Testicular Germ Cell Tumors: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Basic Histopathologic Assessment of Germ Cell Tumors for Clinic and Research
	1 Introduction
		1.1 Normal Macroscopy and Histology of Testis
		1.2 Germ Cell Neoplasia In Situ (GCNIS)
		1.3 Germ Cell Tumors Derived from GCNIS
			1.3.1 Tumors of a Single Histologic Type (Pure Forms)
				Seminoma
				Nonseminomatous Germ Cell Tumors
					Embryonal Carcinoma
					Yolk Sac Tumor, Postpubertal  Type
					Choriocarcinoma and Other Trophoblastic Tumors
					Teratoma, Postpubertal  Type
					Teratoma with Somatic-Type Malignancy
			1.3.2 Tumors of Multiple Histologic Types (Mixed Germ Cell Tumors)
		1.4 Germ Cell Tumors Unrelated to GCNIS
			1.4.1 Spermatocytic Tumor
			1.4.2 Teratoma, Prepubertal  Type
			1.4.3 Yolk Sac Tumor and Mixed Teratoma and Yolk Sac Tumors, Prepubertal  Type
		1.5 Regression of Germ Cell Tumor
		1.6 Concepts for GCT Staging
	References
Chapter 2: Clinical Applications of Immunohistochemistry in Germ Cell Tumors in Men
	Abbreviations
	1 Introduction
	2 Materials
		2.1 Tissue Processing
		2.2 Immunohistochemistry Slide Preparation
		2.3 Immunohistochemistry Staining Using the UltraView Universal DAB Detection Kit by Ventana
	3 Methods
		3.1 Tissue Processing
			3.1.1 Tissue Processing for Tissue Blocks from Orchiectomies and Retroperitoneal Lymph Node Dissections
			3.1.2 Tissue Processing for Tissue Blocks from Biopsies
		3.2 Immunohistochemistry Slide Prepping
		3.3 Immunohistochemistry Staining
		3.4 Hematoxylin and Eosin (H&E) Staining
		3.5 Principles of Immunohistochemistry
	4 Immunohistochemistry in Germ Cell Tumors
		4.1 Commonly Used Immunohistochemical Markers
		4.2 Immunohistochemical Profiles in GCT Subtypes
		4.3 Use of Immunohistochemistry in Specific Clinical Scenarios
	5 Note
	References
Chapter 3: Molecular Characterization of Testicular Germ Cell Tumors Using Tissue Microdissection
	1 Introduction
	2 Laser Microdissection Technology: An Overview
		2.1 LCM, Major Components
		2.2 Types of Specimens Compatible with LCM (Fig. 1)
		2.3 LCM Applications
	3 LCM Technical Procedures and Caveat
		3.1 Tissue Fixation
		3.2 Deparaffinization and Rehydration
		3.3 Staining
		3.4 Dehydration
		3.5 Microdissection (IR Cold Laser Method)
		3.6 Expected Yield of DNA, RNA, and Proteins from  LCM
	4 Target Material Extractions
		4.1 DNA Extraction
		4.2 RNA Extraction
			4.2.1 RNA Extraction Buffer
		4.3 Protein Extraction
	5 Technical Issues
		5.1 Caveat
		5.2 Microdissection Cell Number Required for Various Applications as Reported in Literature
		5.3 Quantity Measurements and Quality Assessment
		5.4 Advantages and Limitations of Laser Capture Microdissection
	6 Summary
	References
Chapter 4: Fluorescence In Situ Hybridization (FISH) Detection of Chromosomal 12p Anomalies in Testicular Germ Cell Tumors
	1 Introduction
	2 Materials
		2.1 Reagents, Supplies, and Equipment
			2.1.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 Quality Control
		3.2 Slide Preparation
		3.3 Results and Interpretation
		3.4 Limitations
	4 Notes
	References
Chapter 5: Germ Cell Tumor Cell Culture Techniques
	1 Introduction
	2 Materials
		2.1 Cryopreservation and Restoration
		2.2 Resazurin Viability Assay
		2.3 Crystal Violet Proliferation Assay
	3 Methods
		3.1 Routine Culture and Subculturing
		3.2 Cryopreservation and Restoration
			3.2.1 Restoration of Cryopreserved Cells
		3.3 Resazurin Viability Assay
		3.4 Crystal Violet Proliferation Assay
	4 Notes
	References
Chapter 6: Three-Dimensional Cultivation of Germ Cell Cancer Cell Lines as Hanging Drops
	1 Introduction
	2 Materials
		2.1 Components for Cultivation of Cell Aggregates from GCC Cell Lines
	3 Methods
		3.1 Preparation of a Single Cell Suspension for the Hanging Drops
		3.2 Cultivation of 3D Cell Aggregates from GCC Cell Lines
		3.3 Harvesting Cell Aggregates for Further Evaluation
	4 Notes
	References
Chapter 7: Cultivation of Testicular Germ Cell Cancer Cell Lines and Establishment of Gene-Edited Subclones Using CRISPR/Cas9
	1 Introduction
	2 Materials
		2.1 Components for Standard Cell Culture
		2.2 Components for Transfection of Cell Lines
		2.3 Gene Editing and Establishment of Clonal Sublines
	3 Methods
		3.1 Standard Cell Culture Protocol for Germ Cell Tumor Cell Lines
		3.2 Transfection of Germ Cell Tumor Cell Lines
		3.3 Protocol for Establishment of Gene-Edited GCT Cell Lines Using CRISPR/Cas9
	4 Notes
	References
Chapter 8: Evaluation of Chemotherapeutic Drugs for Treatment of (Cisplatin-Resistant) Germ Cell Cancer Cell Lines
	1 Introduction
	2 Materials
		2.1 Components for Standard Cultivation of GCC Cell Lines
		2.2 Components for XTT Cell Viability Assay
		2.3 Components for Cell Cycle Analysis by Flow Cytometry
		2.4 Components for Apoptosis Assay by Flow Cytometry
	3 Methods
		3.1 Preparation of Samples
			3.1.1 Seeding GCC Cell Lines for Experiments
			3.1.2 Treatment with Chemotherapeutic Drugs
		3.2 Assessing Cellular Effects of Chemotherapeutic Drugs
			3.2.1 Measurement of Relative Cell Viability by XTT Assay
			3.2.2 Preparation of Samples for Measurement of Cell Cycle Distribution by Flow Cytometry
			3.2.3 Preparation of Samples for Measurement of Apoptosis Induction by Flow Cytometry
	4 Notes
	References
Chapter 9: Assessing Homologous Recombination and Interstrand Cross-Link Repair in Embryonal Carcinoma Testicular Germ Cell Tu...
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Plasmids for DR-GFP Assay
		2.3 Plasmids for TR-OriP-GFP Assay
		2.4 Formation of the  ICL
		2.5 Electroporation
		2.6 Fluorescence-Activated Cell Sorting (FACS)
	3 Methods
		3.1 DR-GFP Assay
		3.2 TR-OriP-GFP Assay
		3.3 Concluding Remarks
	4 Notes
	References
Chapter 10: Production and Analysis of Human Primordial Germ Cell-Like Cells
	1 Introduction
	2 Materials
		2.1 Cell Culture of hiPSCs in the Primed Pluripotency State
			2.1.1 Cell Culture of hiPSCs in the Naive Pluripotency State
		2.2 Generation of hPGCLCs
		2.3 Immunohistochemical Detection of OCT4+ hPGCLCs in FFPE Slides of EBs or on the Surface of Whole EBs
		2.4 FACS Enrichment of CD38+ hPGCLCs from EBs
	3 Methods
		3.1 Cell Culture of hiPSCs in the Primed Pluripotency State
			3.1.1 Starting Culture from Frozen Cell Stock
			3.1.2 Passaging Primed Pluripotency hiPSCs
		3.2 Production of hPGCLCs
			3.2.1 Day 1: Inoculation of Primed Pluripotency hiPSCs into Matrigel-Coated 6-Well Cluster Plates
			3.2.2 Day 2: Medium Change
			3.2.3 Day 3 to Day 4: Conversion of the Primed Pluripotency State of mTeSR1-Maintained hiPSCs to the 4i-Naive (ERK-Independent...
			3.2.4 Day 5: Spin-EB Formation Using AggreWell 400 Microwell Plates
			3.2.5 Day 6: Transfer of EBs to Low Attachment Plates for Rocking Culture
			3.2.6 Day 7 to Day 14: Generation of hPGCLCs on the Surface of EBs
		3.3 Immunohistochemical Staining of EBs
			3.3.1 Embedding EBs in Matrigel for FFPE Immunohistochemistry
			3.3.2 Detecting hPGCLCs on the Surface of EBs by Whole-Mount Immunohistochemistry
		3.4 FACS Enrichment of hPGCLCs from EBs as CD38+ Cells
	4 Notes
	References
Chapter 11: A Genetically Engineered Mouse Model of Malignant Testicular Germ Cell Tumors
	1 Introduction
	2 Materials
		2.1 Animals
		2.2 Mouse Tail DNA Preparation Materials
		2.3 Mouse Genotyping Reagents
		2.4 Mouse Cohort Monitoring Materials
		2.5 Necropsy Materials
		2.6 Chemotherapy Treatments
		2.7 Cell Culture Materials
			2.7.1 Media for Teratocarcinoma Cell Culture
			2.7.2 Consumables, Equipment, and Reagents for Teratocarcinoma Culture
		2.8 Tumor Transplantation Reagents and Equipment
			2.8.1 Tumor Disaggregation Media
			2.8.2 Tumor Collection and Transplantation
	3 Methods
		3.1 Mouse Breeding
			3.1.1 Generating Breeders for gPAK Experimental Crosses
			3.1.2 Maintenance of Breeder Animals (See Note 2)
			3.1.3 Generation of gPAK Experimental Animals
			3.1.4 Generation of gPAK Experimental Animals Containing Oct4-Gfp
		3.2 Genotyping of Experimental and Breeder Animals
			3.2.1 Obtain Genomic DNA from the Mice for Genotyping
			3.2.2 PCR Amplification
			3.2.3 Gel Electrophoresis
			3.2.4 Genotype Interpretation
		3.3 Aging and Monitoring of Experimental Animals
			3.3.1 Manual Monitoring for Tumor Development
			3.3.2 Monitoring Tumor Development by Ultrasound
		3.4 Necropsy
		3.5 Chemotherapeutic Treatment with Bleomycin/Etoposide/Cisplatin (BEP)
			3.5.1 BEP Treatment of  Mice
		3.6 Tumor Transplantation into Secondary Hosts to Assay Tumor-Propagating Activity
		3.7 Teratocarcinoma Cell Culture
	4 Notes
	References
Chapter 12: Targeted Methylation Analyses: From Bisulfite Treatment to Quantification
	1 Introduction
	2 Materials
		2.1 Bisulfite Conversion
		2.2 Real-Time qMSP
		2.3 HRM Methylation-Sensitive Analyses
	3 Methods
		3.1 Bisulfite Conversion
		3.2 Real-Time qMSP
		3.3 HRM Methylation-Sensitive Analyses
	4 Notes
	References
Chapter 13: Integrated Analysis of Germ Cell Tumors
	1 Introduction
	2 Materials
	3 Methods
		3.1 DNA Extraction and Library Creation from FFPE Tissue Samples
		3.2 Preprocessing and Alignment to the Genome
		3.3 Data Processing, Quality Control, and Contamination Estimation
		3.4 Variant Calling
		3.5 Copy Number Analysis
	References
Chapter 14: Use of Genomewide Association Studies to Evaluate Genetic Predisposition to Testicular Germ Cell Tumors
	1 Introduction
	2 Materials
		2.1 DNA Extraction from Saliva Collection Kit
		2.2 DNA Extraction from Assisted Saliva Collection Kit
		2.3 Blood Processing Using Ficoll-Paque Plus
		2.4 DNA Integrity Analysis by Gel Electrophoresis
		2.5 DNA Quantification
		2.6 DNA Dilutions and Plating
		2.7 Illumina Infinium HumanCoreExome Array
		2.8 iPLEX Agena Multiplexed Genotyping (Formerly Sequenom)
	3 Methods
		3.1 DNA Extraction from Saliva Collection Kit
		3.2 DNA Extraction from Assisted Saliva Collection Kit
		3.3 Blood Processing Using Ficoll-Paque Plus
		3.4 DNA Integrity Analysis Using Gel Electrophoresis
		3.5 DNA Quantification by Quant-iT PicoGreen dsDNA Assay
		3.6 DNA Dilutions and DNA Plating
		3.7 Illumina Infinium HumanCoreExome Array
			3.7.1 Preparation of DNA Plate and MSA3 Plate for Amplification
			3.7.2 Fragmentation of the MSA3 Plate
			3.7.3 Precipitation of the MSA3 Plate
			3.7.4 Resuspension of the MSA3 Plate
			3.7.5 Hybridization to Multi BeadChips
			3.7.6 Loading the BeadChips
			3.7.7 Setting Up the Multi BeadChips for Hyb
			3.7.8 Preparation of Reagent for XStain HD BeadChip Process and Washing the BeadChips
			3.7.9 Assembling the Flow-Through Chamber
			3.7.10 Single Base Extension and Staining of the BeadChip
			3.7.11 Washing and Coating of BeadChips
			3.7.12 Imaging of BeadChips Using the iScan System
		3.8 iPLEX Agena Genotyping Using iPLEX Gold Method (Formerly Sequenom)
			3.8.1 PCR Amplification of DNA
			3.8.2 Preparation and Addition of SAP
			3.8.3 Adjustment, Preparation, and Addition of Low-Plex iPLEX Gold Reaction Cocktail
			3.8.4 Cleaning the Low Plex iPLEX Gold Reaction Products
			3.8.5 Transfer of iPLEX Reaction to a SpectroChip
			3.8.6 Analysis of Results Using MassARRAY TYPER 4.0
	4 Notes
	References
Chapter 15: A Circulating MicroRNA Panel for Malignant Germ Cell Tumor Diagnosis and Monitoring
	1 Introduction
	2 Materials
		2.1 Serum Isolation from Whole Blood
		2.2 RNA Extraction
		2.3 Quality Control (QC) Reverse Transcription Step
		2.4 Custom miRNA Panel Multiplex Reverse Transcription (RT) Step
		2.5 Multiplex Preamplification Step
		2.6 Quantitative PCR (qPCR) for Individual miRNAs
	3 Methods
		3.1 Serum Isolation from Whole Blood
		3.2 RNA Extraction
		3.3 Quality Control (QC)
			3.3.1 Quality Control (QC) Reverse Transcription (RT) Step
			3.3.2 Quantitative PCR (qPCR) for Individual miRNAs
			3.3.3 Quality Control (QC) Data Analysis
		3.4 Custom miRNA Panel Multiplex
			3.4.1 Custom miRNA Panel Multiplex Reverse Transcription (RT) Step
			3.4.2 Multiplex Preamplification Step
			3.4.3 Quantitative PCR (qPCR) for Individual miRNAs
			3.4.4 Data Analysis: DeltaDeltaCt Method and Normalization
	4 Notes
	References
Chapter 16: Detection of Circulating Tumor Cells (CTCs) in Patients with Testicular Germ Cell Tumors
	1 Introduction
	2 Materials
		2.1 Cell Lines and Media
			2.1.1 Cell Lines
			2.1.2 Media
		2.2 CTC Detection Using Ficoll-Hypaque Density Gradient Centrifugation
		2.3 Double Immunofluorescence Staining of  CTCs
		2.4 Fluorescence In Situ Hybridization in  CTCs
		2.5 CellSearch Analysis of  CTCs
	3 Methods
		3.1 Cultivation of GCT Cell Line Cells
		3.2 CTC Detection Using Ficoll-Hypaque Gradient Centrifugation
		3.3 Double Immunofluorescence Staining of  CTCs
			3.3.1 SALL4/Keratin
			3.3.2 OCT3/4/EpCAM
		3.4 Fluorescence In Situ Hybridization in  CTCs
			3.4.1 Probe Preparation by Random Priming
			3.4.2 Fluorescence In Situ Hybridization Procedure
		3.5 CellSearch Detection of Circulating Tumor Cells
	4 Notes
	References
Chapter 17: Developing and Using a Data Commons for Understanding the Molecular Characteristics of Germ Cell Tumors
	1 Introduction
	2 Current Cancer Genomic Data Commons Review
		2.1 Overview
		2.2 The National Cancer Institute´s Genomic Data Commons
		2.3 European Genome-phenome Archive
		2.4 University of California Santa Cruz Xena
		2.5 Limitations
	3 Germ Cell Tumor Explorer
		3.1 Data Resources
		3.2 Data Standards
		3.3 Cohort Discovery
		3.4 Online Analysis
		3.5 Data Security
	4 Future Work and Discussion
	5 Summary
	References
Index