Main description:

By integrating technology, supporting infrastructure and efficient application, the all-in-one guide presents molecular diagnostics as an essential component of modern, personalized clinical practice. It considers all important aspects, from the hardware and software needed, to recent improvements in blood- and non-blood-based biomarker tests. Chapters on ethical challenges and a look at current trends and the latest innovations are also included. Bridging the gap between industry and academia, this is a highly useful resource for practitioners as well as for developers of modern, DNA- and RNA-based molecular diagnostics.

Contents:

List of Contributors XIII Preface XIX 1 Next-Generation Sequencing for Clinical Diagnostics of Cardiomyopathies 1 Jan Haas, Hugo A. Katus, and Benjamin Meder 1.1 Introduction 1 1.2 Cardiomyopathies and Why Genetic Testing is Needed 1 1.3 NGS 2 1.4 NGS for Cardiomyopathies 2 1.5 Sample Preparation 3 1.6 Bioinformatics Analysis Pipeline 4 1.7 Interpretation of Results and Translation into Clinical Practice 4 References 6 2 MicroRNAs as Novel Biomarkers in Cardiovascular Medicine 11 Britta Vogel, Hugo A. Katus, and Benjamin Meder 2.1 Introduction 11 2.2 miRNAs are Associated with Cardiovascular Risk Factors 12 2.3 miRNAs in Coronary Artery Disease 13 2.4 miRNAs in Cardiac Ischemia and Necrosis 15 2.5 miRNAs as Biomarkers of Heart Failure 19 2.6 Future Challenges 20 Acknowledgments 20 References 21 3 MicroRNAs in Primary Brain Tumors: Functional Impact and Potential Use for Diagnostic Purposes 25 Patrick Roth and Michael Weller 3.1 Background 25 3.2 Gliomas 26 3.2.1 miRNA as Biomarkers in Glioma Tissue 28 3.2.2 Circulating miRNA as Biomarkers 29 3.3 Meningiomas 30 3.4 Pituitary Adenomas 31 3.5 Medulloblastomas 31 3.6 Other Brain Tumors 32 3.6.1 Schwannomas 32 3.6.2 PCNSLs 33 3.7 Summary and Outlook 33 References 34 4 Genetic and Epigenetic Alterations in Sporadic Colorectal Cancer: Clinical Implications 39 Pawel Karpinski, Nikolaus Blin, and Maria M. Sasiadek 4.1 Introduction 39 4.2 Chromosomal Instability 40 4.3 Microsatellite Instability 43 4.4 Driver Somatic Mutations in CRC 46 4.4.1 APC 46 4.4.2 TP53 47 4.4.3 KRAS 47 4.4.4 BRAF 47 4.4.5 PIK3CA 48 4.4.6 Other Mutations 48 4.5 Epigenetic Instability in CRC 48 4.6 Hypomethylation 49 4.7 CpG Island Methylator Phenotype 50 4.8 Concluding Remarks 51 References 51 5 Nucleic Acid-Based Markers in Urologic Malignancies 63 Bernd Wullich, Peter J. Goebell, Helge Taubert, and Sven Wach 5.1 Introduction 63 5.2 Bladder Cancer 64 5.2.1 Hereditary Factors for Bladder Cancer 65 5.2.2 Single Nucleotide Polymorphisms 65 5.2.3 RNA Alterations in Bladder Cancer 66 5.2.3.1 FGFR3 Pathway 66 5.2.3.2 p53 Pathway 67 5.2.3.3 Urine-Based Markers 67 5.2.3.4 Serum-Based Markers 68 5.2.4 Sporadic Factors for Bladder Cancer 69 5.2.5 Genetic Changes in Non-Invasive Papillary Urothelial Carcinoma 69 5.2.5.1 FGFR 3 69 5.2.5.2 Changes in the Phosphatidylinositol 3-Kinase Pathway 70 5.2.6 Genetic Changes in Muscle-Invasive Urothelial Carcinoma 72 5.2.6.1 TP53, RB, and Cell Cycle Control Genes 73 5.2.6.2 Other Genomic Alterations 74 5.2.7 Genetic Alterations with Unrecognized Associations to Tumor Stage and Grade 75 5.2.7.1 Alterations of Chromosome 9 75 5.2.7.2 RAS Gene Mutations 76 5.3 Prostate Cancer 77 5.3.1 Hereditary Factors for Prostate Cancer 77 5.3.2 Sporadic Factors for Prostate Cancer 80 5.3.2.1 PSA and Other Protein Markers 80 5.3.2.2 Nucleic Acid Biomarkers 81 5.3.3 Prostate Cancer: Summary 87 5.4 Renal Cell Carcinoma 87 5.4.1 Hereditary Factors for RCC 87 5.4.2 Sporadic Factors for RCC 90 5.4.2.1 The Old 90 5.4.2.2 The New 91 5.5 Summary 92 References 96 6 From the Genetic Make-Up to the Molecular Signature of Non-Coding RNA in Breast Cancer 129 Michael G. Schrauder and Reiner Strick 6.1 Introduction 129 6.2 Molecular Breast Cancer Detection 130 6.2.1 Circulating Free DNA 130 6.2.2 Long Intergenic Non-Coding RNA 132 6.2.2.1 HOTAIR 132 6.2.2.2 H19 133 6.2.2.3 GAS5 134 6.2.2.4 LSINCT5 134 6.2.2.5 LOC554202 134 6.2.2.6 SRA1 134 6.2.2.7 XIST 134 6.2.3 Natural Antisense Transcripts 135 6.2.3.1 HIF-1a-AS 136 6.2.3.2 H19 and H19-AS (91H) 137 6.2.3.3 SLC22A18-AS 137 6.2.3.4 RPS6KA2-AS 137 6.2.3.5 ZFAS1 137 6.2.4 miRNAs 138 6.2.4.1 Tissue-Based miRNA Profiling in Breast Cancer 138 6.2.4.2 Circulating miRNAs 141 6.3 Molecular Breast Cancer Subtypes and Prognostic/Predictive Molecular Biomarkers 142 References 144 7 Nucleic Acid-Based Diagnostics in Gynecological Malignancies 155 Sebastian F.M. Hausler, Johannes Dietl, and Jorg Wischhusen 7.1 Introduction 155 7.2 Cervix, Vulva, and Vaginal Carcinoma 155 7.2.1 Background 155 7.2.2 Routine Diagnostics for HPV Infection 157 7.2.2.1 Digene Hybrid Capture 2 High-Risk HPV DNA Test (Qiagen) 158 7.2.2.2 Cervista HPV HR (Holologics) 158 7.2.2.3 cobas 4800 System (Roche) 159 7.2.2.4 APTIMA HPV (Gen-Probe) 159 7.2.2.5 Abbot RealTime High Risk HPV Assay (Abbot) 159 7.2.2.6 PapilloCheck Genotyping Assay (Greiner BioOne) 160 7.2.2.7 INNO-LiPA HPVG enotyping Extra (Innogenetics) 160 7.2.2.8 Linear Array (Roche) 160 7.2.2.9 Recommendations for Clinical Use 160 7.2.3 Outlook DNA Methylation Patterns 161 7.3 Endometrial Carcinoma (Carcinoma Corpus Uteri) 162 7.3.1 Background 162 7.3.2 Routine Diagnostics Microsatellite Instability 162 7.3.3 Emerging Diagnostics miRNA Markers 163 7.4 Ovarian Carcinoma 164 7.4.1 Background 164 7.4.2 Routine Diagnostics 165 7.4.3 Emerging Diagnostics/Perspective miRNA Profiling 166 7.5 Breast Cancer 167 7.5.1 Background 167 7.5.2 Routine Diagnostics 168 7.5.2.1 HER2 Diagnostics 168 7.5.2.2 Gene Expression Profiling 169 7.5.2.3 Hereditary Breast Cancer/BRCA Diagnostics 170 7.5.3 Emerging Diagnostics/Perspectives 173 7.6 Conclusion 175 References 175 8 Nucleic Acids as Molecular Diagnostics in Hematopoietic Malignancies Implications in Diagnosis, Prognosis, and Therapeutic Management 185 Janine Schwamb and Christian P. Pallasch 8.1 Introduction 185 8.2 Methodological Approaches 186 8.3 Cytogenetic Analysis to Molecular Diagnostics 186 8.4 Minimal Residual Disease 186 8.5 Chronic Myeloid Leukemia 187 8.6 Acute Myeloid Leukemia 189 8.7 Acute Lymphocytic Leukemia 191 8.8 Chronic Lymphocytic Leukemia 192 8.9 Outlook and Perspectives 196 References 196 9 Techniques of Nucleic Acid-Based Diagnosis in the Management of Bacterial and Viral Infectious Diseases 201 Irene Latorre, Veronica Saludes, Juana Diez, and Andreas Meyerhans 9.1 Importance of Nucleic Acid-Based Molecular Assays in Clinical Microbiology 201 9.2 Nucleic Acid Amplification Techniques 202 9.2.1 Target Amplification Techniques 203 9.2.1.1 PCR-Based Techniques 203 9.2.1.2 Transcription-Based Amplification Methods 204 9.2.2 Signal Amplification Techniques 204 9.3 Post-Amplification Analyses 205 9.3.1 Sequencing and Pyrosequencing 205 9.3.2 Reverse Hybridization 206 9.3.3 High-Throughput Nucleic Acid-Based Analyses 206 9.3.3.1 DNA Microarrays 206 9.3.3.2 Mass Spectrometry 207 9.3.3.3 NGS 208 9.4 General Overview and Concluding Remarks 209 Acknowledgments 209 References 209 10 MicroRNAs in Human Microbial Infections and Disease Outcomes 217 Veronica Saludes, Irene Latorre, Andreas Meyerhans, and Juana Diez 10.1 Introduction 217 10.2 General Aspects of miRNAs in Infectious Diseases 218 10.2.1 miRNAs in Bacterial Infections 218 10.2.2 miRNAs in Viral Infections 219 10.2.2.1 Cellular miRNAs Control Viral Infections 220 10.2.2.2 Viruses Use miRNAs for Their Own Benefit 221 10.3 miRNAs as Biomarkers and Therapeutic Agents in Tuberculosis and Hepatitis C Infections 222 10.3.1 Tuberculosis: A Major Bacterial Pathogen 222 10.3.1.1 Tuberculosis Diagnosis and the Need for Immunological Biomarkers 222 10.3.1.2 miRNAs Regulation in Response to M. tuberculosis 223 10.3.1.3 Future Perspectives 225 10.3.2 Chronic Hepatitis C: A Major Viral Disease 225 10.3.2.1 Liver Fibrosis Progression and Treatment Outcome 225 10.3.2.2 miRNAs Involved in Liver Fibrogenesis 226 10.3.2.3 Prediction of Treatment Outcome in Chronic HCV-1 Infected Patients 228 10.3.2.4 Future Perspectives 229 10.4 miRNA-Targeting Therapeutics 230 10.5 Concluding Remarks 230 Acknowledgments 231 References 231 11 Towards the Identification of Condition-Specific Microbial Populations from Human Metagenomic Data 241 Cedric C. Laczny and Paul Wilmes 11.1 Introduction 241 11.2 Nucleic Acid-Based Methods in Diagnostic Microbiology 242 11.2.1 Limitations of Culture-Dependent Approaches 242 11.2.2 Culture-Independent Characterization of Microbial Communities 243 11.2.3 Metagenomics 243 11.2.4 Fecal Samples as Proxies to Evaluate Human Microbiome-Related Health Status 244 11.3 Need for Comprehensive Microbiome Characterization in Medical Diagnostics 244 11.4 Challenges for Metagenomics-Based Diagnostics: Read Lengths, Sequencing Library Sizes, and Microbial Community Composition 248 11.5 Deconvolution of Population-Level Genomic Complements from Metagenomic Data 250 11.5.1 Reference-Dependent Metagenomic Data Analysis 251 11.5.1.1 Alignment-Based Approaches 251 11.5.1.2 Sequence Composition-Based Approaches 253 11.5.2 Reference-Independent Metagenomic Data Analysis 254 11.6 Need for Comparative Metagenomic Data Analysis Tools 256 11.6.1 Reference-Based Comparative Tools 257 11.6.2 Reference-Independent Identification of Condition-Specific Microbial Populations from Human Metagenomic Data 257 11.7 Future Perspectives in Microbiome-Enabled Diagnostics 258 Acknowledgments 262 References 262 12 Genome, Exome, and Gene Panel Sequencing in a Clinical Setting 271 Claudia Durand and Saskia Biskup 12.1 Introduction 271 12.1.1 Genetic Inheritance and Sequencing 271 12.1.2 Genetic Testing by DNA Sequencing 272 12.2 Genetic Diagnostics from a Laboratory Perspective From Sanger to NGS 273 12.2.1 Sanger Sequencing 273 12.2.2 NGS 274 12.2.3 Practical Workflow: From a Patient s DNA to NGS Sequencing Analysis 276 12.2.3.1 Preparation of gDNA 277 12.2.3.2 Quality Control 277 12.2.3.3 Library Preparation and Evaluation 277 12.2.3.4 Enrichment 277 12.2.3.5 Quality Control 278 12.2.3.6 Sequencing 278 12.3 NGS Diagnostics in a Clinical Setting Comparison Between Genome, Exome, and Panel Diagnostics 279 12.3.1 Overview 279 12.3.2 Clinical Application of WGS 279 12.3.3 Clinical Application of WES 282 12.3.4 Clinical Application of Diagnostic Panels 284 12.4 Conclusion and Outlook 287 References 289 13 Analysis of Nucleic Acids in Single Cells 291 Stefan Kirsch, Bernhard Polzer, and Christoph A. Klein 13.1 Introduction 291 13.2 Isolating Single Cells 291 13.3 Looking at the DNA of a Single Cancer Cell 292 13.4 Molecular DNA Analysis in Single Cells 294 13.5 Approaches to Analyze RNA of a Single Cell 296 13.6 Expression Analysis in Single Cells and its Biological Relevance in Cancer 299 13.7 Thoughts on Bioinformatics Approaches 300 13.8 Future Impact of Single-Cell Analysis in Clinical Diagnosis 301 References 303 14 Detecting Dysregulated Processes and Pathways 309 Daniel Stockel and Hans-Peter Lenhof 14.1 Introduction 309 14.2 Measuring and Normalizing Expression Profiles 311 14.2.1 Microarray Experiments 311 14.2.2 Normalization 312 14.2.3 Batch Effects 314 14.3 Biological Networks 314 14.4 Measuring the Degree of Deregulation of Individual Genes 315 14.4.1 Microarray Data 316 14.4.2 RNA-Seq Data 317 14.5 Over-Representation Analysis and Gene Set Enrichment Analysis 318 14.5.1 Multiple Hypothesis Testing 320 14.5.2 Network-based GSEA Approaches 320 14.6 Detecting Deregulated Networks and Pathways 321 14.7 miRNA Expression Data 326 14.8 Differential Network Analysis 327 14.9 Conclusion 328 References 328 15 Companion Diagnostics and Beyond An Essential Element in the Puzzle of Transforming Healthcare 335 Jan Kirsten 15.1 Introduction 335 15.2 The Healthcare Environment 335 15.3 What is Companion Diagnostics? 336 15.4 What are the Drivers for Companion Diagnostics? 337 15.5 Companion Diagnostics Market 338 15.6 Partnerships and Business Models for Companion Diagnostics 341 15.7 Regulatory Environment for Companion Diagnostics Tests 342 15.8 Outlook Beyond Companion Diagnostics Towards Holistic Solutions 344 References 348 16 Ethical, Legal, and Psychosocial Aspects of Molecular Genetic Diagnosis 349 Wolfram Henn 16.1 General Peculiarities of Genetic Diagnoses 349 16.2 Informed Consent and Genetic Counseling 350 16.2.1 Testing of Persons with Reduced Ability to Consent 352 16.3 Medical Secrecy and Data Protection 354 16.4 Predictive Diagnosis 355 16.5 Prenatal Diagnosis 356 16.6 Multiparameter Testing 358 References 359 Index 361