Abstract 3585: Determination of gene amplifications with a next-generation sequencing cancer panel

Abstract

Abstract TruSight® Tumor 15* (TST15) uses next-generation sequencing technology to detect somatic point mutations, insertions, and deletions in 15 genes that are commonly mutated in solid tumors. It accurately detects low-frequency mutations with low DNA input and is optimized for formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The test is a multiplexed PCR panel that includes primers to support amplification detection in the EGFR, ERBB2, and MET genes. Here we describe development of a bioinformatics analysis method to enable detection of these gene amplifications. DNA extracted from 31 FFPE samples with amplifications in EGFR, ERBB2, or MET—some with dual amplifications—were diluted with DNA extracted from 18 normal FFPE samples to create a total of 67 unique samples at low-level amplification. Undiluted and diluted samples were assessed for quality based on amplifiability (ΔCq or library yield) and tested with TST15. For comparison, the samples were also tested with droplet digital PCR (ddPCR) to determine the copy ratio of target to wild type. For amplifiable samples, the TST15 algorithm was able to detect amplifications in EGFR and ERBB2 at ddPCR ratios of 1.4 and 1.6, respectively. ddPCR was unable to accurately quantify low MET amplifications; therefore, expected ddPCR ratios were used for comparison. The TST15 algorithm was able to detect amplifications in MET samples at an expected ddPCR ratio of 1.3. Poor sample quality (i.e., high ΔCq or low library yield) impaired amplification detection. In some samples, the presence of multiple amplifications also impaired detection. The addition of this algorithm to the TST15 workflow will allow researchers to more accurately characterize tumor samples by detecting somatic mutations and amplifications simultaneously. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Ina L. Deras, Aaron Wise, Chen Zhao, Christine Glidewell-Kenney, Phillip Le, Elizabeth Upsall Aderhold, Karen Gutekunst. Determination of gene amplifications with a next-generation sequencing cancer panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3585. doi:10.1158/1538-7445.AM2017-3585