Hematopathology (eBook)

Preface 5
Contents 7
Contributors 8
1 Molecular Techniques in Hematopathology 10
Introduction 10
Part I: Specimen Collection and Processing 11
Standard Precautions and Safety 11
Patient Identification and Labeling 11
Cell Enrichment and Selection Techniques 12
Source-Specific Requirements to Ensure Nucleic Acid Integrity 13
Bone Marrow Aspirates, Whole Blood, and Body Fluids 13
Dried Blood Spots (Guthrie Cards) 14
Fresh Tissue 14
Fixed, Paraffin-Embedded Tissue 14
Part II: Nucleic Acid Extraction, Purification, and Storage 15
Overview 15
Extraction Techniques 16
Organic (Phenol--Chloroform) 16
Inorganic (Chaotropic Salt--Silica Column) 17
Stability of DNA in Storage 17
Stability of RNA in Storage 17
Considerations for Long-Term Storage 18
Part III: Assessment of Nucleic Acid Quality and Quantity 18
Part IV: Selected Techniques 19
Electrophoresis 19
Restriction Enzymes 22
DNA Sequencing 23
Sanger Sequencing 23
Pyrosequencing 25
Next-Generation Sequencing (NGS) 26
Polymerase Chain Reaction 27
Reverse Transcription PCR (RT-PCR) 29
Allele-Specific PCR 29
Nested PCR 30
Real-Time PCR 30
Signal Detection Options 31
Quantitative Real-Time PCR 32
Methylation-Specific PCR 33
Restriction Site PCR 34
Asymmetric PCR 34
Clonality Assessment 34
Overview 34
Techniques 37
Limitations 39
References 40
2 Classical and Molecular Cytogenetic Analysis of Hematolymphoid Disorders 48
Cytogenetic Analysis in the Diagnosis of Hematolymphoid Disorders 49
Classical Cytogenetic Analysis of Bone Marrow and Leukemic Blood 50
Historical Perspectives 51
Specimen Collection and Storage 52
Specimen Processing and Tissue Culture 53
Culture Harvesting, Slide Preparation, and Staining 55
Guidelines for Microscopic Analysis of Bone Marrow and Leukemic Blood 55
The Karyotype and Cytogenetic Nomenclature 56
The Molecular Mechanisms Responsible for Chromosomal Rearrangements in Neoplasia 57
Molecular Cytogenetic (FISH) Analysis of Bone Marrow and Leukemic Blood 59
Basic Principles of Fluorescence In Situ Hybridization 59
Clinical Indications for FISH Testing in Hematolymphoid Disorders 60
Types of FISH Probes Routinely Used in Hematological Disorders 61
Advantages and Disadvantages of FISH 63
Diagnostic and Prognostic Cytogenetic Markers in Myeloid Disorders 64
Myeloproliferative Neoplasms (MPN) 64
Myelodysplastic Syndromes (MDS) 68
Acute Myeloid Leukemia 68
Diagnostic and Prognostic Cytogenetic Markers in Lymphoid Disorders 72
B-Lymphoblastic Leukemia/Lymphoma with Recurrent Genetic Abnormalities 72
T-Lymphoblastic Leukemia/Lymphoma 75
Chronic Lymphocytic Leukemia/Small Lymphocytic Leukemia 76
Plasma Cell Myeloma 76
Non-Hodgkin Lymphoma 79
Hodgkin Lymphoma 83
Array-Based Genomic Profiling of Hematolymphoid Disorders 83
References 84
3 Using Cytogenetic and Molecular Tests in Diagnostic Workups with the WHO Classification 2008 88
Diagnostic Workups 89
Chronic Myeloproliferative Disorders 91
Myelodysplastic Disorders 94
Acute Leukemias 95
Acute Leukemias with No Specific Cytogenetic Findings 98
Malignant Lymphomas 99
Some Diagnostic Problems 100
Follicular Lymphoma (FL) 101
Mantle Cell Lymphoma (MCL) 101
Marginal Zone B-Cell Lymphoma (MZL) 102
Diffuse Large B-Cell Lymphoma (DLBCL) 102
Burkitt Lymphoma (BL) 103
Plasma Cell Neoplasms (PCN) 103
T-Cell Lymphomas 104
Anaplastic Large-Cell Lymphomas (ALCL) 104
NK-Cell Lymphomas 105
Tests for Viruses Are Useful in Diagnosis of Hematolymphoid Neoplasms 106
Hodgkin Lymphoma 106
Other Hematolymphoid Neoplasms 107
Summary 107
References 108
4 Update on the Molecular Pathology of Precursor Lymphoid Leukemias 112
Introduction 112
WHO Classification of Precursor Lymphoid Malignancies 113
Overview of Cytogenetic and Molecular Lesions in Precursor Lymphoblastic Malignancies 114
B-lymphoblastic Leukemia with t(9 22)(q34
Down Syndrome-Associated ALL 119
Genetic Factors Predisposing to Precursor Lymphoblastic Leukemia/Lymphoma 122
Cancer Stem Cells in Precursor B-ALL: Definitions and Controversies 122
Altered NOTCH Signaling in T-ALL 126
Role of PTEN and PI3K in Pathogenesis of T-ALL 128
Integration of Whole-Genome Analyses with Other Analytic Approaches to Identify Novel ALL Subtypes 129
Conclusion 129
References 130
5 Molecular Pathology of Acute Myeloid Leukemias 136
Introduction 137
Acute Myeloid Leukemia 138
AML with Recurrent Genetic Abnormalities 139
AML with Balanced Translocations/Inversions 139
Core Binding Factor AML 142
Other Recurrent Translocations 146
Rare Subtypes of AML 148
AML with Gene Mutations 148
Other Gene Mutations in AML with Prognostic Significance 152
Gene Expression and Prognosis in NK-AML 154
Mutations and Translocations Associated with Other Myeloid Neoplasms 155
Future Directions 156
References 156
6 Molecular Pathology of Mature B-Cell and T-Cell Lymphomas 165
Introduction 166
Molecular Testing for B-Cell Non-Hodgkin Lymphoma (B-NHL) 166
B-Cell Biology and Maturation 166
B-Cell Clonality Testing 171
Somatic Hypermutation Testing 174
Follicular Lymphoma 174
Follicular Lymphoma and BCL2 t(14 18)
Other Genetic Abnormalities in Follicular Lymphoma 176
Mantle Cell Lymphoma 177
Detection of Cyclin D1 Dysregulation 178
Other Genetic Abnormalities in MCL 178
Clinical Implications 179
Diffuse Large B-Cell Lymphoma 179
BCL6 Alterations in DLBCL 180
Other Genetic Alterations in DLBCL 182
Marginal-Zone Lymphomas 183
Genetic Abnormalities in MALT Lymphomas 184
Detection of MALT Lymphoma Translocations 185
Clinical Implications 185
Other Marginal-Zone Lymphomas 185
Burkitt Lymphoma 186
Burkitt Lymphoma and MYC 186
Other Genetic Abnormalities in Burkitt Lymphoma 188
Lymphoplasmacytic Lymphoma 189
Molecular Testing for T-Cell Non-Hodgkin lymphoma (T-NHL) 190
T-Cell Biology and Maturation 190
T-Cell Clonality Testing 192
Anaplastic Large-Cell Lymphoma (ALCL) 194
Detection of ALK Dysregulation 197
Other Genetic Abnormalities in ALCL 198
Angioimmunoblastic T-Cell Lymphoma (AITL) 199
Genetic Abnormalities in AITL 199
Mycosis Fungoides (MF) 200
Molecular Staging of Mycosis Fungoides (MF) 201
Genetic Abnormalities in Mycosis Fungoides 201
Sézary Syndrome (SS) 203
Genetic Abnormalities in Sézary Syndrome 203
Hepatosplenic T-Cell lymphoma (HSTL) 204
Enteropathy-Associated T-Cell Lymphoma (EATL) 205
Extranodal Natural Killer-/T-Cell Lymphoma 205
Peripheral T-Cell Lymphoma, Not Otherwise Specified (PTCL-NOS) 206
Hodgkin Lymphoma 207
Nodular Lymphocyte-Predominant Hodgkin Lymphoma (NLPHL) 208
Classical Hodgkin lymphoma (CHL) 208
References 209
7 Molecular Pathology of Myeloproliferative Neoplasms 223
Introduction 224
Chronic Myelogenous Leukemia 225
Historical Perspective and Current Relevance 225
Epidemiology, Clinical, and Laboratory Features 227
The Structure and Pathogenesis of BCR--ABL1 228
Diagnostic Testing 230
Disease Monitoring/Response to Therapy 231
Quantitative RT-PCR in Disease Monitoring -- Optimization, Control Genes, and Reporting 234
Quantitative RT-PCR in Disease Monitoring -- Interlaboratory Standardization Efforts 236
Resistance to Tyrosine Kinase Inhibitor Therapy 237
Summary: Integration of Molecular Diagnostic Testing into CML Management 239
BCRABL1 -Negative Classic Myeloproliferative Neoplasms 240
Epidemiology, Clinical, and Laboratory Features 240
JAK2 V617F's Contribution to Diagnosis of MPNs 241
Other JAK2 Mutations in PV 242
Effect of JAK2 Allelic Burden 243
JAK2 Mutation Detection Methods 244
JAK2 -- Unresolved Issues 245
MPL 246
PDGFR and FGFR1 Abnormalities 246
Spectrum of Eosinophilia-Related Disorders 247
PDGFRA 248
PDGFRB 249
FGFR1 251
Mast Cell Disease 252
References 254
8 Molecular Pathology of Chronic Lymphocytic Leukemia 262
Introduction 262
Cytogenetic Abnormalities 263
Deletion 13q14 264
Deletion 11q22-q23 266
Trisomy 12 267
Deletion 17p13 267
Deletion 6q 268
Trisomy 3q27 268
8q24 gain 269
t(14 19)(q32
Chromosomal Translocations Involving BCL2 270
t(2 14)(p13
Translocations Involving Chromosome 14q32 273
Epigenetic Changes 273
High-Throughput Molecular Methods to Assess CLL 273
Sequence and Mutation Analysis 273
Array Comparative Genomic Hybridization 274
Single Nucleotide Polymorphisms Arrays 275
Multiplex Ligation-Dependent Probe Amplification 276
Gene Expression Profiling 276
MicroRNA 276
B-Cell Receptor 277
Somatic Mutations of Ig Variable Region Genes 278
Stereotypy 281
Pharmacological Inhibition of BCR Signaling 284
Apoptosis 285
Apoptotic Pathway 285
Role of the Microenvironment 286
Conclusions 287
References 288
9 Targeted Therapy in Hematologic Malignancies 299
Introduction 299
Targeted Therapy: Definitions and Classification 300
Therapeutic Monoclonal Antibodies in Targeted Therapy 302
Examples of Investigational Monoclonal Antibodies in Hematologic Malignancies 308
Small Molecule Drugs 309
All-Trans-Retinoic Acid (ATRA) 316
Examples of Small Molecule Investigational Agents for Acute Myeloid Leukemia (AML) 317
Examples of Investigational Agents for the Treatment of Myeloproliferative Neoplasms (MPN) 318
Novel Targeted Agents Under Investigation for the Treatment of CML 320
Angiogenesis-Inhibiting Drugs 320
Apoptosis-Inducing Drugs 321
Cancer Vaccines 321
Gene Therapy 322
Challenges and Changes in Clinical Practice in the Era of Targeted Therapy 323
References 324
10 Micro-RNAs in Hematologic Malignancies 330
Introduction 330
Micro-RNAs in Normal Hematopoiesis 332
Micro-RNAs in Lymphomas 334
Micro-RNAs in Leukemias 337
Concluding Remarks 340
References 340
Index 346