Detection of cytokeratin 20 mRNA in the peripheral blood of patients with colorectal cancer by immunomagnetic bead enrichment and real-time reverse transcriptase-polymeras chain reaction

Background: Detection of circulating cancer cells is a useful indicator for, the risk of recurrence of advanced carcinoma. The aim of the present study was to evaluate the potential value of a novel approach to detect the circulating cancer cells in patients with colorectal cancer. This method is based on a combination of isolation of epithelial cell by a combination of negative and positive immunomagnetic beads with detection of cytokeratin 20 (CK20) mRNA by reverse transcriptase-polymeras chain reaction (RT-PCR). Methods: Peripheral blood samples were collected from 40 patients with colorectal carcinoma on the day before operation or chemical therapy. Mononuclear cells (MNC) were isolated by centrifugation through a Ficoll gradient. Each MNC sample was equally divided into three parts and then CD45 immunomagnetic beads and/or Ber-EP4 immunomagnetic beads were used to enrich colon cancer cells. Finally, the CK20 mRNA was detected by real-time quantitative RT-PCR. As a control, LS174T colon cancer cells were serially diluted with blood from healthy individuals. Results: When CD45 and Ber-EP4 immunomagnetic beads were used successively, a significant correlation between CK20 mRNA levels and the initial cell concentrations was found in the control recovery experiment. The sensitivity of the assay was one cancer cell in 1 mL healthy blood. In the patient group, CK20 mRNA was detected in 80.0%, 82.5% and 72.5% of patients when CD45, Ber-EP4, and CD45/ Ber-EP4 immunomagnetic beads were used, respectively. The positive detection rates of patients with colorectal carcinoma at Dukes A, B, C, and D stage were 0.0% (0/2), 33.3% (3/9), 86.7% (13/15), and 92.9% (13/14), respectively. The CK20 mRNA positive detection rate in peripheral blood was significantly correlated with tumor diameter (P < 0.0 1, chi(2)), lymphatic metastasis (P < 0.05) and hepatic metastasis (P < 0.05), but not with the differentiation of tumor cells. Conclusion: The combined use of negative and positive immunomagnetic beads followed by amplification of CK20 mRNA by means of RT-PCR is a non-invasive, sensitive, and specific assay for the detection of circulating colonic cancer cells. (C) 2005 Blackwell Publishing Asia Pty Ltd.