Development of pyrosequencing methods for the rapid detection of RAS mutations in clinical samples

In advanced colorectal carcinoma (CRC) patients, extended RAS mutations testing (KRAS exons 2 to 4 and NRAS exons 2 to 4) is a prerequisite for patient stratification to anti-EGFr therapy. Accurately distinguishing mutant patients from potential responders has a clinically critical impact, and thus effective and low cost methods are needed for identification of the mutation status. We have developed quantitative pyrosequencing assays for sensitive and rapid detection of mutant RAS alleles in formalin-fixed, paraffin-embedded tissues. Exons 2 to 4 of KRAS and NRAS genes were PCR amplified and analyzed by pyrosequencing. For validation, PCR products were sequenced by conventional Sanger sequencing. Analytical sensitivity of these assays was determined by calculating the limit of detection. The results showed that low levels of mutant RAS alleles (2-13%) can be detected with pyrosequencing assays.