BRAF, K-ras and BAT26 mutations in colorectal polyps and stool

Abstract

Aim: To assess the feasibility of using BRAF, K-ras and BAT26 genes as stool-based molecular markers for detection of colorectal adenomas and hyperplastic polyps (HPs).

Methods: We applied PCR-SSCP and direct sequencing to detect BRAF mutations of polyps and paired stool samples. Primer-mediated restriction fragment length polymorphism (RFLP) analysis and mutant-enriched PCR were used in detection of K-ras mutations of polyp tissues and paired stool samples respectively. BAT26, a microsatellite instability marker was examined by detection of small unstable alleles in a poly (A) repeat.

Results: No genetic alterations were detected in the 36 colonoscopically normal patients in either tissues or stools. BRAF, K-ras and BAT26 mutations were found in 4 (16%), 10 (40%) and 3 (12%) of 25 adenoma tissues and among them, 75%, 80% and 100% of patients were observed to contain the same mutations in their corresponding stool samples. In HPs, mutations of BRAF and K-ras were detected in the tumor DNA of 2 (11.1%) and 8 (33.3%) of 18 patients respectively, all of whom had identical alterations in their stools. Taken together, the three genetic markers detected 15 (60%) of 25 adenomas and 8 (44.4%) of 18 HPs. The sensitivity of stool detection was 80% for adenomas and 100% for HPs with an overall specificity of 92% for adenomas and 100% for HPs.

Conclusion: BRAF, K-ras and BAT26 genes have the potential to be molecular markers for colorectal adenomas and HPs, and can be used as non-invasive screening markers for colorectal polyps.

Figure 1

Example of BRAF mutation analysis in adenoma tissues (T) and paired stool (S) samples. A: SSCP analysis; B: Direct sequencing showing the wild type BRAF; C: BRAF V599E mutation; N: normal. Arrows indicate the new band (A) and the mutation site (C).

Figure 2

Analysis of K-ras mutations at codon 12 (A) and codon 13 (B) in adenoma tissues (T) and paired stool (S) samples. Fragments of 157 bp indicate mutations, and fragments of 128 bp(A) or 125 bp (B) respectively represent wild-type alleles. M: 100 bp ladder marker; P: Positive control; B shows the result that a mutation at codon 13 of K-ras in stool DNA was not observed in tumor counterpart.