Impact of sequencing technology on actionable fusion detection in precision oncology for lung cancer

Precision oncology has transformed the management of non-small cell lung cancer (NSCLC), where identifying targetable genomic alterations is central to selecting targeted therapies and improving outcomes. Among these relevant biomarkers, gene fusions represent key actionable events in NSCLC. Because many fusions generate chimeric transcripts rather than recurrent DNA-level breakpoints, RNA-based next-generation sequencing (NGS) has become essential in the molecular evaluation of lung cancer. These assays may differ substantially in gene content and sequencing technology, factors that influence fusion detection and the identification of clinically actionable biomarkers. Understanding how sequencing strategies affect diagnostic yield is therefore crucial for implementing precision oncology in NSCLC. This study compared two RNA-based NGS assays, TruSight Oncology 500 (TSO-500)-RNA (Illumina) and Oncomine Focus Assay (OFA)-RNA (Thermo Fisher), to evaluate their performance in detecting actionable fusions in NSCLC. RNA extracted from 35 NSCLC tumors (IRB: 2496/18) was analyzed in both platforms. TSO-500-RNA uses a hybrid-capture strategy, interrogating 55 genes and enabling the detection of both known and novel partners, requiring at least 80 ng of RNA input. OFA-RNA employs AmpliSeq amplicon-based technology, targeting 23 predefined genes and requiring only 10 ng of RNA. Fifteen fusions were identified in 13 of 35 tumors (37.2%): thirteen were identified uniquely by TSO-500-RNA, while two ( CD74::ROS1 and KIF5B::RET ) were detected by both panels. Among the 23 genes included in both assays, one actionable fusion ( TRIM33::RET ) was exclusively detected by TSO-500-RNA. Overall, OFA-RNA detected actionable events in 6% of tumors, whereas TSO-500-RNA detected 9%. Most discrepancies were a result of hybrid-capture's greater flexibility to detect novel or noncanonical fusion partners. TSO-500-RNA also detected YAP1::KMT2A , an oncogenic fusion with approved therapies in other cancer types, but outside OFA-RNA's target list. Importantly, no discordances were observed when both assays covered the same gene regions, demonstrating technical robustness of both assays within their designed scope. In conclusion, the sequencing strategy is a major determinant of fusion detection in NSCLC. The hybrid-capture-based TSO-500-RNA enables broader gene coverage and discovery of novel fusion partners, resulting in a higher detection rate. Conversely, the amplicon-based OFA-RNA offers reliable detection of predefined fusions with lower RNA input, a critical advantage when working with limited tumor samples. Both features, comprehensive fusion discovery and compatibility with low-input, are essential to fully support precision oncology. Therefore, panel selection should balance tissue availability with the need for broad fusion detection to guide therapeutic decisions in NSCLC.