Molecular applications in cytology

Description

Preface Contents 1: Why Cytology for Molecular Testing? Pros and Cons 1.1 Introduction 1.2 Little Material for Many Analyses 1.3 Analysis Based on Extracted DNA 1.4 FISH Analysis 1.5 Immunohistochemistry 1.6 Diversity of Cytological Specimens 1.7 Professional Opportunity for Cytotechnicians References 2: How to Prepare Cytological Samples for Molecular Testing 2.1 Introduction 2.2 The Cytopathologist’s Role 2.3 Test Request 2.4 The Integrated and Standardized Cytopathology-­Molecular Report 2.5 Reference Laboratories 2.6 Cell Block 2.7 Direct Smears 2.8 Liquid-Based Cytology 2.9 Fresh Cells 2.10 Nucleic Acid Sample Quantity and Quality Assessment 2.11 DNA Input 2.12 Cytopathology Informatics and Bioinformatics 2.13 Future Directions References 3: Molecular Tests Use in Cytological Material (Analytical Phase) 3.1 Introduction 3.2 qPCR and RT-PCR 3.2.1 Background 3.2.1.1 qPCR with dsDNA-Binding Dyes as Reporters 3.2.1.2 Fluorescent Reporter Probe Method 3.2.2 Data Analysis 3.3 FISH 3.3.1 Background 3.3.2 Process of Preparation and Hybridization 3.3.3 Data Analysis 3.4 Sequencing 3.4.1 DNA Sequencing 3.4.2 RNA Sequencing 3.4.3 Large-Scale Sequencing 3.5 Next-Generation Sequencing (NGS) 3.5.1 Emulsion or Droplet Digital PCR: A Tool for Single Fragment Clonal Amplification 3.5.2 454 Pyrosequencing 3.5.3 Illumina (Solexa) Sequencing 3.5.4 SOLiD Sequencing 3.5.5 Ion Torrent Semiconductor Sequencing 3.5.6 Single-Molecule Real-Time Sequencing (Pacific Biosciences) 3.5.7 Cytological Implementation of NGS 3.6 Others 3.6.1 Comparative Genomic Hybridization (CGH) and Array Comparative Genomic Hybridization (aCGH) 3.6.2 SNP Array 3.6.3 Combined SNP and CGH Array 3.6.4 NanoString Method 3.7 Perspectives References 4: Molecular Cytology Applications on Head and Neck 4.1 Molecular Cytology Applications in the Salivary Gland 4.1.1 Background 4.1.2 Ancillary Studies in Salivary Gland Cytology 4.1.3 Translocations and Fusion Oncogenes in Salivary Gland Tumors 4.1.3.1 Pleomorphic Adenoma and Carcinoma Ex Pleomorphic Adenoma 4.1.3.2 Mucoepidermoid Carcinoma 4.1.3.3 Adenoid Cystic Carcinoma 4.1.3.4 Polymorphous Low Grade Adenocarcinoma 4.1.3.5 Mammary Analogue Secretory Carcinoma 4.1.3.6 Hyalinizing Clear Cell Carcinoma 4.1.3.7 NUT (Nuclear Protein in Testis) Midline Carcinoma (NMC) 4.1.4 Selected Immunohistochemical Stains in the FNA Diagnosis of Salivary Gland Tumors 4.1.4.1 Basaloid Neoplasms 4.1.4.2 Salivary Gland Tumors with Oncocytic Features 4.2 HPV-Associated Head and Neck Squamous Cell Carcinoma 4.2.1 Background 4.2.1.1 Cytologic Evaluation of HPV-Related HNSCC 4.3 Detection Methods for HPV in Cytologic Material 4.3.1 Conventional Methods for HPV Testing Using FFPE Material 4.3.2 p16 Immunohistochemistry 4.3.3 Polymerase Chain Reaction-Based Techniques 4.3.4 DNA and RNA In Situ Hybridization 4.3.5 Combination of Different Testing Methods References 5: Molecular Cytology Applications on the Lung 5.1 Diagnosis 5.2 Sampling 5.3 Pre-Analytical Phase 5.4 Sequencing 5.5 FISH 5.6 RT-PCR 5.7 Next-Generation Sequencing (NGS) 5.8 Immunotherapy References 6: Molecular Cytology of Serous Effusions 6.1 Introduction 6.2 Molecular Tests Applied to Effusion Diagnosis 6.3 Molecular Tests Applied to Effusions as Predictive Test 6.3.1 HER2 Status 6.3.2 EGFR and Related Molecules 6.4 Future Directions References 7: Molecular Cytology Applications on Urine 7.1 Introduction 7.2 General Overview on Molecular Urine Markers 7.3 Ancillary UroVysion FISH Testing for Clarification of Equivocal Urothelial Cell Atypia 7.4 Optimized Criteria Defining a FISH-Positive Result 7.5 Pre-analytic and Analytic Procedures 7.6 Targeted FISH Scoring References 8: Molecular Cytology Applications on Gynecological Cytology 8.1 Introduction 8.2 Classification of Cervical Lesions 8.2.1 Squamous Lesions 8.2.2 Glandular Lesions of the Cervix 8.3 Biomarkers in Cervical Cytology 8.3.1 Biomarkers of the Productive Phase of the HPV-Induced Carcinogenesis 8.3.2 Biomarkers of the Proliferative Phase of the HPV-Induced Carcinogenesis 8.4 Technical Aspects of the Use of Biomarkers in Cervical Cytology 8.5 Molecular Application in Cervical Samples 8.6 HPV Molecular Markers 8.6.1 HPV Genotyping 8.6.2 mRNA 8.7 Molecular Markers Involved in Cell Cycling 8.7.1 Viral Load and Integration 8.8 Molecular Markers Related to Epigenetic Changes 8.8.1 DNA Methylation 8.9 MiRNA 8.10 Cervical Microbiota References 9: Molecular Applications in Hematolymphoid Cytology 9.1 Introduction 9.2 Handling and Triage of Lymphoid Specimens 9.3 Historical Perspective: Cytology and Ancillary Tests 9.4 In Situ Hybridization (ISH) in Hematolymphoid Cytopathology 9.5 Polymerase Chain Reaction (PCR) in Hematolymphoid Cytopathology 9.6 From Discovery Tools to Clinical Use: Next-Generation Sequencing and Other Technologies References 10: Molecular Cytology Application on Thyroid 10.1 Introduction 10.2 Thyroid Lesions 10.2.1 Molecular Markers 10.2.1.1 BRAF 10.2.1.2 RAS 10.2.1.3 RET/PTC 10.2.2 PAX8/Peroxisome Proliferator-Activated Receptor Gamma (PAX8/PPARγ) 10.2.2.1 HMGA2 10.2.2.2 UbcH10 10.2.3 Diagnostic and Prognostic Involvement of Molecular Alterations in the Different Thyroid Lesions 10.2.4 LBC and Molecular Techniques 10.3 The Role of Molecular Testing in Follicular Cell Lesions 10.3.1 From the 7-Gene Panel Test to Next Generation Sequencing (NGS) Tests 10.3.2 Gene Expression Classifiers (GECS) 10.4 The Role of Molecular Testing in Parafollicular Cell Lesions 10.5 The Diagnostic Role of miRNAs in Follicular and Parafollicular Neoplasms 10.6 How to Approach Thyroid Nodules with Ancillary Techniques 10.7 The Future of Molecular Thyroid Cytology References 11: Molecular Cytology Applications on Pancreas and Biliary Tract 11.1 EUS-FNA of Pancreas for Molecular Cytopathology 11.1.1 Molecular Landscape of Solid Pancreatic Neoplasms 11.1.2 Molecular Testing of Solid Pancreatic Neoplasms 11.1.3 Immunocytochemistry in Solid Pancreatic Neoplasms 11.1.4 Evaluation of Pancreatic Cystic Lesions 11.1.5 Molecular Testing of Pancreatic Cystic Lesions 11.1.6 Limitations to Molecular Testing in Pancreatic Lesions 11.1.7 Recommendations 11.1.8 Future Directions 11.2 Biliary Tract Sampling for Molecular Cytopathology 11.2.1 Evaluation of Biliary Tract Specimens 11.2.2 Molecular Testing of Biliary Tract Specimens 11.2.3 Limitations 11.2.4 Recommendations 11.2.5 Future Directions References 12: Molecular Cytology Applications in Soft Tissue 12.1 Introduction 12.2 Overview of Molecular Testing in Soft Tissue Tumors 12.2.1 Fluorescence In Situ Hybridization (FISH) 12.2.2 Real-Time PCR (RT-PCR) 12.2.3 Next-Generation Sequencing (NGS) 12.3 Handling and Triage of Cytology Specimens 12.4 Summary of Molecular Alterations in Different Morphological Patterns of Soft Tissue Tumors 12.4.1 Small Round Blue Cell Tumors 12.4.1.1 Rhabdomyosarcoma Alveolar Rhabdomyosarcoma Embryonal Rhabdomyosarcoma 12.4.1.2 Ewing Sarcoma/PNET 12.4.1.3 Desmoplastic Small Round Cell Tumor 12.4.2 Epithelioid Neoplasms 12.4.2.1 Epithelioid Sarcoma 12.4.2.2 Alveolar Soft Part Sarcoma 12.4.3 Spindle Cell Neoplasms 12.4.3.1 Inflammatory Myofibroblastic Tumor 12.4.3.2 Giant Cell Fibroblastoma/Dermatofibrosarcoma Protuberans 12.4.3.3 Congenital/Infantile Fibrosarcoma 12.4.3.4 Synovial Sarcoma 12.4.3.5 Spindle Cell Melanoma 12.4.4 Myxoid Neoplasms 12.4.4.1 Nodular Fasciitis 12.4.4.2 Extraskeletal Myxoid Chondrosarcomas 12.4.5 Adipocytic Neoplasms 12.4.5.1 Myxoid Liposarcoma 12.4.6 Pediatric Soft Tissue Neoplasms 12.5 Summary References 13: Molecular Cytology Applications in Metastases 13.1 Why Study Metastatic Disease? 13.2 Molecular Techniques Applied to Metastatic Disease Study 13.3 Role of Cytology in the Management of Metastatic Cancer 13.4 Conclusions and Future Directions References 14: Clinical Integration of Molecular Results on Cytology (Post-analytical Phase) 14.1 Why Clinical Integration Is Necessary 14.2 Levels of Clinical Integration 14.3 Clinical Integration: Is the Material Technically Adequate? 14.3.1 Total Cellularity 14.3.2 Percentage of Malignant Cells 14.3.3 Certainties in the Original Cytopathological Diagnosis 14.4 Where Training Is the Solution 14.5 Where There Is a Minimal Report Content References