Abstract 4569: Amplicon-targeted ultra-deep sequencing for the detection of KRAS, BRAF, and PIK3CA actionable mutations

Abstract

Abstract Background: Next-generation deep sequencing techniques have the potential to revolutionize clinical testing, especially in the arena of mutation detection. Molecular diagnostic testing for KRAS, BRAF, and PIK3CA mutations has become an integral part of clinical practice for the determination of treatment eligibility for anti-EGFR therapy. Purpose: Here we investigate the feasibility of ultra-deep advanced sequencing with the 454 GS Junior instrument (454 Life Sciences/Roche, Branford CT) for the simultaneous detection of actionable mutations in KRAS, BRAF, and PIK3CA in formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissue. Methods: Advanced sequencing with a 4-amplicon panel was performed on 17 de-identified residual FFPE colorectal cancer biopsy specimens originally submitted for mutation analysis of one or more genes by standard sequencing. The 4-amplicon panel was multiplexed for up to 4 samples through “barcoding” with molecular identifier (MID) sequences, for a total of 16 amplicons per run. Results: Sequence analysis with ultra-deep coverage of 3000-5000 reads detected KRAS mutations in 5 (29%) tumor specimens, BRAF mutations in 3 (18%), and PIK3CA mutations in 4 (24%). Ten cases had a single mutation and one had two: PIK3CA E545K and BRAF D594G. Coverage was equivalent for each of the four amplicons, and in both the forward and reverse directions, providing a high level of confidence for each mutation detected. Sequencing data was obtained for each sample tested. When sample quantity allowed, Sanger sequencing and/or castPCR (Life Technologies, Grand Island, NY) were performed as method comparisons, and results were concordant. Conclusions: The use of MIDs with ultra-deep advanced sequencing allowed the simultaneous detection of mutations in multiple amplicons from multiple patients in a single sequencing run. This proof-of-concept study confirmed that using advanced sequencing to simultaneously test for multiple biomarkers in one FFPE sample is feasible, and that amplicon-based advanced sequencing is a robust and high-throughput platform for the detection of clinically actionable mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4569. doi:1538-7445.AM2012-4569