Blood Of Colorectal Cancer Patients Research Articles

Early detection of cancer/testis mRNAs in tumor cells circulating in the peripheral blood of colorectal cancer patients

Recent studies have suggested that mRNAs transcribed from cancer/testis genes might be used as biomarkers of cancer cells migrating through the bloodstream. Using RT-PCR we evaluated the expression of several cancer/testis mRNAs (MAGEA1-6, GAGE1-8, NY-ESO-1, SSX1, 2, and 4, XAGE1 and MAGEC1) in primary tumors and peripheral blood samples of colorectal cancer patients. The detection rate of at least one of the transcripts was 95% (37/39 samples) for primary tumors and 81% (52/64 samples) for the peripheral blood. Moreover, selected mRNAs were detectable in cellular fraction of the peripheral blood at all stages the disease (14 out of 14 cases), whereas in extracellular fraction of plasma were found only at stages III and IV. Obtained data open a possibility of early diagnosis of colorectal cancer in people at high risk by peripheral blood analysis.

P-0200 Multiparameter Flow Cytometric Analysis of CD40 Expression on Circulating Colorectal Cancer Cells

ABSTRACT Introduction Circulating tumour cells in the peripheral blood of colorectal cancer patients may be an important indicator of worse prognosis. They include an heterogeneous population of cells with different biological characteristics. The cell surface costimulatory molecule CD40 (a glycosylated phosphoprotein member of Tumour Necrosis Factor, receptor superfamily), is widely expressed by lymphoid cells and by various tumour types. It has been indicated as prognostic/predictive biomarker and as a potential target for therapy in human tumours. In a previous study we showed that the inhibition of colorectal cancer cell proliferation by the soluble CD40 ligand was mainly due to a slowing down of the cell cycle progression thus confirming the multiple anti-proliferative/growth-inhibitory effects of the CD40 pathway stimulation in this tumor. In the present study we wanted to demonstrate the value of a multiparameter flow cytometric (FCM) approach for a further biological characterization (such as the CD40 expression) of circulating colorectal cancer cells in vivo. Methods In the first part of the study, SW48 human colorectal cancer cells (highly positive for CD40) were serially diluted in normal whole blood in order to evaluate the sensitivity of the method in both the cancer cell identification and in their characterization for CD40 expression. Then, we analyzed peripheral blood samples from 9 patients with metastatic colorectal cancer (before starting the medical treatment for their advanced disease) to detect and characterize circulating cancer cells; 3 normal subjects were utilized as negative controls. The study were performed using a Navios FCM (Instrumentation Laboratories) equipped with a dedicated software for multicolor analyses. Results The method was found to have a sensitivity limit of 10(-5) for the detection of circulating tumor cells. The specificity of the method was 100%. In 8 of our patients we were able to differentiate in vivo circulating cancer cells from normal mononuclear cells based on: increased light scatter parameters, cell marker expression (EpCAM+ CD45-) and nuclear stain with 4'6-diamidino-2-2phenylindole, DAPI. A simultaneous analysis of the CD40 expression was also successfully performed on these cells and in 4 out of 8 patients, circulating tumor cells were found to express detectable levels of CD40. Conclusion Multiparameter FCM can effectively detect circulating tumor cells in colorectal cancer patients and allows to evaluate their CD40 expression. The technique has the potential to be a valuable and sensitive tool for prognosis assessment in these patients and for future studies aimed to evaluate new therapeutic strategies such as antitumor immunotherapy approaches in the metastatic setting.

Circulating miR‐34a levels are reduced in colorectal cancer

MicroRNAs (miRNAs) are small, non-coding RNA segments that regulate gene expression via post-transcriptional inhibition and have roles in cell differentiation, proliferation, and apoptosis. Expression differs between tumor and normal tissue in several malignancies. Most work has focused on tissue and cell expression with few reports of circulating miRNAs in colorectal cancer. Available biomarkers for colorectal cancer have limited sensitivity and specificity, thus there is a need for new markers. This study aimed to identify miRNAs that are differentially expressed in the blood of colorectal cancer patients compared to controls and to establish if this is specific to colorectal cancer and thus could be utilized as potential tumor markers. Blood samples were collected from 63 colorectal cancer patients and 45 controls. Expression of 7 target miRNAs (miR-143, miR-145, miR-21, miR-30a-3p, miR-31, miR-34a, and miR-92) was measured using RQ-PCR. Results were correlated with clinicopathological data and analyzed. Analysis of differentially expressed circulating miRNAs was expanded to include 62 patients with prostate, renal, breast, and melanoma cancers. Analysis of the relative quantification of the target miRNAs showed significantly reduced expression (P = 0.004) of miR-34a in colorectal cancer. MiR-34a was also significantly reduced in breast cancer (P = 0.019). This study demonstrates significantly reduced expression of circulating miR-34a in colorectal and breast cancer. This may have future application as part of a biomarker profile.

Comparative Analysis of Tumor Markers and Evaluation of Their Predictive Value in Patients with Colorectal Cancer

Background: We evaluated the feasibility of CEA/CK20 mRNA and CEA/CA19-9 proteins as tumor markers for colorectal cancer by detecting tumor-specific mRNAs in circulating tumor cells and secreted tumor-specific proteins in the peripheral blood of colorectal cancer patients. Patients and Methods: Peripheral blood was obtained from 23 healthy volunteers and 46 colorectal cancer patients on the day of initiation of adjuvant chemotherapy after surgery (stages I–III, n = 27) or on the first day of chemotherapy after diagnosis (stage IV, n = 19). Levels of CEA/CK20 mRNA in peripheral blood mononuclear cells (PBMCs) were determined with quantitative real-time reverse transcription polymerase chain reaction, and serum CEA/CA19-9 protein levels were determined by radioimmunoassay. Results: The detection sensitivity of CK20 mRNA was approximately 1 tumor cell in 1 × 10⁷ PBMCs, and that of CEA mRNA was approximately 1 tumor cell in 1 × 10⁶ PBMCs. Patients with stage IV colorectal cancer had higher levels of CEA mRNA, CK20 mRNA, and serum CEA than patients at stages I–III. Peripheral blood CEA mRNA levels were predictive of overall survival, while serum protein levels of CEA and CA19-9 had no predictive value. Conclusion: Peripheral blood CEA mRNA is a useful marker of overall survival in colorectal cancer patients, that is sensitive and specific.

Quantitative analysis of TEM-8 and CEA tumor markers indicating free tumor cells in the peripheral blood of colorectal cancer patients

Colorectal cancer (CRC) remains the third most common cancer in the world. Approximately in 50 percent of patients, metastatic disease is a major cause of death. Therefore, early diagnosis of CRC is crucial for a successful outcome. For the detection of circulating cancer cells, this study applied a sensitive method that employed specific tumor markers for early detection. A total of 80 blood samples from 40 CRC patients and 40 age-matched healthy controls were collected for the study. The circulating mRNA levels of two CRC tumor markers, tumor endothelial marker 8 (TEM-8) and carcinoembryogenic antigen (CEA) were evaluated using an absolute quantitative real-time PCR assay in a Stratagene Mx-3000P real-time PCR system. GAPDH was used as the endogenous control. TEM-8 and CEA were primarily detected more in the CRC patients rather than in the controls: 22/40 vs 9/40, p=0.009 and 30/40 vs 11/40, p=0.00054, respectively. In the CRC patients, the mRNA level of these markers was significantly higher in comparison to the normal controls (p=0.018 and 0.01). The overall sensitivity of this panel was 65% with a specificity of 75%. Statistical analysis for demographic variants did not reach significant values. TEM-8 and CEA markers were detected more frequently and in significantly higher levels in the blood samples of patients compared with samples from age-matched healthy controls. The copy number of CEA and TEM-8 mRNA, as detected by a real-time quantitative PCR, appears to be a promising marker for evaluating the risk of tumor spread.

Comparison of three distinct methods for the detection of circulating tumor cells in colorectal cancer patients

The detection of circulating tumor cells (CTCs) has considerable utility in the clinical management of patients with solid cancers. However, the phenotypic heterogeneity of CTCs and their low numbers in the bloodstream of patients means that no standardized detection method currently exists for these cells. This, together with differences in pre-analytical sample processing, has led to the collection and accumulation of inconsistent data among independent studies. Here, we compare the ability of three methods to detect CTCs in the blood of colorectal cancer patients. Specifically, different aliquots of the same blood sample were screened for the presence of CTCs by a multimarker RT-PCR assay, the standardized CellSearch assay and dHPLC-based gene mutation analysis. In the population tested, none of the blood samples analysed appeared to be positive by all three methods. Of the samples, 75% were positive for the presence of CTCs by the RT-PCR method. Only 20% were positive by the CellSearch assay, while 14.3% of samples displayed gene mutations consistent with the presence of CTCs when the dHPLC method was applied. The samples which were positive for CTCs by the CellSearch assay did not overlap with those that were positive by dHPLC. Interestingly, however, all of these samples were positive when assessed by RT-PCR. Conversely, of the samples that resulted negative by RT-PCR analysis, none appeared to be positive by either of the other methods. These data, therefore, indicate that of the three methods tested, the multimarker RT-PCR assay provides maximal probability of CTC detection. Here, we present the preliminary results of an ongoing clinical study. Future follow-up involving detection of CTCs in the blood of colorectal cancer patients using these three distinct methods will allow us to verify whether either a single method, or a combination of different assays, is necessary to uncover further prognostic significance of circulating tumor cells.

Assessment of Activity of an Adhesion Molecule CD134 and CD137 in Colorectal Cancer Patients

Epidemiological studies prove that incidence of colorectal cancer is increasing. The first line therapy of colorectal cancer is surgical resection of the primary tumor and elimination of regional and remote metastases. THE AIM OF THE STUDY was to determine expression of adhesion molecules CD134 and CD137 in the peripheral blood in colorectal cancer patients, depending on clinical cancer stage, size and invasion of the tumor. The study enrolled 72 patients with primary colorectal adenocarcinoma. An average patient age was 64.55 years. Clinical tumor stage was assessed using two scales: Dukes: A and Astler-Coller scale. Expression of adhesion molecules was determined in the peripheral blood collected on the day of the procedure and 10 days after the procedure. An average activity of CD134 molecules (12.66%) was significantly higher than that of CD137 (6.26%) (p<0.001). Clinical tumor stage was assessed on Dukes scale and was unrelated to CD134 activity, while activity of CD137 was related to clinical cancer stage. CD137 activity is directly proportional to colorectal cancer stage. Surgical resection of the tumor results in increased CD134 and CD137 expression. Long term studies, enrolling larger groups of patients, including their subdivision to colon and rectal cancer, are required to utilize CD134 and CD137 in immune therapy of colorectal cancer.

Cytokeratin 20 and vascular endothelial growth factor as molecular markers in Egyptian patients with colorectal cancer

To investigate the expression of cytokeratin 20 (CK20) and vascular endothelial growth factor (VEGF) in the peripheral blood of colorectal cancer (CRC) patients, and correlate the findings with the pathologic data of the patients. This study was carried out on 50 subjects, 40 patients with histologically confirmed colorectal carcinoma undergoing elective surgery and 10 healthy individuals matched for age and sex. Total RNA extraction followed by real time quantitative RT-PCR and real time TaqMan quantitative assay for peripheral blood expression of CK20 and VEGF was done for both patients and controls. (1) Statistically significant high levels of CK20,VEGF, CEA (p = 0.000 each) and CA19-9 (p = 0.002) in CRC patients when compared with controls; (2) Statistically significant increase in the expression of CK20 in advancing CRC stage C (p = 0.001) and with LN metastasis (p = 0.000); (3) Statistically significant increase in the expression of VEGF in advancing CRC stage C (p = 0.002), pathologic grade (p = 0.038), and with LN metastasis (p = 0.004); and (4) statistically positive correlation between CK20 and VEGF expressions, and also between these markers and CEA level. CK20 and VEGF expressions in peripheral blood of CRC patients are promising molecular markers for CRC progression and metastasis.

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.

Antigen-specific Tregs control T cell responses against a limited repertoire of tumor antigens in patients with colorectal carcinoma.

Spontaneous antitumor T cell responses in cancer patients are strongly controlled by Tregs, and increased numbers of tumor-infiltrating Tregs correlate with reduced survival. However, the tumor antigens recognized by Tregs in cancer patients and the impact of these cells on tumor-specific T cell responses have not been systematically characterized. Here we used a broad panel of long synthetic peptides of defined tumor antigens and normal tissue antigens to exploit a newly developed method to identify and compare ex vivo the antigen specificities of Tregs with those of effector/memory T cells in peripheral blood of colorectal cancer patients and healthy subjects. Tregs in tumor patients were highly specific for a distinct set of only a few tumor antigens, suggesting that Tregs exert T cell suppression in an antigen-selective manner. Tumor-specific effector T cells were detectable in the majority of colorectal cancer patients but not in healthy individuals. We detected differences in the repertoires of antigens recognized by Tregs and effector/memory T cells in the majority of colorectal cancer patients. In addition, only effector/memory T cell responses against antigens recognized by Tregs strongly increased after Treg depletion. The selection of antigens according to preexisting T cell responses may improve the efficacy of future immunotherapies for cancer and autoimmune disease.

On the origin of serum CD26 and its altered concentration in cancer patients.

Dipeptidyl peptidase IV (DPP-IV), assigned to the CD26 cluster, is expressed on epithelial cells and lymphocytes and is a multifunctional or pleiotropic protein. Its peptidase activity causes degradation of many biologically active peptides, e.g. some incretins secreted by the enteroendocrine system. DPP-IV has, therefore, become a novel therapeutic target for inhibitors that extend endogenously produced insulin half-life in diabetics, and several reviews have appeared in recent months concerning the clinical significance of CD26/DPP-IV. Biological fluids contain relatively high levels of soluble CD26 (sCD26). The physiological role of sCD26 and its relation, if any, to CD26 functions, remain poorly understood because whether the process for CD26 secretion and/or shedding from cell membranes is regulated or not is not known. Liver epithelium and lymphocytes are often cited as the most likely source of sCD26. It is important to establish which tissue or organ is the protein source as well as the circumstances that can provoke an abnormal presence/absence or altered levels in many diseases including cancer, so that sCD26 can be validated as a clinical marker or a therapeutic target. For example, we have previously reported low levels of sCD26 in the blood of colorectal cancer patients, which indicated the potential usefulness of the protein as a biomarker for this cancer in early diagnosis, monitoring and prognosis. Through this review, we envisage a role for sCD26 and the alteration of normal peptidase capacity (in clipping enteroendocrine or other peptides) in the complex crosstalk between the lymphoid lineage and, at least, some malignant tumours.

Detection of CD4+ , CD8+ and NK in peripheral Mood of patients with colorectal cancer and its relationship with micrometastasis in regional lymph nodes

Objective To detect the positive rate of CK20 mRNA in regional lymph nodes and CD4+ ,CD8+ and NK cells in peripheral blood of colorectal cancer patients, and intestigate their relation-ship. Methods The positive rate of CD4+, CD8+ and NK cells in peripheral blood of 21 patients with colorectal cancer was detected by flow cytometry. The positive rate of CK20 mRNA in lymph nodes was tested by real time-polymerase chain reaction. The lymph node metastasis was examined pathologically. Re-suits For the opitive micrometastasis in regional lymph nodes patients,it was 16 nodes(6% ,16/281 ) by conventional pathological and 140 nodes (50%. 140/281 ) by real time-polymerase chain reaction, accord-ing to the opitive micrometastasis in regional lymph nodes restage the Dukes stage ,before the operation,the opitive rate of CD4+ , CD4+/CD8+ of patients in Dukes A and B were higher then Dukes C (P <0.05) ; and CD8+ in Dukes A patients were lower then Dukes C ( P < 0.05 ) ; and NK in Dukes B patients were higher then Dukes C ( P < 0. 05 ). The new Dukes stage was significantly correlated with CD4+ ( r = -0.497,P <0.01) ;CD4+/CD8+ (r = -0.714,P <0.01) ;CD8+ (r =0. 945,P <0.01 ). Conclusion For detecting the micrometastasis of lymph nodes, the method of realtime-pelymerase chain reaction were better than anventional pathological significantly, and the opitive micrometastasis in lymph nodes were cor-related significantly with the low immunity function of colorectal cancer patients. Key words: Colorectal carcinoma; Lymph node ; Micrometastasis ; Cytokeratin 20

Quantitative Real-time RT-PCR Detection for Survivin, CK20 and CEA in Peripheral Blood of Colorectal Cancer Patients

To establish a sensitive method for the early detection of circulating tumor cells (CTCs) in peripheral blood (PB) of colorectal cancer (CRC) patients. 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. 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. 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.

Quantitative RT-PCR Detection of Colorectal Tumor Cells in Peripheral Blood—A Systematic Review

Twenty percent to 40% of patients with node-negative colorectal cancer die of metastatic disease. Detection of cancer cell dissemination has been proposed as a tool to select patients at highest risk for recurrence. In this review, we summarize the evidence for detection with quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assays of circulating tumor cells (CTCs) in peripheral blood of colorectal cancer patients. Pubmed and Embase were systematically searched for all English publications relevant to circulating cancer cells, peripheral blood, quantitative RT-PCR (q-RT-PCR), and colorectal cancer. Cross-references and the "related articles" function were used to broaden the search. Manuscripts reporting on the results of nonquantitative RT-PCR assays were excluded. The study methodology, CTCs detection rates in peripheral blood, and prognostic value were reviewed. Twelve manuscripts on qRT-PCR were retrieved. Stage dependence was found for detection of CTCs in four of 10 studies. From univariate analysis performed for disease-free survival and overall survival in 4 of 12 studies, there was evidence (P < 0.05) for an effect of the detection of CTCs with qRT-PCR. None of the included trials identified detection of CTCs in peripheral blood as an independent predictor of survival. Quantification of CTCs in peripheral blood holds promise in predicting stage and outcome in colorectal cancer patients. At present, evidence from the literature is too scarce to integrate quantitative RT-PCR assays to detect CTCs into the management of colorectal cancer.

Quantitative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood of colorectal cancer patients

This study is aimed at establishing a sensitive approach to detect disseminated tumor cells in peripheral blood and evaluate its clinical significance. A total of 198 blood samples including 168 from colorectal carcinoma (CRC) patients and 30 from healthy volunteers were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to evaluate the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and cytokeratin 19 (CK19) mRNA. CEA mRNA was detected in 35.8% of patients and 3.3% of controls, CK20 mRNA in 28.3% of patients and 6.7% of controls, and CK19 mRNA in 41.9% of patients and 3.3% of controls. CEA and CK20 mRNA positive ratio increased with the advancing Dukes stages, but there was no significant difference in positive ratio between any two stages (P>0.05). Also, relatively high positive ratio of CEA, CK20 and CK19 mRNA expression was observed in some CRC patients with earlier Dukes stages. A higher positive ratio was obtained when two or three detection markers were combined compared to a single marker. Our study indicates that quantitative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood is a valuable tool for monitoring early stage dissemination of CRC cells in blood circulation.

Long-term prognostic value of detection of circulating colorectal cancer cells using CGM2 reverse transcriptase-polymerase chain reaction assay

The criteria commonly used for prognosis of colorectal cancer remain histoprognostic and are based on primarily TNM classification. The lack of discrimination of purely histoprognostic criteria is evidenced by the development of different outcomes in similarly staged patients. The aim of this work was to study the long-term prognostic value of preoperative detection of circulating enterocytes in the blood of colorectal cancer patients using the CGM2 reverse transcriptase-polymerase chain reaction (RT-PCR) assay. A nested RT-PCR with specific primers for CGM2 was used preoperatively to detect circulating enterocytes in 121 patients (64 men, 57 women; mean age, 70 years) with colorectal neoplasms. Circulating enterocytes were detected in 58/121 (48%) patients. The positivity rate was not correlated with American Joint Committee on Cancer (AJCC) staging (stage I, 11/28 (39%); stage II, 13/34 (38%); stage III, 15/23 (65%); stage IV, 17/32 (53%); sterilized (after radiotherapy, no residual neoplasm) 2/4 (50%); not significant [NS]), but circulating enterocytes were detected more frequently in patients with metastatic lymph nodes (60% vs 41%, P = .06). Overall 5-year survival rates (mean +/- SD) were 40 +/- 13% and 45 +/- 13% for patients without and with circulating enterocytes, respectively (P = NS). Similarly, recurrence-free survival rates were 71 +/- 4% versus 72 +/- 14% (P = NS). Using univariate analysis, AJCC stage (P < .0001) was correlated with survival. AJCC stage (P = .007) and obstructive neoplasms (P = .043) were correlated with recurrence-free survival. Using multivariate analysis, AJCC stage was correlated with survival and recurrence-free survival. Preoperative detection of circulating enterocytes using CGM2 RT-PCR assay provides no specific prognostic information and cannot be used as a decision criterion for adjuvant therapy.

Immunobead multiplex RT-PCR detection of carcinoembryonic genes expressing cells in the blood of colorectal cancer patients

Circulating cell detection using reverse transcriptase-polymerase chain reaction (RT-PCR) techniques has been studied as a new prognostic factor in colorectal cancer patients. With the view of enhancing detection sensitivity, we developed a new multiplex RT-PCR assay for circulating cell detection based on the expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5; formerly CEA) and CEACAM7 (formerly CGM2). Between November 2002 and December 2003, 45 stage III-IV, 39 stage I-II colorectal cancer patients, 32 non-colorectal cancer patients and 41 healthy individuals were included. Positive selection using HEA-125 immunobeads was applied to blood samples before mRNA extraction, cDNA synthesis and a multiplex CEACAM5/CEACAM7 RT-PCR assay. For both CEACAM5 and CEACAM7, the limit of detection was found to be as low as 1 expressing cell in 10(6) nucleated blood cells. The multiplex RT-PCR assay was negative for the 41 healthy individuals and the 32 non-colorectal cancer patients. The test was positive in 53/84 (63%) of the colorectal cancer patients for CEACAM5 and/or CEACAM7, whereas 32/84 (38%) were positive for both markers. Colorectal cancer patients were positive for one of the two markers in 80% of cases (36/45) for stage III-IV patients (CEACAM5 73%, CEACAM7 51%) and in 44% of cases (17/39) for stage I-II patients. This multiplex RT-PCR assay with two markers proved to be more sensitive than use of a single marker in detecting circulating tumour cells. The discrepant expression of CEACAM5 and CEACAM7 may label circulating tumour cells that have different levels of differentiation and subsequent aggressive behaviour.

Significance of CK20 mRNA expression in peripheral blood of colorectal cancer patients by real-time fluorescent quantitative RT-PCR

To detect the expression of cytokeratin 20 (CK20) mRNA in peripheral blood of colorectal carcinoma and to discuss its clinical value. Real-time fluorescent quantitative RT-PCR was used to detect the CK20 mRNA expression in the peripheral blood of 51 patients with colorectal carcinoma and 30 healthy volunteers. 27.45% of the patients showed CK20 mRNA expression, while it was 6.67% for the control group (P<0.025). With the progress of Dukes' stages, the expression level of CK20 mRNA increased, but there was no statistic significance (P<0.05). More samples in Dukes'C and D than in Dukes'A and B stages showed >10 copies/ml. The detection of CK20 mRNA expression in peripheral blood of patients with colorectal carcinoma may be helpful to identify early shedding tumor cells. It is also useful to monitor the progression of the disease and observe the effect of clinical treatment.

Impact of circulating free tumor cells in the peripheral blood of colorectal cancer patients during laparoscopic surgery.

Despite widespread use of laparoscopic surgery for colorectal operations, its application for curative resection of colorectal cancer is still controversial. One of the major concerns is the impact of the laparoscopic procedure on dissemination of tumor cells. The main purpose of this study was to investigate the impact of laparoscopic surgery on circulating tumor cells in colorectal cancer patients. Quantitation of circulating free tumor cells (FTCs) was performed preoperatively, during the operation, and 14 days later by means of real-time quantitative reverse transcriptasepolymerase chain reaction (RT-PCR) targeting guanylyl cyclase C (GCC) mRNA in 42 colorectal cancer patients undergoing laparoscopic resections. Despite an increasing trend of FTC detection in patients with advancing stage, there is no significant difference in the preoperative FTC level by disease stage. No elevation in FTC level was found during the laparoscopic procedure in most patients compared with their preoperative FTC value. Patients with a persistently high FTC load [per nucleated blood cells (NBCs)] (> 10(2) FTCs/10(6) NBCs) 2 weeks postoperatively portends a poor prognosis regarding disease recurrence and tumor-related mortality when compared to those with an undetectable or low FTC load (< or = 10(2) FTCs/10(6) NBCs). We concluded that the laparoscopic procedure itself had no significantly deleterious effect on circulating FTCs and that the detection of FTCs by real-time qRT-PCR might be of clinical importance during the postoperative follow-up for colorectal cancer patients.

Quantitative detection of CEA expressing free tumor cells in the peripheral blood of colorectal cancer patients during surgery with real-time RT-PCR on a LightCycler

We applied novel real-time reverse transcriptase-polymerase chain reaction (RT-PCR) with a LightCycler for quantitative detection of carcinoembryonic antigen (CEA) mRNA expressing tumor cells in the peripheral blood of colorectal cancer patients. Analysis of peripheral blood samples from 99 potentially curative colorectal cancer patients revealed a significantly higher mean CEA mRNA value in post-operative bloods (18.71) than in pre-operative blood (1.03) (P=0.003). Kaplan–Meier analysis demonstrated disease free survival of patients with positive CEA mRNA in post-operative blood to be significantly shorter than in cases negative for CEA mRNA (P=0.03). These results suggest that tumor cells could be shed into the bloodstream during surgical procedures, and these free tumor cells are accompanied by a poor patient outcome. Real-time quantitative RT-PCR is a useful technique for quantitative assessment of free tumor cells in the peripheral blood of colorectal cancer patients.