Targeted RNA-based next-generation sequencing as a robust diagnostic tool for non-small cell lung cancer genetic testing

<bold>Background:</bold> Management of advanced non-small cell lung cancer (NSCLC) requires accurate molecular testing for predictive biomarkers. We have evaluated the performance of RNA-based targeted next-generation sequencing (tNGS) for detection of actionable molecular variants in various types of NSCLC specimens. <bold>Methods:</bold> In total, 603 specimens from 588 NSCLC patients (~72% adenocarcinoma) were tested for relevant single nucleotide variants (SNVs incl. indels) and gene fusions using FusionPlex Lung assay (Archer) on Illumina platform, with reference to IVD-tests (qPCR, FISH) and DNA-based tNGS. <bold>Results:</bold> We obtained valid results for 507 samples from 501 patients. 16% of specimens did not meet criteria required for NGS procedure (83%) or data analysis (17%), mostly bronchoscopic material, fine-needle biopsies, archival samples (~78%). Validation process established technical and analytical borderline quality values for laboratory procedures and data reporting what resulted in ~98% concordance with reference methods (n=203). In 280 (55%) samples a pathogenic molecular variant was detected: SNVs in <italic>KRAS</italic>, <italic>EGFR</italic>, <italic>BRAF</italic>, <italic>MET</italic>, <italic>ERBB2</italic>, <italic>PIK3CA</italic> (n=260) and gene fusions of <italic>ALK</italic>, <italic>ROS1</italic>, <italic>RET</italic>, <italic>NTRK1</italic>, <italic>FGFR2</italic>, <italic>FGFR3</italic>, <italic>NRG1</italic> (n=24). Variants detected in 158 (56%) specimens were druggable. In 12 cases co-occurrence of 2 alterations was found. Novel <italic>TMEM79::NTRK1</italic> fusion variant and <italic>EGFR</italic> p. Glu746_Thr751delinsIle mutation were identified. <bold>Conclusion:</bold> RNA-based tNGS has proven to be a reliable, informative diagnostic tool for simultaneous detection of actionable SNVs and gene fusions (known and novel) in multiple genes, providing pivotal data for therapeutic decisions in NSCLC. <bold>Funding:</bold> IGiChP 3.3/22.