Clinical Cancer Research The Science of Cancer Health Disparities Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 13, 2406-2413, April 15, 2007. Published Online First April 3, 2007;
doi: 10.1158/1078-0432.CCR-06-2054
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
1078-0432.CCR-06-2054v1
13/8/2406    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, J.-Y.
Right arrow Articles by Uen, Y.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, J.-Y.
Right arrow Articles by Uen, Y.-H.

Cancer Therapy: Clinical

Multiple Molecular Markers as Predictors of Colorectal Cancer in Patients with Normal Perioperative Serum Carcinoembryonic Antigen Levels

Jaw-Yuan Wang1,2, Shiu-Ru Lin3, Deng-Chyang Wu2, Chien-Yu Lu2, Fang-Jung Yu2, Jan-Sing Hsieh1,2, Tian-Lu Cheng4, Lok-Beng Koay5 and Yih-Huei Uen6

Authors' Affiliations: 1 Department of Surgery, Kaohsiung Medical University Hospital; 2 Faculty of Medicine, College of Medicine, 3 Graduate Institute of Medical Genetics, and 4 Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan; and 5 Department of Internal Medicine and 6 Division of General Surgery, Department of Surgery, Chi Mei Foundation Medical Center, Taipei Medical University, Taipei, Taiwan

Requests for reprints: Yih-Huei Uen, Chi Mei Foundation Medical Center, 901 Chung-Hua Road, Yung-Kang City, Tainan 710, Taiwan. Phone: 886-6-2812811; Fax: 886-6-2828928; E-mail: cy61412{at}ms14.hinet.net and cmh7500{at}chimei.org.tw.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: In this study, a high-sensitivity colorimetric membrane array method was used to detect circulating tumor cells (CTC) in the peripheral blood of colorectal cancer (CRC) patients with normal perioperative serum carcinoembryonic antigen (CEA) levels. This membrane array method was evaluated as a potential diagnostic and postoperative surveillance tool.

Study Design: Membrane arrays consisting of a panel of mRNA markers that include human telomerase reverse transcriptase, cytokeratin-19, cytokeratin-20, and CEA mRNA were used to detect CTCs in the peripheral blood of 157 postoperative CRC patients with normal perioperative serum CEA levels and in 80 healthy individuals. Digoxigenin-labeled cDNA were amplified by reverse transcription-PCR from the peripheral blood samples, which were then hybridized to the membrane array. The sensitivity, specificity, and accuracy of membrane arrays for the detection of CTCs were then calculated.

Results: Using the four markers in combination, expression of any three markers or all the four markers in this panel was significantly correlated with the clinicopathologic characteristics, including depth of tumor invasion, lymph node metastasis, tumor-node-metastasis stage, and postoperative relapse (all P < 0.05). The interval between the detection of all four positive molecular markers and subsequent elevated CEA ranged from 3 to 8 months (median 6 months). The expression of all four mRNA markers was an independent predictor for postoperative relapse. CRC patients with all four mRNA markers expression showed a significantly poorer survival rate than those with less than four positive markers.

Conclusions: The constructed membrane array method was helpful in the early prediction of postoperative relapse in CRC patients with normal perioperative serum CEA levels.

Relapses have an important meaning in relation to survival for curative surgical intervention of cancer patients. Since its discovery by Gold and Freedman in 1965 (1), serum carcinoembryonic antigen (CEA) has been used extensively in clinical practice for tumor follow-up to detect recurrence in colorectal cancer (CRC) patients. An elevated preoperative CEA is a poor prognostic sign and correlates with a reduced overall survival after surgical resection. Many studies have established the usefulness of serum CEA-oriented serial monitoring for predicting recurrence and prognosis of CRC (2–7). However, few studies have thus far investigated the surveillance of patients with normal perioperative serum CEA levels.

Because early detection is an important factor in reducing cancer mortality, the development of a sensitive, specific, and convenient diagnostic method for detecting CRC at a very early stage is an issue of utmost importance, especially for CRC patients with normal perioperative serum CEA levels. Models of metastasis indicate that primary tumor cells spread to other organs via circulation (blood and lymphatics). Sensitive detection of occult carcinoma cells in the peripheral blood of CRC patients has important prognostic and therapeutic implications. Such neoplastic cells may be present in the bloodstream in very low numbers and would hardly be detected by conventional methods. With recent developments in molecular technology, the use of PCR, reverse transcription-PCR (RT-PCR), or real-time quantitative-PCR (Q-PCR) assays now permit sensitive detection of circulating tumor cells (CTC) in peripheral blood.

Accumulated reports have described the use of human telomerase reverse transcriptase (hTERT), cytokeratin-19 (CK-19), cytokeratin-20 (CK-20), and CEA mRNA as biomarkers for the detection of occult residual disease for CRC (8–13). Our recent investigations also indicate that CEA mRNA for detection of CTCs in peripheral blood using RT-PCR is a rational approach for the surveillance of postoperative CRC patients (14). In addition, our developed membrane array–based multimarker assay can detect CTCs in the peripheral blood of various cancer patients (15–19). Thus, the aim of this study was to analyze CRC patients with normal perioperative serum CEA levels by a panel of molecular markers using a constructed membrane array method and evaluate their role in postoperative surveillance.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients. Two hundred and eighty-nine stage I to III CRC patients were admitted to the Department of Surgery of Kaohsiung Medical University Hospital for elective surgery between May 2002 and June 2004. Of these, 157 (54.3%) with perioperative serum CEA levels <5 ng/mL were enrolled in the present study. Seventy-eight patients were males and 79 were females. The mean age was 65.8 years (range 20-91 years).

All of the 157 patients underwent radical resection for the primary lesion. Preoperative serum CEA was sampled within 1 week before surgery and postoperative serum CEA was obtained at least 4 weeks later. Simultaneously, an additional 4-mL sample of peripheral blood was obtained from each CRC patient postoperatively for total RNA isolation. Peripheral blood samples were taken from 80 healthy individuals to serve as controls. To prevent contamination of epithelial cells, the blood samples were obtained through a catheter inserted into a peripheral vessel, and the first 5 mL of blood were discarded. Written informed consent was obtained from each subjects and/or guardian. Sample acquisition and subsequent use were also approved by the hospital institutional review board.

The clinical stage and pathologic features of the primary tumors were defined according to the sixth edition of the tumor-node-metastasis (TNM) staging system of the International Union Against Cancer (20). As to the histologic types, 21 were well-differentiated, 112 were moderately differentiated, and 24 were poorly differentiated carcinoma. Of the 157 CRC patients, 15 were subsequently diagnosed with stage I, 70 with stage II, and 72 with stage III. The clinicopathologic characteristics of these patients were listed in Table 1 .


View this table:
[in this window]
[in a new window]

  		Table 1. Characteristics of colorectal cancer patients

 
mRNA isolation and first-strand cDNA synthesis. Total RNA was extracted from the fresh whole blood of CRC patients and healthy volunteers using a QIAamp RNA Blood Mini Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's instructions. The RNA concentration was determined spectrophotometrically on the basis of absorbance at 260 nm.

Membrane arrays. The procedure for the design and preparation of the membrane array was in accordance with our recent work (16). Oligonucleotide probes were then synthesized according to the design sequences, purified, and controlled before being grafted onto the substrates (Table 2 ). The newly synthesized oligonucleotide fragments were then dissolved in di-water to a concentration of 20 mmol/L, applied to a BioJet Plus 3000 nanoliter dispense system (BioDot, Inc., Irvine, CA), which blotted sequentially the four target DNAs, one housekeeping gene (ß-actin), and one tuberculosis gene (50 nL per spot and 1.5 mm between spots) on a Nytran SuperCharge nylon membrane (Schleicher and Schuell, Dassel, Germany) in triplicate, and then cross-linked to the membrane using a UV Stratalinker 1800 (Stratagene, La Jolla, CA). Each spot contained 20 ng of PCR-amplified DNA derived from sequence-verified cDNA clones. DMSO was also dispensed onto the membrane as a blank control.


View this table:
[in this window]
[in a new window]

  		Table 2. Oligonucleotide probe sequences of mRNA markers

 
Digoxigenin-labeled cDNA targets and hybridization. Four hundred nanograms of first-strand cDNA targeted for hybridization were made by reverse transcription of mRNA from the peripheral blood of the study subjects and control in the presence of digoxigenin-labeled UTP (Roche Diagnostics GmbH, Penzberg, Germany) using SuperScript II reverse transcriptase (Life Technologies Inc., Rockville, MD). The lifts were covered with ExpressHyb Hybridization Solution (BD Biosciences, Palo Alto, CA) containing DIG-11-UTP–labeled cDNA probes, and then incubated with antidigoxigenin alkaline phosphatase–conjugated antibody (Roche Diagnostics). The arrays were then incubated for hybridization at 42°C for 6 h in a humid chamber.

For signal detection, the membranes were incubated for 15 min in a chromogen solution containing nitroblue tetrazolium and 5-bromo-4-chloro3-indolyl-phosphate. The hybridized membrane arrays were then scanned using an Epson Perfection 1670 flat bed scanner (SElKO EPSON Corp., Nagano-ken, Japan). Subsequent quantification analysis of the intensity of each spot was carried out using AlphaEaseFC software (Alpha Innotech Corp., San Leandro, CA). The density ratio of each mRNA marker was divided with ß-actin mRNA as an internal control. The spots that consistently displayed signal intensity higher than the cutoff value calculated from the receiver-operating characteristic (ROC) curves by at least one factor were considered to be positive or overexpressed.

Diagnostic accuracy of the membrane arrays. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of the membrane array for CRC were analyzed. Investigators were blinded to the groups of blood samples.

Detection of serum CEA. Serum CEA levels were determined by an enzyme immunoassay test kit (DPC Diagnostic Product Co., Los Angeles, CA) with the upper limit of normal as 5 ng/mL according to the manufacturer's instructions.

Follow-up. All of the patients were carefully and regularly followed-up until their death or until June 2006 (median follow-up, 36 months; range, 24-49 months). At each monthly visit, physical examinations, routine blood work, serum CEA measurement, and liver function tests were conducted where appropriate. Abdominal ultrasonography was done every 6 months, as well as annual computed tomography studies for the chest and abdomen. The development of new or recurrent metastatic lesions after surgery is defined as a postoperative relapse. During the follow-up period, 53 (33.8%) patients suffered from local recurrence or distant metastasis and 38 (24.2%) expired.

Statistical analysis. All of the data were analyzed using the Statistical Package for the Social Sciences version 12.0 software (SPSS, Inc., Chicago, IL). Data were presented as mean ± SD. ROC curve analyses were done to analyze the membrane array data of hTERT, CK-19, CK-20, and CEA gene expression in the peripheral blood of the subjects. The area under ROC curve and the corresponding 95% confidence intervals (95% CI) were calculated for each marker. The cutoff value at the highest accuracy (minimal false-negative and false-positive results) was determined.

Two-sided Pearson {chi}2 test and the Fisher exact test were used to analyze the potential correlation between the expression of molecular markers used in combination and the clinicopathologic features of the study subjects. To clarify the clinical significance of these mRNA markers combined into a diagnostic panel as the predictor of postoperative relapse, a multivariate adjustment was done by logistic regression analysis. The cumulative overall survival rates were calculated by the Kaplan-Meier method, and the differences in survival rates were analyzed by log-rank test.

To minimize the interpretation bias, the overall survival analysis was applied. A P value <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
ROC curve analyses on the membrane array data of the 237 subjects (157 CRC patients and 80 normal individuals) were done. The ROC curves for each mRNA marker are shown in Fig. 1 . Accordingly, the optimal cutoff value and area under ROC curve for each particular single mRNA marker was as follows: 0.50 and 0.885 (95% CI, 0.829-0.941) for hTERT, 0.48 and 0.868 (95% CI, 0.809-0.927) for CK-19, 0.62, and 0.866 (95% CI, 0.808-0.924) for CK-20, and 0.55 and 0.903 (95% CI, 0.853-0.952) for CEA.


Figure 1
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 1. ROC curves for hTERT, CK-19, CK-20, and CEA mRNA from the analysis of membrane array data of 237 subjects. Sensitivity (Y axis) was plotted against the false-positive fraction (1-specificity; X axis) for various cutoff values. The plot is highlighted with the numbers in parenthesis that indicate the cutoff value, sensitivity, and specificity. The area under the ROC curve for hTERT, CK-19, CK-20, and CEA mRNA is 0.885 (95% CI, 0.829-0.941), 0.868 (95% CI, 0.809-0.927), 0.866 (95% CI, 0.808-0.924), and 0.903 (95% CI, 0.853-0.952), respectively.

 
The examination of single markers with membrane array displayed degrees of sensitivity, specificity, and diagnostic accuracy ranging from 77.1% to 82.8%, 77.5% to 81.3%, and 77.2% to 82.3% (Table 3 ), respectively. The false-positive rates of hTERT, CK-19, CK-20, and CEA mRNA for healthy individuals were 20%, 21.2%, 22.5%, and 18.7%, respectively. When combined with all four mRNA markers, the false-positive rate was reduced to 0%.


View this table:
[in this window]
[in a new window]

  		Table 3. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of each mRNA marker and the combination between CRC patients and healthy individuals

 
Table 4 shows the correlation of the sensitivity of each molecular marker in CRC patients with various TNM stages. Sensitivity was associated with advanced stages (all P < 0.05). Figure 2 shows the schematic representation (Fig. 2A) and representative results (Fig. 2B) of membrane array hybridization for four CRC patients and a normal control. Significant overexpression of one to four markers was observed in the peripheral blood of the four CRC patients, respectively, but not in the control sample.


View this table:
[in this window]
[in a new window]

  		Table 4. One hundred and fifty-seven colorectal cancer patients with or without each mRNA expression stratified according to TNM stage

 

Figure 2
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 2. A, schematic representation of the membrane array with four target genes, one housekeeping gene (ß-actin), one tuberculosis gene (negative plant), and one blank control. A triplicate set of four mRNA markers for CRC was blotted on the nylon membrane. In addition, a housekeeping gene and a tuberculosis gene that served as positive and negative controls were also blotted on the membrane. B, comparing gene expression patterns between CRC patients and a healthy control. Spots within the red circle, ß-actin (positive control). The overexpression of one to four markers was observed in four individual CRC patients, respectively, but not in the healthy individual.

 
As analyzed in combining these four markers with member arrays, expression of any three markers or all of the four in this panel was more significantly correlated with the clinicopathologic characteristics than any single or two markers (Table 5 ), including depth of tumor invasion (P = 0.002 and P < 0.001), lymph node metastasis (P = 0.009 and P < 0.001), TNM stage (both P < 0.001), and postoperative relapse (P = 0.037 and P < 0.001). Forty-five (50%) of the 90 patients with an overexpression of all four molecular markers and 8 (11.9%) of the 67 patients without overexpression of any molecular marker had postoperative relapse. Among the 53 patients with postoperative relapse, 45 (84.9%) overexpressed all four molecular markers.


View this table:
[in this window]
[in a new window]

  		Table 5. Correlation between clinicopathologic features of colorectal cancer patients and detection of four mRNA markers in combination

 
To find out the lead time among all of the four positive molecular markers and elevated serum CEA levels (>5 ng/mL), we further analyzed 90 CRC patients with all four positive molecular markers. Of the 90, 45 developed postoperative relapse whereas 40 had elevated serum CEA levels in subsequent follow-up period (data not shown). Thirty-seven of the 40 patients with persistently elevated serum CEA levels developed postoperative relapse, whereas eight postoperative relapse patients still had normal serum CEA levels (data not shown). The interval between the 40 patients with elevated CEA levels and the presence of all four positive molecular markers ranged from 3 to 8 months (median, 6 months; Fig. 3 ).


Figure 3
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 3. The interval between subsequently elevated CEA levels and the presence of all four positive molecular markers in the 40 CRC patients.

 
The predictive values of the mRNA molecular markers or clincopathologic features for postoperative relapse were analyzed using multivariate logistic regression analysis (Table 6 ). The CRC patients with all four markers had a relative risk of 18.749 of developing postoperative relapse compared with those without any of the markers (P = 0.019). Furthermore, statistically significant difference was observed in terms of survival rate between CRC patients with expression of all four markers and those with less than four positive markers using the log-rank test (Fig. 4 ; P = 0.006).


View this table:
[in this window]
[in a new window]

  		Table 6. Association between postoperative relapse and combinations of mRNA molecular markers or clincopathologic features from colorectal cancer patients using multivariate logistic regression analysis

 

Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. Cumulative overall survival rate of 157 CRC patients by Kaplan-Meier analysis. Patients with all four mRNA markers expression in the peripheral blood showed a significantly poorer survival rate than those with less than four positive mRNA maker expression (P = 0.006).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The only method of treatment that offers a favorable prognosis for CRC is radical resection of a part of the colon or rectum, including the pertinent lymph glands, and a radical removal of metastases. However, even such presumably curative surgery does not ensure full recovery, as recurrences are frequent and, according to several analyses, the 5-year survival rate is <50%. The most significant reason for this poor therapeutic success is residual micrometastases.

CEA has been the most extensively investigated tumor marker for CRC. In fact, serum CEA protein is currently the most widely used marker in the surveillance or follow-up of CRC. In addition, patients with preoperative CEA levels higher than 5 ng/mL have been regarded as a high-risk group for recurrence after curative resection (5). Again, an initially elevated CEA level that fails to fall to within normal limits in the early postoperative period after curative resection may signify an additional relevant risk factor (3, 21). Nevertheless, the monitoring of recurrence or metastasis in asymptomatic patients without accompanying serum CEA elevation after curative resection has rarely been addressed.

McCall et al. (22) indicated that the diagnosis of recurrent disease for CRC patients may be made several months earlier by investigating the first abnormal CEA level. In the current investigation, we showed that our constructed colorimetric membrane array could detect CTCs in peripheral blood of CRC patients with normal perioperative serum CEA levels at an earlier stage, with a median time of 6 months earlier than the elevation of serum CEA levels. Therefore, these molecular markers might have an advantage in detecting postoperative relapse earlier for patients with normal perioperative serum CEA levels.

Indeed, 6 months is a good lead time for the introduction of new therapeutic strategies to possibly cure these patients. On the other hand, the panel of overexpressed molecular markers is prominently correlated not only to the clinicopathologic characteristics but also to postoperative relapse and overall survival rates, particularly in patients who express with all four molecular markers. These data suggest that the molecular markers have the clinical potential to be important prognostic factors for CRC patients with normal perioperative serum CEA levels after radical resection.

Likewise, Ishikawa et al. (23) has disclosed that tumor angiogenesis could predict recurrence in rectal cancer patients with normal serum CEA levels. Consistent with the findings of the angiogenesis marker from Ishikawa's group, we also observed that our multimarker panel is superior to conventional pathologic variables, such as tumor depth and lymph node involvement, for the prediction of postoperative relapse. Accordingly, concomitant molecular detection of CTCs with a multimarker panel is a justifiable supplementary approach to the current pathology staging system, which may help physicians make accurate judgment on clinical treatment and in predicting prognosis for CRC patients. A more intensive follow-up plan may be recommended for these patients despite normal perioperative serum CEA levels after radical resection.

Recently, using an RT-PCR assay for the detection of CEA mRNA has been shown to be feasible and promising as a tool for the early detection of micrometastatic CTCs (14). A multimarker panel is now the trend to enhance the sensitivity of methods for CTC detection, compared with using a single marker (24–26). The present study shows that detection sensitivity (77.1-82.8%) of the colorimetric membrane array method is considerably higher than that of our previous RT-PCR assay (52.8-72.2%) for the detection of CTCs in each corresponding molecular marker (14). This membrane array assay is more accurate in discriminating CRC patients and normal subjects.

Several recent RNA-based approaches focused only on the clinical significance of single-marker analysis. Although these methods provide useful data, one of the limitations was that the methodology could analyze only one molecular target at a time. Due to the heterogeneity of the expression of tumor-related genes, a multimarker assay is regarded as more reliable and sensitive than a single-marker assay (24–27). As expected, the detection rate and diagnostic accuracy for CTCs of CRC patients were significantly increased, as a panel of multiple markers was used, compared with single markers. Our highly sensitive and high-throughput assay was able to detect simultaneously a panel of informative molecular markers for the presence of CTCs, with time saving and cost-effectiveness. Therefore, a combined panel of these four mRNA markers may not only be a promising tool for the detection of CTCs but also a significant powerful predictor for prognosis, despite normal perioperative serum CEA levels.

Half of the all marker-positive patients developed postoperative relapse, which is significantly higher than the 11.9% of the all marker-negative patients. The false-negative rate (11.9%) of the membrane array method in predicting postoperative relapse, at least in part, might result from CTCs intermittently flowing into the bloodstream of the bowel, the heterogeneous character of the tumor itself, or even the relatively short follow-up period (17, 28, 29). Alternatively, this may be quite reasonable because few carcinoma cells shed into the bloodstream succeed in establishing metastatic disease (30). Using multiple blood sampling (12) or a refined normalization procedure (31) might improve the sensitivity of the membrane array method.

Our results indicated that only 50% of 90 CRC patients with four positive markers developed postoperative relapse. One possible explanation for the high false-positive rate may be that some of our patients received adjuvant chemotherapy (high-risk stage II and stage III CRC), which might affect the postoperative relapse rate. Furthermore, extending the follow-up period may decrease the false-positive rate in predicting relapse. On the other hand, designing new probes to replace the false-positive probes would be useful in improving the false-positive rate of this membrane array assay.

In summary, the constructed membrane array method for the detection of CTCs has been shown to be complementary to the surveillance of CRC patients with normal perioperative serum CEA level. The highly sensitive and high-throughput assay has the clinical potential for early detection of postoperative relapse, with a lead median time of 6 months before the measurement of abnormal CEA levels. However, additional work in a larger patient population by means of serial blood sampling and long-term follow-up is needed to confirm the clinical significance of membrane arrays as adjuvant surveillance.


   Footnotes
 
Grant support: Chi Mei Medical Center and Kaohsiung Medical University Research Foundation grant 95-CM-KMU-01.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/17/06; revised 10/25/06; accepted 11/ 9/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gold P, Freedman SO. Specific carcinoembryonic antigens of the human digestive system. J Exp Med 1965;122:467–81.[Abstract]
  2. Hohenberger P, Schlag PM, Gerneth T, Herfarth C. Pre- and postoperative carcinoembryonic antigen determinations in hepatic resection for colorectal metastases. Predictive value and implications for adjuvant treatment based on multivariate analysis. Ann Surg 1994;219:135–43.[Medline]
  3. Slentz K, Senagore A, Hibbert J, Mazier WP, Talbott TM. Can preoperative and postoperative CEA predict survival after colon cancer resection? Am Surg 1994;60:528–31.[Medline]
  4. McCall JL, Black RB, Rich CA, et al. The value of serum carcinoembryonic antigen in predicting recurrent disease following curative resection of colorectal cancer. Dis Colon Rectum 1994;37:875–81.[CrossRef][Medline]
  5. Wiratkapun S, Kraemer M, Seow-Choen F, Ho YH, Eu KW. High preoperative serum carcinoembryonic antigen predicts metastatic recurrence in potentially curative colonic cancer: results of a five-year study. Dis Colon Rectum 2001;44:231–5.[CrossRef][Medline]
  6. Wichmann MW, Muller C, Hornung HM, Lau-Werner U, Schildberg FW; the Colorectal Cancer Study Group. Results of long-term follow-up after curative resection of Dukes A colorectal cancer. World J Surg 2002;26:732–6.[CrossRef][Medline]
  7. Louhimo J, Carpelan-Holmstrom M, Alfthan H, et al. CA 72-4 and CEA are independent prognostic factors in colorectal cancer. Int J Cancer 2002;101:545–8.[CrossRef][Medline]
  8. Guadagni F, Kantor J, Aloe S, et al. Detection of blood-borne cells in colorectal cancer patients by nested reverse transcription-polymerase chain reaction for carcinoembryonic antigen messenger RNA: longitudinal analyses and demonstration of its potential importance as an adjunct to multiple serum markers. Cancer Res 2001;61:2523–32.[Abstract/Free Full Text]
  9. Ito S, Nakanishi H, Hirai T, et al. 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. Cancer Lett 2002;183:195–203.[CrossRef][Medline]
  10. Lledo SM, Garcia-Granero E, Dasi F, et al. Real time quantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA in patients with colorectal cancer. Colorectal Dis 2004;6:236–42.[CrossRef][Medline]
  11. Castells A, Boix L, Bessa X, Gargallo L, Pique JM. Detection of colonic cells in peripheral blood of colorectal cancer patients by means of reverse transcriptase and polymerase chain reaction. Br J Cancer 1998;78:1368–72.[Medline]
  12. Wharton RQ, Jonas SK, Glover C, et al. Increased detection of circulating tumor cells in the blood of colorectal carcinoma patients using two reverse transcription-PCR assays and multiple blood samples. Clin Cancer Res 1999;5:4158–63.[Abstract/Free Full Text]
  13. Wong IH, Yeo W, Chan AT, Law WY, Johnson PJ. Quantitative relationship of the circulating tumor burden assessed by reverse transcription-polymerase chain reaction for cytokeratin 19 mRNA in peripheral blood of colorectal cancer patients with Dukes' stage, serum carcinoembryonic antigen level and tumor progression. Cancer Lett 2001;167:183–91.[CrossRef][Medline]
  14. Wang JY, Wu CH, Lu CY, et al. Molecular detection of circulating tumor cells in the peripheral blood of patients with colorectal cancer using RT-PCR: significance in the prediction of postoperative metastasis. World J Surg 2006;30:1007–13.[CrossRef][Medline]
  15. Chen YF, Wang JY, Wu CH, et al. Detection the circulating cancer cells with K-ras oncogene using membrane array. Cancer Lett 2005;229:115–22.[CrossRef][Medline]
  16. Wu CH, Lin SR, Yu FJ, et al. Development of a high-throughput membrane-array method for molecular diagnosis of circulating tumor cells in patients with gastric cancers. Int J Cancer 2006;119:373–9.[CrossRef][Medline]
  17. Wang JY, Yeh CS, Chen YF, et al. Development and evaluation of a colorimetric membrane-array method for the detection of circulating tumor cells in the peripheral blood of Taiwanese patients with colorectal cancer. Int J Mol Med 2006;17:737–47.[Medline]
  18. Yeh CS, Wang JY, Wu CH, et al. Molecular detection of circulating cancer cells in the peripheral blood of patients with colorectal cancer by using membrane array with a multiple mRNA marker panel. Int J Oncol 2006;28:411–20.[Medline]
  19. Sheu CC. Development of a membrane array-based multimarker assay for detection of circulating cancer cells in patients with non-small cell lung cancer. Int J Cancer 2006;119:1419–26.[CrossRef][Medline]
  20. International Union Against Cancer. TNM classification of malignant tumors. 6th ed. New York: Wiley-Liss, Inc.; 2002.
  21. Compton C, Fenoglio-Preiser CM, Pettigrew N, Fielding LP. American Joint Committee on Cancer Prognostic Factors Consensus Conference: Colorectal Working Group. Cancer 2000;88:1739–57.[CrossRef][Medline]
  22. McCall JL, Black RB, Rich CA, et al. The value of serum carcino-embryonic antigen in predicting recurrent disease following curative resection of colorectal cancer. Dis Colon Rectum 1994;37:875–81.[CrossRef][Medline]
  23. Ishikawa H, Fujii H, Yamamoto K, et al. Tumor angiogenesis predicts recurrence with normal serum carcinoembryonic antigen in advanced rectal carcinoma patients. Surg Today 1999;29:983–91.[CrossRef][Medline]
  24. Racila E, Euhus D, Weiss AJ, et al. Detection and characterization of carcinoma cells in the blood. Proc Natl Acad Sci U S A 1998;95:4589–94.[Abstract/Free Full Text]
  25. Hoon DS, Wang Y, Dale PS, et al. Detection of occult melanoma cells in blood with a multiple-marker polymerase chain reaction assay. J Clin Oncol 1995;13:2109–16.[Abstract/Free Full Text]
  26. Conzelmann M, Linnemann U, Berger MR. Molecular detection of clinical colorectal cancer metastasis: how should multiple markers be put to use? Int J Colorectal Dis 2005;20:137–46.[CrossRef][Medline]
  27. Sher YP, Shih JY, Yang PC, et al. Prognosis of non-small cell lung cancer patients by detecting circulating cancer cells in the peripheral blood with multiple marker genes. Clin Cancer Res 2005;11:173–9.[Abstract/Free Full Text]
  28. Mori M, Mimori K, Ueo H, et al. Molecular detection of circulating solid carcinoma cells in the peripheral blood: the concept of early systemic disease. Int J Cancer 1996;68:739–43.[CrossRef][Medline]
  29. Molnar B, Ladanyi A, Tanko L, Sreter L, Tulassay Z. Circulating tumor cell clusters in the peripheral blood of colorectal cancer patients. Clin Cancer Res 2001;7:4080–5.[Abstract/Free Full Text]
  30. O'Sullivan GC, Collins JK, Kelly J, et al. Micrometastases: marker of metastatic potential or evidence of residual disease? Gut 1997;40:512–5.[Abstract/Free Full Text]
  31. Schuchhardt J, Beule D, Malik A, et al. Normalization strategies for cDNA microarrays. Nucleic Acids Res 2000;28:E47.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Ann. Surg. Oncol.Home page
Y.-H. Uen, C.-Y. Lu, H.-L. Tsai, F.-J. Yu, M.-Y. Huang, T.-L. Cheng, S.-R. Lin, and J.-Y. Wang
Persistent Presence of Postoperative Circulating Tumor Cells is a Poor Prognostic Factor for Patients with Stage I-III Colorectal Cancer after Curative Resection
Ann. Surg. Oncol., August 1, 2008; 15(8): 2120 - 2128.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
1078-0432.CCR-06-2054v1
13/8/2406    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, J.-Y.
Right arrow Articles by Uen, Y.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, J.-Y.
Right arrow Articles by Uen, Y.-H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online