Lymphoma: Methods and Protocols (Methods in Molecular Biology, 2865)

Preface
Contents
Contributors
Chapter 1: B Cell Differentiation and the Origin and Pathogenesis of Human B Cell Lymphomas
	1 B Cell Development and Differentiation
		1.1 Introduction
		1.2 B Cell Diversity and Antibody Structure
		1.3 B Cell Development and Differentiation
		1.4 The Germinal Center Reaction
		1.5 B Cell Memory
		1.6 T Cell-Independent Immune Responses
	2 Cellular Origin of Human B Cell Lymphomas
	3 Genetic Lesions in the Pathogenesis of B Cell Lymphomas
	4 Epigenetic Alterations
	5 Viruses in B Cell Lymphomas
	6 Microenvironmental Interactions in B Cell Lymphomas and the Role of the BCR
	7 Conclusions
	References
Chapter 2: Flow Cytometry for Non-Hodgkin and Hodgkin Lymphomas
	1 Introduction
	2 Materials
		2.1 Buffers and Cell Staining Reagents
	3 Methods
		3.1 Sample Preparation
			3.1.1 Disaggregation of Tissue Specimens
			3.1.2 Cell Surface Labeling of Cell Suspensions
			3.1.3 Cytoplasmic or Nuclear Labeling of Cell Suspensions (for BCL2, BCL6)
		3.2 Gating Strategies, Data Analysis, and Interpretation
			3.2.1 B-Cell Analysis
			3.2.2 T- and NK-Cell Analysis
			3.2.3 CHL Analysis
			3.2.4 Nodular Lymphocyte-Predominant Hodgkin Lymphoma Analysis
			3.2.5 T-Cell/Histiocyte-Rich Large B-Cell Lymphoma Analysis
			3.2.6 Contribution of Reactive T Cells to the Diagnosis of CHL, NLPHL, and THRLBCL
	4 Notes
	References
Chapter 3: Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Gene...
	1 Introduction
	2 Materials
		2.1 Histological Stainings
		2.2 Laser-Assisted Microdissection
		2.3 Proteinase K Digestion
		2.4 First Round of  PCR
		2.5 Second Round of  PCR
			2.5.1 VH  PCR
			2.5.2 Vκ  PCR
		2.6 PCR Product Purification and Direct Sequence Analysis
	3 Methods
		3.1 Histological Staining
		3.2 Microdissection
		3.3 Proteinase K Digestion
		3.4 First Round of  PCR
		3.5 Second Round of  PCR
			3.5.1 VH  PCR
			3.5.2 Vκ  PCR
		3.6 PCR Product Purification and Direct Sequence Analysis
		3.7 Sequence Evaluation
	4 Notes
	References
Chapter 4: PCR GeneScan Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes for Clonality Diagnostics in Suspect Ly...
	1 Introduction
	2 Materials
		2.1 DNA Extraction
		2.2 PCR Setup
		2.3 GeneScan Fragment Analysis
	3 Methods
		3.1 Samples and Quality Controls
		3.2 DNA Extraction
			3.2.1 Extraction from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue
			3.2.2 Extraction from Fresh Tissues or Human Fluids
			3.2.3 Extraction from Peripheral Blood or Bone Marrow
		3.3 PCR Setup
			3.3.1 Suspect B-Cell Proliferations
			3.3.2 Suspect T-Cell Proliferations
		3.4 GeneScan Fragment Analysis
		3.5 Post-analytical Data Interpretation
	4 Notes
	References
Chapter 5: Expression Cloning of Antibodies from Single Human B Cells
	1 Introduction
	2 Materials
		2.1 Isolation of Single Human B Cells by Flow Cytometry
		2.2 Reverse Transcription-PCR (RT-PCR) and Ig Gene Amplification
		2.3 Expression Vector Cloning
		2.4 Expression of Recombinant Monoclonal Antibodies
	3 Methods
		3.1 Isolation of Single Human B Cells by Flow Cytometry
		3.2 RT-PCR and Ig Gene Amplification
			3.2.1 cDNA Synthesis
			3.2.2 Ig Gene Amplification
		3.3 Expression Vector Cloning
			3.3.1 Ig Gene-Specific PCR for DNA Assembly
			3.3.2 Directional Cloning into Expression Vectors Using DNA Assembly
		3.4 Expression of Recombinant Monoclonal Antibodies
			3.4.1 Recombinant Antibody Production
			3.4.2 Recombinant Antibody Purification
	4 Notes
	References
Chapter 6: Stereotyped B-Cell Receptor Immunoglobulins in B-Cell Lymphomas
	1 Introduction
		1.1 Antigen Selection Shapes the B-Cell Receptor Immunoglobulin Gene Repertoire of Mature B-Cell Malignancies
		1.2 Discovery of B-Cell Receptor Immunoglobulin Stereotypy in CLL
			1.2.1 Prevalence of BcR IG Stereotypy in CLL
			1.2.2 Relevance of Major Stereotyped Subsets
			1.2.3 BcR IG Stereotypy in the Context of CLL Ontogeny
		1.3 B Cell Receptor Immunoglobulin Stereotypes in Other Mature B-Cell Neoplasms
			1.3.1 Mantle Cell Lymphoma
			1.3.2 Splenic Marginal Zone Lymphoma
	2 Identification of BcR IG Stereotypes: Methodologies and Criteria
		2.1 Identifying BcR IG Stereotypes
		2.2 Major Stereotyped Subsets
		2.3 Major Subset Satellites
	3 Future Developments and Perspective
	References
Chapter 7: Flow Cytometric MRD Detection in Selected Mature B-Cell Malignancies
	1 Introduction
	2 Materials
		2.1 Staining for MRD  Flow
		2.2 Photomultiplier Tube Voltage Setup
		2.3 Light Scatter and Compensation Setup
		2.4 Data Analyses for MRD Flow in MM and CLL
	3 Methods
		3.1 Staining for MRD Flow in CLL
		3.2 Staining for MRD Flow in  MM
		3.3 Standardized Instrument PMT Voltage Setup
		3.4 Standardized Light Scatter and Compensation Setup
		3.5 Data Acquisition
		3.6 Data Analysis for MRD Flow in CLL
		3.7 Data Analysis for MRD Flow in  MM
	4 Notes
	References
Chapter 8: MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Target...
	1 Introduction
	2 Materials
		2.1 Bone Marrow and Peripheral Blood Sample Processing at Diagnosis
		2.2 Detection of Clonal IG Targets or Chromosomal Translocations at Diagnosis
			2.2.1 Agarose Gel Electrophoresis and Genescan Analysis
			2.2.2 PCR-Heteroduplex Analysis
			2.2.3 Sanger Sequencing of Clonal Rearrangements
		2.3 RQ-PCR Sensitivity Testing
			2.3.1 Design and Testing of Allele-Specific Oligonucleotide Primers
			2.3.2 RQ-PCR Analysis of Dilutions of Diagnostic Sample
		2.4 ddPCR for MRD Quantification
		2.5 Bone Marrow and Peripheral Blood Sample Processing During Follow-Up
		2.6 MRD Analysis of Follow-Up Samples
	3 Methods
		3.1 Bone Marrow and Peripheral Blood Sample Processing at Diagnosis
		3.2 Detection of Clonal IG Gene Rearrangements and Determination of Chromosomal Translocations at Diagnosis
			3.2.1 Genescan Analysis
			3.2.2 PCR-Heteroduplex Analysis
			3.2.3 PCR Product Purification and Sanger Sequencing of Clonal Rearrangements
			3.2.4 Sequence Interpretation
			3.2.5 Selection of MRD-PCR Targets
		3.3 RQ-PCR Sensitivity Testing
			3.3.1 Design of Allele-Specific Oligonucleotide Primers
			3.3.2 Application of Consensus Primers and Probes for t(14;18) RQ-PCR and ddPCR
			3.3.3 RQ-PCR Analysis of Dilution Series of Diagnostic Sample
			3.3.4 RQ-PCR Data Interpretation: Quantitative Range and Sensitivity
		3.4 Bone Marrow and Peripheral Blood Sample Processing During Follow-Up
		3.5 MRD Analysis of Follow-Up Samples
			3.5.1 Control Gene RQ-PCR Analysis
			3.5.2 MRD Analysis
			3.5.3 RQ-PCR MRD Data Interpretation
		3.6 ddPCR
			3.6.1 Target ddPCR Analysis
			3.6.2 ddPCR Data Interpretation
		3.7 Concluding Remarks
	4 Notes
	References
Chapter 9: FISH and FICTION in Lymphoma Research
	1 Introduction
	2 Materials
		2.1 Preparation of Locus-Specific FISH Probes
			2.1.1 DNA Preparation from BAC/PAC Clones
			2.1.2 DNA Labeling by Random Priming
			2.1.3 Probe Precipitation
		2.2 Sample Preparation
			2.2.1 Preparation of Cell Suspensions Form Cultured Tumor Cells from Bone Marrow or Blood
			2.2.2 Preparation of FISH Slides from Cell Suspensions
		2.3 Hybridization
		2.4 Washing
		2.5 Evaluation
		2.6 FISH to Paraffin-Embedded Tissue Sections
		2.7 FICTION
			2.7.1 FICTION on Cryosections
	3 Methods
		3.1 Preparation of Locus-Specific FISH Probe
			3.1.1 DNA Preparation from BAC/PAC Clones
			3.1.2 DNA Labeling by Random Priming
			3.1.3 Probe Precipitation
		3.2 Sample Preparation
			3.2.1 Preparation of Cell Suspensions from Cultured Tumor Cells, from Bone Marrow, or from Blood
			3.2.2 Preparation of FISH Slides from Cell Suspensions
		3.3 Hybridization
		3.4 Washing (See Note 9)
		3.5 Evaluation
		3.6 FISH to Paraffin-Embedded Tissue Sections
		3.7 FICTION on Cryosections
		3.8 FICTION ``Rescue´´
		3.9 FICTION on Paraffin-Embedded Tissue
	4 Notes
	References
Chapter 10: Genotype from Phenotype: Using CRISPR Screens to Dissect Lymphoma Biology
	1 Introduction
	2 Materials
		2.1 Plasmids
		2.2 Generation of Cas9-Expressing Cell Lines
		2.3 Library Virus Preparation and Titration
		2.4 Transduction, Cell Culture, and Pellet Collection
		2.5 Library Preparation and Detection of sgRNA Abundance
	3 Methods
		3.1 Obtain Plasmids
		3.2 Generate Cas9 Efficient Clonal Cell Lines
		3.3 Library Virus Preparation and Titration
		3.4 Transduction, Cell Culture, and Pellet Collection
		3.5 Library Preparation and Detection of sgRNA Abundance
		3.6 Adaptations to Methods
			3.6.1 Sorted Screens: Transduction and Cell Culture
			3.6.2 Sorted Screens: Isolation of Phenotypically Distinct Populations by FACS Sorting
			3.6.3 Sorted Screens: NGS Library Preparation
			3.6.4 Epistasis CRISPR Screens
	4 Notes
	References
Chapter 11: Lymphoma and Leukemia Cell Vulnerabilities and Resistance Identified by Compound Library Screens
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Inhibitor Library
		2.3 Single Agent Screen
		2.4 Combination Screen
		2.5 Cell Viability Assay
	3 Methods
		3.1 Cell Culture
		3.2 Inhibitor Library
		3.3 Single Agent Screen
		3.4 Combination Screen
		3.5 Cell Viability Assay
	4 Notes
	References
Chapter 12: Use of Proximity Ligation Assay to Study Lymphoid Malignancies
	1 Introduction
	2 Materials
		2.1 Cell Preparation
		2.2 Antibodies, Ligation, and Amplification
	3 Methods
		3.1 Slide Preparation
		3.2 Cell Preparation
		3.3 Antibody Staining
		3.4 Ligation and Amplification
		3.5 Confocal Imaging
		3.6 Data Analysis
	4 Notes
	References
Chapter 13: Phosphoproteomic Analysis of Signaling Pathways in Lymphomas
	1 Introduction
	2 Materials
		2.1 Sample Preparation (See Note 1)
		2.2 In-Solution Protein Digestion
		2.3 Peptide Purification
		2.4 Isobaric Peptide Labeling (Optional)
		2.5 Phosphopeptide Enrichment
		2.6 Sample Fractionation
		2.7 MS Analysis and Data Processing
	3 Methods
		3.1 Sample Preparation
		3.2 In-Solution Protein Digestion
		3.3 Peptide Purification
		3.4 Isobaric Peptide Labeling (Optional)
		3.5 Enrichment of Phosphopeptides
		3.6 High pH Reversed Phase Fractionation
		3.7 MS Analysis and Data Processing
	4 Notes
	References
Chapter 14: Single Cell VDJ Sequencing of Normal and Malignant B and T Cells
	1 Introduction
	2 Materials
		2.1 Sample Preparation for Processing Human B Cells for Single Cell Sequencing
		2.2 10x Genomics scVDJ Sequencing
		2.3 10x Genomics Spatial scVDJ Sequencing
		2.4 BD Rhapsody
	3 Methods
		3.1 Sample Preparation
			3.1.1 Starting Material
			3.1.2 Sample Cleanup
			3.1.3 Enrichment of Cell Types
			3.1.4 Accurate Cell Counting in 10x Genomics Experiments
			3.1.5 Using Cell Hashing for Sample Pooling
			3.1.6 Processing of Human B Cells for scVDJ Sequencing
		3.2 10x Genomics
			3.2.1 Sequencing
			3.2.2 CellRanger Pipeline
			3.2.3 Output (Generated by CellRanger 7.2.0)
			3.2.4 Clonal Analysis
		3.3 Spatially Resolved scVDJ Sequencing
		3.4 BD Rhapsody
			3.4.1 Sequencing
			3.4.2 Running the BD Rhapsody Pipeline
			3.4.3 Output (Generated by Pipeline v2.0)
			3.4.4 Clonal Analysis
	4 Notes
	References
Chapter 15: Single-Cell Transcriptomic Analysis of Normal and Malignant B Cells
	1 Introduction
	2 Materials
		2.1 Preparation of Single Cell Suspensions
		2.2 Sc-Gene Expression and V(D)J Immune Profiling
	3 Methods
		3.1 Single-Cell Gene Expression and V(D)J Immune Profiling of B Cells
			3.1.1 Sample Selection and Cell Isolation
			3.1.2 GEM Generation, RT-Cleanup, and cDNA Amplification
			3.1.3 5′ GEX Library Construction
			3.1.4 V(D)J cDNA Amplification and Library Construction
			3.1.5 Library Quantification, Pooling, and Sequencing
		3.2 A Computational Pipeline for sc-Transcriptomic Analysis
			3.2.1 Alignments
			3.2.2 Quality Controls
			3.2.3 Cluster Identification, Dimensional Reduction, and Visualization
			3.2.4 Differential Gene Expression Analysis and Visualization
			3.2.5 Trajectory Inference and Pseudotime Analysis
			3.2.6 VDJ Data Annotations and Integration
	4 Notes
	References
Chapter 16: Multiplexed Immunophenotyping of Lymphoma Tissue Samples
	1 Introduction
	2 Materials
		2.1 Tissue Samples
		2.2 Equipment
		2.3 Reagents
		2.4 Chemicals
		2.5 Buffers
	3 Methods
		3.1 Sample Selection and Tissue Slide Preparation
		3.2 Antibody Panel Design
		3.3 Antibody Conjugation and Validation
			3.3.1 Prepare Antibody for Conjugation
			3.3.2 Antibody Reduction
			3.3.3 Prepare Oligo Solution
			3.3.4 Incubation of Reduced Antibody with Maleimide-Modified DNA Oligonucleotides
			3.3.5 Elution of the Conjugated Antibody
		3.4 CODEX Antibody Staining for FFPE Tissue
			3.4.1 Tissue Deparaffinization and Hydration
			3.4.2 Antigen Retrieval
			3.4.3 Tissue Bleaching
			3.4.4 Blocking and Antibody Staining
			3.4.5 Post-staining Antibody Fixation
			3.4.6 Preparation of Reporter Plate
			3.4.7 Equipment Set-Up and Start of Multicycle Experiment
		3.5 Post-experiment Hematoxylin-Eosin (H&E) Staining
		3.6 Downstream Data Analysis Approaches
	4 Notes
	References
Chapter 17: Genetic Manipulation and Extended Culture of Human Germinal Center B Cells to Model Lymphomagenesis
	1 Introduction
	2 Materials
		2.1 Enrichment of GC B Cells from Discarded Human Tonsil Tissue
		2.2 In Vitro Culture of Human GC B Cells
		2.3 Viral Transduction and Immortalization of Human GC B Cells
		2.4 Nucleofection of Human GC B Cells
	3 Methods
		3.1 Enrichment of GC B Cells from Discarded Human Tonsil Tissue
		3.2 In Vitro Culture of Human GC B Cells
		3.3 Viral Transduction and Immortalization of Human GC B Cells
		3.4 Nucleofection of Human GC B Cells
	4 Notes
	References
Chapter 18: Genetic Mouse Models of Lymphomas
	1 Introduction
	2 Mouse Genetics and Allele Design
	3 Mouse Models of Diffuse Large B Cell Lymphoma
		3.1 Cluster BN2/C1
		3.2 Cluster 2/A53
		3.3 EZB/C3 DLBCL
		3.4 ST2/C4 DLBCL
		3.5 MCD/C5 DLBCL
	4 Breeding Strategy, Colony Maintenance
	5 Model Characterization
	6 Mouse Models as Preclinical Avatars
	7 Concluding Remarks
	References
Chapter 19: Establishing Patient-Derived Xenograft (PDX) Models of Lymphomas
	1 Introduction
	2 Materials
		2.1 Preparation of Single Cell Suspensions
		2.2 Injection of Single Cell Suspensions
		2.3 Tumor Monitoring and Harvesting
	3 Methods
		3.1 Sample Preparation
		3.2 Solid Tissue Disaggregation
			3.2.1 Mechanical Disaggregation
			3.2.2 Enzymatic Disaggregation
		3.3 Liquid Sample Preparation
		3.4 Preparation of Cells for Injection
		3.5 Sample Administration
			3.5.1 Subcutaneous Injection of Cell Suspensions
			3.5.2 Intraperitoneal Injection of Cell Suspensions
		3.6 Monitoring Tumor Growth
			3.6.1 Subcutaneously Injected Animals
			3.6.2 Intraperitoneally Injected Animals
		3.7 Harvest, Passaging, and Storage of Cells
			3.7.1 Harvest
			3.7.2 Passaging and Cryo-storage of PDX Cells
	4 Notes
	References
Chapter 20: Detection and Characterization of Clonal Hematopoiesis
	1 Introduction
		1.1 Clonal Hematopoiesis
		1.2 Detection of Clonal Hematopoiesis Variants
	2 Materials
		2.1 DNA Isolation from Peripheral Blood or Bone Marrow
		2.2 Pre-Capture Library Preparation for Detection of Clonal Hematopoiesis
		2.3 Hybridization of Target Capture Probes and Capture of Custom Targets for Detection of Clonal Hematopoiesis
		2.4 Next-Generation Sequencing
		2.5 Analysis of Error-Corrected Next-Generation Sequencing  Data
	3 Methods
		3.1 DNA Isolation from Peripheral Blood or Bone Marrow
		3.2 Pre-Capture Library Preparation for Detection of Clonal Hematopoiesis
			3.2.1 DNA Fragmentation
			3.2.2 Ligation of xGen UDI-UMI Adapters
			3.2.3 Purification of Indexed Libraries
			3.2.4 Pre-Capture PCR Amplification
			3.2.5 Purification of Amplified Pre-Capture Libraries
			3.2.6 Quality Control of Pre-Capture Libraries
		3.3 Hybridization of Target Capture Probes and Capture of Custom Targets for Detection of Clonal Hematopoiesis
			3.3.1 Prepare and Pool Samples for Hybridization
			3.3.2 Hybridize Capture Probes with Library Pools
			3.3.3 Prepare Streptavidin Beads for Capture
			3.3.4 Capture of Hybridized Targets to Streptavidin Beads
			3.3.5 Post-Capture PCR Amplification
			3.3.6 Purification of Amplified Post-Capture Libraries
			3.3.7 Quality Control of Post-Capture Libraries
		3.4 Next-Generation Sequencing
			3.4.1 Preparation of Twist Custom Libraries for Pooling and Sequencing
			3.4.2 Illumina Sequencing Run Set-Up
		3.5 Analysis of Error-Corrected Next-Generation Sequencing
			3.5.1 Preprocessing of Sequencing Data and Variant Calling
			3.5.2 Filtering of Somatic Variants
	4 Notes
	References
Chapter 21: Detection of Circulating Tumor DNA in Lymphoma Patients
	1 Introduction
	2 Materials
		2.1 Blood Collection and Processing
		2.2 cfDNA, gDNA Extraction, Quantification, and Quality Control
		2.3 Library Preparation and Sequencing
	3 Methods
		3.1 Pre-analytics
		3.2 Plasma Separation and Blood Processing
			3.2.1 Plasma Separation
			3.2.2 Blood Processing
		3.3 DNA Extraction
			3.3.1 cfDNA Extraction
			3.3.2 DNA Quality Control
		3.4 gDNA Extraction from Blood Cells and Quantification
			3.4.1 Maxwell
			3.4.2 Qiagen
		3.5 Library Preparation
			3.5.1 gDNA Library
			3.5.2 cfDNA Library
		3.6 Libraries Quality Checks and Storage
		3.7 Gene Panel (Panel20042021)
		3.8 Library Enrichment
		3.9 Ultra-deep Sequencing
		3.10 Bioinformatic Pipeline for Variant Calling
			3.10.1 Files Creation
			3.10.2 Statistical Analysis
			3.10.3 ctDNA Load Calculation
			3.10.4 Analytical Sensitivity of CAPP-seq
		3.11 Variant Quality Assessment by Integrative Genomics Viewer
	4 Notes
	References
Index