An improved assay for detection of theranostic gene translocations and METexon 14 skipping in thoracic oncology

Theranostic translocations may be difficult to detect by routine techniques, especially when specimens are exiguous. We recently demonstrated in a series of translocated lung adenocarcinomas that LD-RT-PCR (ligation-dependent reverse transcription polymerase chain reaction) assay could identify ALK, ROS1and RETrearrangements with 64% sensitivity and 100% specificity. Here, we report an upgraded version of this assay used in a routine prospective cohort of lung carcinomas. Newly diagnosed lung carcinomas referred to the Rouen molecular platform between 15/05/2018 and 15/05/2019 for ALK and ROS1 IHC, genotyping (SNaPshot© +/− high-throughput genotyping) and sometimes FISH (standard routine process) were tested prospectively in parallel with the LD-RT-PCR assay designed to detect at one go ALK, ROS1and RETtranslocations and METexon 14 skipping. 413 tumors from 396 patients were included. LD-RT-PCR had a global sensitivity of 91.43% (standard routine process: 80%), with a specificity of 100%. It detected 15/18 ALKand 4/4 ROS1translocated tumors, but also 6/6 tumors with METexon 14 skipping retrieved by genotyping. In addition, it retrieved 7 alterations missed by the routine process, then confirmed by other means: 5 METexon 14 skipping and 2 RETtranslocated tumors. Finally, it allowed to deny an effect on METexon 14 skipping for 8 mutations detected by routine genotyping. We successfully implemented LD-RT-PCR in routine analysis. This technique is cheap, fast, sensitive, specific, and easily upgradable (e.g., NTRKtranslocations), but still requires IHC to be performed in parallel. Owing to its advantages, we recommend considering it, in parallel with IHC and genotyping, as an excellent cost-effective alternative, for the systematic testing of lung adenocarcinoma, to FISH and to more expensive and complex assays such as RNA-seq.