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
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
 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
 Cost of NGS

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

Transformative step
 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 Pacific Biosciences
and laborious- serial “gene by Helicos Biosciences
gene” analysis NABsys
VisiGen Biotechnologies
Complete Genomics
Oxford Nanophore Technologies
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
 Inherited Conditions-
Challenges and Opportunities
Challenges Opportunities

Example: Example:
Monogenic disorders Multifactorial disease

Risk loci more often in Pathogenicity of variants

Novel missense mutations Germ line mosaicism
non-coding often unclear- less testing
or inter-gene regions vs. monogenic disease
Structural aberrations Imprinting effects
Reference human genome
Epigenetic factors cataloguing of variants =
more test offerings
 NGS Application Examples-
Neoplastic Conditions
Cancer susceptibility genes
Patient stratification
Risk assessment
Risk management Predictions of therapeutic
response
Somatic/driver mutations personalized treatment

Therapeutic monitoring
Micro-RNAs

Prognosis
Methylation
Epigenetic changes
Alterations in gene expression

Molecular profiling

Tumor sub-typing
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
 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
 NGS Application Examples-
Other Considerations
Different NGS platforms have different capabilities

RNA and DNA DNA copy number DNA

sequence changes variations rearrangements

RNA expression
Methylation
profiles

A single method usually provides

only part of this variety of
information - cost , specimen type,
and application considerations
important
 NGS Application Examples-
Other Considerations
Mutation confirmation
NGS- significant false Usually by Sanger sequencing-will
positive rate platform evolution eliminate?

Variable % tumor cells May overlap with NGS

false positive rate
and variable % tumor
cells with (presumably) Low level mutations- not easily
confirmed by Sanger sequencing
secondary mutation (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
 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
 Clinical Utility-Challenges

Which variants are

clinically actionable?

Development of evidence-based
NGS data density = scientific standards to evaluate
utility in in different patient
frequently encountered populations for accurate
variants of unknown risk estimation

significance Risk of over interpretation

unnecessary medical action
unwarranted psychological stress

Careful selection of patients for

genome sequencing and
genetic counseling-crucial
 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
 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
 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
 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)
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 QC of image processing,

(alignment and assembly) base calling, filtering,
alignment, SNP
finding/application steps
archiving
 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?)
 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
 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?
 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
 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…….