The-765 G > C Polymorphism in the Cyclooxygenase-2 Gene and Digestive System Cancer : a Meta-analysis

Digestive system cancer is a sophisticated disease that results from complex interactions between environmental factors, genetic variants, and gene-environment interactions (Berlau et al., 2004; Pharoah et al., 2004; Yaghoobi et al., 2004). Helicobacter pylori (H. pylori) infection, environmental tobacco smokes and lifestyle represent the most important exogenous risk factors (Cook et al., 2010). Although these factors have been documented to influence the risk of cancer, not all individuals develop the disease, even though they are exposed in the same environment. This indicates that genetic differences, such as variants, may contribute to cancer pathogenesis. Therefore, numerous published studies have focused on the association of genetic variants with cancer susceptibility. And among them, cyclooxygenase (COX) gene has been extensively studied. The COX gene, mapped to chromosome 1q25.2-q25.3 in human, is 8.3 kb in size, contains 10 exons and produces


Introduction
Digestive system cancer is a sophisticated disease that results from complex interactions between environmental factors, genetic variants, and gene-environment interactions (Berlau et al., 2004;Pharoah et al., 2004;Yaghoobi et al., 2004). Helicobacter pylori (H. pylori) infection, environmental tobacco smokes and lifestyle represent the most important exogenous risk factors (Cook et al., 2010). Although these factors have been documented to influence the risk of cancer, not all individuals develop the disease, even though they are exposed in the same environment. This indicates that genetic differences, such as variants, may contribute to cancer pathogenesis. Therefore, numerous published studies have focused on the association of genetic variants with cancer susceptibility. And among them, cyclooxygenase (COX) gene has been extensively studied.
The COX gene, mapped to chromosome 1q25.2-q25.3 in human, is 8.3 kb in size, contains 10 exons and produces

RESEARCH ARTICLE
The -765G>C Polymorphism in the Cyclooxygenase-2 Gene and Digestive System Cancer: a Meta-analysis Fen Zhao 1,2& , Yue Cao 3& , Hong Zhu 4& , Min Huang 1 , Cheng Yi 4 , Ying Huang 1 * an mRNA of 4.6 kb, which encodes a constitutive isoenzyme (COX-1) and an inducible isoenzyme (COX-2) (Tazawa et al., 1994). COX-1 is constitutively expressed and is involved in the homeostasis of various physiological functions (Dubois et al., 1998), while COX-2, known as rate-limiting enzyme produced during the production of prostaglandins, is often undetectable in normal tissue, whereas in tumor tissue specimens its expression is observably higher (Harrison et al., 1994;Seibert et al., 1994;Wu et al., 1996;Bakhle et al., 2001;Cao et al., 2002;Wang et al., 2007). This gene is polymorphic, and a large number of single nucleotide polymorphisms (SNPs) have been identified, such as -765G>C (reference SNP ID, rs20417), -1195G>A (rs689466), -8473T>C (rs5275), -1759G>A (rs3218625), -202C>T (rs2745557), and -1290A>G (rs689466). Among all of these polymorphisms, the -765G>C polymorphisms in COX-2 gene were the most widely studied for their implication in cancer risk. Several meta-analyses investigating this -765G>C polymorphism of COX-2 gene with digestive system cancer risks were performed; however, the results were inconsistent for different ethnicity and cancer types (Dong et al., 2010;Liu et al., 2010;Wang et al., 2013;Yan et al., 2013). Therefore, we conducted this meta-analysis on all eligible case-control studies to estimate the overall digestive system cancer risk associated with this polymorphism.

Selection of studies
A systematic literature search of the Pubmed, Medline (Ovid), Embase, CNKI, Weipu database, Wanfang database and CBM database was carried out to identify studies involving association between the -765G>C polymorphism of COX-2 gene and digestive system cancer risk (updated on Feb 10, 2014). The search terms were used as follows: (cyclooxygenase-2 or COX-2 or PTGS2) in combination with (polymorphism or variant or mutation) and (cancer or carcinoma or neoplasm). The search results were limited to English and Chinese languages. Studies included in our meta-analysis met the following inclusion criteria: (1) evaluation of the -765G>C polymorphism of COX-2 gene and digestive system cancer risk, (2) the design had to be a case-control design published in a journal, (3) genotype distributions in both cases and controls were available for estimating an odds ratio with 95% confidence interval (CI) and P value, and (4) genotype distributions in control group should be consistent with Hardy-Weinberg equilibrium (HWE). Studies were excluded if one of the following existed: (1) no controls, (2) genotype frequencies or number not reported, and (3) abstracts, reviews. For duplication or overlapping publications, the studies with larger number of cases and controls or been published latest were included.

Data extraction
Two independent reviewers (FZ and CY) collected the data and reached a consensus on all items. In case of disagreement, a third author (HZ) would assess these articles. A standardized data form was used and included: first author's name, year of publication, original country, ethnicity, cancer type, study design, total number of cases and controls, genotyping method, and genotype distribution in cases and controls.

Statistical analysis
Odds ratios (ORs) with 95% CI was used to assess the strength of association between the COX-2-765G>C polymorphism and digestive system cancer risk. We first estimated with the risk of genetic model (CC+GC vs GG), and then estimated the risk of (C vs G) model. The pooled OR was calculated by a fixed-effects model or a random-effects model according to the heterogeneity. Heterogeneity was checked by a χ 2 -based Q statistic and P <0.10 was considered statistically significant. If the result was p>0.10, OR was pooled according to the fixed-effect model; otherwise, the random effect model was used. The statistical significance of OR was analyzed by Z test, and p<0.05 was considered as statistically significant. To evaluate the ethnicity-specific, cancer type-specific, study design-specific effects, we performed stratification analyses on ethnicity, cancer type, and study design. For the subgroup analysis by ethnicity, the study populations were stratified into four groups: Asians, Caucasians, Americans, Africans, Mixed (if it was difficult to discriminate the ethnicity of participants according to the data presented, the study was termed "Mixed"). Subgroup analysis by cancer type were preformed if one cancer type contained three and more than three individual studies (if 1 cancer type was investigated <3 individual case-control studies, then it was combined into the group of "others"). In addition, subjects were categorized into different classifications according to study design: population-based case-control study (PCC), hospital-based case-control study (HCC), and family-based case-control study (FCC).
Sensitivity analysis was also performed by sequence excluding individual study to check the robustness of the result . The possible publication bias was examined visually in a Begg's funnel plot and the degree of asymmetry was tested by Egger's test (Begg et al., 1994;Egger et al., 1997). HWE was tested by Pearson's χ 2 test . Statistical analysis was performed using Reman5.2 software (The Cochrane Collaboration, www.cochrane.org).
The characteristics of each case-control study are listed in Table 1. Genotype and allele distributions for each case-control study are shown in Table 2.
Quantitative data synthesis All studies: As shown in Figure 2, the heterogeneity of (CC+ GC vs GG) for all 38 studies was assessed and the value of χ 2 was 259.68 with 37 degrees of freedom and p<0.00001 in a random-effects model. Additionally, the I-square, which is another index of the test of heterogeneity,   Table 3. Subgroup analyses: In the subgroup analysis by ethnicity (GC+CC vs GG, Figure 3) DOI:http://dx.doi.org/10.7314/APJCP.2014.15.19.8301 The -765G>C Polymorphism and Digestive System Cancer -a Meta-analysis CI=0.29-1.37 and p=0.24). Thus, the Asians and Africans C carriers (CC+GC) may have higher risk of digestive system cancer than others ethnicity. Summary results of other genetic comparisons are listed in Table 3. In the subgroup analysis by cancer type (GC+CC vs GG, Figure  4), the analysis was stratified into five subgroups: gastric cancer (1732 cases and 2855 controls), colorectal cancer (5058 cases and 7687 controls), oral cancer (651cases and 581 controls), esophageal cancer (2099 cases and 3101 controls), and others (1274 cases and 1950 controls).
Significantly increased risks were found among gastric cancer (OR=1.64, 95% CI=1.03-2.61, and p=0.04), but not among colorectal cancer (OR=1.01, 95% CI=0.89-1.14, and p=0.92), oral cancer (OR=0.66, 95% CI=0.29-1.50, and p=0.32), esophageal cancer (OR=1.32, 95% CI=0.77-  therefore, the comparisons of C vs G did not include the two studies. a All studies excluding the studies without HWE. b If it was difficult to discriminate the ethnicity of participants according to the data presented, the study was termed "mixed". c Cancers studied if 1 cancer type was investigated by<3 individual case-control studies, then it was combined and termed "others". population-based case-control study (PCC), hospital-based case-control study (HCC), and family-based case-control study (FCC). 2.25, and p=0.32), and others (OR=1.89, 95% CI=0.96-3.72, and p=0.06). Thus, the gastric cancer C carriers (CC+GC) may have a higher risk than others cancer type. Summary results of other genetic comparisons are listed in Table 3. For subgroup analysis by study design (GC+CC vs GG, Figure 5), no significant association between the -765G>C polymorphism of COX-2 gene and digestive system cancer risk was found in HCC (4409 cases and 5423 controls: OR=1.27, 95% CI=0.92-1.76, and p=0.15), PCC (6100 cases and 10392 controls: OR = 1.20, 95% CI = 0.99-1.46, and P = 0.16), or FCC (305 cases and 359 controls: OR = 0.93, 95% CI = 0.67-1.29, and p=0.67). Thus, the polymorphism may not increase cancer risk in different study design. Summary results of other genetic comparisons are listed in Table 3.

Sensitivity analysis
In order to assess the stability of the results of the meta-analysis, we performed a sensitivity analysis through sequentially excluded individual studies. After sequentially excluding each case-control study, statistically similar results were obtained for (GC+CC vs GG) (all P values were <0.05), suggesting the stability of this meta-analysis (data not shown).

Publication bias
The publication bias was assessed by Begg's funnel plot and Egger's test. The graphical funnel plot of 38 studies of the -765G>C polymorphism of COX-2 gene appeared to be asymmetrical in the (CC+GC vs GG) (Figure 6), which suggested that the combined ORs were not stable, possible explanation may be that probably due to limited number of eligible studies included. Therefore, more studies with large sample size were required to minimize the likelihood of bias.
Given the important roles of COX-2 in cancer etiology, it is possible that genetic variations of the COX-2 gene may affect the susceptibility to cancer development. Genetic variants, such as SNPs in the promoter region of the COX-2 encoding gene, is the most extensively studied polymorphism, which features guanine (G) converting to cytosine (C) at position -765 bp of the promoter region, affecting transcription activity of -765G>C polymorphism of COX-2 gene and its functional activity (Papafili et al., 2002;Szczeklik et al., 2004;Sitarz et al., 2008). To date, conclusions of the association of COX-2-765G>C polymorphism with digestive system cancer is still uncertain; thus, we performed a meta-analysis of 38 case-control studies, including 10814 cases and 16174 controls, to evaluate the associations between the COX-2-765G>C polymorphism and digestive system cancer risks. Considering the genetic background, cancer type and study design may affect the results of meta-analysis, subgroup analyses was performed by ethnicity, cancer type, and study design.
Our results showed that the COX-2-765G>C polymorphism was significantly associated with digestive system cancer risks in the (GC+CC vs GG) genetic model. In addition, in the (C vs G) model, we found significant associations between this polymorphism and digestive system cancer. These results indicated that this polymorphism may contribute to cancer risks. Consistent with the previous meta-analysis (Cao et al., 2010;Liang et al., 2011;Wang et al., 2013), we found significant increased risk -765G>C polymorphism with digestive system cancer, strongly suggesting that this polymorphism may contribute to digestive system cancer pathogenesis and help to explain individual differences of host susceptibility.
Considering the property of genetic background may affect the results of genetic association studies, we performed subgroup analysis by ethnicity. In this meta-analysis , we found that the variant C allele carriers (CC+GC) had increased risk of digestive system cancer in Asians and Africans, but not in Caucasians, Americans, and Mixed, suggesting a possible role of ethnic differences in genetic backgrounds and the environment they lived in. Thus, further studies are demanded to assess the effect of gene-environment interactions in different ethnicities and to validate these findings. In addition, significantly increased risks were found in Africans only one casecontrol study included, it may be due to chance because studies with small sample size may have insufficient statistical power to detect a slight effect. Therefore the results should be explained with great caution. When stratified separately according to cancer type, we found that this polymorphism was significantly associated with increased risks of gastric cancer. Possible explanation may be that differences in etiology may exist in difference cancer type. Subgroup analysis was also performed by study design, no significant increased risk of digestive system cancer was found among in HCC, PCC, and FCC subjects. Based on our study, it is worth noting that, selection bias was well avoided by performing rigorous and scientific inclusion criteria and exclusion criteria.
Heterogeneity is one of the important issues when performing meta-analysis. We found that heterogeneity between studies existed in overall comparisons. After subgroup analysis by ethnicity or cancer type, the heterogeneity was effectively removed or decreased among Americans and colorectal cancer, possible explanations may be that differences in genetic backgrounds and environmental exposures existed among different ethnicities, and differences in etiology may exist in difference cancer type. Another important factor contributing to heterogeneity was that homogeneity in either the case or control groups was uncertain. Ideally, all cases and controls should be matched for age, sex, and environmental exposures. In this meta-analysis, these issues could not all be explained precisely because of insufficient information for individual person.
Some limitations of this meta-analysis should be acknowledged when explaining our results. Firstly, all eligible studies were published reports written in English and Chinese indexed by the selected databases. It is possible that some potential published studies in other languages or unpublished studies could be missed, which might bias the results. Secondly, some studies were excluded due to lack of original data by email from the corresponding author, we could not evaluate the potential interactions between this polymorphism and digestive system cancer risks, which may lead to a selection bias. Thirdly, this meta-analysis included data from Asians, Caucasians, Americans, Africans and Mixed, no studies from Dutch populations; thus, our study may be applicable to these ethnic groups only. And the last, data were not stratified by other factors such as age, gender, family history, lifestyle variables, because insufficient information could be extracted from the primary publication. It is worth mentioning a study published by Saad et al (Saad et al., 2013), the study indicated potential value of PA extracted from rice bran in reducing colonic cancer risk in rats. Which may have implications for further medical research concerning digestive system cancer and personalized therapy for digestive system cancer patients.
In conclusion, To our knowledge, this is the most comprehensive meta-analysis conducted to date with respect to the associations between the -765G>C polymorphism of COX-2 gene and digestive system cancer risks. Our results indicated the COX-2-765G>C polymorphism was significantly associated with increased risk of digestive system cancer, especially for Asians, Africans and gastric cancer. These results may have implications for further medical research concerning digestive system cancer and personalized therapy for digestive system cancer patients. Regarding some limitations for this study, future large-scale studies will be needed to clarify the gene-gene and gene-environment interactions to better display the association between the -765G>C polymorphisms in COX-2 gene and digestive system cancer risks.