Carcinoembryonic Antigen mRNA In Peripheral Blood Research Articles

Detection and quantification of circulating tumor cells in esophageal squamous cancer by real-time fluorescent quantitative reverse transcription-polymerase chain reaction

Objective To evaluate circulating tumor cells (CTCs) in peripheral blood of patients with primary esophageal squamous cancer by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (fqRT-PCR). Methods Carcinoembryonic antigen (CEA) mRNA was detected in the peripheral blood samples of 43 patients with primary esophageal squamous cancer and 10 controls by real-time fqRT-PCR. Results Twenty of 43 (46.51%) patients with esophageal squamous cancer had detectable CEA mRNA in peripheral blood samples and there was no detectable CEA mRNA in control group.The positive expression rate of CEA mRNA was significantly higher in patients with TNM stage Ⅲ, Ⅳ than that in stage Ⅰ , Ⅱ (P＜0.05), but there was no significant difference between stage Ⅰ and Ⅱ , neither between stage Ⅲ and Ⅳ (P＞0.05). CEA mRNA copies were significantly higher in patients with stage Ⅳ than that in stage Ⅰ , Ⅱ , Ⅲ (P＜0.01), but there were no significant differences among stage Ⅰ , Ⅱ and Ⅲ (P＞0.05). There was no correlation between the expression of CEA mRNA and cancer cell differentiation. The follow-up data showed the specificity for predicting metastasis of high CEA mRNA level in peripheral blood was 81.25% (13/16). Conclusion The detection of CEA mRNA in peripheral blood by fqRT-PCR is a sensitive, specific and reliable method to monitor the CTCs in patients with esophageal squamous cancer. Key words: Esophageal cancer; Carcinoembryonic antigen; Peripheral blood

Dissemination profile of perioperative tumor cells in peripheral blood of colorectal cancer patients detected by multiple marker genes

This work proposes a method to assess the molecular profile of perioperative circulating tumor cells in peripheral blood (PB) of colorectal cancer patients for differentiating the dissemination process of tumor cells. Two-point quantification of multiple marker genes was designed for describing the profile. The expression levels of cytokeratin 20 (CK20), carcino-embryonic antigen (CEA) and survivin mRNA in PB and tumor tissue samples in 37 colorectal cancer patients from 1 d pre-operation to 2 h post-operation were detected with real-time quantitative reverse transcription-polymerase chain reaction. β-Actin mRNA was used as internal control to standardize the results of different mRNA expression levels. The data analysis using Stata statistical packages, Chi-Square test and Mann-Whitney test indicated the expression level of CEA mRNA in PB increased significantly, while those of CK20 and survivin mRNA decreased significantly. Quantitative comparison with tumor tissues indicated that the increase of CEA mRNA level in PB coincided with the decrease of CK20 and survivin mRNA levels in different tumor cells. These results showed surgical manipulation caused tumor cells shedding into blood from primary tumor tissue and significant increase of CEA mRNA level, while occult tumor cells with high expression levels of CK20 and survivin mRNA before surgery decreased after surgery.

Carcinoembryonic Antigen Messenger RNA Expression in Blood Predicts Recurrence in Esophageal Cancer

The clinical significance of isolated tumor cells (ITC) in blood has not been clearly established, particularly during follow-up in cancer patients. We conducted a longitudinal analysis of the relationship between ITC in blood during follow-up and clinicopathologic findings in patients with esophageal squamous cell carcinoma. Blood samples obtained from 106 patients were examined by real-time RT-PCR assay targeting carcinoembryonic antigen (CEA) mRNA. Follow-up examination every 3 months after surgery included testing for CEA mRNA and tumor markers, as well as imaging. Thirty-nine (36.8%) patients were positive for CEA mRNA expression. CEA mRNA positivity significantly correlated with tumor depth, lymph node metastasis, stage, and venous invasion. Recurrent disease was found in 34 of 106 (32.1%) cases. CEA mRNA was found in 28 (76.5%) patients experiencing relapse. Of these 28 patients, the number positive of CEA mRNA before detection by imaging, at the same time of detection by imaging, and after detection by imaging was 18 (52.9%), 8 (23.5%), and 2 (5.9%), respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for CEA mRNA were higher than those for serum CEA or squamous cell carcinoma. Patients positive for CEA mRNA experienced significantly shorter disease-free interval than those with negative CEA mRNA (P < 0.001). According to multivariate analysis, CEA mRNA positivity was an independent factor for disease-free interval. Examination of CEA mRNA in peripheral blood during follow-up is useful for early detection of occult recurrence. We believe that CEA mRNA in blood will be a new marker for recurrence in esophageal squamous cell carcinoma.

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

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. 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. 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. 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.

Immunobead RT-PCR versus regular RT-PCR amplification of CEA mRNA in peripheral blood.

The reverse transcription polymerase chain reaction (RT-PCR) amplification of tumor-specific mRNA has been used for the detection of cancer cells in peripheral blood. More recently, an immunomagnetic isolation and reverse transcription polymerase chain reaction (immunobead RT-PCR) was developed which has reportedly significant advantages over the previous RT-PCR analysis. In our study, we compared these two methods using a model set of peripheral blood containing tumor cells under standardized conditions. In order to compare the false positive rate, normal peripheral blood samples from five volunteers were analyzed by both methods. A model set of peripheral blood containing tumor cells was established by adding SNUC4 human colon cancer cells to peripheral blood collected from normal volunteers not showing any nonspecific bands upon electrophoresis of the PCR products. RT-PCR amplification of carcinoembryonic antigen (CEA) mRNA was done with total RNA and mRNA prepared from this model sample. In immunobead RT-PCR analysis, mRNA was prepared from the cells isolated with anti-CEA antibody-coated magnetic beads or anti-Ber-EP4 antibody-coated magnetic beads before the RT-PCR analysis. The immunobead RT-PCR yielded no non-specific band, while the regular RT-PCR using total RNA did show non-specific band formation in all five samples. When mRNA rather than total RNA was used, nonspecific bands were formed in three of the five samples. Immunobead RT-PCR allowed the detection of 10(1) tumor cells in 1 ml of peripheral blood. The regular RT-PCR analysis had a detection limit of 10(2) tumor cells in 1 ml of peripheral blood. The immunobead RT-PCR proved to be more sensitive and specific than the regular RT-PCR at least in our model system.

Detection of colonic cells in peripheral blood of colorectal cancer patients by means of reverse transcriptase and polymerase chain reaction.

Circulating tumour cells play a central role in the metastatic process, but little is known about the relationship between this cellular subpopulation and the development of secondary disease. This study was aimed at assessing the presence of colonic cells in peripheral blood of patients with colorectal cancer in different evolutionary stages, by means of reverse transcriptase polymerase chain reaction (RT-PCR) targeted to carcinoembryonic antigen (CEA) mRNA. In vitro sensitivity was established in a recovery experiment by preparing serial colorectal cancer cell dilutions. Thereafter, 95 colorectal cancer patients and a control group including healthy subjects (n=11), patients with other gastrointestinal neoplasms (n=11) or inflammatory bowel disease (n=9) were analysed. Specific cDNA primers for CEA transcripts were used to apply RT-PCR to peripheral blood samples. Tumour cells were detected down to five cells per 10 ml blood, thus indicating a sensitivity limit of approximately one tumour cell per 10(7) white blood cells. CEA mRNA expression was detected in 39 out of 95 colorectal cancer patients (41.1%), there being a significant correlation with the presence of distant metastases at inclusion. None of the healthy volunteers and only 1 of 11 patients (9.1%) with other gastrointestinal neoplasms had detectable CEA mRNA in peripheral blood. By contrast, CEA mRNA was detected in five of the nine patients (55.6%) with inflammatory bowel disease. These results confirm that it is feasible to amplify CEA mRNA in the peripheral blood, its presence being almost certainly derived from circulating malignant cells in colorectal cancer patients. However, CEA mRNA detectable in blood of patients with inflammatory bowel disease suggests the presence of circulating non-neoplastic colonic epithelial cells.