Next Generation Sequencing (NGS) in the Clinic – Considerations for

Molecular Pathologists

Jane Gibson, Ph.D., FACMGProfessor of Pathology

Director of Molecular DiagnosticsUniversity 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

Transformative step

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

solutions

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 BiosciencesHelicos BiosciencesNABsysVisiGen BiotechnologiesComplete GenomicsOxford Nanophore Technologies

NGS Application Examples-Inherited Conditions

Discovery tool: Single gene disordersi.e. AD – Kabuki syndrome (MLL)

Causative mutations for multigenicdiseases –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

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 variantsoften unclear- less testing

vs. monogenic disease

Reference human genomecataloguing of variants =

more test offerings

NGS Application Examples-Neoplastic Conditions

Cancer susceptibility genes

Risk assessmentRisk 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

MethylationEpigenetic changes

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 tumorDNA 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 DNAsequence changes

DNA copy number variations

DNA rearrangements

RNA expressionprofiles Methylation

A single method usually provides only part of this variety of

information - cost , specimen type, and application considerations

important

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

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

NGS data density = frequently encountered

variants of unknown significance

Which variants are clinically actionable?

Development of evidence-basedscientific standards to evaluate

utility in in different patient populations for accurate

risk estimation

Risk of over interpretationunnecessary medical action

unwarranted psychological stress

Careful selection of patients forgenome 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 (alignment and assembly)

QC of image processing, 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…….