Cited 30 times since 2009 (1.9 per year) source: EuropePMC Cellular oncology : the official journal of the International Society for Cellular Oncology, Volume 31, Issue 3, 1 1 2009, Pages 161-167 A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations. Kramer D, Thunnissen FB, Gallegos-Ruiz MI, Smit EF, Postmus PE, Meijer CJ, Snijders PJ, Heideman DA
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.
