Abstract 5359: An ultrasensitive and highly reproducible hybridization capture-based Next-Generation Sequencing clinical assay to enable precision oncology in patients with solid tumors

Abstract

Abstract Sequencing whole cancer exome and whole genome provides useful information to study cancer evolution. However, cost and amount of data may become overwhelming. Enrichment capture-based methods to design custom targeted gene panel have rapidly evolved recently in cancer genomics arena. Custom targeted gene sequencing entails several advantages (i.e., better quality, ethical, affordable, technically suitable, personalized and reimbursable) for cancer patients over whole exome and whole genome sequencing. With this in mind, and to enable precision oncology in patients with solid tumors, we developed ICG100 2.0 panel, a hybridization capture-based next-generation sequencing assay for targeted sequencing of all exons and flanking introns of 162 commonly mutated cancer genes in formalin-fixed, paraffin-embedded (FFPE) tumors. To establish ICG100 2.0 panel, we compared three commercially available capture based technologies and evaluated reproducibility, sensitivity, specificity and the detection limit for low-frequency variants using internally developed bioinformatics pipeline. Cell lines, reference standards/synthetic DNA and solid tumor samples with the known genetic information was utilized in this analysis. Results were derived from MiSeq and NextSeq platforms and cross-compared on other platforms, including MassArray and ddPCR for establishing concordance and uniformity. Intra and interrun replicates were utilized to assess the quality, precision and reproducibility of variant calling. Mean depth of coverage was observed at >300X with >99% sensitivity and specificity. These findings and observations will guide other clinical laboratories to establish new assay that require less DNA input, enzyme based fragmentation and reduced preparation time. While we show that the three capture based methods (after comparison) had an overall accuracy in SNP and CNV detection similar to each other with minor differences, we describe an approach to assess and establish the best assay from the clinical standpoint to guide treatment decisions and match cancer patients to the most appropriate clinical trials. Citation Format: Moises Hernandez, Sara King, Sharanya Raghunath, Christopher Johnson, David Loughmiller, Lincoln Nadauld, Pravin J. Mishra. An ultrasensitive and highly reproducible hybridization capture-based Next-Generation Sequencing clinical assay to enable precision oncology in patients with solid tumors [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 5359. doi:10.1158/1538-7445.AM2017-5359