Urothelial Carcinoma: Methods and Protocols

Preface
Contents
Contributors
Part I: Molecular Characterization
Chapter 1: Scoring Systems for Immunohistochemistry in Urothelial Carcinoma
1 Introduction
2 Methodical Prerequisites
2.1 Staining Procedure
2.2 Determination of Dimensions under a Microscope
3 Markers and Applicable Scoring Systems in Urothelial Carcinoma
3.1 Membranous Markers
3.1.1 PD-L1
3.1.2 Receptor Tyrosine Kinases (RTKs)
3.1.3 Fibroblast Growth Factor Receptors (FGFRs)
3.1.4 Epidermal Growth Factor Receptor (EGFR)
3.1.5 Epidermal Growth Factor Receptor 2 (EGFR2) or ErbB2/Her2neu
3.1.6 Other Membranous Markers
3.2 Cytoplasmic Markers
3.2.1 Keratins
3.2.2 Markers of Neuroendocrine Differentiation
3.2.3 Vimentin
3.3 Nuclear Markers
3.3.1 Hormone Receptors
3.3.2 Mismatch Repair (MMR) Proteins
3.3.3 p53
3.3.4 Ki67
3.3.5 GATA3
4 Conclusion
References
Chapter 2: A Panel-Based Method for the Reproduction of Distinct Molecular Subtype Classifications of Muscle-Invasive Urotheli...
1 Introduction
2 Materials
3 Methods
3.1 Selection of a Marker Set for Molecular Subtyping
3.2 Sample Selection
3.3 Preparation and Gene Expression Analysis of Fresh Frozen Tumor Samples
3.4 Preparation and Gene Expression Analysis of FFPE Tumor Samples
3.5 Rule Sets for the Gene Panel-Based Molecular Classifications
3.5.1 TCGA Classification
3.5.2 MDA Classification
3.5.3 GSC Classification
3.5.4 LundTax Classification
3.5.5 Consensus Classification
4 Notes
References
Chapter 3: Analysis of Mutational Signatures Using the mutSignatures R Library
1 Introduction
2 Materials
3 Methods
3.1 Importing Data from Bladder Cancer VCF Files and Data Pre-processing
3.2 De Novo Extraction of Mutational Signatures
3.3 Estimating Activity of Known Mutational Signatures
3.4 Building Plots, Inspecting, and Exporting Results
4 Notes
References
Chapter 4: A Drug Repurposing Pipeline Based on Bladder Cancer Integrated Proteotranscriptomics Signatures
1 Introduction
2 Materials
2.1 Transcriptomics Resources
2.2 Proteomics Resources
2.3 Databases for Acquiring BC-Associated Features from the Literature
2.4 Databases for Pathway Analysis
2.5 Resources for Signature Matching Drug Repurposing
2.6 Drug Databases and Relevant Resources for Compound Annotation and Prioritization
3 Methods
3.1 Acquisition and Reanalysis of Transcriptomics Data from Repositories
3.2 Acquisition and Reanalysis of Proteomics Data from Repositories
3.3 Acquisition of BC-Associated Features from the Literature
3.3.1 Acquisition of Core Molecular Features for BC Using the DisGeNET Database
3.3.2 Acquisition of Core Molecular Features Specific for BC Using the BioKB Platform
3.4 Data Integration
3.4.1 N-integration (Paired Omics Datasets)
3.4.2 P-integration: Multi-dataset Integration on the Feature Level
3.4.3 Late-Stage Integration of Results
3.5 Functional/Pathway Enrichment Analysis
3.6 Applying the Connectivity Map for Drug Repositioning
3.7 Annotation and Prioritization of the Repurposed Drugs
3.7.1 Manual Investigation of Your Drug List for Drug Annotation and Prioritization
3.7.2 Inspect for Drugs that Have Been Previously Associated with BC and Perform Drug Annotation Using the Open Targets Platfo...
4 Notes
References
Chapter 5: Characterization of Native COMPASS Complex in Urothelial Carcinoma Cells by Size Exclusion Chromatography
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Fractionation for Nuclear Protein Extraction
2.3 Size Exclusion Chromatography
2.4 Immunoblot Analysis
3 Methods
3.1 Cell Culture and Harvest
3.2 Cell Fractionation for Nuclear Protein Extraction
3.3 Size Exclusion Chromatography
3.4 Immunoblot Analysis
4 Notes
References
Part II: Urothelial Carcinogenesis
Chapter 6: Reconstructing Phylogenetic Relationship in Bladder Cancer: A Methodological Overview
1 Previous Work on Bladder Cancer
2 Evaluating Genomic Data for Phylogenetic Reconstruction
3 The Choice of Sequencing Technique
3.1 Whole Genome Sequencing
3.2 Whole Exome Sequencing
3.3 Unique Molecular Identifiers
4 The Choice of Samples
5 Processing Your Data
5.1 Quality Control
5.2 Adapter Trimming
5.3 Sequence Alignment
5.4 Variant and Copy Number Calling
6 Phylogenetic Reconstruction
6.1 Calculating the Cancer Cell Fraction
6.2 Subclonal Deconvolution
6.3 Advantages and Disadvantages of Different Deconvolution Methods
6.4 Phylogenetic Tree Visualization
7 Concluding Remarks and Recommended Literature
References
Chapter 7: Using Sister Chromatid Exchange Assay to Detect Homologous Recombination Deficiency in Epigenetically Deregulated U...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cisplatin Treatment
2.3 BrdU Incorporation
2.4 Preparation of Metaphase Chromosome Spreads
2.5 Differential Sister Chromatid Staining
2.6 SCE Scoring and Analysis
3 Methods
3.1 Cell Culture, Cisplatin Treatment, and BrdU Incorporation
3.2 Preparation of Metaphase Chromosome Spreads
3.3 Differential Sister Chromatid Staining
3.4 SCE Scoring and Analysis
4 Notes
References
Chapter 8: Identification of STAG2-Mutant Bladder Cancers by Immunohistochemistry
1 Introduction
2 Materials
2.1 Bladder Tumors
2.2 Antibodies and Staining Dyes
2.3 Buffers and Reagents
3 Methods
3.1 Immunohistochemistry
3.2 Pathologic Evaluation
3.2.1 Classification of STAG2-Positive Tumors
3.2.2 Classification of STAG2-Negative Tumors
3.2.3 Classification of STAG2 Mosaic Tumors
4 Notes
References
Part III: Cellular and Animal Models
Chapter 9: Genome-Wide CRISPR Screening for the Identification of Therapy Resistance-Associated Genes in Urothelial Carcinoma
1 Introduction
1.1 The CRISPR-Cas9 System for Gene Editing
1.2 CRISPR-Cas9-Based Screens
2 Materials
2.1 Cells
2.2 Libraries
2.3 Bacteria and Media
2.4 Antibiotics
2.5 Antibodies, Primer, and Reagents
2.6 Production of Lentivirus in HEK293T Cells and Lentiviral Infection
2.7 Preparation of Cellular DNA for NGS
3 Methods
3.1 Generation of T24 SAM Cells
3.2 Amplification of Libraries
3.3 Production of Lentiviruses Using Library DNA from Subheading 3.2
3.4 Titration of sgRNA Library Lentivirus
3.5 Infection of T24 SAM Cells with sgRNA Library DNA and Generation of CDK4/6 Inhibitor-Resistant Cells
4 Notes
References
Chapter 10: Tissue Slice Culture and Analysis of Tumor-Associated Hyaluronan in Urothelial Carcinoma
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents and Supplies
2.2.1 Tissue Slice Cultures
2.2.2 Polyacrylamide Gel Electrophoresis
2.2.3 Immunofluorescent Staining
3 Methods
3.1 Tissue Slicing Using a Compresstome
3.2 Tissue Slice Culture and Preparation of Tissue-Conditioned Medium for Hyaluronan Analysis
3.3 Analysis of Tissue-Associated Hyaluronan Using Electrophoresis
3.4 Visualization of Tissue-Associated Hyaluronan by Immunofluorescent Staining
4 Notes
References
Part IV: Biomarkers
Chapter 11: NGS-Based Tumor-Informed Analysis of Circulating Tumor DNA
1 Introduction
2 Materials
2.1 Liquid Biopsies and CfDNA/ctDNA Extraction
2.2 Tumor and Germline NGS Data, NGS Library, and Custom Panel Capture
2.3 Specialized Laboratory Equipment
3 Methods
3.1 Preparation of Plasma and Urine
3.1.1 Blood in EDTA Tubes
3.1.2 Blood in Streck Tubes
3.1.3 Urine
3.2 CfDNA/ctDNA Extraction
3.3 Design of Customized Capture Panels
3.4 NGS Library and Custom Panel Capture Protocol
3.4.1 Perform End Repair and dA-Tailing
3.4.2 Ligate UMI Adapters to dA-Tailed Fragments
3.4.3 SPRI Beads Purification of Libraries (1.8x)
3.4.4 Amplify Libraries
3.4.5 SPRI Beads Purification of Libraries (1.8x)
3.4.6 Double Capture of Custom Panel
3.4.7 Bind Hybridized Targets to Streptavidin Beads
3.4.8 Post-capture PCR Amplify, Purify, and Perform QC
3.5 NGS Sequencing for Captured Libraries from cfDNA/ctDNA
4 Notes
References
Chapter 12: Considering the Effects of Modern Point-of-Care Urine Biomarker Assays in Follow-Up of Patients with High-Risk Non...
1 Introduction
1.1 Target Population
1.2 Performance of WLC
1.3 Performance of Urine Markers: Urine Cytology
2 Materials
3 Methods
3.1 BTA Stat
3.2 NMP22 BladderChek
3.3 CancerCheck UBC Rapid VISUAL and UBC Rapid Test
3.4 Uromonitor
3.5 Data Work-Up
4 Notes
5 Discussion
6 Conclusions
References
Chapter 13: Simplex Droplet Digital PCR Assays for the Detection of TERT Promoter Mutations in Urine Samples for the Non-invas...
1 Introduction
2 Materials
2.1 DNA Isolation for Urine Samples
2.2 uTERTpm Mutation Screening
3 Methods
3.1 Urine Cell-Free DNA Isolation from Urine Supernatants
3.2 DNA Isolation from Cell Pellet
3.3 DNA Isolation from Whole Urine Samples (Cell-Free DNA and Cell DNA Within the Same Fraction)
3.4 TERT Probes and Primers Design
3.5 TERT-Mutated Cell Lines for Experimental Positive Controls
3.6 Generation of ddPCR Droplets Using the AutoDG Platform
3.7 Amplification Conditions
3.8 Scan of the 96-Well Plate by the QX200 Droplet Reader
3.9 Count of Numbers of ddPCR Droplets and Calculations of Mutant Allelic Fraction (MAF)
3.10 Determination of the Threshold Number of Minimum Mutated Droplets to Call a Mutation
3.11 Determination of the Limit of Detection (LOD) of the ddPCR Assays
4 Notes
5 Conclusion
References
Chapter 14: Predictive Biomarkers of Response to Neoadjuvant Therapy in Muscle Invasive Bladder Cancer
1 Introduction
2 Biomarkers Predicting Outcome After Neoadjuvant Chemotherapy
2.1 DNA Damage Repair Alterations
2.2 Gene Expression Profiling and Molecular Subtypes
2.3 COXEN (Co-expression Extrapolation) Algorithm
2.4 Receptor Tyrosine Kinase
3 Predicting Response to Neoadjuvant Immune Checkpoint Inhibition
4 Monitoring Response to Neoadjuvant Therapy with Biomarkers in Blood
5 Conclusion
References
Chapter 15: Assessment of PD-L1 Status in Urothelial Cancer
1 Introduction
2 Materials
3 Methods
4 Notes
5 Conclusive Remarks
References
Part V: Therapy Development
Chapter 16: Epigenetic Priming and Development of New Combination Therapy Approaches
1 Introduction
2 Materials
2.1 Urothelial Cancer Cell Lines, Control Cell Lines, Cell Culture Media, and Materials
2.2 Pharmacological Compound Treatment
2.3 Assessing Treatment Effects in UCC and Control Cells
2.3.1 MTT Assay for Analysis of Cell Viability and IC50 Values
2.3.2 Colony Forming Assay
2.3.3 Analysis of Caspase-Dependent Induction of Apoptosis
2.3.4 Assessment of Senescence Induction by Flow Cytometry (FACS)
2.3.5 Quantification of Cells Undergoing Mitosis by Flow Cytometry
2.3.6 Immunofluorescence Staining of DNA Damage
2.4 Assessing Treatment Effects In Vivo
2.4.1 Establishment of the Subcutaneous Cell Line-Derived Xenograft Model
2.4.2 Treatment of the Mice
2.4.3 Collection of the Tumors
3 Methods
3.1 Cell Culture and Seeding for Experiments
3.2 Pharmacological Compound Treatment
3.3 Assessing Treatment Effects in UCC and Control Cells
3.3.1 MTT Assay for Analysis of Cell Viability and IC50 Values
3.3.2 Colony Forming Assay
3.3.3 Analysis of Caspase-Dependent Induction of Apoptosis
3.3.4 Assessment of Senescence Induction by Flow Cytometry (FACS)
3.3.5 Quantification of Cells Undergoing Mitosis by Flow Cytometry
3.3.6 Immunofluorescence Staining DNA Damage
3.4 Assessing Treatment Effects In Vivo
3.4.1 Establishment of the Subcutaneous Cell Line-Derived Xenograft
3.4.2 Treatment of the Mice
3.4.3 Collection of the Tumors
4 Notes
References
Chapter 17: Evaluation of FGFR Alteration Status in Urothelial Tumors
1 Introduction
2 Materials
2.1 SNaPshot Analysis of Nine Hot Spot FGFR3 Point Mutations
2.1.1 Multiplex Polymerase Chain Reaction (PCR)
2.1.2 First Digestion
2.1.3 SNaPshot PCR
2.1.4 Second Digestion
2.1.5 Preparation for Analysis Using the Applied Biosystems 3500 Genetic Analyzer
2.2 QIAGEN Therascreen FGFR RGQ RT-PCR Kit
3 Methods
3.1 SNaPshot Analysis of Nine Hot Spot FGFR3 Point Mutations
3.1.1 Multiplex PCR
3.1.2 First Digestion
3.1.3 SNaPshot PCR
3.1.4 Second Digestion
3.1.5 Analysis Using ABI 3500 Genetic Analyzer
3.2 QIAGEN Therascreen FGFR RGQ RT-PCR Kit
4 Notes
5 Conclusive Remarks
References
Chapter 18: Antibody-Drug-Conjugates (ADC): A Novel Treatment Option in Urothelial Carcinoma
1 Introduction
2 Structure and Mode of Action
3 Monoclonal Antibody
4 Linker
5 Payload
6 Clinical Use of ADCs in Urothelial Carcinoma
7 Considerations for Preclinical and Translational Research
References
Chapter 19: Intravesical Infusion of Oncolytic Virus CG0070 in the Treatment of Bladder Cancer
1 Introduction
1.1 CG0070
1.2 Retinoblastoma (Rb) Pathway
1.3 GM-CSF
1.4 Xenograft Models
1.5 Clinical Trials Using CG0070
2 Materials
2.1 General Supplies for Storage and Preparation
2.2 General Supplies for Administration
2.3 Syringes for Administration
3 Methods
3.1 Using a 2-Way Catheter
3.2 Using a 3-Way Catheter
4 Notes
5 Conclusions
References
Chapter 20: Analysis of ICAM-1 Expression on Bladder Carcinoma Cell Lines and Infectivity and Oncolysis by Coxsackie Virus A21
1 Introduction
1.1 Current Treatment for Bladder Cancer
1.2 Novel Treatments for Bladder Cancer
1.3 Oncolytic Viruses as Novel Treatments for Bladder Cancer
1.4 The Oncolytic Virus Coxsackie A (CVA21) Requires ICAM-1 Receptors on the Cell Surface for Entry into Bladder Cancer Cells
2 Materials
2.1 Bladder Cancer Cell Lines
2.2 Reagents
2.3 Virus
3 Methods
3.1 QuantiBRITE: Expression of ICAM-1 Receptors on Cancer Cell Lines
3.2 Dilution of Working Virus Concentration
3.3 Incubation of Cells with Oncolytic Virus and Assessment of Cell Viability Using MTS Assay
4 Notes
References
Index