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 < 0.01, chi(2)), lymphatic metastasis (P < 0.05) and hepatic metastasis (P < 0.05), but not with the differentiation of tumor cells.


CONCLUSIONS
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.
Guo
Junming
J
School of Medicine, Ningbo University, Ningbo 315211, China. junmingguo@yahoo.com
Xiao
Bingxiu
B
Jin
Zhijin
Z
Qin
Lijun
L
Chen
Jian
J
Chen
Hui
H
Zhang
Xinjun
X
Liu
Zhong
Z
D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000097:blood / Q000175:diagnosis*; D005260:Female; D006801:Humans; D018189:Immunomagnetic Separation; D053554:Keratin-20; D007633:Keratins / Q000097:blood*; D017493:Leukocyte Common Antigens; D008297:Male; D008875:Middle Aged; D009360:Neoplastic Cells, Circulating; D012333:RNA, Messenger / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001613:beta 2-Microglobulin / Q000032:analysis; D000951:Antigens, Neoplasm
D014408:Biomarkers, Tumor; D053554:Keratin-20; D007633:Keratins; D017493:Leukocyte Common Antigens; D012333:RNA, Messenger; D001613:beta 2-Microglobulin; C507499:KRT20 protein, human; C115012:human epithelial antigen-125; D000951:Antigens, Neoplasm