Rare RAS Mutations in Metastatic Colorectal Cancer Detected During Routine RAS Genotyping Using Next Generation Sequencing

Overall survival of metastatic colorectal cancer (mCRC) patients has been improved with the addition of targeted therapy such as anti-epithelial growth factor receptor monoclonal antibodies (anti-EGFR mAbs) to standard chemotherapy. Retrospective studies and randomized trials showed that the presence of RAS mutations was linked to the absence of clinical response to anti-EGFR mAbs. Patients harboring KRAS and NRAS mutations on exons 2, 3 or 4 have little or no benefit from anti-EGFR therapies. Polymerase chain reaction (PCR)-based assays are routinely used to assess KRAS and NRAS status, whereas deep sequencing with next generation sequencing (NGS) currently represents an alternative method. The objective of our study was to identify KRAS and NRAS non-hotspot mutations using NGS of mCRC tumor samples. DNA was extracted from 188 consecutive formalin-fixed paraffin embedded samples of histologically proven colorectal cancer tumor tissue from patients with mCRC. Following amplification, DNA was sequenced by ultra-deep pyrosequencing. Non-hotspot mutations identified by NGS (frequency of mutated allele range [1.8-70.6 %]) were confirmed by Sanger direct-sequencing when possible. NGS procedure was applicable in 94 % of the cases and detected mutations in 62 % of the samples. Nine uncommon mutational profiles were found with a frequency of mutated allele > 1 %. Silent mutations were found in 3.6 % of the samples. Mutations at or near functional domains of RAS proteins, other than defined hotspots, were found in 3.6 %. NGS proved to be accurate, sensitive and suitable for routine RAS genotyping. Clinical responses to anti-EGFR mAbs are potentially impaired in the presence of these uncommon RAS mutations.