Detection of gene fusions and exon skipping events in lung FFPE samples with Oncomine Precision Assay on Ion Torrent Genexus(TM) System

Abstract Introduction: Gene fusions and exon skipping events play an important oncogenic role in non-small cell lung cancer (NSCLC). Here we employed the Oncomine Precision Assay (OPA) for sequencing of 998 clinical research FFPE (Formalin-Fixed Paraffin-Embedded) lung samples using the Genexus࣪ integrated sequencing platform. The RNA assay strategy is aimed at providing a wide scope for studying known oncogenic fusions and exon skip variants, as well as a method for detection of novel fusion combinations and detection of fusions in a partner agnostic manner. We summarize the findings that include detected samples with oncogenic fusions in tyrosine kinase genes ALK, RET, ROS1 as well as MET exon14 skipping, and demonstrate the novel fusion detection capabilities of the panel. Methods: The Oncomine Precision Assay panel developed using the Ion AmpliSeq HD technology for use on both tissue and liquid biopsy samples to detect fusions with high sensitivity in low input RNA. The panel features 981 known fusion isoforms in 16 oncogenic drivers as well as assays for exon skipping and deletion in MET and EGFR. The amplicons are strategically designed around known fusion breakpoints and generate reads only if the variant is present. Results are assessed bioinformatically with a framework that includes detailed genomic annotations of the fusion breakpoint, and generates interpretable fusion calls and report in the Genxus࣪ software. In addition, the panel contains an algorithm for novel fusion detection, and 78 exon-junction amplicons for partner agnostic fusion detection in ALK, RET, NTRK1,2,3 and FGFR1,2,3 with an exon tiling expression imbalance assay. We used the Genexus࣪ instrument to sequence 998 unique lung FFPE samples and analyzed the results with the Genexus࣪ fusion analysis workflow. Results: A total of 998 unique lung FFPEs were sequenced, of which 906 (91%) resulted with >5000 aligned reads and at least 5 of the 7 RNA expression controls at the assay threshold. A total of 25 samples with ALK fusions were detected by either the targeted fusions or the expression imbalance assays. In addition, we detected 10 samples with RET fusions, 4 with FGFR3 fusions, 3 with ROS1 fusions, and additional single observations of NRG1, RSPO3 and FGFR1 fusions. Exon 14 skipping in MET was detected in 14 samples, of which we were able to detect a likely splice site variant SNV or indel at, or near the boundaries of exon 14, with the DNA assays of the panel. These variants were observed in a mutually exclusive set of 59 total samples (6.5% of the samples). Conclusions: We used the OPA panel for fusions and intragenic rearrangements that retains the simple workflow and fast turn-around time of previous Oncomine fusion, and demonstrated known and novel fusion detection as well exon skip variant detection capabilities in a research cohort of lung FFPE samples. For research use only. Not for use in diagnostic procedures. Citation Format: Amir Marcovitz, Jeoffrey Schageman, Jian Gu, Stephen Wunsch, David Chi, Paul D. williams, Scott P. Myrand, Fiona C. Hyland, Seth Sadis, Kelli S. Bramlett. Detection of gene fusions and exon skipping events in lung FFPE samples with Oncomine Precision Assay on Ion Torrent Genexus࣪ System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 78.