Japanese Journal of Clinical Oncology Advance Access published online on October 8, 2008
Japanese Journal of Clinical Oncology, doi:10.1093/jjco/hyn105
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© The Author (2008). Published by Oxford University Press. All rights reserved
Quantitative Real-time RT–PCR Detection for Survivin, CK20 and CEA in Peripheral Blood of Colorectal Cancer Patients
1 Clinical Laboratory Medicine Department, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan
2 Department of Blood Transfusion, ZhongNan hospital of Wuhan University, Wuhan, China
For reprints and all correspondence: LiHua Hu, Clinical Laboratory Medicine Department, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. E-mail: shencx1975{at}sina.com
Received August 10, 2008; accepted September 3, 2008
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Abstract |
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 TOP Abstract INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONFundingConflict of interest statementReferences |
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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 ampli
cation 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.
Key Words: colorectal cancer • survivin • CK20 • CEA
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONFundingConflict of interest statementReferences |
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Colorectal cancer (CRC) is one of the world's most common malignancies. Despite recent advances in various diagnostic and therapeutic methodologies, distant metastases remain a major cause of death for CRC patients (1). It has been reported that
30% of the CRC patients who undergo a curative resection nevertheless subsequently develop metastatic disease, the existence of micrometastasis may play a key role in relapse (2). Therefore, there is urgent need to establish sensitive methods for early detection of circulating tumor cells (CTCs) or micrometastasis in peripheral blood (PB) of CRC patients.
Several studies have reported that cytokeratin-20 (CK20) and carcinoembryonic antigen (CEA) are reliable target genes for the detection of disseminated CRC cells (1,3,4). However, some papers reported that these marker genes have relatively low sensitive and specific test (5). The well-known markers CEA and CK20 are probably expressed in CTCs but devaluated by the relatively high background level expression. Survivin, a novel inhibitor of the apoptosis protein family, has been found to be expressed in many common human cancers such as breast cancer. However, it is not found in normal tissues (6). Numerous studies have been conducted which suggest that survivin might be an ideal tumor marker for the diagnosis and prognosis of cancers (7,8). Tumor cell detection by RT–PCR may be hampered by insufcient or absent expression of tumor markers, resulting in false-negative detection. It has therefore been proposed that the assessment of multiple tumor markers in one blood sample would enhance the sensitivity of tumor cell detection (9). Multiple marker assays may significantly improve the sensitivity of detecting heterogeneous tumor cells compared with single marker assays (10,11). However, there have been no previous reports simultaneously analyzing the mRNA molecular markers of survivin, CK20 and CEA for the detection of CTCs in the PB of CRC patients. Recently, immunomagnetic beads were used to isolate epithelial cells followed by RT–PCR analysis of expression of CK20 in patients with CRC (12).
In the present study, we used CD45 immunomagnetic beads and Ber-EP4 immunomagnetic beads to enrich the colon cancer cells from PB. Subsequently, qRT–PCR was used to detect the expression levels of CK20, survivin and CEA mRNA. We supposed that the combined analysis of these three markers with different functional molecular targets could improve the sensitivity for early detection of CTCs of CRC diagnosis in PB, and explore the correlation between the expression of these molecular markers and a variety of clinicopathological features. Eventually, these mRNA molecular markers could be involved in a potentially non-invasive approach, and used to identify patients at a higher risk of postoperative metastasis.
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PATIENTS AND METHODS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONFundingConflict of interest statementReferences |
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Patient Characteristics
A total of 156 CRC patients were involved in this study. Their ages ranged from 30 to 82, with a mean age of 66 years. All of the patients were examined and monitored in Union Hospital and ZhongNan Hospital located in Wuhan, Hubei, P.R. China, from 2006 to 2008. The diagnosis of CRC, and Clinical stages and pathological features of primary tumors were defined according to the criteria of the American Joint Commission on Cancer. The clinicopathological characteristics of all CRC patients enrolled, including age, sex, tumor size, location, Dukes stage and lymph node metastasis, were summarized in Table 1. Informed consent was obtained from all patients and the research protocol was approved by the institutional Ethics of Human Research Committee. PB samples were collected from the patients before surgery and chemotherapy or radiotherapy. In addition, 40 patients with benign colorectal disease and no previous history of neoplastic disease served as benign control, 40 healthy individuals served as controls and 45 patients with other solid tumors (including five hepatic cancers, 10 lung cancers, 20 breast cancers and 10 gastric cancers) were involved in the study.
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Mononuclear Cells Collection
To prevent contamination of epithelial cells, PB samples were obtained through a catheter inserted into a peripheral vessel, and the first 5 ml of blood was discarded, then 5 ml of whole blood was drawn into a tube with EDTA. The mononuclear cells were collected by centrifugation through a Ficoll density gradient. The combination of negative and positive immunomagnetic bead methods was used to enrich cancer cells. First, CD45 immunomagnetic beads (Ningbo Sientz Biotechnology, China) were used to deplete the leukocytes from mononuclear cells. Then, Ber-EP4 immunomagnetic beads (Dynabeads Epithelial Enrich, Dynal, Oslo, Norway) were used to enrich cancer cells.
Cell Line
Human CRC cell line LoVo was preserved and cultured in an RPMI1640 medium (Sigma Chemical Co., St Louis, MO, USA) with 10% fetal bovine serum (Gibco, USA) at 37°C in a 5% CO2 environment. Log-phase cells were collected at <90% confluence by trypsin digestion, resuspended in a phosphate-buffered saline (PBS) and counted using a hemocytometer. These cells were used for the sensitivity test.
Total RNA Extraction and cDNA Synthesis
Total RNA was extracted from CRC cell line LoVo and whole blood cells using a Trizol Kit (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. RNA was analyzed by gel electrophoresis and its concentration was determined by a spectrophotometer reading at 260 and 280 nm.
One microgram of total RNA was reverse transcribed with Oligo(dT) 18 primers and Super-Script III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions.
Standards for qRT–PCR
The cDNA of the CRC cell line LoVo was subjected to PCR amplification using specific primers of survivin, CK20 and CEA. The amplified products were cloned into pGEM Teasy T-vector (Promega, MD, USA). Ligated fragments were transformed into DH5a competent cells. The exact sequence of the inserted plasmids was analyzed by sequencing with M13 universal primers. Serial dilutions from the resulting plasmids were used as standard curves, each containing a known amount of input copy number.
Quantitative Real-Time Reverse Transcription–Polymerase Chain Reaction
Primers were designed for survivin, CK20, CEA and GAPDH using Premier 5.0 software (National Bioscience). To avoid amplification of contaminating genomic DNA, one of the two primers was placed at the junction between the two exons or in a different exon. The oligonucleotides were synthesized by Invitrogen (ShangHai, China). The final sequences and amplification conditions used were shown in Table 2.
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qRT–PCR of the three transcripts was performed using a LightCycler instrument (Roche Diagnostics, Netherlands). PCR amplifications were performed in a total volume of 20 µl, each reaction contained 10x buffer 2 µl, dNTP mixture 0.4 µl (10 mmol/l), each primer 1 µl (10 µmol/l), cDNA 2 µl, SyberGreen I 1 µl (x20), MgCl2 3.2 µl (25 mmol/l), Taq DNA polymerase 1 U, BSA 2.0 µl (1 mg/ml) and ddH2O 6.4 µl. All samples were measured in duplicate. The average value of both duplicates was used as quantitative value. If only one of the duplicates gave a positive signal, the positive result was taken. For all samples, positive, negative and no template controls were performed.
Data Analysis
With the LightCycler software, crossing points (beginning of the PCR exponential phase) were assessed by the second-derivative maximum algorithm and plotted against the concentrations of the standards. To correct for the differences in both RNA quality and quantity between samples, data were normalized using the ratio of the target cDNA concentration to that of GAPDH. After PCR, melting curve was constructed by increasing the temperature from 65 to 95°C with a temperature transition rate of 0.1°C/s. To ensure that the correct product was amplified in the reaction, all samples were separated by 2% agarose gel electrophoresis. Sample concentration was calculated using the plasmid standard curve. The relative sample amount was expressed as ratio marker [100x Survivin, CEA, CK20 (copies/µl)]/[GAPDH (copies/µl)] (13). If the mean Ct value for a gene of interest was higher or equal to 39, the gene expression was considered to be undetectable. To determine the cut-off value for cancer-associated gene-expression in PB, the relative sample amount was set at 95% confidence intervals of the control group. For a particular marker, a test was regarded to be positive if the expression ratio was higher than the cut-off value. For a combined marker, a test was positive if any of the three markers was positive.
Statistical Analysis
All data were analyzed using the Statistical Package for the Social Sciences software (Version 13.0, SPSS, Chicago, IL, USA). The two-sided Pearson 
2 test and the Fisher exact test were used to compare the clinicopathologic parameters between mRNA marker-positive patients and mRNA marker-negative patients. A probability of <0.05 was considered to be statistically significant. Real-time PCR was calculated using linear regression and Pearson's correlation.
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RESULTS |
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Sensitivity and Specificity Tests of Real-Time PCR
Single and sharply defined melting curves with narrow peaks were obtained for PCR products of the analyzed genes. Bands visible after electrophoresis in 2% agarose gel and ethidium bromide staining correlated well with the obtained quantitative PCR results. To establish the limit of sensitivity tests of qRT–PCR assays, 2–2 x 105 LoVo human CRC cells were added to 5 ml whole blood samples obtained from healthy volunteers. The serial dilutions of human CRC cells LoVo in PB samples were subjected to the RNA extraction and RT–PCR procedure. Thus, in this system, the highest detection sensitivity observed was two LoVo cells in 5 ml of whole blood.
Real-Time PCR Amplification Efficiencies and Linearity
Real-time PCR efficiencies were calculated from the given slopes in LightCycler software. The corresponding real-time PCR efficiency (E) of one cycle in the exponential phase was calculated according to the equation: E= 10–1/slope (13). Investigated transcripts showed high real-time PCR efficiency rates; for CK20, 2.05; survivin, 2.04; and CEA, 2.14 in the investigated range from 2 to 2 x 105 copies/µl cDNA input with high linearity (Pearson correlation coefficient r > 0.95).
Expression of Survivin, CK20 and CEA mRNA in PB of CRC Patients
We used the combination negative and positive immunomagnetic bead method to isolate the cancer cells in PB from 156 patients with CRC. The survivin, CK20 and CEA mRNA were then amplied. Representative results of qRT–PCR curve for amplifying mRNA were obtained with the LightCycler (Fig. 1). The positive rates of survivin, CK20 and CEA mRNA in the PB of CRC patients were 57.7, 47.4 and 39.1%, respectively. The expression of survivin, CK20 and CEA mRNA in CRC patients are significantly higher than that in benign control and healthy volunteers (P < 0.001) (Table 3). For examining the sensitivity of multimarker genes, we combined survivin, CK20 and CEA mRNA expressions as mRNAs panel, and the results of sensitivity and specificity for detection were shown in Fig. 2. The positive rates (60.9%) of the three combined markers showed a significant increase compared with that of survivin, CK20 and CEA single marker detection (57.7, 47.4 and 39.1%, respectively). The three marker genes were also detected in the PB patients with solid tumors other than CRC. The expression of survivin (P = 0.605) and CK20 (P = 0.537) was not significantly higher than that of patients with solid tumors. However, the expression of CEA (P < 0.001) in the PB of CRC patients was significantly higher than that of patients with solid tumors (Table 3).
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Clinicopathological Features of CRC Patients with or without mRNA Expression in PB
There were no significant difference in expression of survivin, CK20 and CEA mRNA with respect to age (P = 0.409, 0.244 and 0.656, respectively), sex (P = 0.424, 0.553 and 0.704, respectively), tumor size (P = 0.417, 0.591 and 0.406, respectively) and location (P = 0.900, 0.798 and 0.231, respectively). However, it was found that the expression of survivin, CK20 and CEA mRNA significantly correlated with Dukes stages (all P < 0.001) and lymph node metastasis (P < 0.001, P = 0.011 and P < 0.001, respectively) (Table 4).
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DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONFundingConflict of interest statementReferences |
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At present, sampling of the lymph nodes or bone marrow for the detection of regions of metastatic disease in patients with solid cancer can only be undertaken at the time of initial diagnosis and surgery. However, the sampling of these tissues and the methods used are inaccurate and time consuming, and cannot be used for easy routine screening to determine disease recurrence and response to treatment. Compared with lymph nodes or bone marrow, the sampling of PB is quick, simple and non-invasive. To date, a number of highly sensitive techniques for the detection of disseminated cancer cells in the blood are available, including immunohistochemistry (14), flowcytometry (15) and RT–PCR (16,17). Although being powerful tools for implementing this purpose, these techniques have some demerits (10). In contrast, qRT–PCR combines the high-efficiency of PCR, the high sensitivity and accurate quantification of spectral analysis. In the present study, we developed a qRT–PCR method to detect CTCs of CRC. For the first time, we combined survivin, CEA and CK20 mRNA to detect CTCs of CRC. This assay was designed to use only SyberGreen I, without any need for expensive hybridization probes. The specificity of the amplification products were verified by melting curve analysis and electrophoresis of PCR products. The assay sensitivity demonstrated that target molecules can be detected at a sensitivity of at least two LoVo cells in 5 ml of whole blood. This sensitivity is sufficiently high to determine very low levels of survivin, CK20 and CEA mRNA in PB.
A successful qRT–PCR assay for the detection of CTCs in PB relies on the balance of sensitivity and specicity, i.e. the assay must be sensitive enough to enable detection of very small numbers of tumor cells in an abundance of nucleated blood cells, and must be specic enough not to detect illegitimate transcripts or to mistake illegitimate transcripts for those derived from tumor cells. Different marker genes and various detection techniques, alone or in combinations, have been used for the detection of tumor cells in PB from CRC patients (18).The detection rate of CTCs in PB from CRC patients is in the range from 27 to 88% (1,3,19–21). To improve the reliability of CTCs by qRT–PCR, we applied special experimental modalities such as preanalytical enrichment techniques and used multiple tumor markers. We combined the use of survivin, CK20 and CEA mRNA detection in PB of CRC patients, the sensitivity increased from 39.1% for CEA mRNA alone to 60.9% for the combined panel. However, it is not ignored that the significant increase in positive rate involves the decrease in specificity for detection. Survivin, CK20 and CEA mRNA are widely expressed in other cancers, such as gastric, breast, lung cancers. As a result, the three markers can be combined to screen micrometastatic tumor cells in CRC patients. Lack of a specific gene for reliable detection of CRC patients remains a fundamental problem.
In solid malignancies, metastasis is the most important factor affecting the prognosis and overall survival of patients. Before becoming clinically overt metastases, tumor cells have to spread from the primary tumor through body 
uids such as lymph or blood in single cells or small cell clusters. Thus, from a merely theoretical point of view, the existence of disseminated tumor cells in these uids can be regarded as a necessary appearance on the way to clinically detectable metastases. Disseminated tumor cells in blood are not all capable of growing out and forming metastases, it can be anticipated that a respectable number of tumor cells survive and leave the circulation successfully (18). The existence of disseminated tumor cells in the blood may be established as an additional staging parameter or prognostic factor for carcinoma patients. Recently, it was reported that they found no significant relationship between the results of CEA mRNA expression in preoperative PB and clinicopathological factors using quantitative real-time RT–PCR (22,23). In contrast, Miura et al. (24) reported that the CEA mRNA level was significantly higher in Dukes D patients than in the other clinical stages. In the present study, we have shown a significant relationship between the each of the three mRNA markers transcripts in preoperative PB and different Dukes stages (P < 0.001), Lymph node metastasis (P < 0.05). The detection rate increased with the stage of the tumor in the present investigation. Relatively high positive rate of survivin, CK20 and CEA mRNA expression was also observed in some CRC patients with earlier Dukes stages. This indicates that the analysis of the molecular marker panel does not only reflect the biological activity of CRC in cell spreading, but also provides valuable prognostic information, which can be readily monitored throughout the disease course. However, it needs to be studied in larger patient cohorts to precisely define the clinical relevance.
In conclusion, qRT–PCR detection for survivin, CK20 and CEA mRNA in PB of CRC patients can have a clinical significance in monitoring early stage hematogenous spreading that may further develop into metastasis or recurrence. Multiple marker assays may significantly improve the sensitivity of detecting heterogeneous tumor cells compared with single marker assays. This analysis can offer a simple, non-invasive and promising tool for the early detection of micrometastatic tumor cells in CRC patients. However, a further study with long-term follow-up in a larger number of patients is required to confirm the clinical application of this molecular marker.
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Funding |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONFundingConflict of interest statementReferences |
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This study was supported by a grant from the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20060487045).
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Conflict of interest statement |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONFundingConflict of interest statementReferences |
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None declared.
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References |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONFundingConflict of interest statementReferences |
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