Persistence of K-ras Mutations in Plasma after Colorectal Tumor Resection

Abstract

Free DNA in the circulation is increased five-to ten-fold in patients with solid tumours compared to healthy controls. A range of tumor-specific mutated DNA has been shown to be readily extractable and possible to analyse from plasma and serum in these patients. K-ras oncogene mutations are an early event in a subset of colorectal tumors and have been found in 30-60% of patients with colorectal carcinoma (CRC). The presence of tumor-derived k-ras gene mutations in the circulation has previously been described before surgery. The aim of this study was to characterize the presence of mutant k-ras in plasma in the short-term postoperative period after radical surgery of CRC patients, and further to characterize this in relation to relapse of the disease. Tumors and corresponding plasma pre- and postoperatively on day three after surgery were collected from 25 patients with CRC (Dukes' stage A-D). Biopsies for DNA extraction from the tumors were collected from the most invasive parts microscopically. After PCR amplification of the k-ras gene (codon 12 and 13), the presence of mutations was analysed by TGGE (temperature gradient gel electrophoresis). Twenty four/25 patients underwent putatively curative resections. Sixteen of the 25 patients (64%) expressed k-ras mutations in their tumor. Of these, 9 patients (56%) also had detectable k-ras mutations in preoperative plasma samples. On day three postoperatively, 8 of these patients persistently were found to have mutant k-ras in the plasma. This was not correlated with tumor stage. None of the 9 tumor mutation-negative cases expressed mutated k-ras in their plasma pre- or postoperatively. The results indicate that plasma mutant k-ras can be detected pre- and early postoperatively in all stages of colorectal neoplasia. No correlation between short-term postoperative persistence of mutant plasma-DNA and disease recurrence at follow-up was found. However, the use of k-ras as a marker during postoperative follow-up and as a possible tool for early detection of recurrent disease must be further characterized.