Clinical validation of coexisting driver mutations in colorectal cancers.

Mutational profiling is recommended for selecting targeted therapy and predicting prognosis of metastatic colorectal cancer (CRC). Detection of coexisting mutations within the same pathway, which are usually mutually exclusive, raises the concern for potential laboratory errors. In this retrospective study for quality assessment of a next-generation sequencing assay, we examined BRAF, KRAS, and NRAS genes within the mitogen-activated protein kinase (MAPK) pathway and the PIK3CA gene within the phosphatidylinositol 3-kinase (mTOR) pathway in 744 CRC specimens submitted to our clinical diagnostics laboratory. Although coexistence of mutations between the MAPK and mTOR pathways was observed, it rarely occurred within the MAPK pathway. Retrospective quality assessments identified false detection of coexisting activating KRAS and NRAS mutations in 1 specimen and confirmed 2 activating KRAS mutations in 2 specimens and coexisting activating KRAS and NRAS mutations in 2 specimens, but no coexisting activating RAS and BRAF mutations. There were 15 CRCs with a kinase-impaired BRAF mutation, including 3 with a coexisting activating KRAS mutation, which may have therapeutic implications. Multiregional analysis based on different histologic features demonstrated that coexisting KRAS and NRAS mutations may be present in the same or different tumor populations and showed that invasion of adenomas by synchronous adenocarcinomas of different clonal origin may result in detection of coexisting mutations within the MAPK pathway. In this study, we proposed an operating procedure for clinical validation of unexpected coexisting mutations. Further studies are warranted to elucidate the biological significance and clinical implications of coexisting mutations within the MAPK pathway.