Rapid and Cost-Efficient Detection of RET Rearrangements in a Large Consecutive Series of Lung Carcinomas

-kinase-activating gene rearrangements occur in approximately 1-2% of non-small-cell lung carcinomas (NSCLCs). Their reliable detection requires next-generation sequencing (NGS), while conventional methods, such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) or variant-specific PCR, have significant limitations. We developed an assay that compares the level of RNA transcripts corresponding to 5'- and 3'-end portions of the gene; this test relies on the fact that translocations result in the upregulation of the kinase domain of the gene and, therefore, the 5'/3'-end expression imbalance. The present study included 16,106 consecutive NSCLC patients, 14,449 (89.7%) of whom passed cDNA quality control. The 5'/3'-end unbalanced expression was observed in 184 (1.3%) tumors, 169 of which had a sufficient amount of material for the identification of translocation variants. Variant-specific PCR revealed rearrangements in 155/169 (91.7%) tumors. RNA quality was sufficient for RNA-based NGS in 10 cases, 8 of which carried exceptionally rare or novel ( and ) translocations. We also applied variant-specific PCR for eight common rearrangements in 4680 tumors, which emerged negative upon the 5'/3'-end unbalanced expression test; 33 (0.7%) of these NSCLCs showed fusion. While the combination of the analysis of 5'/3'-end expression imbalance and variant-specific PCR allowed identification of translocations in approximately 2% of consecutive NSCLCs, this estimate approached 120/2361 (5.1%) in / / / / / -negative carcinomas. -rearranged tumors obtained from females, but not males, had a decreased level of expression of thymidylate synthase ( < 0.00001), which is a known predictive marker of the efficacy of pemetrexed. The results of our study provide a viable alternative for testing in facilities that do not have access to NGS due to cost or technical limitations.