Differences in the frequencies of K-ras c12-13 genotypes by gender and pathologic phenotypes in colorectal tumors measured using the allele discrimination method.

The frequencies of different genotypes of the K-ras oncogene in colorectal cancer (CRC) reveal complex relationships among gender, age, and tumor aggression, however, differences among these studies could also be attributed to a lack of standardization of the detection methods used. We developed the allele discrimination assay, which uses dual-color real-time polymerase chain reaction (qPCR) as a fast K-ras genotyping method, and demonstrated higher sensitivity and specificity than DNA sequencing with formalin-fixed paraffin tissues. The assay detected K-ras mutations among 83 of 204 patients with CRC (40.7%); 20.6% of these mutations were G12D (GAT) mutations, 7.4% were G13D (GAC) and G12V (GTT), and 5.3% were other types. A higher proportion of females was observed overall in tumors with K-ras mutations (60.2%, P = 0.01), codon 12 mutations (63.2%, P = 0.005), and transversions (69.6%, P = 0.02), which reflected the higher prevalence of females among the well- to moderately differentiated tumors (29% in males vs. 53% in females; interaction P = 0.03). The opposite was observed for poorly differentiated tumors (47% in males vs. 35% in females). No significant influence of age was found on the prevalence of K-ras mutation. Males with pathological changes and females with poorly differentiated tumors displayed GAT as a less common genotype compared with most other prevalence studies. In conclusion, allele discrimination, with no additional amplification step, is a fast and reliable genotyping method for detecting K-ras c12-13 mutations. Using this method, we demonstrate differences in the frequencies of K-ras genotypes by gender and pathologic phenotypes of CRC.