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© 2003 American Society for Clinical Oncology Soluble E-Cadherin is an Independent Pretherapeutic Factor for Long-Term Survival in Gastric Cancer
From the Departments of Medicine and Surgery, University of Hong Kong Medical Center, Queen Mary Hospital, Hong Kong, China. Address reprint requests to Kent-Man Chu, MD, Division of Upper Gastrointestinal Surgery, Department of Surgery, University of Hong Kong Medical Center, Queen Mary Hospital, Pokfulam, Hong Kong, China; email: chukm{at}hku.hk.
Purpose: To evaluate whether pretherapeutic serum soluble E-cadherin is an independent factor predicting long-term survival in gastric cancer. Gastric cancer remains the second leading cause of cancer-related deaths in the world, but a satisfactory tumor marker is currently unavailable for gastric cancer. Soluble E-cadherin has recently been found to have prognostic value in gastric cancer. Patients and Methods: One hundred sixteen patients with histologically proven gastric adenocarcinoma were included in the trial. Pretherapeutic serum was collected, and soluble E-cadherin was assayed using a commercially available enzyme-linked immunosorbent assay kit. The patients were followed up prospectively at the outpatient clinic. Results: There were 75 men and 41 women, with a mean (± SD) age of 66 ± 14 years. Forty-eight percent of tumors were located in the gastric antrum. The median survival time was 11 months. The mean pretherapeutic value of soluble E-cadherin was 9,159 ng/mL (range, 6,002 to 10,025 ng/mL), and the mean pretherapeutic level of carcinoembryonic antigen was 11 ng/mL (range, 0.3 to 4,895 ng/mL). On multivariate analysis, soluble E-cadherin is an independent factor predicting long-term survival. Ninety percent of patients with a serum level of E-cadherin greater than 10,000 ng/mL had a survival time of less than 3 years (P = .009). Conclusion: Soluble E-cadherin is a potentially valuable pretherapeutic prognostic factor in patients with gastric cancer.
GASTRIC CANCER remains the second leading cause of cancer-related deaths in the world.1 At present, there is no satisfactory tumor marker for the diagnosis or monitoring of the disease. The most frequently used tumor marker in gastric cancer is carcinoembryonic antigen (CEA), but only a modest proportion of patients have elevated levels of the marker. Prognostic indicators are important for the selection of therapeutic approach, especially with regard to chemotherapy or aggressive surgery. Therefore, the identification of an accurate prognostic factor is seriously needed. The cadherins are a major class of adhesion molecules that play an important role in the homotypic cell-cell adhesion and, hence, cancer cell metastasis and invasion. E-cadherin is a member of the cadherin family and is expressed on all epithelial cells. The invasiveness of epithelial tumor cell lines could be inhibited in vitro by transfection with E-cadherin cDNA, and the invasiveness of these cell lines were induced again by exposure to anti–E-cadherin monoclonal antibodies.2–4 Underexpression of the E-cadherin molecule has been found in various malignancies, and it has the potential value of being a prognostic factor.5 In addition to its role in metastasis, E-cadherin is one of the most important candidate genes in gastric carcinogenesis. Somatic mutations of the E-cadherin gene have been identified in more than 50% of diffuse types of gastric cancer.6 According to Knudson’s two-hits theory, somatic mutation of E-cadherin is the first of the two hits mechanisms for the silencing of the molecule, whereas methylation of E-cadherin has been shown recently to be the second hit.7,8 In fact, methylation of E-cadherin has recently been shown to be the second genetic hit9 in gastric carcinogenesis. Serum soluble E-cadherin is the degradation product of the cellular E-cadherin molecule. It is found in the circulation of normal individuals but is particularly elevated in patients with malignancies. Gofuku et al10 showed that the concentration was significantly elevated in 67% of patients with gastric cancer. We have also found previously that high concentration of serum soluble E-cadherin was associated with inoperability/palliative treatment and lymph node metastasis.11 The result has prompted us to further investigate whether soluble E-cadherin could predict long-term survival accurately. We have also studied the correlation between serum level of soluble E-cadherin and the protein expression by immunohistochemical staining.
Patient Selection One hundred sixteen patients with gastric cancer recruited for the previous soluble E-cadherin study were enrolled onto this study.11 Tumor was staged according to the criteria of the Japanese Research Society for Gastric Cancer and classified histologically according to the World Health Organization and the Lauren’s classification.12 Patients were followed up prospectively the fourth week after discharge from the hospital, and every 3 months thereafter in the clinic. Any further investigations were directed by clinical suspicion of recurrence. Curative resection was defined as International Union Against Cancer R0 resection. Palliative treatment included International Union Against Cancer R1 or R2 resection, gastrojejunostomy, or palliative chemotherapy. Conservative treatment referred to treatment in which patients received symptomatic support only. Recurrence was defined as the reappearance of tumor after curative surgery. There were 75 men and 41 women in the study, with a mean (± SD) age of 66 ± 14 years. Forty-eight percent of tumors were located in the gastric antrum. Sixty percent of the tumors were intestinal type, and 32% were diffuse type, according to the Lauren’s classification. The percentages of patients having stage I, II, III, and IV diseases were 12.9%, 13.8%, 26.7%, and 40.5%, respectively, whereas the staging data were inadequate in seven patients (6%). To study the correlation between serum level of soluble E-cadherin and the protein expression, we collected surgical specimens from 17 patients who underwent gastrectomy for gastric cancer from October 2001 to April 2002. Immunostaining was performed on these specimens. The correlation between the staining patterns and the soluble E-cadherin levels was studied. Because of the lack of long-term follow-up in these patients, they were not included in the survival analysis.
Assay of Soluble E-Cadherin and CEA The levels of pretherapeutic E-cadherin were determined in our previous study. The method of assay was described elsewhere.13 In brief, the first monoclonal antibody, HECD-1, was coated onto the microtiter-plate wells to create the solid phase. Nonspecific binding was blocked by a blocking buffer. Serum samples from patients and the standard solutions supplied were incubated in the microtiter-plate wells. The second monoclonal antibody, SHE 13–1, labeled with peroxidase was added. During incubation, human E-cadherin molecule was trapped by the two monoclonal antibodies as a sandwich. The reaction between the peroxidase and the substrate solution (H2O2 and tetramethylbenzidine) resulted in color development with intensities proportional to the concentration of human E-cadherin present in the samples and standards. The color developed was measured with the microtiter-plate reader for measurement of absorbance at 450 nm. Accurate sample concentrations of human E-cadherin were determined by comparing the specific absorbances with those obtained from the standards plotted on a standard curve. CEA was also measured pretherapeutically by a commercial enzyme immunoassay kit (CEA; Dainabbott, Tokyo, Japan). The recommended cutoff level is 5 ng/mL for CEA.14
Immunohistochemical Staining for E-Cadherin
Statistical Methods
At the time of writing, the median time of follow-up was 15 months (range, 1 to 58 months). Twenty-seven percent of patients (31 of 116 patients) remained alive and disease-free, 2% (two of 116 patients) were alive with disease, 60% (70 of 116 patients) died of disease, 9% (10 of 116 patients) died of unrelated causes, and 3% (three of 116 patients) were lost to follow-up. The median survival was 11 months (range, 0.7 to 58 months). The 13 patients who died of causes other than gastric cancer and who defaulted follow-up were excluded from the survival analysis. Another five patients were excluded because they did not receive surgery because of poor premorbid state and eventually died of gastric cancer. Among the remaining 98 patients, 52 received palliative treatment/chemotherapy or conservative management, and 46 underwent curative operation. Ninety-four percent of patients (49 of 52 patients) who received palliative/conservative treatment died at follow-up. Thirty-seven percent of the patients (17 of 46 patients) who underwent curative operation died because of gastric cancer at follow-up. Among the patients who received curative surgery, 65% (30 of 46 patients) had no recurrence; in the remaining 16 patients, recurrence developed in the liver (n = 2; 4%), peritoneum (n = 7; 15%), lymph node (n = 4; 9%), ovary (n = 2; 4%), and bone (n = 1; 2%).
Soluble E-Cadherin and CEA as Predictor of Survival
We arbitrarily defined the cutoff level of soluble E-cadherin at 10,000 ng/mL and the cutoff level of CEA at 5 ng/mL. The survival curves according to the levels of E-cadherin and CEA are shown in Fig 1  . Patients with a soluble E-cadherin level less than 10,000 ng/mL or CEA less than 5 ng/mL had significantly better survival than patients whose soluble E-cadherin or CEA was above these levels (log-rank test, P = .0001 and .0002, respectively). Moreover, 90% of patients (18 of 20 patients) with a soluble E-cadherin level greater than 10,000 ng/mL survived less than 3 years, whereas 39% of patients (29 of 75 patients) with levels less than 10,000 ng/mL survived more than 3 years (P = .009). Among the 20 patients with soluble E-cadherin levels greater than 10,000 ng/mL, 16 received palliative treatment, and the rest received curative operation.
Eighty-eight percent of patients (21 of 24 patients) with CEA levels greater than 5 ng/mL survived less than 3 years, whereas 46% of patients (26 of 56 patients) with CEA levels less than 5 ng/mL survived more than 3 years (P = .005). Among the 24 patients with CEA levels greater than 5 ng/mL, 19 received palliative treatment, and the rest received curative operation. When the values of E-cadherin and CEA were considered together, 63% of patients (32 of 51 patients) who survived less than 3 years had either E-cadherin or CEA levels higher than the cutoff values, whereas 83% of patients (25 of 30 patients) who survived more than 3 years had both values below the cutoffs (P < .00001). There was no difference in soluble E-cadherin level between patients with recurrence and those without recurrence (P = .18) or in CEA level (P = .35).
Correlation Between Soluble E-Cadherin Level and E-Cadherin Immunostaining
Tumor factors such as tumor-node-metastasis staging, differentiation, histologic classification, and tumor size are known to carry strong prognostic value. However, a prognostic indicator that could predict the operability, survival rate, and recurrence rate before the pathology of a resected specimen is available (and, hence, before operation) would be particularly helpful. This would provide important guidance and clues for the selection of therapeutic approach. In this study, we identified that, by using a simple enzyme-linked immunosorbent assay test, the pretherapeutic level of soluble E-cadherin could serve such a purpose. In patients with colorectal cancers, a significant inverse relationship between preoperative elevated plasma CEA levels and patient survival has been observed.16–20 Despite disagreement among various reports regarding the relationship between preoperative CEA and prognosis, most investigators noted that a high preoperative CEA level was indicative of a poor prognosis. Nevertheless, the prognostic significance of CEA is controversial in patients with gastric cancer. Contradictory results were found in the biomedical literature.21–23 The value of CEA in predicting long-term survival in patients with gastric cancer has not been well documented. Nakane et al21 showed a significant correlation between preoperative CEA levels and poor prognosis for patients who had resection, but Kodera et al14 found no difference in CEA among patients at any stage. Wang et al19 pointed out that studies published to date might have used different analytic methods or, in other words, different CEA kits to measure CEA. Watine and Friedberg20 suggested that not only the analytic methods but also the preanalytic methods might also be heterogeneous in the various studies. Preanalytic methods include the patients’ preparation before blood sampling (diet duration, smoking, and physical exercise), time of blood sampling (in relation to well-known CEA circadian variations24), materials and methods used for blood sampling, and duration and conditions of sample transportation and storage before the measurements (freezing and thawing of serum).24,25 Various markers, including CA 19-9,26 CA 72-4,27 and CA-125,28 had been tested for their prognostic value in gastric cancer. However, to date, none of these markers has been shown to be an independent prognostic marker. The present study demonstrated that soluble E-cadherin is a significant independent survival indicator when only pretherapeutic factors were considered, indicating that E-cadherin could be a prognostic marker, in addition to CEA, in patients with gastric cancer. It is probable that soluble E-cadherin is a valid survival indicator because of its relationship with staging, which has been demonstrated in our previous study,11 and that staging is an independent prognostic factor. A concentration of soluble E-cadherin greater than 10,000 ng/mL had a high predictive value for a survival time of less than 3 years. However, a concentration of soluble E-cadherin less than 10,000 ng/mL was associated with a survival time of 3 or more years in only 38% of patients. The reason the level of tumor markers at the time of surgery influences long-term survival is still not clear. An elevated serum level may be correlated with the presence of micrometastasis because E-cadherin is a metastasis suppressor gene. A significant prognostic value of soluble E-cadherin has not been reported in other types of tumors. This could be because of a different underlying molecular pathway in gastric cancer compared with other tumors. Mutation or hypermethylation of E-cadherin have been demonstrated as important mechanisms in the early carcinogenesis of gastric and breast cancer but not in other cancers. This might account for the difference in the prognostic value of soluble E-cadherin in different tumors. We observed a correlation between serum soluble E-cadherin level and immunohistochemical expression similar to what has been described by Gofuku et al10; preserved or lost E-cadherin expression was associated with low serum levels, but partially reduced expression was associated with high serum levels. The loss of E-cadherin expression could be a result of gene mutation6 or promoter hypermethylation7,8 in gastric cancer. However, the partial reduction in E-cadherin expression in tumors and the increase in serum level, as discussed by Gofuku, could be a result of the impaired stability of the E-cadherin protein caused by trypsin-like activity29 or a change in extracellular calcium level.30 A statistically significant difference was not seen in the concentration of soluble E-cadherin between patients with and without recurrence; this might be because of the small sample size (only 16 patients had recurrence among the 46 patients who received curative operation). The CEA gene family belongs to the immunoglobulin super-gene family, which is one of the classes of adhesion molecules. In humans, it is clustered on the long arm of chromosome 19 and consists of approximately 20 genes.31 E-cadherin belongs to the cadherin family, which is also a class of adhesion molecules. It is well known that adhesion molecules play important roles in invasion and metastasis.5 Hence, it is not surprising that both CEA and soluble E-cadherin have similar prognostic property. In conclusion, soluble E-cadherin is a potentially valuable pretherapeutic factor in predicting long-term survival in patients with gastric cancer. By using a pretherapeutic level of greater than 10,000, we were able to predict that 90% of patients would have a survival time of less than 3 years. Patients with pretherapeutic levels higher than this should perhaps receive more aggressive treatment, such as extended lymphadenectomy and adjuvant therapies. Further prospective study is required to investigate the value of soluble E-cadherin to predict recurrence.
The work is supported by the University Department of Medicine Grant and the Gastric Cancer Research Fund of the Department of Surgery, University of Hong Kong Medical Center, Hong Kong, China.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
