Transcript Document

Clinical Application of Next Generation
Sequencing for Personalized Medicine in Solid
Tumors
Presented by: Ryan Bender, PhD FACMG
Cancer Statistics
Evolution of Knowledge in NSCLC
• Traditionally, non-small-cell lung cancers
have been classified according to
histological features.
• Various driver mutations have been
associated with these cancers over time.
• The mutations are mutually exclusive,
except for those in PIK3CA.
New driver mutations in non-small-cell lung cancer
William Pao, Nicolas Girard, Lancet Oncol 2011; 12: 175–80
• Mutations associated with drug sensitivity
EGFR Gly719X, exon 19 deletion, Leu858Arg,
Leu861Gln
• Mutations associated with primary drug resistance
EGFR exon 20 insertions
• Mutations associated with acquired drug resistance
EGFR Thr790Met, Asp761Tyr, Leu747Ser, Thr854Ala
Lung Genetic Profiles Today (by Histology)
Next-Generation Sequencing
Greater volume of clinically-actionable information
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Additional markers analyzed
Pathways more fully interrogated
Clinical trials opportunities expanded
Additional service and reporting options
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Expanded NGS panel for Select and Comprehensive profiles
45 gene pan-tumor NGS panel
Enhanced specimen-friendly requirements
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Smaller specimens accepted (8-10 unstained, unbaked, positively charged slides)
FFPE or Formalin samples accepted
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Illumina MiSeq
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Illumina MiSeq TruSeq Amplicon Cancer Hotspot
Panel Validation
• Used 80 FFPE samples (70 FFPE tissue samples, 6 cell lines and
4 HapMap samples)
• >98% accuracy when compared to Sanger.
• One discordant result stemming from misalignment of
A502_Y503dup mutation in KIT (Using Pindel and validating
bioinformatics solution)
• >97% precision for inter-operator, inter-lot and inter-machine
tests (Most sources of discordance involved indeterminate
results)
• Linear and reliable variant detection down to 5%
• All reported mutations have >99% confidence at a sensitivity
of 10%
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NGS Use in Pathway Analysis For Personalized
Medicine
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Current Biomarkers for Colorectal Cancer
Anti-EGFR Monoclonal Antibodies
• Cetuximab (Erbitux)
– Manufactured and marketed by ImClone and Bristol-Myers
Squibb (~$30,000/ 8 week cycle)
– Approved in 2006 for treatment of squamous cell
carcinomas of the head and neck
• Panitumamab (Vectibix)
– Manufactured by Amgen (~$100,000/year)
– Approved for treatment of colorectal cancer in 2006
• 2009 – Discovered KRAS mutations are negative indicators of
response to EGFR mabs in colorectal cancer
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The EGFR Pathway and Colon Cancer
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The “Real” EGFR Pathway
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The EGFR Pathway and Colon Cancer
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Benefit of Molecular Profiling on Therapy Response
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Adapted from DeRooke et al. 2010
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Current Commercial and Caris Offerings
Gene
ASR Commercial Kits
Current NGS Offering
KRAS
7 most frequent mutations
affecting codons 12 and 13
All mutations at codons 12, 13 and 61 as well
as additional mutations (A146T)
BRAF
V600E/K
All V600 mutations as well as mutations in
exon 11 and other mutations near V600
(D594, L597 and K601)
E542K, E545K, E545D and
H1047R
All mutations in exons 9 and 20 as well as
select mutations in exons 1, 5 and 7
NRAS
None
All codon 12, 13 and 61 mutations
PTEN
None
Protein expression by IHC and hotspot
mutation analysis
PIK3CA
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NGS as a Companion to Companion Diagnostics
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Roche cobas BRAF Mutation Assay
• FDA approved companion
diagnostic for Zelboraf use
• Specific to the p.V600E
mutation
• Can pick up other
mutations – p.V600K,
p.V600D, p.V600E(2),
V600_K601delinsD
• Does not detect p.V600R
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BRAF Mutation Analysis
• Sequencing
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V600E
Other V600 mutations
Exon 15 insertions/deletions (V600_K601delinsE/D)
L597 and K601 mutations
Exon 11 mutations
• FDA Approved Method – BRAF cobas 4800 V600 mutation test
– PCR based
– FDA approved companion diagnostic for Zelboraf and Debrafenib
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BRAF Mutation Analysis: PCR vs Sequencing
Sample
PCR Result
Sequencing Result
Patient 1
Wild Type
V600E(2)
Patient 2
Mutated
V600K
Patient 3
Wild Type
V600K
Patient 4
Wild Type
V600R
Patient 5
Mutated
Wild Type
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V600E
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Detection of BRAF V600E(2) by NGS
7/16/2015
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Detection of BRAF V600E(2) by NGS
7/16/2015
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Cobas BRAF Mutation Assay
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NGS and Incidental Findings
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Case #1
• 64 y/o patient presenting with growth on spinal cord
• Biopsy reveal heavily pigmented nodule positive for MART-1,
HMB-45 and MITF
• Malignant melanoma with unknown primary favored as Dx
• Sample tested for standard melanoma markers
• NGS performed with a request to report BRAF and KIT
mutation status
• GNA11 Q209L mutation detected
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Case #1 H&E
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Melanoma vs Melanocytoma
Melanocytoma
Melanoma
Metastatic
Not typical
5-yr Survival
~80% complete resection
Stage II – 45-80%
~40% incomplete resection Stage IV – 5-20%
Typical Therapy
Surgical resection and/or
radiation
Surgical resection and/or
chemotherapy
Typical Mutated
Oncogenes
GNAQ and GNA11
BRAF, NRAS and KIT
Targeted Therapies
None Currently Available –
MEK inhibitor trials
Vemurafenib, Imatinib,
Sunitinib
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Typical
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Conclusions
• NGS sequencing is a high throughput method for
interrogation of the mutation status of a large number of
biomarkers
• Pathway analysis is expensive and laborious by previous
methods – becoming standard of care for cancer therapy
• NGS adds additional information that some companion
diagnostics do not provide
• Potential benefit of incidental findings to clarify diagnosis or
detect rare finding
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Questions?
7/16/2015
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