Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer

A new method for detecting circulating tumor cells that is based on magneti c-activated cell separation (MACS) and nested mutant allele-specific amplif ication (nested MASA) was evaluated in patients with colorectal cancer usin g the p53 and K-ras genes as genetic markers. By negative selection with an ti-CD45 monoclonal antibody-conjugated supermagnetic microbeads, the propor tion of tumor cells was enriched 9-fold. By the combination of MACS and nes ted MASA, 10 tumor cells in 10(7) normal peripheral blood mononuclear cells could be detected without false-positives. Using this method, we examined blood taken from the tumor drainage veins of 23 patients with colorectal ca ncer. Eighty-seven percent (20/23) of primary tumor tissues showed p53 and/ or K-ras gene mutations. Forty-five percent (9/20) of patients with p53 and /or K-ras mutations in the primary tumor showed the same mutated genes in t he blood samples. There was a significant association between the presence of p53 and K-ras gene mutation in the blood and tumor size, depth of invasi on, and venous invasion. Blood gene mutation was detected in 80% (4/5) of s amples from patients with synchronous liver metastases. Sixty percent (3/5) of patients with mutant genes in the blood developed asynchronous liver me tastases after surgery. The overall survival of patients with p53 and/or K- ras gene mutation-positive findings in blood was significantly shorter than that of patients testing negative on Kaplan-Meier analysis. Our results su ggest that the method may be useful for reliable detection of tumor cells c irculating in the blood and may help to identify patients at high risk for relapse. (C) 2000 Wiley-Liss, Inc.