Clinical Cancer Genotyping Using Next-Generation Sequencing
Long Phi Le, MD, PhD
Department of Pathology Center for Integrated Diagnostics Massachusetts General Hospital
Boston, MA [email protected]
Disclosures
Co-Founder of ArcherDx Enzymatics: Equity holder, Consultant Broad Clinical Research Sequencing Platform: Medical Director
EGFR: Erlotinib/ Gefitinib 20% Lung adenocarcinomas
ALK: Crizotinib 3-5% Lung adenocarcinoma
BRAF V600E: PLX4032 60% Melanoma
BRAF
1799 T>A
V600E
KRAS: Cetuximab resistance 36-50% Colon adenocarcinoma
Molecular Markers and Targeted Therapy
Identification of PF2341066
PF2341066 Inhibits ALK activity
PF2341066 demonstrates cytocidal activity in cells exhibiting ALK fusion (Pfizer in house)
PF2341066 activity in cells exhibiting ALK fusion in broad screen (MGH-McDermott)
Discovery of EML4-ALK fusions in NSCLC (CREST) Japan Science & Technology Agency)
2007
PF2341066 FIP
May
2005 2006 2008 2009
Objective responses demonstrated in ALK fusion positive NSCLC and IMT
Phase III study of PF02341066 in ALK positive NSCLC starts
Slide Courtesy of Ross Camidge
Timeline for PF2341066 and ALK in NSCLC
Considerations in Clinical Genotyping Platform
Logistics • Clinical patient coordinator • Accessioning • Tracking • Automation • Test all tumors
Clinical Test • Archived FFPE tissue • Analytical sensitivity (5%) • 2 week turnaround time • Report in patient’s record • Performed in a CLIA lab
Actionable Targets • Predicts response/resistance • Clinches diagnosis • Yields prognosis • Stratify patients for trials • Adaptability for new targets
Other • Economics • Insurance and billing • Translational research • Bioinformatics • Patient Consent
ddNTP
ddNTP
ddNTP
loci of interest
Multiplex PCR Single Base Extension Reaction Capillary Electrophoresis
Electrophoretic Output
Increasing
molecular weight
Rela
tive
flu
ore
sce
nce
A B D C F E
SNaPshot Genotyping
KRAS 23%
No Mutation 42%
EGFR 15%
TP53 5%
IDH1 <1%
NRAS 1% BRAF 2%
HER2 2%
PIK3CA 4%
ALK 3%
CTNNB1 2%
AKT 1%
N=650
Lung Cancers: SNaPshot
More Than Just Point Mutations
copy number indels
SNVs
epigenetics
non-coding RNAs
proteomics
gene expression
rearrangements
MGH Center for Integrated Diagnostics
• HER2, EGFR, MET, PDGFRA, FGFR1, PIK3CA (many tumors)
• Oligodendroglioma 1p/19q FISH analysis (glioma)
• Sarcoma translocation FISH: Synovial Sarcoma, Ewing’s Sarcoma, Alveolar Rhabdomyosarcoma, Myxoid Liposarcoma • ALK, RET, ROS1 FISH (lung) • MYC FISH (lymphoma) (all FISH: 60-100)
• Hereditary Hemochromatosis/HFE (heme, 5-10)
• MGMT promoter methylation (glioma, 1-5) • Microsatellite instability (colon, 1-3) • SNAPSHOT-NGS (many tumors, 40-60)
• Chimerism (BMT transplant, 8-15)
• Array CGH (ASD, MCA, ID, 4-8)
• NGS AMP-Translocation V1 (10-15)
• Cancer genetics has rapidly expanded with high complexity • Demands for clinical molecular profiling face some challenges: many targets, limited sample quantity/quality, fast turnaround time, uncertain reimbursement • Continued need for higher-throughput cancer genotyping
Summary
Started in 1990, initially led by James Watson and then Francis Collins took over in 1993
International effort (US, China,
France, Germany, Japan, UK)
Public: $2.7 billion effort (FY 1991) funded by the NIH, Wellcome Trust, and other groups (13 years)
Private: $300 million effort led by
Craig Venter and Celera Genomics
(3 years)
The Human Genome Project
NGS Assays Comparison
Assay Sample Input Cost Turnaround
Time Informatics Interpretation Discovery Potential
Hotspot + TSP + $ 1-2 weeks + + +
Rearrangement + $ 1-2 weeks + + +
100-200 Gene Panel ++ $$ 2-4 weeks +++ +++ ++
1000+ Gene Panel ++ $$$ 2-4 weeks ++++ ++++ ++++
Exome ++ $$$$$ 3-4 weeks +++++ ++++++ ++++++
Whole Genome +++ $$$$$$$ 4-8 weeks ++++++++ +++++++++++ ++++++++
Tiered Clinical Cancer Genotyping
Disease Progression
2nd Tier Result 1st Tier
Hotspot+
Rearrangement
$
Positive None
Comprehensive Panel
$$$
Negative
Comprehensive Panel
$$$
Comprehensive Panel
$$$
Target Capture Technologies
Solution-phase Capture
Microdroplet PCR
Reaction Array
TruSeq Amplicon
Haloplex
Ion Torrent AmpliSeq
Cancer: More Than Just Point Mutations
copy number indels
SNVs
epigenetics
non-coding RNAs
proteomics
gene expression
rearrangements
Detecting Rearrangements Status Quo
Immunohistochemistry
- Antigen expression
in tumor but not in
normal
- Ab availability
- Ab specificity
- Qualitative analysis
FISH
- Technically
challenging
- Expert analysis
- Poor scalability and
multiplexing ability
RT-PCR
- Requires knowledge
of both partners
- Limited scalablity
and multiplexing
ability
Hotspot+ Panel
Clinical targeted sequencing of FFPE DNA
• ~370 amplicon targets w/ bi-directional coverage (hotspots + TP53 + PTEN + CDKN2A)
• 1000X median coverage
• 4-500 Mb data per tumor
• 2-5% analytical sensitivity
• 2-week turnaround time
Desired
• Sensitivity for hotspots
• SNV, indel, copy number
The Problem
Report
Results
Worksheets
Path Review
Requisition
Consent
Path Report
* 1 Patient with 1 surgical specimen may have 6+ different tests
Office/Lab
MGH MDX and TRL Informatics 2005-2012
PowerPath
CoPath TRL DB
FISH SpreadSheet
GeneInsight
NAE SpreadSheet
File Maker Post-it Notes
Clinical NGS Workflow
Order Sample
Submission Pre-
Analytical Wet Lab Sequencing
Analysis Variant Store
Variant Filtering
Variant Vetting
Variant Curation
Reporting Decision Support
Action Outcome New
Knowledge
Clinical NGS Workflow
Order Sample
Submission Pre-
Analytical Wet Lab Sequencing
Analysis Variant Store
Variant Filtering
Variant Vetting
Variant Curation
Reporting Decision Support
Action Outcome New
Knowledge
CIDWikiLIMS
Pipeline VarVault VarVetter WikiVar
CIDPortal
4th Interpretation
Variant1 1st
Interpretation 2nd
Interpretation 3rd
Interpretation
NGS Variant Interpretation Complexity
Variant2
Variant3
(Versioned)
• Next-generation sequencing offers the throughput and scalability to achieve broad-based clinical cancer genotyping
• Challenges in broad deployment:
• Single gene tests vs. panel testing
• Complex assay validation for multi-gene/target tests
• Complex variant assessment/interpretation with dynamic evidence
• Lack of end-to-end informatics solution
• Complex reporting
• NGS reimbursement still not clear
• FDA regulation of laboratory developed tests (LDTs)
Summary