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Next Generation Sequencing (NGS) in the Clinic
- 1. Next Generation Sequencing(NGS) in the Clinic – Considerations for Molecular Pathologists Jane Gibson, Ph.D., FACMG Professor of Pathology Director of Molecular Diagnostics University of Central Florida College of Medicine Chair, AMP Whole Genome Analysis Working Group
- 2. Opportunities and Challengesassociated with Clinical Diagnostic Genome Sequencing: A Report of the Association for Molecular Pathology **Iris Schrijver , Nazneen Aziz, Daniel H. Farkas, Manohar Furtado, Andrea Ferreira- Gonzalez, Timothy C. Greiner, Wayne W. Grody, Tina Hambuch, Lisa Kalman, Jeffrey A. Kant, Roger D. Klein, Debra G.B. Leonard, Ira M. Lubin, Rong Mao, Narasimhan Nagan, Victoria M. Pratt, Mark E. Sobel, Karl V. Voelkerding, Jane S. Gibson **The Whole Genome Analysis Working Group is a working group of the AMP Clinical Practice Committee
- 3. Goals • Key opportunitiesand challenges associated with clinically diagnostic genome sequencing • Application examples • Aspects of clinical utility, ethics and consent • Analytic and post-analytic considerations • Professional implications
- 4. Cost of NGS Transformativestep Innovations in chemistry, optics, fluidics computational hardware, and bioinformatics solutions
- 5. NGS Platforms • Differin design and chemistries • Fundamentally related-sequencing of thousands to millions of clonally amplified molecules in a massively parallel manner • Orders of magnitude more information-will continue to evolve • Attractive for clinical applications – individual sequencing assays costly and laborious- serial “gene by gene” analysis Pacific Biosciences Helicos Biosciences NABsys VisiGen Biotechnologies Complete Genomics Oxford Nanophore Technologies
- 6. NGS Application Examples- InheritedConditions Discovery tool: Single gene disorders i.e. AD – Kabuki syndrome (MLL) Causative mutations for multigenic diseases –superior to “one by one” approach of traditional sequencing Diagnostic advancements for diseases with overlapping symptoms, multiple possible syndromes/genes
- 7. Inherited Conditions- Challenges andOpportunities Challenges Example: Monogenic disorders Novel missense mutations Structural aberrations Germ line mosaicism Imprinting effects Epigenetic factors Opportunities Example: Multifactorial disease Risk loci more often in non-coding or inter-gene regions Pathogenicity of variants often unclear- less testing vs. monogenic disease Reference human genome cataloguing of variants = more test offerings
- 8. NGS Application Examples- NeoplasticConditions Cancer susceptibility genes Risk assessment Risk management Tumor sub-typing Micro-RNAs Prognosis Alterations in gene expression Molecular profiling Patient stratification Predictions of therapeutic response personalized treatment Therapeutic monitoring Somatic/driver mutations Methylation Epigenetic changes
- 9. NGS Application Examples- NeoplasticConditions • Mutation panel screening • Exome and transcriptome screening • Genome sequencing-comparison to normal tissue/reference sample Human genome project – reference genome and massive cataloguing of variants from different tumor sources (http://cancercommons.org, www.icgc.org and http://cancergenome.nih.gov/ Cost effective profiling of patient tumor DNA vs. mutation screening or profiling studies
- 10. NGS Analysis AndNeoplastic Conditions • Quantitative nature of NGS- improvement vs. chip technology • Gene expression tests- Mammaprint (70 genes), Oncotype DX (21 genes) and Rotterdam signature (76 genes) – replaced by NGS analysis of signature transcripts? • Germ line DNA characterization and somatic changes, transcriptome and methylation profiles - using a single, rapid and cost effective platform
- 11. NGS Application Examples- OtherConsiderations Different NGS platforms have different capabilities RNA and DNA sequence changes DNA copy number variations DNA rearrangements RNA expression profiles Methylation A single method usually provides only part of this variety of information - cost , specimen type, and application considerations important
- 12. NGS Application Examples- OtherConsiderations NGS- significant false positive rate Mutation confirmation Usually by Sanger sequencing-will platform evolution eliminate? Variable % tumor cells and variable % tumor cells with (presumably) secondary mutation May overlap with NGS false positive rate Low level mutations- not easily confirmed by Sanger sequencing (higher detection threshold ≈ 15-20%) without more sensitive mutation screening - DGGE, dHPLC, pyrosequencing or mutation enrichment- i.e. COLD PCR Numerous heterogeneous aberrations- i.e. oncologic applications need algorithm development
- 13. Clinical Utility • Balanceof net health benefits vs. harm • NGS –transformative for personalized treatment of disease • Clinical indication - includes test rationale, patient population and clinical scenarios • Principles of comparative effectiveness- requires individualized evidence-based approach for each patient
- 14. Clinical Utility-Challenges NGS datadensity = frequently encountered variants of unknown significance Which variants are clinically actionable? Development of evidence-based scientific standards to evaluate utility in in different patient populations for accurate risk estimation Risk of over interpretation unnecessary medical action unwarranted psychological stress Careful selection of patients for genome sequencing and genetic counseling-crucial
- 15. Informed Consent andEthical Considerations • Create patient awareness of benefits and harms • No specific guidance exists- institutional policies vary • Potential for anxiety and uncertainty exist especially for variants of unknown significance • Discovery of incidental findings unrelated to the disease in question
- 16. Analytical Considerations-Regulation, Assay Validation,and Reference Materials • FDA-lab developed tests (LDT)-validation • FDA-approved/cleared tests-verification • No FDA-cleared NGS tests at present-validation (LDT) must document that targeted analyte(s) can be detected in a robust and consistent manner CLIA regulations (CFR§493.1253) – accuracy, precision, analytical sensitivity, analytical specificity, reportable range, reference intervals, and other characteristics necessary for assay performance Considerable uncertainty regarding regulatory pathway for NGS tests
- 17. Analytical Considerations-Regulation, Assay Validation,and Reference Materials • Challenges: sequences are not truly complete – gaps in reads, GC rich regions, bioinformatics limitations with indel variant calling • “gold standard” comparison- Sanger sequencing, however the technical capabilities are dwarfed by NGS • Regardless - all NGS steps must be evaluated, and quality control metrics must be in place- is sequencing portions of a reference genome(s) sufficient? • Development of reference materials (RMs) for meaningful validation is key
- 18. Development of NGSGuidelines • Division of Laboratory Science and Standards (CDC) • Genetic Testing Reference Material Coordination Program (Get-RM) (CDC) http://www.cdc.gov/dls/genetics/rmmaterials/default.aspx • Clinical Laboratory Standards Institute (CLSI) • American College of Medical Genetics (ACMG) • College of American Pathologists (CAP) • Association For Molecular Pathology (AMP)
- 19. Bioinformatics NGS diagnostics -shifted towards data analysis rather than the technical component NGS infrastructures must consist of appropriate expertise and computational hardware Unprecedented amounts of medical data and various processing algorithms necessitate adequate tools for Data management (alignment and assembly) QC of image processing, base calling, filtering, alignment, SNP finding/application steps archiving
- 20. Bioinformatics-Other Considerations • Evaluationof the variant positions “called” involves queries of all known relevant databases • Lack of databases curated to accept clinical standards likely the most significant challenge in managing and reporting genome sequencing data • EHR considerations – test ordering, archiving of NGS reports, patient consent, data (reinterpretation?)
- 21. NGS-Post-Analytical Considerations • Expertinterpretation and guidance- correlation of age, gender, clinical presentation, family hx • Team approach ideal -pathologists, geneticists, other providers • Proficiency testing and alternative assessment are challenging • Proficiency testing schemes based on NGS methods vs. specific genes are likely
- 22. Professional Considerations- Reimbursement andGene Patents • Challenging reimbursement issues • AMA CPT editorial panel- proposed tier system of category 1 codes to replace stacking codes (83890-83914) • Genome sequencing may potentially involve numerous patented gene sequences • Development of an affordable system of common access to genes?
- 23. Genomics Education • Goal:provide trainees with solid grasp of current concepts within broad range of opportunities • AMP, CAP, ACMG and others working in this area • Training Residents in Genomics (TRIG)- curriculum designed to be adopted by any Pathology residency • Training needed outside the fields of Pathology and Genetics is needed
- 24. No longer anabstract concept for the future, the exciting reality of powerful genome testing has decisively arrived……. No longer an abstract concept for the future, the exciting reality of powerful genome testing has decisively arrived…….