Molecular techniques for the detection of colorectal cancer cells in the peritoneal cavity
Colorectal cancer is a major health problem, and many patients relapse despite apparently curative treatment. Local recurrence is an important factor, and is more common when cancer cells are present on the free serosal surface or circumferential resection margin. We hypothesised that detecting tumour cells in the peritoneal cavity during surgery (in peritoneal washings) or post-operatively (in drain fluids) would act as a marker for risk of local recurrence, and that the use of molecular biological techniques would allow for the sensitive detection of such cells. We collected samples of colorectal tumours, with washings from the peritoneal cavity at the start and end of surgery, and fluid from the surgical drain on the first and second postoperative day. Epithelial cells in the peritoneal samples were enriched using magnetic cell separation (MACS). Using mutation specific PCR or a Mismatch Ligation Assay we detected common mutations of K-ras, TP53, APe and BRAF in primary tumour samples, and when such a mutation was found, we studied the peritoneal samples from that patient for the same mutation. We detected 23 mutations in 22 (out of 46) tumours from 21 patients. In 16 patients, at least one of the peritoneal samples gave a positive result for the same mutant DNA. Using MACS increased the proportion of positive samples. Half of the patients with positive peritoneal samples had Dukes' stage A or B tumours. Follow up is not yet long enough to allow conclusions to be drawn on the significance of these results. We have described techniques allowing mutations to be characterised in half of colorectal tumours and have demonstrated the presence of cells with the same mutation in peritoneal samples from three-quarters of patients. Longer follow up will show whether our tests are too sensitive, or whether they provide useful information about likely local recurrence.