title: The use of COLD-PCR and high-resolution melting analysis improves the limit of detection of KRAS and BRAF mutations in colorectal cancer.
abstract: 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. In addition, the BRAF V600E is mutated in about 10% of CRCs, and the development of a specific inhibitor of mutant BRAF kinase has prompted a growing interest in BRAF (V600E) detection. Traditional methods, such 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 BRAF(V600E) in 117 sporadic CRCs based on coamplification at lower denaturation temperature PCR (COLD-PCR) and high-resolution melting (HRM). Using traditional PCR and direct sequencing, we found KRAS mutations in 47 (40%) patients and BRAF(V600E) in 10 (8.5%). The use of COLD-PCR in apparently wild-type samples allowed us to identify 15 newly mutated CRCs (10 for KRAS and 5 for BRAF (V600E)), raising the percentage of mutated CRCs to 48.7% for KRAS and to 12.8% for BRAF (V600E). Therefore, 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 time-consuming procedures.
authors.affiliation: Division of Clinical Biochemistry, University of Florence, Florence, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Mancini
authors.forename: Claudio
authors.initials: C
authors.lastname: Santucci
authors.forename: Roberta
authors.initials: R
authors.lastname: Sestini
authors.forename: Lisa
authors.initials: L
authors.lastname: Simi
authors.forename: Nicola
authors.initials: N
authors.lastname: Pratesi
authors.forename: Fabio
authors.initials: F
authors.lastname: Cianchi
authors.forename: Rosa
authors.initials: R
authors.lastname: Valanzano
authors.forename: Pamela
authors.initials: P
authors.lastname: Pinzani
authors.forename: Claudio
authors.initials: C
authors.lastname: Orlando
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics*; D017931:DNA Primers; D011905:Genes, ras; D006801:Humans; D057230:Limit of Detection; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*
chemical_list: D017931:DNA Primers; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf