Coupling of serum CK20 and hyper-methylated CLIP4 as promising biomarker for colorectal cancer diagnosis: from bioinformatics screening to clinical validation.

Colorectal cancer (CRC) is one of the most common and lethal malignancies. The identification of minimally invasive and precise biomarkers is an urgent need for the early diagnosis of CRC. Through bioinformatics analysis of 395 CRC tissues and 63 CRC cell lines, CK18, CK20, de-methylated HPDL and hyper-methylated CLIP4 were identified as candidate serum biomarkers. Then, a training cohort consisting of 60 CRC, 30 colorectal adenomas (CA) and 33 healthy controls and a validation cohort consisting of 60 CRC, 30 CA and 30 healthy controls were enrolled. In the training cohort, enzyme-linked immunosorbent assay (ELISA) showed that CK18 and CK20 were all significantly higher in CRC and CA. CK18 diagnosed CRC with 46.67% sensitivity and 87.3% specificity; CK20 diagnosed CRC with 28.33% sensitivity and 90.47% specificity. Methylation-specific PCR (MSP) indicated that de-methylated HPDL and hyper-methylated CLIP4 were significantly detected in CRC and CA. De-methylated HPDL diagnosed CRC with 36.67% sensitivity and 93.65% specificity and hyper-methylated CLIP4 with 73.33% sensitivity and 84.13% specificity. Random combined analysis suggested that CK20/hyper-methylated CLIP4 diagnosed CRC with 91.67% sensitivity and 82.54% specificity. In the validation cohort, CK20 diagnosed CRC with 36.7% sensitivity and 88.3% specificity and hyper-methylated CLIP4 with 80% sensitivity and 85% specificity. CK20/hyper-methylated CLIP4 diagnosed CRC with 95% sensitivity and 81.7% specificity. Compared with serum biomarkers reported before, CK20/hyper-methylated CLIP4 possessed the potential to be a new effective and precise diagnostic biomarker for CRC.

Aberrant Cytokeratin 20 mRNA Expression in Peripheral Blood and Lymph Nodes Indicates Micrometastasis and Poor Prognosis in Patients With Gastric Carcinoma.

Several studies suggest that peripheral blood and lymph node micrometastases may be a causative factor for gastric cancer recurrence. Cytokeratin 20 shows enriched expression in intestinal epithelial cells. This study aimed to evaluate the clinical utility of monitoring cytokeratin 20 levels in peripheral blood and lymph nodes of patients with gastric cancer for detecting micrometastasis and predicting prognosis. We detected messenger RNA levels of cytokeratin 20 in gastric cancer cell lines and in the peripheral blood of 125 patients (85 patients with gastric cancer and 40 patients with benign neoplasm) by fluorescence quantitative real-time polymerase chain reaction both before and after radical resection. In all, 1586 lymph node samples from 85 patients with gastric cancer were evaluated for cytokeratin 20 expression using real-time polymerase chain reaction, as well as by immunohistochemistry staining with anti-pan-keratin and anti-cytokeratin 20 antibodies. All patients underwent follow-up until cancer-related death or for more than 3 years after tumor resection. We found that elevated cytokeratin 20 expression in peripheral blood as detected by quantitative real-time polymerase chain reaction closely correlates with poor clinicopathological characteristics. Detecting cytokeratin 20 messenger RNA in the lymph nodes by quantitative real-time polymerase chain reaction enabled more accurate determination of the clinicopathological staging of gastric cancer, best treatment approach, and prognosis. Our findings show that patients with increased cytokeratin 20 messenger RNA expression in the peripheral blood or lymph nodes have a shorter time to recurrence and poorer overall survival.

Circulating uPA as a potential prognostic biomarker for resectable esophageal squamous cell carcinoma.

Previous research showed that the 4 genes of matrix metallopeptidase 9 (MMP9), cyto-keratin 20 (CK20), cyto-keratin 19 (CK19) and urokinase type plasminogen activator (uPA) are detectable in the peripheral blood. All the 4 genes are related to tumor invasion and metastasis. However, whether their expression is associated with clinicopathologic factors and the prognosis of patients with esophageal squamous cell carcinoma (ESCC) is still confused. Expression levels of MMP9, CK20, CK19, and uPA were evaluated by quantificational real-time polymerase chain reaction (qRT-PCR) in peripheral blood of 205 ESCC patients who received radical resection. The cut-off value was 1000 copy numbers. Their impacts on clinicopathologic factors and survival were investigated. The uPA expression positively correlated with gender (P = .046) and tumor size (P = .046). Meanwhile, CK19 expression positively correlated with tumor size (P = .029), vascular invasion (P = .024), and CK20 expression positively correlated with tumor size (P = .035) and degrees of differentiation (P = .032). Moreover, the overexpression of MMP9 has a correlation with postoperative radiotherapy (P = .041) and chemotherapy (P = .012). Among the 4 genes, only uPA is a prognostic indicator for disease-free survival and overall survival both in univariate analysis and multivariate analysis (P = .015). This study suggests that circulating uPA mRNA in peripheral blood can serve as a potential unfavorable prognosis biomarker in ESCC. Further perspective, multi-center and large-scale study is still needed.

Prediction of breast cancer metastasis risk using circulating tumor markers: A follow-up study.

Distant organ tumor dissemination is a major cause of breast cancer-related deaths. In 2010, we analyzed the prognostic importance of the circulating tumor markers (CTMs) cytokeratin 19 (CK19), CK20, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in relation to the clinical and pathological characteristics of patients with breast cancer (BC). To assess the clinical utility of CK19, CK20 and EGFR in predicting distant metastasis in BC, here we report 7-year follow-up results of 77 patients. The patients with at least one positive CTM were classified as CTM(+) and those negative for all CTMs were assigned to CTM(-) group. In patients who received no treatment following CTM analysis, 25.0% had metastasis in CTM(+) and 10.0% in CTM(-) group. In patients who received one of the following therapies: chemotherapy, radiotherapy or hormone therapy, or the combinations of these therapies, the rate of metastasis was 33.3% in CTM(+) and 20.0% in CTM(-) group. Disease-free time was shorter in CTM(+) patients compared to CTM(-) group (28.83 ± 10.76 and 41.38 ± 9.5 months, respectively). According to multivariate Cox proportional hazard regression analysis, the presence of regional lymph node metastasis, Ki-67 expression, higher tumor grade and CTM expression status were predictors of poor prognosis associated with distant metastasis (p < 0.05). Also, CTM positivity was a factor associated with metastasis-related poor prognosis (HR = 0.492, p = 0.026). The mean survival for CTM(+) patients was shorter than that for CTM(-) patients (90.671 ± 2.66 and 101.23 ± 3.92 months, respectively; p > 0.05). Our findings demonstrate that CTM positivity may indicate a high metastasis risk; however, CTM negativity does not guarantee low metastasis risk. These results may encourage further preclinical investigation of CTMs, to evaluate the possible implications of these findings to the clinical setting.

Cytokeratin 20 positive cells in blood of colorectal cancer patients as an unfavorable prognostic marker.

OBJECTIVES: Cytokeratin 20 (CK20) is one of the most investigated markers for the detection of circulating colorectal cancer (CRC) cells by reverse transcription polymerase chain reaction (RT-PCR). The aim of this study was to evaluate prognostic value of RT-PCR detection of circulating CRC cells using CK20 as a marker, and to compare the value of preoperative and postoperative blood sample analysis for that purpose. METHODS: Ribonucleic acid (RNA) was isolated from mononuclear cell fraction of blood samples taken from 95 CRC patients before and after tumor resection and from 23 healthy volunteers and assayed by real-time RT-PCR for CK20 expression. RESULTS: In patients positive for CK20 postoperatively both progression-free survival (PFS) and overall survival were significantly shorter than in patients negative for CK20 postoperatively, while the difference between patients positive and negative for CK20 preoperatively was not statistically significant in terms of neither PFS nor overall survival. CONCLUSION: Our results have shown prognostic value of circulating cancer cells detected in postoperative blood samples from CRC patients using CK20 as marker for RT-PCR, which has potential implications for treatment of these patients. In clinical practice, CK20 expression profile could be a factor in weighting treatment options in CRC patients. In cases where multiple treatment options are possible, patients with positive postoperative CK20 expression could be candidates to receive more aggressive treatment.

Predictive value of CK20 in evaluating the efficacy of treatment and prognosis after surgery for colorectal cancer.

Herein, correlations between expression levels of CK20 and efficacy of treatment and postoperative prognosis of colorectal cancer were evaluated to elucidate the clinical value of CK20. Postoperative follow-up was performed on 62 patients who underwent surgery for colorectal cancer between January 2010 and December 2010. Samples of tumor tissues and intraperitoneal drainage fluids were collected. Blood samples were obtained during the 2-year follow-up period. The expression of CK20 in surgical specimen, intraperitoneal drainage fluids, and postoperative serum samples was quantified by enzyme-linked immunosorbent assay, RT-PCR, and western blotting. Correlation between the levels of CK20 and postoperative outcomes was investigated by Spearman correlation analysis. In both tumor specimens and intraperitoneal drainage fluids, CK20 levels were lower in patients with earlier cancer stages than in those at later stages. During postoperative follow-up, serum negative CK20 patients had significantly higher 3-year survival rates than serum positive CK20 patients. All differences were statistically significant (P < 0.05). CK20 levels can provide clinically valuable information on the postoperative prognosis of patients with colorectal cancer.

Cytokeratin 20 improves the detection of circulating tumor cells in patients with colorectal cancer.

Cytokeratin 20 (CK20) is a well-established marker for colon epithelium. Herein, we suggest that CK20 is a biomarker for detecting circulating tumor cells (CTCs) in patients with metastatic colorectal cancer. Blood specimens (7.5 mL) were collected during surgery after liver mobilization from 25 patients with colorectal cancer. The FDA approved CellSearch™ system and two panels of antibodies against cytokeratins, cytokeratin 8, 18 and 19 (CK8/18/19) and CK8/18/19/20, were used for the detection of CTCs. All the patients' samples were processed using the anti-CK8/18/19 panel. The number of detected CTCs was low, 52% of the patients lacked CTCs and 40% had ≤ 2 CTCs/7.5 mL blood. Nine of the patients' blood samples were processed with both antibody panels. The detection rate of CTCs was significantly higher using the anti-CK8/18/19/20 panel compared with the anti-CK8/18/19 panel, p-value 0.0078. Our data show that inclusion of CK20 as a biomarker efficiently improves the detection of CTCs in colorectal cancer patients. The finding in our study is of clinical importance since a new prognostic biomarker would provide an important tool in individual clinical decision-making for colorectal cancer patients.

Detection of circulating tumor cells in bladder cancer using multiplex PCR assays.

AIM: The aim of this study was to develop multiplex-PCR assays for the detection of circulating tumor cells in peripheral blood and urine samples of patients with bladder cancer. MATERIALS AND METHODS: Peripheral blood and urine samples were collected from 208 patients (169 patients and 39 healthy volunteers). After RNA extraction and cDNA synthesis, the samples were analyzed for the expression of cytokeratin 19 (CK19), CK20 and epidermal growth factor receptor (EGFR) mRNA in blood and for SURVIVIN, human telomerase reverse transcriptase (hTERT), cytokeratin 20 (CK20) mRNA in urine, using multiplex-PCR assays. RESULTS: EGFR and CK20 alone or in combination as well as all urine markers correlated well with histological grade. hTERT correlated well with primary tumor size T≥3. Patients with positive urine markers had significantly worse progression-free survival. CONCLUSION: Multiplex-PCR assays can be a useful tool for staging and monitoring purposes in patients with bladder cancer.

[Detection of guanylate cyclase C mRNA and cytokeratin 20 mRNA in peripheral blood and analysis of prognosis in early to moderate colorectal cancer patients].

OBJECTIVE: To investigate the associations of guanylate cyclase C (GC-C) mRNA and cytokeratin 20 (CK20) mRNA with metastasis and prognosis in early to moderate colorectal cancer patients. METHODS: GC-C mRNA and CK 20 mRNA in peripheral blood of 74 colorectal cancer patients without distant metastasis were detected by fluorescent quantitative PCR (FQ-PCR). Based on their clinicopathological and postoperative follow-up data, the relationship and clinical significance of these data with metastasis hazards and prognosis factors were analyzed. RESULTS: The positive rate of GC-C mRNA in 74 colorectal cancer patients was 33.8% (25/74), and CK20 mRNA was 31.1% (23/74). The 1-, 2-, 3- year disease-free survival rates of patients were 94.6%, 82.4% and 78.4% respectively. There were significant differences in positive rates of GC-C mRNA and CK20 mRNA, tumor differentiation, mesentery lymph node metastasis, tumor embolus in vessel and postoperative chemotherapy associated with 3-year disease free survival rate by Kaplan-Meier analysis (all P<0.05). While mesentery lymph node metastasis and tumor embolus in vessel were independent risk factors of 3-year disease-free survival (P<0.05). CK20 mRNA and tumor embolus in vessel were independent risk factors of 3-year disease-free survival by analysis stratified with clinical stage (P<0.05). CONCLUSIONS: Detection of CK20 mRNA and GC-C mRNA in peripheral blood may be important for early detection of early metastasis of colorectal cancer.

Detection of cytokeratin-20-positive cells in preoperative and postoperative blood samples from colorectal cancer patients by real-time RT-PCR.

BACKGROUND: Detection of circulating cancer cells by reverse transcription polymerase chain reaction (RT-PCR) has been studied as a prognostic marker in patients with colorectal cancer (CRC) but so far with conflicting results regarding specificity and prognostic value. In this study cytokeratin-20 (CK20) was evaluated by real-time RT-PCR as a marker for circulating CRC cell detection and the influence of surgical tumor resection on the presence of circulating CRC cells was analyzed. METHODS: RNA was isolated from the mononuclear cell fraction of blood samples taken from 95 CRC patients before and after tumor resection and from 23 healthy volunteers and assayed by real-time RT-PCR for CK20 expression. RESULTS: Among 23 healthy volunteers one was positive for CK20. Among 95 CRC patients, 25 were positive for CK20 before and 23 after surgery. Sixteen patients positive before surgery became negative after surgery, while 14 patients negative before surgery became positive after surgery. An increase in the proportion of CK20-positive samples with increasing stage of disease was observed for preoperative but not postoperative blood samples. CONCLUSIONS: Its association with clinical stage indicates that CK20 might have prognostic value as a marker for detection of circulating CRC cells. Surgical tumor resection can both reduce and induce the presence of circulating CRC cells.

Expression and prognostic significance of CEACAM6, ITGB1, and CYR61 in peripheral blood of patients with gastric cancer.

BACKGROUND: We examined CEACAM6, ITGB1, and cyr61 concentrations from patients with gastric cancers (GCs) to assess their clinical application for diagnosing and monitoring diseases. METHODS: Real-time reverse transcription-polymerase chain reaction was used to detect the expressions of CEA, CEACAM6, ITGB1, IGF1R, CK20, cyr61, and S100A4 in peripheral blood karyocyte from 82 patients with GC, 24 patients with recurrence, and 37 healthy volunteers. Receiver operating characteristics (ROC) curves were constructed. RESULTS: There were significant association between these CEACAM6, ITGB1, and cyr61 and TNM Stages and distant metastasis. The AUC of CEACAM6 was 0.884 ± 0.044 (P = 0.0001), the AUC of cyr61 was 0.833 ± 0.047 (P = 0.0001), and the AUC of ITGB1 was 0.838 ± 0.042 (P = 0.0001) by differentiating preoperative GC patients from healthy volunteers from ROC curve analysis. The AUC of CEACAM6 was 0.761 ± 0.066 (P = 0.001), the AUC of CYR61 was 0.762 ± 0.063 (P = 0.001), and the AUC of ITGB1 was 0.824 ± 0.051 (P = 0.0001), by differentiating recurrence of GC from healthy volunteers from ROC curve analysis. CONCLUSION: The method of detecting the expression of CEACAM6, ITGB1, and CYR61 in peripheral blood of GC patients was more sensitive than CEA, IGF1R, CK20, and S100A4 for the early diagnosis of metastasis and recurrence.

The detection of disseminated tumor cells in bone marrow and peripheral blood of gastric cancer patients by multimarker (CEA, CK20, TFF1 and MUC2) quantitative real-time PCR.

OBJECTIVE: To investigate the suitability of multimarker detection of DTCs in PB and BM of GC patients. DESIGN AND METHOD: A qRT-PCR assay was developed to estimate the number of CEA, CK20, TFF1 and MUC2 transcripts in PB and BM samples of 35 GC patients prior to the initiation of therapy. PB samples from healthy volunteers and BM from patients with hematological malignancies were used as negative controls. RESULTS: In PB analysis; 22.9%, 37.1%, 31.4%, and 22.9% of GC patients and in BM analysis; 20%, 28.6%, 45.7%, and 22.9% of GC patients were positive for CEA, CK20, TFF1 and MUC2 mRNAs, respectively. Samples from the control group were negative for the expression of all the markers tested in this study. A higher positive ratio was obtained with the multimarker detection in comparison to the single marker detection. There was a significant correlation between the PB and BM samples for DTC detection. CONCLUSION: Multimarker detection assay is a reliable and powerful tool for the early detection of DTCs in GC patients.

Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients.

AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting circulating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P < 0.001) and preoperative serum levels of CEA (P = 0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented significant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RT-PCR assay can provide useful information concerning disease stage and overall survival of CRC patients.

[Detection of lymph node micrometastasis for patients with extrahepatic cholangiocarcinoma and its prognostic significance].

OBJECTIVE: To observe the expression of antibodies of cytokeratin 19 and 20 in lymph node micrometastasis in patients with extrahepatic cholangiocarcinoma (EHCC), evaluate the prognostic significance of lymph node (LN) micrometastasis and study the correlation between lymph node micrometastasis and clinicopathological features, CA19-9 and CEA. METHODS: A total of 279 lymph nodes was intra-operatively collected from 59 EHCC patients and routine histological examination performed. Immunohistochemical staining was performed on all samples by the murine antibodies of anti-CK19 and anti-CK20 respectively. Then the micrometastasis was identified microscopically according to the color of cells. The results were analyzed according to clinical, pathological and follow-up data. And the relation of micrometastasis with clinical pathological factors and its impact upon survival rate were analyzed. RESULTS: Among 59 EHCC patients, 14 (23.72%) LN metastasis were found with HE staining and 21 micrometastases with CK staining. The incidence of nodal involvement in 59 EHCC patients increased from 5.37% (15/279) by HE staining to 13.98% (39/279) by CK staining. Among 45 patients not positive for LN metastases with HE staining, CK staining was positive in 7 patients and the incidence of micrometastasis was 15.56%. The preoperative serum CA19-9 levels in patients with LN micrometastasis was higher than that those without LN metastasis (P < 0.05). And there was a positive correlation between occult nodal micrometastasis and serum concentrations of CA19-9 (r(s) = 0.371, P < 0.05). The histological type and lymphatic vessel infiltration of tumor were the most importance factors for LN micrometastasis through Logistic regression analysis (P < 0.05). CONCLUSION: The CK immunohistochemical staining can detect the micrometastases in HE negative LN. And LN micrometastasis can more accurately predict the prognosis of EHCC patients.

Clinical significance of cytokeratin 20-positive circulating tumor cells detected by a refined immunomagnetic enrichment assay in colorectal cancer patients.

PURPOSE: Current immunomagnetic enrichment method can only detect general epithelial antigens of circulating tumor cells (CTC). Further characterization of the CTCs to provide specific information on the tumor type is not possible. We attempted to overcome this drawback by developing the methodology for using a gastrointestinal-specific anti-cytokeratin (CK) 20 antibody to detect CTCs in colorectal cancer patients' blood. EXPERIMENTAL DESIGN: The protocol was validated using a colorectal cancer SW480 cell line. The clinical significance of findings in colorectal cancer was investigated by detecting CK20-positive CTCs (pCTC) in patients with colorectal cancer, other common cancers, colorectal adenoma, benign colorectal diseases, and normal subjects. Moreover, the malignant nature of CK20 pCTCs was examined by comparing chromosome 17 aberration patterns with those from the corresponding primary tumors. RESULTS: The assay successfully showed CK20-positive SW480 cells. When applied in patient samples, the detection rates were 62% (132 colorectal cancer patients; median number = 11 CTCs), 0% (120 patients with other common cancers), 6% (50 colorectal adenoma patients), 0% (120 patients with benign colorectal diseases), and 0% (40 normal subjects). Furthermore, statistical analysis showed that CK20 pCTC numbers were associated with tumor-node-metastasis stage and lymph node status. Using the median CK20 pCTC numbers as the cutoff points, stratified groups of colorectal cancer patients had significant differences in their recurrence, metastasis, and survival. Finally, chromosome 17 aneusomy in 90% of colorectal cancer patients with CK20 pCTCs matched with those from the primary tumors. CONCLUSIONS: Detection of CK20 pCTCs using the new protocol could generate clinically important information for colorectal cancer patients.

[Diagnostic significance of cytokeratin 19 and 20 expression on micrometastasis of colorectal cancer].

OBJECTIVE: To inquire into the diagnostic significance of cytokeratin 19(CK19) and cytokeratin 20(CK20) expression on hematogenous micrometastasis of colorectal cancer. METHODS: Forty-four patients with colorectal cancer were collected as colorectal cancer groups, and another 18 patients treated with abdominal surgical operations because of benign diseases were collected as benign disease group. Blood of all the patients was harvested from their portal and peripheral veins, and CK19 and CK20 levels in the blood were determined by RT-PCR. RESULTS: There were no positive expression of CK19 and CK20 in the portal and peripheral blood of all the patients in benign disease group. Of the colorectal cancer group, 34 patients(77.3%) appeared positive expressions of CK19 and/or CK20 in portal and peripheral blood, and there was significant difference in the expressions of CK19 and CK20 between the two groups(P<0.05). Within the colorectal cancer group, the positive expression rates of CK19 and CK20 in peripheral blood were 36.4% and 52.3%, and the rates in portal blood were 59.1% and 72.7%. The rates of portal blood were significantly higher than those of peripheral blood(P<0.05). The positive expression rate in patients at stage III( was significantly higher than that in patients at stage I( orII( (P<0.05). The postoperative metastasis and recurrence rate of colorectal cancer in patients with positive expression of CK19 and CK20 in peripheral blood was 61.5%, which was significantly higher than that(25.0%) in patients with positive expression in portal blood only(P<0.05). CONCLUSIONS: In patients with colorectal cancer, the expressions of CK19 and CK20, which are determined by RT-PCR in blood from portal and peripheral veins, are the sensitive and specific indexes for diagnosing hematogenous micrometastasis of the cancer.

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.

[Cytokeratin 20 as a marker of tumor progression or dissemination in patients with colorectal cancer]. / Detección de micrometástasis por RT-PCR de citokeratina 20 y su correlación con la sobrevida global en pacientes portadores de cáncer colorrectal.

BACKGROUND: Colorectal cancer relapses or metastasizes in 30% of cases. Cytokeratin 20 is present in 95% of colorectal tumors and their metastases and could be used as a marker to detect tumor cells. AIM: To assess the usefulness and prognostic value of peripheral blood and bone marrow cytokeratin 20 determinations in patients with colorectal cancer. MATERIAL AND METHODS: Blood and bone marrow samples were obtained from 56 patients with colorectal cancer aged 26 to 77 years (31 females) before surgical procedure. They were followed for a mean of 22 months (range 2.9 to 72 months) after surgery. Blood and bone marrow from 45 patients without cancer and 35 healthy subjects were used as negative controls. Messenger RNA expression of cytokeratin 20 was studied by real time and nested polymerase chain reaction. RESULTS: Cytokeratin 20 was detected in 6% of controls and 41% of patients. There was no relation between cytokeratin 20 expression and age, gender, overall survival, tumor relapse, progression, localization or stage. CONCLUSIONS: Cytokeratin 20 determination is not useful as a marker of tumor progression or dissemination in patients with colorectal cancer.

Molecular signature detection of circulating tumor cells using a panel of selected genes.

Circulating tumor cell (CTC) detection in peripheral blood of colon and other epithelial cancer patients is becoming a scientifically recognised indicator for the presence of primary tumors and/or metastasis. The resulting need to further develop CTC detection-based systems for improved diagnosis, prognosis and assessment of therapy efficacy in tumour patients has prompted the application of different approaches, including expression analysis of tissue-specific and epithelial genes. In this context, lack of specificity of the analysed genes remains a fundamental problem for reliable CTC detection. In this study, we have selected a panel of highly specific epithelial genes: cytokeratin 20 (CK20), cytokeratin 19 (CK19), carcinoembryonic antigen (CEA) and guanylyl cyclase C (GCC), and performed RT-PCR analysis to assess their expression in total blood and in different cell fractions of peripheral blood (PBMC and CD45-negative population) of cancer patients and healthy controls. Our results demonstrate that analysis of a single gene in a CTC-enriched population (CD45(-) peripheral blood cells) of cancer patients allows detection of a CTC molecular signature in at most 63.3% of cases, while analysis of all four genes performed in all three sample types increases the detection of positive patient samples to 87.7%. Healthy controls did not show positivity for any combination of these genes, although positivity was observed for the CEA marker alone, which was detected in 3 (6.6%) out of 45 donors, and only in the CD45(-) fraction. Here, we demonstrate that combined analysis of the genes above, in multiple blood fractions, results in a highly specific and sensitive CTC detection system in patients with metastatic solid tumors. Therefore, we believe that validation on a large scale of this approach, which demonstrates higher specificity in patients compared to controls, could become a relevant CTC screening test in patients with established metastatic disease, and furthermore, may also be useful for evaluating the possible presence of CTCs before the onset of clinically manifested metastatic spreading.