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 Challenges associated 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 opportunities and 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
Innovations in chemistry, optics, fluidics computational hardware, and bioinformatics solutions
Transformative step

5. NGS Platforms Differ in 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- Inherited Conditions
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 and Opportunities
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- Neoplastic Conditions
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- Neoplastic Conditions
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 ( and
Cost effective profiling of patient tumor
DNA vs. mutation screening or profiling studies

10. NGS Analysis And Neoplastic 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- Other Considerations
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- Other Considerations
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 Balance of 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 data density = 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 and Ethical 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. Considerable uncertainty regarding regulatory
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§ ) – 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 NGS Guidelines
Division of Laboratory Science and Standards (CDC)
Genetic Testing Reference Material Coordination Program (Get-RM) (CDC)
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
Evaluation of 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
Expert interpretation 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 and Gene Patents
Challenging reimbursement issues
AMA CPT editorial panel- proposed tier system of category 1 codes to replace stacking codes ( )
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 an abstract 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…….