RET FISH analysis is a sensitive but highly unspecific screening method for RET fusions in lung cancer.

Abstract Introduction RET gene fusions are established oncogenic drivers in 1% of non-small cell lung cancer (NSCLC). Accurate detection of advanced patients with RET fusions is essential to ensure optimal therapy choice. We investigated performance of fluorescence in situ hybridization (FISH) as diagnostic test for detecting functional RET fusions. Methods Between January 2016 and November 2019, 4873 NSCLC patients were routinely screened for RET fusions using either FISH (n=2858) or targeted RNA next generation sequencing (NGS) (n=2015). If sufficient material was available, positive cases were analyzed by both methods (n=39) and multiple FISH assays (n=17). In an independent cohort of 520 NSCLC patients, whole genome sequencing (WGS) data were investigated for disruptive structural variations and functional fusions in the RET and compared to ALK and ROS1 loci. Results FISH analysis showed a RET rearrangement in 48/2858 cases; of 30 rearranged cases double tested with NGS, only 9/30 had a functional RET fusion. RNA NGS yielded RET fusions in 14/2015 cases; all 9 cases double tested by FISH showed RET locus rearrangement. Of these 18 verified RET fusion cases, 16 showed a split signal and two a complex rearrangement by FISH. By WGS the prevalence of functional fusions compared to all disruptive events was lower in the RET (4/9, 44%) compared to the ALK (27/34, 79%) and ROS1 (9/12, 75%) loci. Conclusions FISH is a sensitive but unspecific technique for RET screening, always requiring a confirmation using an orthogonal technique, due to frequently occurring RET rearrangements not resulting in functional fusions in NSCLC.