Abstract 3007: Validation of an NGS panel assay for detection of hotspot cancer somatic mutations

Abstract

Abstract Background The gene tests to predict whether cancers respond to specific targeted therapies or prediction of prognosis has quickly increased over the last decade. Next-generation sequencing (NGS) has become a powerful tool for high-throughput and sensitive gene mutation testing, which is required for development of targeted therapies, clinical trials, and further research. The aim of this study was to validate the AmpliSeq for Illumina Cancer Hotspot Panel v2 on the Illumina MiSeq platform and simultaneously analyze 2800 somatic mutations across the hotspot regions of 50 genes, including SNP and small INDEL, with known associations to cancer, as identified in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. This panel enables the study of the genes associated with different cancer types, including the cancers of the lung, colon, breast, thyroid, oral, bladder, prostate, liver, kidney, ovarian and cervix, etc. Methods and findings We validated this assay using the genomic DNA extracted from human paraffin-embedded (FFPE) samples and peripheral blood with negative control DNA (NA12878), positive control DNA (HD701), blank control as well as the global proficiency testing samples. Following two lab scientists parallel NGS library preparation, sequenced on Miseq platform (2x150 PE) and analyzed using a pipeline centered on Illumina Basespace, the somatic mutations were called at an average number of reads coverage >2500X, variant frequency cutoff 1%. An integrated test report can be generated within five working days. Panel sequencing analysis showed that the mutations are 100% concordance with known mutations and consistent in four runs, between lab personal performances, different input DNA concentrations (1ng-100ng), etc. Briefly, 25 mutations were detected in the FFPE samples and 20 mutations detected in proficiency testing samples. The variant types, HGVS ID and frequency are 100% match with its standard results which are a prove of precision and sensitivity. Conclusions A sensitive, high-throughput, pan-cancer mutation panel for NGS analysis of cancer hot-spot mutations in 50 genes was highly repeatable and reproducible and validated for routine application in a gene testing lab. Note: This abstract was not presented at the meeting. Citation Format: Wenzhi Li, Henry She, Xiaohua Tian, Margaret Neja, Adrian Harris, Yuhua Li. Validation of an NGS panel assay for detection of hotspot cancer somatic mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3007.