Next-Generation Pathology: Deep DNA Sequencing and Targeted Therapy for Cancer
Christine Sheehan, Alex Parker, Mirna Jarosz, Sean Downing, Roman Yelensky, Doron Lipson, Gary Palmer, Maureen Cronin, Jeffrey Ross. Albany Medical College, Albany, NY; Foundation Medicine Inc., Cambridge, MA
Background: Gene sequencing currently used to select therapy in non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and melanoma (MM) employs traditional standard-of-care (SOC) “hotspot” single gene mutation analysis. Massively parallel (next-generation sequencing (NGS)) has now been adapted to formalin-fixed (FFPE) specimens to provide high sensitivity detection for point mutations, insertion/deletions, translocations and copy number alterations (CNA).
Design: FFPE specimens from 83 solid tumors (50 CRC, 29 NSCLC, 4 MM) with results available from SOC genotyping by allele-specific PCR (KRAS codons 12/13, EGFR exons 17-20, or BRAF V600E) were fully sequenced for 145 genes by NGS. Hybridization-capture of 2574 exons across 145 oncogenes, tumor suppressor genes and ADME-related genes was performed to produce libraries appropriate for paired-end sequence analysis on the Illumina HiSeq2000 platform (Illumina, Inc., San Diego, CA).
Results: NGS recapitulated the SOC test results in all cases. In-depth sequence analysis with median coverage averaging 213-fold (range 8 to 461) detected a per-sample average of 2 previously-described mutations, 7 novel mutations and 2 CNAs in the CRC, including frequent alterations in TP53 (33), APC (27), KRAS (12) and BRAF (6). The NSCLC averaged 1 previously described mutation, 8 novel mutations and 1 CNA per sample, most frequently KRAS (10), TP53 (7), JAK2 (3), EGFR (2) and BRAF (2). The MM exhibited on average 1 previously described mutation, 7 novel mutations and 3 CNAs including TP53 (4) and BRAF (2). In addition to validated clinically actionable mutations in EGFR, KRAS, and BRAF, and multiple alterations in well-known cancer genes (TP53, STK11, APC, MLH1, BRCA2, SMAD4), a significant number of additional genomic alterations that have potential therapeutic implications were also detected including activating mutations in the PI3 kinase subunit gene PIK3CA; mutations in MET, KIT, ERBB2 and CDKN2A; driver mutations not usually associated with solid tumors, such as the lymphoma-associated JAK2 V617F mutation in two NSCLCs; and in 1 CRC, a novel chromosome 2 rearrangement adjacent to the ALK kinase domain confirmed by analyzing a cDNA library constructed from extracted tumor RNA.
Conclusions: NGS of hundreds of cancer-related genes can be reliably performed at a high level of sensitivity and specificity in clinical FFPE samples of solid tumors, can reproduce SOC single gene traditional sequencing results and shows great potential to inform on therapeutic decisions for patients with CRC, NSCLC and MM.
Tuesday, March 20, 2012 1:00 PM
Platform Session: Section E, Tuesday Afternoon