Concurrent targeted resequencing for translocations and mutations in tumor samples.

Abstract

e14597 Background: Genetic variations are diverse, and proper nucleic acid templates should be analyzed for optimal outcomes. Previous NGS assays separately treat DNA and/or RNA, and are costly and produce limited information. Here we present a concurrent assay simultaneously converting both RNA and DNA templates in a single-tube format to streamline variant detection process. Methods: We developed a high-throughput targeted resequencing assay utilizing both DNA and RNA templates for mutation and fusion detection, and applied to a cohort of over 1000 lung tumor samples. Total nucleic acids from tissue samples were processed in a single-tube format throughout, and post-assay data analysis split RNA and DNA signals for corresponding variant calling. Sensitivity and specificity were sufficient for tissue samples. Results: In addition to common EGFR, KRAS, BRAF, PIK3CA mutations and ALK, ROS1, RET fusions and MET Exon 14 skipping, we also identified rare HLA-DRB1---MET and MSN---NTRK2 fusions. We found that MET exon 14 skipping was abundant, and that EGFR T790M is more associated with Exon 19 deletion than with L858R. We also found baseline mutation frequency for FFPE samples at 4-5%. Conclusions: Overall, this method is robust and convenient for clinical molecular diagnosis for tissue samples where variant types are diverse and time and budgets are constrained. Extreme caution is suggested for making positive calls below 5% MAF.