Comprehensive next-generation sequencing for clinically actionable mutations from formalin-fixed cancer tissues.

Abstract

10564 Background: With the increasing availability of genomically targeted therapies, the comprehensive description of patient tumor genomes can be an effective diagnostic approach that informs optimal therapy selection. Whereas individual gene sequencing (e.g., EGFR) is widely used in cancer diagnosis, the feasibility of genome-scale testing in routinely collected formalin-fixed paraffin-embedded (FFPE) cancer specimens has not previously been demonstrated. Methods: Sequences of 176 oncogenes, tumor suppressor genes and drug-metabolizing enzyme genes were analyzed from FFPE cancer biopsies. Two 20 micron sections were obtained from 101 specimens of colorectal cancer, non-small cell lung cancer or melanoma. Extracted DNA was subjected to hybrid capture and paired-end sequencing of 2574 exons encompassing the 176 genes. Results: We report sequence analysis of 176 genes in the first 33 cancer specimens that were previously subjected to conventional diagnostic sequencing or genotyping of EGFR, KRAS and/or BRAF. Our 176-plex assay at an average median coverage of 301 (range 24 to 461) automatically recovered 9 of 10 previously reported mutations, with the 10th observable following manual review. This result indicates that standard-of-care single-gene sequencing results are recapitulated by our multiplexed next generation sequencing assay. Overall, at least one previously described somatic mutation documented in the COSMIC database was observed in 30/33 cancer specimens (91%). In addition to mutations in well-known cancer genes such as TP53, STK11, APC, MLH1, BRCA2, and SMAD4 we detected many mutations that are plausibly clinically actionable beyond well-known expected mutations in EGFR, KRAS and BRAF. These included activating mutations in the PI3 kinase subunit gene PIK3CA along with mutations in MET, KIT, ERBB2 and CDKN2A. Conclusions: Our results indicate that comprehensive, high quality sequencing of hundreds of genes is feasible from routinely collected FFPE tissues, and that plausibly actionable mutations will be discovered by this approach. We suggest that clinical-grade sequencing should become a routine part of all clinical trials, and increasingly, of clinical care.