Study On Mutation Traits Of KRAS And BRAF Gene In Colorectal Tumor And Comparative Analysis Of Test Methods

Objective: To study the traits of KRAS and BRAF gene mutations in distal colorectal adenoma and adenocarcinoma. Then to get the clinic value of molecular diagnosis in guiding theory of colorectal cancer preliminarily; At the same time,Comparative analysis of Competitive Allele-Specific Taq Man PCR(cast PCR) and DNA direct sequencing for detection of KRAS G12 D and BRAF V600 E in endoscopic biopsy samples.Methods: Genomic DNAs were extracted from the lesions tissues, which were obtained in distal colorectal adenoma(32 cases) and adenocarcinoma(20 cases) with endoscopic biopsy forceps,and then were sequenced by DNA direct sequencing.At the same time,KRAS G12 D and BRAF V600 E mutations were detected by cast PCR.Results: 1?KRAS mutations were detected in 7 cases of 32 adenoma patients(21.9%) and 7 cases of 20 distal colorectal adenocarcinoma patients(35%) by DNA direct sequencing. KRAS mutations were most typed with G12 D and G13 D mutations in both distal colorectal adenocarcinoma patients and adenoma patients. No significant differences were observed for KRAS mutations between adenoma and adenocarcinoma in distal colorectum(P>0.05) and between KRAS mutation rate and gender?adenoma differentiation degree and differentiation types in both groups. Moreover, BRAF V600 E mutation was detected in these patients. 2?In both colorectal adenoma and adenocarcinoma, the mutation rate of KRAS G12 D by Cast PCR was higher than DNA direct sequencing(28.1% vs 12.5% for colorectal adenoma; 30% vs 15% for colorectal adenocarcinoma). Negative coincidence rate of these two methods is 100%. Cast PCR was enabled to detect mutant allele as little as 0.1% according to Mutation Detector system. However, statistics analysis indicated that the mutation rate of KRAS G12 D is no significantly differences by Cast PCR and DNA direct sequencing in our samples(P>0.05). The overall coincidence rate was 87.5%(Kappa value 0.6429) and 85%(Kappa value 0.5833) in colorectal adenoma and adenocarcinoma, respectively. Moreover, BRAF V600 E mutation was detected in these patients.Conclusions: 1?Among patients with distal colorectal adenocarcinoma and adenoma, mutation rate of BRAF V600 E is much lower than western countries but that of KRAS G12 D or G13 D is high which is almost the same with western countries. So, there may be more clinic value of KRAS mutations in anti-epidermal growth factor receptor(EGFR)monoclonal antibody-based therapy for colorectal cancer. We can be further detect mutated BRAF gene mutation on the base of anti-EGFR monoclonal antibody resistance in wild type of KRAS colorectal cancer. 2?Our data suggested that cast PCR was clinic practical, with highly sensitive to detect mutation in biopsy samples, as well as simple operation and good repeatability.