Abstract 4261: Oncomine® Cancer Panel: simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in solid tumors

Introduction: The Oncomine® Cancer Panel (OCP) is designed to simultaneously detect and report hotspot mutations, Copy Number Variants (CNVs) and gene fusions in 143 genes with minimal DNA/RNA sample input. The OCP utilizes the AmpliSeq™ library preparation chemistry for the Ion Torrent™ Next Generation Sequencing (NGS) platform, in combination with data annotations by the Oncomine® Knowledgebase. Here we report analytical validation of OCP. Methods: DNA and RNA, extracted from the FFPE processed GM12878, was used as the negative control to evaluate the specificity of OCP. A RNA sample containing multiple oncogenic gene fusions, and a DNA sample containing multiple hotspot SNV and indels were used as the positive control. Fresh DNA from cancer cell lines (HCC1143 or NCI-H2122) along with DNA from matched normal cell lines, HorizonDx NGS standards TruQ-1, TruQ-2, several engineered FFPE samples with gene fusions or copy number changes, and 26 clinical FFPE samples of a variety of solid tumor types (lung, breast, colon, ovary, stomach, uterus and larynx) were used to evaluate the OCP performance. The analysis of the sequencing data was primarily performed with the OCP pipeline integrated with Oncomine® Knowledgebase from Life Technologies, supplemented by the MolecularMD proprietary pipeline. Only the genetic alterations with clinical utility were selected as the final output from the data pipeline. Of these detected by OCP, the SBS and indels were confirmed by the Illumina TruSeq, Ion Torrent AmpliSeq commercial cancer panels, or by Sanger sequencing. Any CNVs detected were confirmed by FISH, if possible, and detected fusions were confirmed by RT-PCR or FISH. Results: No clinically relevant genetic alterations were detected from the negative control FFPE-GM12878, indicating the high specificity of the OCP. High specificity was achieved, in part, using stringent filters, which removed error prone regions from analysis. The LOD for SBS and short indel detection was 5%, as assessed by TruQ-1 and TruQ-2, each containing 15 variants. While the exact LOD for gene fusion detection is currently under evaluation, the 100%, 50% and 20% fusion were detected in the RNA samples from serially diluted EML4-ALK or SL34A2-ROS1 fusion into the FFPE-GM12878 RNA. Four known gene amplifications (MYC, CCND1, MDM2, AKT1) with CNV from 5.4X to 14.5X in HCC1143, and a 15.7X MYC amplification in NCI-H2122 were detected. In addition, an EML4-ALK.E13A20 fusion was detected in a lung cancer FFPE sample, and confirmed by FISH. The analytical sensitivity and the specificity of the OCP are currently assessed with 26 clinical samples and three engineered FFPE samples. Conclusions: These results demonstrate the high sensitivity and specificity of OCP. With as little as 10 ng RNA and 20 ng DNA, OCP provides comprehensive screening of patient FFPE tumor specimens for the detection of a broad spectrum of clinically relevant genetic alterations. Citation Format: Peng Fang, Zhenyu Yan, Weihua Liu, Jennifer Biroschak, Paul Labrousse, Jennifer Wright, Cindy Spittle, Chad Galderisi, Li Jin. Oncomine® Cancer Panel: simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4261. doi:10.1158/1538-7445.AM2015-4261