Abstract 645: Targeted next generation sequencing in molecular diagnostics of solid tumors
Authors: Grassini Greta , Pascali Valeria De , Francaviglia Ilaria , Magliacane Gilda , Cin Elena Dal , Talarico Anna , Iacona Chiara , Doglioni Claudio , Pecciarini Lorenza , Cangi Maria Giulia 
Published: August 2018

Abstract
Cancer genomics role in patients management underlines the importance of rigorous validation of molecular diagnostics approaches to guide treatment decisions. Nowadays only next generation sequencing (NGS) assays can satisfy the need to match a comprehensive tumor molecular profile to the right therapies. Nevertheless, given the complexity, cost, and outcome implications for patients, a clinically valid NGS assay requires careful consideration of gene panel size, inclusion of appropriate markers, ability to detect multiple genomic aberration types, performance with low quality/quantity of nucleic acids, and workflow feasibility. In this study we describe the introduction of the Oncomine Comprehensive Assay (OCA) into a Molecular Pathology diagnostic framework. The OCA analyzes 143 genes of which 73 oncogenes are interrogated for mutational hotspots and 26 tumor suppressor genes for all exons. In addition, the OCA detects copy number variations in 49 genes and fusion drivers in 22 genes. Multiplexed PCR-based DNA and RNA libraries were sequenced on Ion S5 and data analysis was performed by the Torrent Suite using the Oncomine pipeline. The OCA was clinically validated analyzing a cohort of 200 tumor samples of 4 solid tumor types (100 Non Small Cell Lung Cancers, 40 Colon Adenocarcinomas, 40 Pancreatic Adenocarcinomas, 20 Esophageal Adenocarcinomas), for which routine molecular analysis was available. We successfully identified relevant somatic point mutations, indels, and high-level CNAs: TP53, KRAS and EGFR were the genes most frequently mutated though alteration frequencies varied among different tumor types, as expected. OCA DNA profiling demonstrated 95% concordance with standard methods for detecting commonly targeted somatic variants with highly concordant observed variant allele frequencies for detection of KRAS, EGFR and BRAF mutations. Also high-level CNAs strongly correlated with available routine standard tests. The concordance of OCA analysis and standard tests at the RNA level was definitely lower (80%): a subset of 45 lung samples were tested for ALK, RET, ROS1 rearrangements by OCA RNA assay and we successfully detected 15 of the 24 expected fusions, indicating the feasibility of identifying them by this NGS panel, but also stressing the limits of a targeted approach to detect widely variable breakpoints in the analyzed gene fusions. In order to detect all the ALK, ROS1, and RET fusions variants, we perform the parallel validation of the Archer FusionPlex RNA-based ALK, RET, and ROS1 gene fusion assay in 8 lung adenocarcinomas. Results are under evaluation. We conclude that the OCA DNA analysis can be a valuable tool in solid tumor molecular diagnostics being able to identify both common and additional relevant variants beyond current routine practice, and represents a broadly applicable targeted NGS assay which allows advanced precision oncology for different solid tumors types.
Citation Format: Greta Grassini, Valeria De Pascali, Ilaria Francaviglia, Gilda Magliacane, Elena Dal Cin, Anna Talarico, Chiara Iacona, Claudio Doglioni, Lorenza Pecciarini, Maria Giulia Cangi. Targeted next generation sequencing in molecular diagnostics of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 645.