Current Evidence on the Association between Cytotoxic T-Lymphocyte Antigen 4 +49G > A Polymorphism and Digestive System Cancer Risks: a Meta-analysis Involving 11,923 Subjects

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) plays an important role in downregulating T cell activation and proliferation. The CTLA-4 +49G > A polymorphism is one of the most commonly studied polymorphisms in this gene due to its association with many cancer types, but the association between CTLA-4 +49G > A polymorphism and digestive system cancer risks remain inconclusive. An updated meta-analysis based on 17 independent case-control studies consisting of 5,176 cancer patients and 6,747 controls was performed to address this association. Overall, there was no statistically increased risk of digestive system cancers in every genetic comparison.In subgroup analysis, this polymorphism was significantly linked to higherrisks for pancreatic cancer (GG vs. AA, OR=1.976, 95% CI = 1.496-2.611; GA vs. AA, OR=1.433, 95% CI = 1.093-1.879; GG/GA vs. AA, OR=1.668, 95% CI = 1.286-2.164; GG vs. GA/AA, OR = 1.502, 95% CI = 1.098-2.054; G vs. A, OR=1.394, 95%CI = 1.098-1.770). We also observed increased susceptibility of hepatocellular cell carcinoma in homozygote comparison (OR=1.433, 95% CI = 1.100-1.866) and dominant model (OR=1.360, 95% CI = 1.059-1.746). According to the source of controls, significant effects were only observed in hospital-based studies (GA/ AA vs. GG, OR=1.257, 95% CI = 1.129-1.399). In the stratified analysis by ethnicity, No significantly increased risks were found in either Asian or Caucasian. Our findings suggest that the CTLA-4 +49G > A polymorphism may be not associated with an elevated digestive system cancer risks. Current Evidence on the Association between Cytotoxic T-Lymphocyte Antigen 4 +49G > A Polymorphism and Digestive System Cancer Risks: a Meta-analysis Involving 11,923 Subjects


Introduction
CTLA-4, a member of the immunoglobulin super-family, is a costimulatory molecule expressed by activated T cells and has the function of down-regulating T-cell activation [1]. CTLA-4 can also induce FAS-independent apoptosis of activated T cells, which may further inhibit immune function of T lymphocytes. A list of mechanisms of CTLA-4 function have been indicated, such as ligand competition with the positive T-cell co-stimulatory CD28 molecule, interference of TCR signaling, and inhibition of cyclin D3 and cyclin-dependent kinases production [2]. In tumor-transplanted mice, injection with antibodies that block CTLA-4 function enhanced T cell activation [3], rejected a variety of different tumors, and had long-lasting anti-tumor immunity [4], suggesting that the CTLA-4 may play an important role in carcinogenesis.
The CTLA-4 gene is located on chromosome 2q33, consisting 4 exons that encode separate functional domains: a leader sequence, an extracellular domain, a transmembrane domain, and a cytoplasmic domain [5][6][7]. This gene is polymorphic and more than 100 single nucleotide polymorphisms have been identified [8]. An common polymorphism at position 49 in CTLA-4 exon 1 (rs231775), which causes an amino acid change (threonine to alanine) in the peptide leader sequence of the CTLA-4 protein [9]. Recent studies indicated that this polymorphism may influence the ability of CTLA-4 to bind with B7.1 and affect T-cell activation subsequently [10,11].
Previous studies have identified that this polymorphism is associated with different cancers including lung cancer, breast cancer, and cervical cancer [10,12]. However, the results of studies on the association between the +49 A > G polymorphism and the risk of digestive system cancers remain inconsistent [10,[13][14][15][16][17][18][19][20][21][22][23][24][25][26]. To improve the efficiency of meta-analysis on digestive cancers and reduce the potential between-study heterogeneity which might derive from various cancers in diverse systems, we focused on digestive system cancers only and added more recent studies in this meta-analysis.

Search Strategy
In this meta-analysis, a comprehensive literature research of the US National Library of Medicine's Pub Med database, ISI Web of Knowledge, Medline, Embase and Google Scholar Search (update to November, 2012) were conducted using the search terms including ''CTLA-4'', ''polymorphisms'', ''cancer'', and the combined phrases in order to obtain all genetic studies on the relationship of CTLA-4 + 49G/A polymorphism and cancer. We also used a hand search of references of original studies or reviewed articles on this topic to identify additional studies. The following criteria was used to select the eligible studies: (1) a case-control study on the association between CTLA-4 + 49G/A polymorphism and cancer; (2) detailed number of different genotypes for estimating an odds ratio (OR) with 95% confidence interval; (3) when several publications reported on the same population data, the largest or most complete study was chosen.

Data Extraction
Data extraction was carried out independently by two investigators after the concealment of authors, journals, supporting organizations and funds to avoid investigators' bias. For each eligible study, the following information was recorded: the first author's name, the year of publication, country of origin, cancer type, genotyping method, sources of controls, racial descent of the study population, number of cases and number of controls with different allele frequencies.

Statistical Analysis
The strength of relationship between CTLA-4 + 49G/A polymorphism and cancer was assessed by using Crude OR with 95% CI. We examined the association between the CTLA-4 + 49G/A polymorphism and digestive cancer risks using the following genetic contrasts: homozygote comparison (GG vs. AA), heterozygote comparison (GA vs. AA), dominant genetic model (GG + GA vs. AA), recessive genetic model (GG vs. GA + AA) and allelic comparison (G vs. A). Between-study heterogeneity was evaluated by Q-test. Fixed effects model was used to pool the data when the P-value of Q-test ≥ 0.05, otherwise, random-effects model was selected. Both funnel plot and Egger's test were used to assess the publication bias. (P<0.05 was considered representative of statistical significance). All statistical analyses were performed using STATA11.0 software and Review Manage (v.5; Oxford, England).

Eligible studies
By the inclusion and exclusion criteria, 17 relevant studies involving 5,176 cases and 6,747 controls were selected in this metaanalysis. The main characteristics of these studies are shown in table 1. Genotype distribution of the CTLA-4 + 49G/A polymorphism among cancer cases and controls of the 17 studies are shown in table 2. All studies were case-control studies, including five colorectal cancer studies, four gastric cancer studies, two esophageal cancer studies, two hepatocellular cell carcinoma studies, two oral cancer studies and two pancreatic cancer studies. There were 12 studies of Asian descent and five studies of Caucasian descent. Hospital based controls were carried out in 12 studies, while population based controls were carried out in 5 studies. The genotyping method contains the classic polymerase chain reaction-restriction fragment length polymorphism assay(PCR-RFLP), RFLP and Taqman. The distribution of genotypes in the controls was all in agreement with HWE.

Meta-analysis
The association strength between CTLA-4 + 49G/A polymorphism and the susceptibility for digestive system cancers are shown in

Publication bias
Both Begg's funnel plot and Egger's test were performed to assess the publication bias of the literature. The shape of the funnel plots did not reveal any evidence of obvious asymmetry in the overall metaanalysis ( Figure 1 shows the funnel plot of overall GG vs. AA). Then, Egger's test was used to provide statistical evidence of funnel plot symmetry. The results still did not present any obvious evidence of publication bias in the subgroup analyses.

Discussion
The result of this meta-analysis suggested that CTLA-4 + 49G/A polymorphism was not overall significantly associated with digestive system cancer risk. In stratified analysis by ethnicity, we also failed to detect any significant association in either Asian or Caucasian. However, in subgroup analysis, this polymorphism was significantly linked to higher risks for pancreatic cancer. Besides, when stratified according to study design, positive associations were observed in hospital-based studies.
The CTLA-4 49G>A SNP has been linked to elevated risk of breast cancer in an Iranian population [6], and non-Hodgkin's lymphoma in an European Caucasian population [26]. In addition, two more studies suggested that this polymorphism is associated with different cancers including lung cancer and cervical cancer [10,21]. A metaanalysis conducted by Zheng et al. suggested that the CTLA-4 + 49G/A polymorphism was associated with an increased risk of developing solid tumors (including lung cancer, breast cancer, colorectal cancer, gastric cancer, skin cancer, thymoma, nasopharyngeal carcinoma, cervical squamous cell carcinoma, esophageal cancer, oral squamous cell carcinoma, HBV-related hepatocellular carcinoma, and renal cell cancer [27]. Interestingly, Zhang et al. conducted a meta-analysis and the results indicated that the polymorphism is associated with a decreased risk of lung cancer and breast cancer but not of cervical cancer, colorectal cancer, or gastric cancer [28].
In our analysis, we first reported that there was no statistically increased risk between the CTLA-4 + 49G/A polymorphism and digestive system cancers. In subgroup analysis, we observed this polymorphism was significantly linked to higher risks for pancreatic cancer. We also observed the CTLA-4 + 49G/A polymorphism was associated with an increased risk of developing hepatocellular cell carcinoma but not gastric cancer, colorectal cancer and oral cancer. However, all of these results should be interpreted with caution. On condition that, for some cancer types, only two case-control studies were included, which may have limited power to reveal a reliable association. Furthermore, we observed inconsistent results between hospital-based studies and population-based studies, which may be explained by the biases brought by hospital-based studies, controls in hospital-based studies may be less representative of general population than controls from population-based studies.
There were some limitations in our meta-analysis. Firstly, sample size in any given cancer was not sufficiently large. It might be difficult to get a concrete conclusion if the number of included studies in subgroup was few. Secondly, due to the original data of the eligible studies were unavailable, it is difficult for us to evaluate the roles of some special environmental factors and lifestyles such as diet, alcohol consumption, and smoking status in developing cancer. And thirdly, language bias might derive from the screened references of English documents only.
In conclusion, our meta-analysis suggested that the CTLA-4 + 49G/A polymorphism may be not associated with an elevated digestive system cancer risks. Large well-designed epidemiological studies are needed to validate our findings.