Molecular detection of circulating tumor cells in the peripheral blood of patients with colorectal cancer using RT-PCR: significance of the prediction of postoperative metastasis.
BACKGROUND
Approximately 20%-45% of colorectal cancer (CRC) patients ultimately develop local recurrence or metastasis following curative surgical resection. The latter is caused by tumor cells shed from the primary carcinoma prior to or during operation, currently undetected by standard clinical staging. Fortunately, the presence of tumor cells in peripheral blood can be detected by molecular methods and is being regarded increasingly as a clinically relevant prognostic factor.


METHODS
To detect the presence of circulating tumor cells and evaluate their relationship to postoperative metastatic relapse, we simultaneously examined human telomerase reverse transcriptase (hTERT), cytokeratin-19 (CK-19), cytokeratin-20 (CK-20), and carcinoembryonic antigen (CEA) mRNA (messenger RNA) in the peripheral blood of 72 CRC patients and 30 healthy individuals. Using a reverse-transcriptase polymerase chain reaction (RT-PCR), these tumor-related mRNAs were amplified; in addition, analyses were carried out for their correlation with patients' clinicopathologic features, as well as the occurrence of postoperative metastasis.


RESULTS
In RT-PCR analysis of the peripheral blood, 69.4% (50 out of 72), 66.7% (48 out of 72), 52.8% (38 out of 72), and 72.2% (52 out of 72) of CRC patients were positive for hTERT, CK-19, CK-20, and CEA mRNA respectively. All 30 healthy individuals were negative for hTERT and CEA mRNA expression, while 2 were positive for either CK-19 mRNA or CK-20 mRNA expression. The detection of CEA mRNA was significantly correlated with depth of tumor invasion (P=0.012), vessel invasion (P=0.035), TNM stage (P<0.0001), and postoperative metastasis (P<0.0001), while positive hTERT mRNA was correlated with TNM stage (P=0.037) and CK-19 was correlated with depth of tumor invasion (P=0.039) and postoperative metastasis (P=0.017). In addition, multivariate logistic regression showed that only CEA mRNA was an independent and significant predictor of postoperative metastasis (P=0.006). Our findings suggest that CEA mRNA may be a more reliable marker than hTERT, CK-19, and CK-20 for the detection of circulating cancer cells in the peripheral blood of CRC patients.


CONCLUSIONS
Using RT-PCR for the detection of CEA mRNA is feasible and may be a promising tool for early detection of micrometastatic circulating tumor cells in CRC patients. CRC patients expressing positive CEA mRNA in peripheral blood have a significantly higher risk of postoperative metastasis. Nevertheless, confirmation of CEA mRNA as a prognostic predictive factor requires the continuation of patient follow-up.
Wang
Jaw-Yuan
JY
MedicoGenomic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan. srlin@ms2.hinet.net
Wu
Chan-Han
CH
Lu
Chien-Yu
CY
Hsieh
Jan-Sing
JS
Wu
Deng-Chyang
DC
Huang
Sung-Yu
SY
Lin
Shiu-Ru
SR
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis*; D002272:Carcinoembryonic Antigen / Q000032:analysis / Q000235:genetics; D002277:Carcinoma / Q000556:secondary* / Q000601:surgery; D015179:Colorectal Neoplasms / Q000473:pathology* / Q000601:surgery; D005260:Female; D006801:Humans; D053539:Keratin-19 / Q000032:analysis / Q000235:genetics; D053554:Keratin-20 / Q000032:analysis / Q000235:genetics; D008207:Lymphatic Metastasis; D008297:Male; D008875:Middle Aged; D009364:Neoplasm Recurrence, Local; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000032:analysis / Q000235:genetics
D014408:Biomarkers, Tumor; D002272:Carcinoembryonic Antigen; D053539:Keratin-19; D053554:Keratin-20; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D019098:Telomerase