecules
	Blood group antigen proteins with other functions
		Duffy blood group system
		Kidd blood group system
	Summary
	Further reading
		AE1
		Rh
		Glycophorins C and D
		Adhesion proteins
		Other blood group antigen proteins
Chapter 21 Autoimmune hematological disorders
	Introduction
	The immune system
		The innate immune system
		The adaptive immune system
		B- and T-cells possess antigen receptors on their surface
		The major histocompatibility complex
		Soluble molecules: cytokines orchestrate the immune response
		Cytokine profiles of Th1 and Th2 responses (Table 21.1)
		T-cells (Figure 21.2)
		Immunological tolerance prevents damage to self-antigens
	The spectrum of autoimmune diseases
	Role of genetic factors
		Some autoimmune diseases have strong association with cytokine gene polymorphisms
		Antibody Fc receptor gene polymorphisms
	Mouse models of autoimmune disease
	Human studies
		Critical events in the generation of autoreactivity
		Autoimmune disease is complex and multifactorial
		How do autoreactive cells induce tissue damage in autoimmune disease?
	Immune thrombocytopenia as a hematological model of autoimmune disease
		Etiology
		Clinical features
		Diagnosis
		Standard treatment
	Targeted versus untargeted therapies for autoimmune disease
		Campath-1H
		Anti-CD20 monoclonal antibody therapy
		Costimulatory blockade
		Other options: Helicobacter pylori eradication
		Thrombopoietin receptor agonists
	Novel therapies for the treatment of ITP
	Conclusions
	Further reading
		General
		Immune system and HLA
		Immune tolerance
		Autoimmunity
		Role of genetic and other factors in autoimmune disease
		Immune thrombocytopenia (ITP)
		Therapeutic advances
Chapter 22 Molecular therapeutics in hematology: gene therapy
	Introduction
	General comments on gene transfer/therapy
	Viral vectors for gene transfer
	Oncoretroviral gene transfer
	Lentiviral gene transfer
	Adenoviral gene transfer
	Adeno-associated virus gene transfer
	Herpes simplex virus gene transfer
	Genetic immunotherapy
	Methods to improve gene therapy safety and suicide gene therapy
	Conclusions
	Further reading
		Introduction
		Viral vectors for gene transfer
		Oncoretroviral gene transfer
		Lentiviral gene transfer
		Adenoviral gene transfer
		Adeno-associatedvirus gene transfer
		Genetic immunotherapy
		Methods to improve gene therapy safety, and suicide gene therapy
Chapter 23 Pharmacogenomics
	Introduction
	Principles of pharmacogenomics
		Variation in the human genome
		Approaches for establishing pharmacogenomic models
	Pharmacogenomics to improve childhood ALL therapy
		Rasburicase and G6PD deficiency
		Optimization of thiopurine therapy
		Relapsed ALL and drug resistance, thiopurine resistance and variants in NT5C2 and PRPS1
		Optimization of therapy in children with TCF3-HLF ALL
	Pharmacogenomics to optimize oral antithrombotic therapy
		Coumarins and variants in CYP 2C9 and VKORC1
		Clopidogrel and variants in CYP2C19
	Summary and challenges for the future
	Further reading
		Principles of pharmacogenomics
		Pharmacogenomics to improve childhood acute lymphoblastic leukemia therapy
		Pharmacogenomics to improve oral antithrombotic therapies
Chapter 24 Cancer stem cells
	The cancer stem cell concept
	The cell of origin
	Pre-leukemic stem cells
	CSC-targeted therapies
		Targeting cell surface antigens
		Targeting CSC self-renewal
		Targeting CSC survival
		Targeting LSC dormancy
		Targeting the CSC niche
		Targeting epigenetic modifiers
		Other CSC-targeted therapies
	Conclusion
	Further reading
		The cancer stem cell concept
		The cell of origin
		Pre-leukemic stem cells
		Targeting cell surface antigens
		Targeting CSC self-renewal
		Targeting CSC survival
		Targeting LSC dormancy
		Targeting the CSC niche
		Targeting epigenetic modifiers
		Other CSC-targetedtherapies
		Conclusion
Chapter 25 CAR-T cell therapy
	History of CAR-T cell therapy
	CAR structure and function
		Extracellular scFv and linker
		Hinge domain
		Transmembrane domain
		Intracellular co-stimulatory domains
	CAR-T cell manufacturing
		Cell isolation method
		Construct integration method
		Expansion method
		Other considerations
	CAR-T disease-specific clinical data
		Acute lymphoblastic leukemia
		B-cell lymphomas
		Chronic lymphocytic leukemia
		Multiple myeloma
	Sources of CAR-T resistance
		CAR-T-cell exhaustion
		Epitope shedding
	Toxicities of CAR-T therapy
		Cytokine release syndrome
		Immune effector cell-associated neurotoxicity syndrome
		B-cell aplasia, hypogammaglobulinemia, and infections
		Cytopenias
		Coagulopathy
	CAR-T cells in the tumor microenvironment
		Tumor microenvironment
		Cytokine signaling in the TME
		TME structure
		Therapeutic approaches to mitigate the effects of the TME
	References
Chapter 26 Molecular basis of transplantation
	Introduction and definition
	Principles and clinical indications of HCT
	Exploiting HCT for resetting the immune system
	Basic concepts in the immunology of allogeneic HCT
		The major histocompatibility complex
		Mechanisms of allorecognition: lessons from solid organ transplantation
		Transplantation tolerance: clonal deletion
		Transplantation tolerance: peripheral regulation
	Graft-versus-host and graft-versus-leukemia
		The immunological targets of GvHD and GvL
		Graft-versus-leukemia
		Are tumor-specific T-cells generated after allografting?
		Graft-versus-host disease
	The pathogenesis of GvHD
		Acute GvHD and the “cytokine storm”
		Acute GvHD and T-cell trafficking
		Chronic GvHD
	Can GvHD and GvL be dissected?
		Prevention and treatment of GvHD
		Induction of selective GvL
	New directions
	Conclusions
	Further readings
		Clinical use of HCT
		Transplantation tolerance
		GvHD and GvL
Index
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