The use of COLD-PCR and High Resolution Melting Analysis improves the limit of detection of KRAS and BRAF mutations in colorectal cancer

Fast and reliable tests to detect mutations in human cancers are required to better define clinical samples and orient targeted therapies. KRAS mutations occur in 30-50 % of colorectal cancers (CRCs) and represent a marker of clinical resistance to cetuximab therapy. BRAF V600E is mutated in about 10% of CRCs. The development of a specific inhibitor of mutant BRAF kinase has prompted a growing interest in BRAF V600E detection. Traditional methods, as PCR and direct sequencing, do not detect low-level mutations in cancer resulting in false negative diagnoses. In this study we designed a protocol to detect mutations of KRAS and BRAFV600E in CRCs based on coamplification at lower denaturation temperature PCR (COLD-PCR) and high resolution melting (HRM). The combined methods were applied to the evaluation of 117 sporadic CRCs, in which we found KRAS mutations by PCR and direct sequencing in 47 (40%) patients and BRAFV600E in 10 (8.5%). The use of COLD-PCR in apparently wild type samples allowed us to identify 15 new mutated CRCs (10 for KRAS and 5 for BRAF V600E). The percentage of mutated CRCs increased to 48.7% for KRAS and to 12.8% for BRAF V600E. COLD-PCR combined with HRM permits the correct identification of less represented mutations in CRC and better selection of patients eligible for targeted therapies, without requiring expensive and timeconsuming procedures.