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

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
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-
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
(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 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. 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 NGS Guidelines
• 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
• 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
(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 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…….