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

Background: 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.

Objective: The objective of our study was to identify KRAS and NRAS non-hotspot mutations using NGS of mCRC tumor samples.

Method: 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.

Results: 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.

Conclusion: Clinical responses to anti-EGFR mAbs are potentially impaired in the presence of these uncommon RAS mutations.