A Fast, Sensitive and Accurate High Resolution Melting (HRM) Technology-Based Assay to Screen for Common K-ras Mutations

BACKGROUND Increasing evidence points to a negative correlation between K-ras mutations and patient's response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid and reliable assays for mutational analysis of the K-ras gene are strongly needed. METHODS We designed a high resolution melting (HRM) technology-based approach followed by direct sequencing to determine K-ras exon 1 (codons 12/13) tumour genotype. RESULTS Reconstruction experiments demonstrated an analytical sensitivity of the K-ras exon 1 HRM assay following sequencing of 1.5-2.5% of mutated DNA in a background of wild-type DNA. Assay reproducibility and accuracy were 100%. Application of the HRM assay following sequencing onto genomic DNA isolated from formalin-fixed paraffin-embedded tumour specimens of non-small cell lung cancer (n=91) and colorectal cancer (n=7) patients revealed nucleotide substitutions at codons 12 or 13, including a homozygous mutation, in 33 (34%) and 5 (5%) cases, respectively. Comparison to conventional nested-PCR following cycle-sequencing showed an overall high agreement in genotype findings (kappa value of 0.96), with more mutations detected by the HRM assay following sequencing. CONCLUSIONS HRM allows rapid, reliable and sensitive pre-screening of routine diagnostic specimens for subsequent genotyping of K-ras mutations, even if present at low abundance or homozygosity, and may considerably facilitate personalized therapy planning.