Detection of BRAF mutations in colorectal tumours and peritoneal washings using a mismatch ligation assay
- Correspondence to: Dr A Morris Department of Biological Sciences, University of Warwick, Coventry CV4 AL, UK; a.g.morriswarwick.ac.uk
- Accepted 20 September 2004
Abstract
Aims: To detect cells bearing BRAF mutations in colorectal tumour samples and peritoneal washings, using a mismatch ligation assay (MLA).
Methods: DNA from 46 colorectal tumours was studied. Part of exon 15 of the BRAF gene was amplified using the polymerase chain reaction, and T→A mutations at codon 600 were detected using MLA. When a mutation was detected, the same mutation was sought in peritoneal washings from that patient.
Results: BRAF mutations were detected in five of the 45 analysable tumour samples. In four cases, this result was confirmed by sequencing analysis. More tumours with BRAF mutations were Dukes’ stage C or D rather than A or B (p < 0.02). Dilution experiments revealed that one mutant cell could be detected in 1000 normal cells. Cells with the same BRAF mutation were present in the peritoneal washing taken at the start of the operation in four of the five patients.
Conclusions: MLA is a suitable technique for the detection of BRAF mutations, and allows the detection of small numbers of isolated tumour cells shed from the primary tumour.
- MACS, magnetic activated cell separation
- MLA, mismatch ligation assay
- PCR, polymerase chain reaction
