Quantitative real-time RT-PCR detection for survivin, CK20 and CEA in peripheral blood of colorectal cancer patients.
OBJECTIVE
To establish a sensitive method for the early detection of circulating tumor cells (CTCs) in peripheral blood (PB) of colorectal cancer (CRC) patients.


METHODS
PB samples were collected from 156 CRC patients, 40 benign colorectal disease patients, 40 healthy individuals and 45 patients with other solid tumors. The combination of negative and positive immunomagnetic bead method was used to enrich cancer cells. Then, cytokeratin-20 (CK20), survivin and carcinoembryonic antigen (CEA) mRNA were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). In addition, analyses were carried out for their correlation with patients' clinicopathologic features.


RESULTS
The positive rates of survivin, CK20 and CEA mRNA in the PB of CRC patients were 57.7, 47.4 and 39.1%, respectively, and the sensitivity increased from 39.1% of CEA mRNA alone to 60.9% of the combined panel. The expression of the three mRNAs in CRC patients was significantly higher than that in benign control and healthy volunteers, and the expression of survivin and CK20 was not significantly higher than that of patients with other solid tumors. However, the expression of CEA mRNA was significantly higher than that of patients with other solid tumors. The expression of survivin, CK20 and CEA mRNA was significantly correlated with Dukes stages and lymph node metastasis.


CONCLUSIONS
The combined use of negative and positive immunomagnetic beads followed by amplification of survivin, CK20 and CEA mRNA by means of qRT-PCR is a non-invasive and sensitive assay for the detection of circulating CRC cells. The combined panel improved the sensitivity of detection in CRC patients.
Clinical Laboratory Medicine Department, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. shencx1975@sina.com
ChangXin
C
Shen
LiHua
L
Hu
Lin
L
Xia
YiRong
Y
Li
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D002272:Carcinoembryonic Antigen / Q000097:blood* / Q000235:genetics; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000097:blood* / Q000175:diagnosis*; D005260:Female; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D053554:Keratin-20 / Q000097:blood / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000166:cytology; D008297:Male; D008869:Microtubule-Associated Proteins / Q000097:blood* / Q000235:genetics; D008875:Middle Aged; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000097:blood* / Q000235:genetics; D009360:Neoplastic Cells, Circulating / Q000737:chemistry*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin
C107489:BIRC5 protein, human; D002272:Carcinoembryonic Antigen; D051034:Inhibitor of Apoptosis Proteins; C507499:KRT20 protein, human; D053554:Keratin-20; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D000077022:Survivin