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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 • • • Additional markers analyzed Pathways more fully interrogated Clinical trials opportunities expanded Additional service and reporting options • • Expanded NGS panel for Select and Comprehensive profiles 45 gene pan-tumor NGS panel Enhanced specimen-friendly requirements • • Smaller specimens accepted (8-10 unstained, unbaked, positively charged slides) FFPE or Formalin samples accepted 5 Illumina MiSeq ©2013 Caris Life Sciences and affiliates. 6 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% ©2013 Caris Life Sciences and affiliates. 7 NGS Use in Pathway Analysis For Personalized Medicine ©2013 Caris Life Sciences and affiliates. 8 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 ©2012 Caris Life Sciences, Ltd. and Affiliates 10 The EGFR Pathway and Colon Cancer ©2012 Caris Life Sciences, Ltd. and Affiliates 11 The “Real” EGFR Pathway ©2012 Caris Life Sciences, Ltd. and Affiliates 12 The EGFR Pathway and Colon Cancer ©2012 Caris Life Sciences, Ltd. and Affiliates 13 Benefit of Molecular Profiling on Therapy Response ©2013 Caris Life Sciences and affiliates. Adapted from DeRooke et al. 2010 14 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 ©2012 Caris Life Sciences, Ltd. and Affiliates 15 NGS as a Companion to Companion Diagnostics ©2013 Caris Life Sciences and affiliates. 16 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 ©2012 Caris Life Sciences, Ltd. and Affiliates 17 BRAF Mutation Analysis • Sequencing – – – – – 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 ©2012 Caris Life Sciences, Ltd. and Affiliates 18 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 ©2012 Caris Life Sciences, Ltd. and Affiliates V600E 19 Detection of BRAF V600E(2) by NGS 5/23/2017 20 Detection of BRAF V600E(2) by NGS 5/23/2017 21 Cobas BRAF Mutation Assay 22 ©2012 Caris Life Sciences, Ltd. and Affiliates 23 NGS and Incidental Findings ©2013 Caris Life Sciences and affiliates. 24 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 ©2013 Caris Life Sciences and affiliates. 25 Case #1 H&E 5/23/2017 26 ©2013 Caris Life Sciences and affiliates. 27 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 5/23/2017 Typical 28 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 ©2013 Caris Life Sciences and affiliates. 29 Questions? 5/23/2017 30