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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.

KRASgenotyping is mandatory before anti-epidermal growth factor receptor monoclonal antibody therapy in metastatic colorectal cancer, which is the second leading cause of cancer-related death in the United States and in Europe. Thus, large-scale KRASmutation screening is needed for efficient patient management and in the future metastatic colorectal cancer genotyping might also include the detection of the BRAFV600E mutation, which is a very strong negative prognostic factor in colorectal cancer. We report our experience of routine KRAS/BRAFmutation screening practice performed on 1130 formalin-fixed paraffin-embedded tumor samples from 992 colorectal cancer patients. DNA was extracted from macrodissected tumor areas highlighted by a pathologist, KRAScodons 12/13 and BRAFV600E mutations were assessed in a single SNaPshot® multiplex assay and each mutation was confirmed by an independent analysis. KRASand BRAFmutations were, respectively, present in 41.8 and 6.5% of the tumor samples. If KRASand BRAFmutations were mutually exclusive, four samples presented two concomitant KRASmutations. Genotyping of paired primary tumors and metastases from 44 patients indicated that 5 patients (11.4%) presented discordant KRASmutational status. KRASgenotype heterogeneity was also observed within primary tumor sites in seven cases. Non-reproducible KRASartefactual mutations were detected in 53 samples (4.7%). We found that the prominent mechanism leading to these artefactual mutations was the fragmentation of DNA occurring during tissue processing. Routine KRASgenotyping performed on formalin-fixed paraffin-embedded tissues requires, therefore, the development of quality control scheme for molecular pathology, especially because of DNA damages induced by formalin fixation. The tumor heterogeneity observed in some patients indicates that it should be more appropriate to perform KRASgenotyping on metastases if sample is available.