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Journal of Clinical Oncology, Vol 25, No 31 (November 1), 2007: pp. 4887-4894 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.09.8921 Epigenetic Silencing of Cyclooxygenase-2 Affects Clinical Outcome in Gastric Cancer
From the Department of Molecular Oncology and Division of Gastrointestinal Surgery, John Wayne Cancer Institute, Santa Monica, CA; Departments of Surgery and Medical Statistics and Bioinformatics, Leiden University Medical Center; and the Department of Pathology, Vrije Universiteit Medical Center, Amsterdam, the Netherlands Address reprint requests to Dave S.B. Hoon, Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA 90404; e-mail: hoon{at}jwci.org
Purpose: Overexpression of cyclooxygenase-2 (COX-2) in gastric cancer has been shown to enhance tumor progression. We investigated whether silencing by promoter region hypermethylation of the COX-2 gene contributes to disease outcome in gastric cancer. Materials and Methods: COX-2 methylation status was initially assessed by capillary array electrophoresis methylation–specific polymerase chain reaction (CAE-MSP) and COX-2 protein expression by immunohistochemistry (IHC) in 40 primary gastric cancer tissues in a pilot study. Prognostic end points of correlative studies of COX-2 methylation status were time to recurrence, overall survival, and standard clinicopathologic features. CAE-MSP analysis was then validated in a second independent gastric cancer population (n = 137). Results: COX-2 methylation was detected in 23% and 28% of the pilot and validation patient groups, respectively. COX-2 expression (IHC) in gastric tumors inversely correlated with COX-2 gene methylation status in the pilot study (P = .02). COX-2 methylation in tumors was significantly associated with lower T, N, and TNM stage in the validation patient group (P = .02, P = .006, and P = .008, respectively). Patients with COX-2 methylated tumors had significantly longer time to recurrence and improved overall survival in a multivariate analysis in the validation patient group (hazard ratio[HR], 0.49; 95% CI, 0.24% to 0.99%; HR, 0.62; 95% CI, 0.38% to 0.99%, respectively). Conclusion: Hypermethylation of COX-2 gene promoter was identified as an independent prognostic factor in gastric cancer patients. The results suggest promoter hypermethylation to be an important regulatory mechanism of COX-2 expression in gastric cancer and an important prognostic biomarker.
Despite decreased incidence, gastric cancer remains the second most common cause of cancer death and the third most common cancer worldwide.1 Recently, gastric cancer therapy has received more attention, since neoadjuvant modalities have been shown to improve outcome for resectable tumors.2-4 Molecular surrogate marker(s) of disease outcome could benefit the management of gastric cancer patient treatment. Approximately 60% of human genes are associated with clusters of CpG dinucleotides or CpG islands.5 Clustered methylation of CpG islands at a gene promoter or transcription start site is associated with gene silencing.6 Hypermethylation of tumor-related regulatory genes may play a significant role in tumor transformation and progression, impacting the clinical course of disease. Recent studies focus on hypermethylation of specific tumor-related genes with suppressing capacities on growth and proliferation of gastric cancer.7-11 Methylation status of gene promoter(s) can have tumor suppressor functions or tumor-inducing capacities. Epigenetic inactivation of genes related with tumor progression has not been well studied in gastric cancer as related to disease outcome. COX-2 (cyclooxygenase-2/PTGS2, prostaglandin-endoperoxide synthetase-2) expression is upregulated in gastrointestinal cancers.12-15 In gastric cancer, COX-2 expression is involved in several tumor progression-related mechanisms, such as angiogenesis,16 inhibition of apoptosis,17 and invasiveness.18 Song et al19 demonstrated regulation of COX-2 mRNA and protein expression by hypermethylation of the COX-2 promoter region in gastric cancer lines. Most gastric cancers overexpress COX-2, and COX-2 expression assessed by immunohistochemistry (IHC) was identified to impact disease survival.20 Because of the reported epigenetic regulation and predictive value of COX-2 expression, we hypothesized that COX-2 promoter hypermethylation status could be used as a biomarker for the clinical outcome of patients with gastric cancer. To study this, we first assessed COX-2 promoter methylation status by quantitative methylation–specific polymerase chain reaction (MSP), as well as its relation to COX-2 protein expression in paraffin-embedded archival tissue (PEAT) of gastric cancer patients with known disease outcome initially in a pilot study,21,22 and then a validation study.23,24 The clinical impact of COX-2 methylation in the cancer trial patients was studied by correlating disease outcome to tumor COX-2 methylation status. The MSP findings were confirmed in a large, independent validation patient group, selected from a multicenter randomized trial comparing primary tumor resection with limited versus extended nodal dissection.23,24
Tumor Specimens The pilot study group contained patients (n = 59) accrued in the fluorouracil, doxorubicin, and methotrexate (FAMTX) trial conducted by the Dutch Gastric Cancer Group21,22 evaluating preoperative chemotherapy with FAMTX for gastric cancer. As a validation set, patients were used from the D1D2 trial by the Dutch Gastric Cancer Group.23,24 The trial evaluated (sub) total gastrectomy for gastric cancer with D1 to D2 lymph node dissection, of which the latter included partial removal of spleen and pancreas. All tumors were classified and staged according to the revised guidelines set by the International Union Against Cancer. PEAT specimens of the primary tumor were collected from both trials. PEAT specimens from gastric tissue biopsies for benign conditions were also collected as controls from 18 patients without a history of malignancy. The study was approved by the human subjects institutional review boards of both participating institutions (Saint John's Health Center/John Wayne Cancer Institute; Leiden University Medical Center).
Tissue and DNA Preparation
Analysis of CpG Island Methylation Status
IHC
MSP Assay Validation
Study Design and Statistical Analyses
MSP Assay Validation Regulation of COX-2 expression by promoter region methylation has previously been shown in gastric cancer cell lines.19 We first verified whether the CAE-MSP assay on COX-2 methylation status associated with regulation of COX-2 mRNA expression. Two representative gastric cancer lines assessed by the CAE-MSP assay as completely methylated (KATO-III, MI = 1.0) and completely unmethylated (FN-0028, MI = 0). The individual CpG-dinucleotide methylation status of the target region of the promoter transcription site was confirmed by bisulfite sequencing in RL-0380 and FN-0028 to be, respectively, completely methylated and nonmethylated. This established these cell lines as suitable positive and negative controls for the CAE-MSP assay. RL-0380 and FN-0028 cell lines were than treated by 5-aza and TSA to evaluate COX-2 gene re-expression. In KATO-III, the demethylating effect was confirmed by CAE-MSP, and expression of COX-2 mRNA was induced (COX-2/GAPDH ratio was 0 v 4.46 E-02, respectively, before and after treatment). In FN-0028, COX-2 mRNA was present before treatment and did not significantly change after treatment (COX-2/GAPDH ratio was 1.69 E-04 v 3.45 E-04, respectively). Results confirmed that promoter region methylation affects COX-2 expression, and validated the CAE-MSP assay.
Primary Tumor COX-2 Methylation Status
Primary Tumor COX-2 Methylation Status and COX-2 IHC
COX-2 Methylation and Clinical Outcome We evaluated gastric cancer patients' clinical prognostic factors between COX-2 methylated and nonmethylated FAMTX trial tumors in a pilot study. COX-2 methylation status showed no relation to sex, age, Lauren type, T, N, or TNM stage, or resectability in this patient group (Table 1). Univariate analysis of Kaplan-Meier survival curves (Fig 3A and 3B) demonstrated that COX-2 methylation status gave significant differences in overall survival and time to recurrence (TTR). Five patients that did not receive a curative (R0) resection were excluded for the analysis of TTR. Multivariate analysis (Table 2) showed that methylation of the COX-2 gene was a favorable independent prognostic factor for TTR and overall survival. Nodal status showed the strongest predictive value, as expected. The multivariate analysis also showed predictive value for the FAMTX trial randomization arm, indicating improved outcome for patients that received surgery alone.
Based on the discussed pilot studies, we validated the utility of COX-2 methylation status in an independent gastric cancer population that received no neoadjuvant treatment. To establish sample size, we initially performed power calculations using the results of the test set. Based on these values, we calculated that 129 patients were sufficient to obtain significance with an  level of .05 and 90% power. Because of expected difficulties in retrospectively analyzing (average) 12-year-old tissue blocks, we added 40 extra patients to account for patients for whom no results could be generated. To obtain a homogenous study group, we used specimens from patients without postoperative mortality and that received R0 resection (resection margin microscopically free of tumor; likely to be node negative). An unbiased selection was performed to assure sufficient events for survival analysis. Thirty-seven percent of patients analyzed were node negative in the validation group (similar to the test set). Overall, 178 specimens were assessed, whereby CAE-MSP could be generated from 137 patients. The patients in analysis were then assessed for differences from the nonanalyzed patients (Table 3). As expected, the selected study group showed a significantly higher proportion of node-positive patients. Older gastric cancer patients are more likely to be node positive at time of surgery, explaining the higher age of the 137 analyzed patients. Thirty-eight patients (28%) were classified as COX-2 methylated using the pilot study cutoff (MI, 0.24). Mean MI was 0.71 (standard deviation [SD], 0.29; range, 0.26 to 1.00) in methylated tumors and 0.01 in nonmethylated primaries (SD, 0.04; range, 0 to 0.19). In this validation group, a significant correlation was observed for COX-2 methylated tumors with lower TNM stage, node negativity, and lower T stage (Table 2). The predictive value of methylation status was confirmed in univariate and multivariate analysis for overall survival and TTR (Figs 3C and 3D and Table 2, respectively). Nodal involvement was the strongest prognostic variable and was present in all patients with methylated COX-2 who relapsed within 1.8 years after surgery. Patients with COX-2 methylation had improved TTR when only node-negative patients were analyzed (P = .03). Lauren type, showing borderline significance in the FAMTX trial, was selected as an independent factor in the larger study group. T stage was not a prognostic factor and this may be due to the tight association with nodal status (P < .0001). Because of this dependence, TNM stage was not a significant factor in multivariate analysis. Trial randomization to extended or limited nodal dissection had no predictive value, as shown in the initial trial analyses.24
In gastrointestinal cancers, the role of COX-2 in tumor-promotion has been shown.34,35 Numerous stimulatory factors, such as growth factors and cytokines, have been reported to cause overexpression of COX-2 in cancer.34,36-38 Studies have suggested methylation as a regulatory mechanism of COX-2 expression in gastric cancer in vitro and in primary tumors.19,33,39-41 Methylated COX-2 gene status may abolish the effect of regulatory factors, such as CCAAT/enhancer-binding protein β, and growth factors, due to the inactivation of binding elements at the promoter region.42 COX-2 methylation status was independent in multivariate analyses of nodal status, which is a highly important predictor of disease recurrence. This independence may be explained by tumor-enhancing processes intrinsic to gastric adenocarcinoma associated with increased COX-2 expression. Examples of such events are angiogenesis, reduced apoptosis, and enhanced inflammation in the microenvironment.16,18,43 Also, our data suggests that COX-2 is involved in Lauren's histologic classification. In the pilot study, gastrointestinal-type tumors correlated with decreased IHC staining intensity (P = .03), and absence of COX-2 expression in the intestinal tumor type has previously been reported.20 In the D1D2 trial patient group, diffuse-type tumors had a significantly decreased MI compared with intestinal-type tumors (P = .03). The outcome of both trials, for which patient specimens were used in this study, was negative. The FAMTX trial did not show improved resectability rate of gastric cancer by preoperative chemotherapy, and extended D2 nodal dissection did not reduce recurrence rates, indicating the problem of managing gastric cancer disease. Development of molecular biomarkers for primary gastric cancer may allow for better management strategies. Long-term analyses of the D1D2 trial suggested that node-positive gastric cancer patients may benefit from more extensive nodal dissection at primary tumor surgery,24 and two randomized trials have now shown the benefit of (neo-) adjuvant treatment for gastric cancer.3,4 The D1D2 trial data showed that 20% of node-negative patients show disease recurrence within 3 years after surgery. Biomarker(s) to select poor prognosis, node-negative patients could be potentially useful for stratification in patients receiving adjuvant or neoadjuvant therapy. COX-2 methylated tumors showed significantly less recurrence in univariate analysis in node-negative patients only; however, our study was underpowered to show independence of COX-2 methylation status as a predictor of poor prognosis in node-negative patients. The actual advantage of our study for clinical applicability is that COX-2 methylation status is a primary tumor characteristic and can be assessed in standard diagnostic tumor biopsy. Primary tumor COX-2 methylation status may therefore be used as a preoperatively assessable biomarker to tailor treatment modalities at time of surgery to gastric cancer patients. To date, no such biomarkers are preoperatively available in gastric cancer. DNA is much more stable in archived tissues as compared with proteins. Our approach to assess gene COX-2 methylation instead of protein expression by IHC analysis may be more promising as a biomarker in assessment of PEATs. Furthermore, our study is the first in gastric cancer to show that DNA methylation is an independent prognostic factor predicting a favorable effect on patient outcome when downregulated. Our results shift the paradigm that tumor-acquired DNA methylation of promoter region(s) of gene(s) results in an adverse outcome in gastric tumors.
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment: N/A Leadership: N/A Consultant: N/A Stock: N/A Honoraria: Anton J. Bilchik, Sonofi Aventis, Valley Lab, Boston Scientific Research Funds: N/A Testimony: N/A Other: N/A
Conception and design: Michiel F.G. de Maat, Peter J.K. Kuppen, Rob A.E.M. Tollenaar, Cornelis J. H. van de Velde, Dave S.B. Hoon Financial support: Cornelis J.H. van de Velde, Dave S.B. Hoon Administrative support: Cornelis J.H. van de Velde, Dave S.B. Hoon Provision of study materials or patients: Cornelis J.H. van de Velde, Anton J. Bilchik Collection and assembly of data: Michiel F.G. de Maat, Cornelis J.H. van de Velde, Pieter de Heer, Anneke Q. van Hoesel, Gerrit A. Meijer, Nicole C. van Grieken, Peter J.K. Kuppen Data analysis and interpretation: Michiel F.G. de Maat, Cornelis J.H. van de Velde, Naoyuki Umetani, Pieter de Heer, Hein Putter, Rob A.E.M. Tollenaar, Dave S.B. Hoon Manuscript writing: Michiel F.G. de Maat, Cornelis J.H. van de Velde, Naoyuki Umetani, Pieter de Heer, Hein Putter, Rob A.E.M. Tollenaar, Dave S.B. Hoon Final approval of manuscript: Cornelis J.H. van de Velde, Naoyuki Umetani, Hein Putter, Gerrit A. Meijer, Rob A.E.M. Tollenaar, Dave S.B. Hoon
Supported in part by funding from the Martin H. Weil Laboratory (John Wayne Cancer Institute), the Rod Fasone Memorial Fund (John Wayne Cancer Institute) and the Drie Lichten Foundation (Leiden, the Netherlands). Presented in part on at the Dutch Society for Gastroenterology meeting, Veldhoven, the Netherlands, October 5, 2006. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test. t test evaluated differences in age between groups. The Mann-Whitney U test was used for ordinal variables. Survival length was determined from the day of primary tumor surgery to the date of death or last clinical follow-up. The Kaplan-Meier method was used for survival analysis grouping with COX-2 methylation status. Differences between curves were analyzed using the log-rank test. Cox's proportional hazard regression model was used in a backward stepwise method for variable selection in multivariate analyses. T stage, N stage, TNM stage, trial randomization, Lauren classification, and complete resection were included in the model. Kruskal-Wallis test was used to assess the relation between COX-2 MI and the different staining-intensity categories. The statistical package SPSS, version 12.0.1 (SPSS Inc, Chicago, IL) was used; a value of P < .05 (two tailed) was considered significant. 
