Association between the TERT Genetic Polymorphism rs2853676 and Cancer Risk: Meta-Analysis of 76 108 Cases and 134 215 Controls

Background Several recent studies have identified that the TERT genetic polymorphism rs2853676 is associated with cancer risk, but presented inconsistent results. We investigated these inconclusive results by performing a meta-analysis to systematically evaluate the association. Methods We conducted a search in PubMed, Google Scholar and ISI Web of Science to select studies on the association between TERT rs2853676 and cancer risk. We conducted a stratified analysis using cancer type, ethnicity and source of controls. We calculated the odds ratios (OR) and 95% confidence intervals (CI). Article quality, heterogeneity, sensitivity, publication bias and statistical power were also assessed. Results 26 articles covering 76 108 cases and 134 215 controls met our inclusion criteria. A significant association between TERT rs2853676 allele A and cancer susceptibility was demonstrated under a per-allele risk analysis (OR = 1.08, 95% CI = 1.04-1.13). Stratification analysis revealed an increased cancer risk in subgroups of glioma, lung cancer and ovarian cancer. No significant increase was found in melanoma, breast cancer, pancreatic cancer and colorectal cancer. In a subgroup analysis of lung cancer, a statistically significant increase was only observed in adenocarcinoma. Moreover, a stratified analysis performed for ethnic groups revealed that the significant increase was only observed in Caucasians, whereas a non-significant increase was found in Asians. Conclusions This meta-analysis suggests that the TERT genetic polymorphism rs2853676 is associated with increased risk of glioma, lung adenocarcinoma and ovarian cancer among Caucasians. Further functional studies are warranted to validate this association and investigate further.


Introduction
Cancer is a major global public health problem. Fourteen million people were diagnosed with cancer worldwide in 2012. By 2032, the global cancer incidence is predicted to reach to 25 million [1]. In the United States, cancer is the second leading cause of death following heart disease and the leading cause of death among adults aged between 40 and 79 years [2]. Although the causes of cancer are multi-factorial, genetic and environmental factors play an important role in cancer pathogenesis. Recent epidemiological studies have identified several genetic polymorphism loci on chromosome 5p15.33 that are associated with the risk of many types of cancer [3][4][5]. Chromosome 5p15.33 contains two key genes, cleft lip and palate transmembrane 1-like (CLPTM1L) and telomerase reverse transcriptase (TERT).
As the main catalytic subunit of telomerase, TERT is essential for the maintenance of telomere DNA length in chromosomes [6]. Telomerase is an RNA-dependent DNA polymerase that synthesizes repetitive DNA (TTAGGG repeats) sequences, which bind abundant specialized proteins onto the chromosome ends [7]. The telomeres prevent coding sequence erosion and protect chromosomes from rearrangements, fusion and genome instability by conducting chromosomal complete replication and regulating gene expression [8]. The expression of telomerase is extremely low in most normal human somatic cells, but is present in over 90% of human malignancies. In vitro immortalized cells and the stem cell lines of actively proliferating tissues show a high level of telomerase expression [9,10]. The activation of telomerase is a vital step during cellular immortalization and the malignant transformation of human cells. This activation requires the TERT catalyst [11].
A series of important cancer-related polymorphisms have been reported within the TERT gene using a meta-analysis approach and have been identified as contributing to the risk of several cancers, such as the susceptibility to rs2736098 for lung and bladder cancer [12] and that to rs2736100 for lung cancer and glioma [13]. The rs2853676 polymorphism has been mapped to intron 2 of the TERT gene, which was implicated in an increased risk of glioma in 2009 [14]. Since then, several studies have assessed the association between the polymorphism and cancer risk, but have presented inconclusive results. We performed a meta-analysis to summarize the available evidence and more precisely characterize the relationship between the TERT rs2853676 polymorphism and cancer risk.

Search strategy
According to the Meta-analysis of Observational Studies in Epidemiology guidelines [15], we conducted systematic searches in PubMed, Google Scholar and ISI Web of Science, up to September 20, 2014. We used the systemic literature search terms "TERT or rs2853676," "polymorphism or variant" and "cancer or carcinoma or tumor or neoplasm." All related reference articles and review articles were searched to identify additional relevant eligible publications. Unpublished data were also obtained from the authors by e-mail.

Inclusion and exclusion criteria
Identified studies meeting all of the following criteria were included: (1) articles about the TERT polymorphism rs2853676 and cancer risk that were published in English; (2) a case-control or case-cohort design addressing race and the numbers of affected and unaffected human control subjects; and (3) sufficient data to calculate an odds ratio (OR) with a 95% confidence interval (CI). The exclusion criteria were: (1) investigations in subjects with family cancer risk; (2) published as an abstract, summary, case report, comment letter, review or editorial; and (3) in overlapped case series, in which case all but the latest or largest study were excluded.

Data extraction
Data were extracted independently by two investigators, according to the inclusion and exclusion criteria listed above. Discrepancies were resolved by discussion and consensus. We extracted the first author, year of publication, cancer type, patient ethnicity, source of control group (population-based, hospital-based, multiple or nested-in-cohort controls), number of cases and controls, genotyping method, histological subtype, minor allele frequency, genotype and/or per-allele risk OR and 95% CI from each study. The data were extracted separately by population or cancer type, if these were explicitly given. The quality of each study was evaluated using previously published quality assessment criteria [16]. The quality scores of the studies ranged from 0 to 15. Scores 9 were considered to indicate low quality, while those > 10 were considered to indicate high quality.

Statistical analysis
Statistical analyses were performed using Stata 12.0 software (Stata Corporation, College Station, Texas). All of the tests were two-sided with a p-value. The Hardy-Weinberg equilibrium among the control subjects was assessed with a chi-square test, in which p < 0.05 suggested a significant deviation from equilibrium. The OR and 95% CI were calculated to assess the strength of the association between the rs2853676 polymorphism and cancer risk. The significance of the combined OR was determined with a Z test, in which p < 0.05 was considered statistically significant. Stratified analyses based on cancer type, ethnicity, histological subtype and source of controls were quantified with ORs and 95% CIs. Ethnicity data sets were categorized as Caucasian, Asian, African or multiple. If a cancer type contained only one data source, it was combined into the "other cancers" group.
The heterogeneity between the studies was calculated by Cochran's Q-test, in which p < 0.10 indicated significant heterogeneity. If the heterogeneity was significant, the randomeffects model (DerSimonian and Laird method) was applied [17], otherwise the fixed-effects model was used (Mantel-Haenszel method) [18]. The I 2 was calculated to quantitatively estimate the heterogeneity, with I 2 < 25%, I 2 = 25-75% and I 2 > 75% representing low, moderate and high heterogeneity, respectively [19].
Sensitivity analyses were performed by sequential removal of each study to assess the stability of the results. Begg's funnel plots and Egger's linear regression tests were used to examine the publication bias, in which p < 0.05 indicated statistical significance [20]. Moreover, we estimated the statistical power of each subgroup analysis. Power analyses of the meta-analyses were all conducted using PS (Power and Sample Size Calculations) software version 3.0.5. To guard against Type I errors, α was typically set at 0.05, while β was set at 0.20 to guard against Type II errors, thus a sufficient power of the statistical test would be greater than 80% [21].
A stratified analysis by source of controls indicated a significantly increased risk associated with population-based, hospital-based, and nested-in-cohort controls, with ORs of 1.15 (95% CI = 1.06-1.25), 1.14 (95% CI = 1.03-1.28), and 1.09 (95% CI = 1.00-1.18), respectively. No significant increase was found in multiple-source controls. A stratified analysis by cancer type  was also performed in Caucasians and Asians. The results for glioma and breast cancer were the same in Caucasians as in the overall population, but a non-significant increase in risk was found for glioma and lung cancer in Asians (Table 2).

Heterogeneity test and sensitivity analyses
Significant heterogeneity existed, mainly in all cancer and the subgroups of ethnicity, population-based controls, hospital-based controls and multiple-source controls. However, most of the heterogeneity disappeared, except melanoma and "other cancers," in the analysis of cancer type subgroups (Table 2). A sensitivity analysis was conducted to assess the influence of each study, by sequential omission of each eligible study. The results showed that the significance of the OR was not affected by any single study (Fig 3). In addition, after the removal of a study that resulted in a departure from the Hardy-Weinberg equilibrium, no significant alteration was found in the OR.

Publication bias
Publication bias was assessed with Begg's funnel plots and Egger's test. The shapes of the funnel plots did not show any evidence of publication bias (Fig 4). No significant publication bias was found by Egger's test in the overall or subgroup analyses (Table 2).

Discussion
The TERT gene is the main catalytic subunit of telomerase, which is encoded by a single-copy gene, mapped on chromosome 5p15.33 and contains 16 exons and 15 introns spanning about 35 kb [48]. The gene consists of three distinct structural domains: an RNA-binding domain, a reverse transcriptase domain and a carboxy-terminal extension, which is thought to represent the putative thumb domain of TERT [49]. A high level of TERT expression is involved in a variety of human malignancies. TERT may play an important role in the pathogenesis of cancer [6,9,50]. The TERT gene sequence has been proposed as a general mechanism affecting individual susceptibility to cancer risk [4,5,47]. A growing number of epidemiological studies have been conducted in response, which have provided evidence that TERT polymorphisms contribute to cancer development [4,5].
The polymorphism rs2853676 is located in intron 2 of the TERT gene. The association between this polymorphism and cancer risk has been assessed in several studies, which showed inconclusive results. Only one meta-analysis demonstrated strong evidence that rs2853676 increased the risk of central nervous system tumors, but the evidence for the risk of lung cancer was weak [51]. A recent study from the Population Architecture using the Genomics and Epidemiology and Transdisciplinary Research in Cancer of the Lung consortia identified that rs2853676 was associated with an increased risk of lung adenocarcinoma [4]. Our meta-analysis suggested that the TERT genetic polymorphism rs2853676 allele A increased several cancer risk, based on 76 108 cases and 134 215 controls. The association mainly existed in the Caucasian population, especially for glioma, lung cancer and ovarian cancer. No significant association was found in melanoma, breast cancer, pancreatic cancer or colorectal cancer. In a subgroup analysis of lung cancer, a statistically significant association was only observed in adenocarcinoma. Interestingly, the Asian population showed no significant result in any type of cancer. Notably, one study described an increased risk for prostate cancer [41], whereas another described a reduced risk for testicular germ cell cancer [29]. Further studies are required to validate these associations in urogenital system tumors.
The heterogeneity between our studies was significantly reduced in the analysis of the cancer type subgroups, indicating that the effect of TERT polymorphisms may be modified by tumor origin. The effect may be cancer-type specific and play a different role in the etiology of different tumors [47]. However, the exact functional mechanisms underlying the association between the rs2853676 polymorphism and cancer remains unclear. Several studies have suggested that telomere length alters cancer risk [5,33,52]. However, this alteration has not been observed in rs2853676 [28,38,43], except by Melin et al., who detected potential relevance at higher ages in a small sample [53]. The other plausible mechanisms underlying the association between the rs2853676 polymorphism and cancer risk may be attributable to environmental risk factors or genetic background. The modification of the TERT function is likely to also play an important role. A high linkage disequilibrium with other nearby biologically potential functional polymorphisms or disease-causing mutations may also exist. In National Cancer Institute controls, rs2853676 was in modest linkage disequilibrium (r 2 = 0.25, D' = 0.82) with rs2736100 and showed a similar pattern of association with lung adenocarcinoma (OR = 1.16, p = 3.44×10 -4 ) [54]. A recent Japanese study also identified that TERT rs2853677 (European ancestry: r 2 = 0.59) was associated with lung adenocarcinoma (p = 3.1×10 -40 ) [55]. Park and collaborators speculated that the association between rs2853676 and adenocarcinoma may be influenced by rs2736100 and rs2853677 [4].
Several limitations in this meta-analysis should be addressed. First, our meta-analysis only presented limited studies that were available to adjust the estimates, and more individual data would be required to draw a more precise conclusion. Second, gene-gene and gene-environment interactions may have influenced our results, as cancer is mainly caused by genetic and environmental factors. However, no appropriate information was available to test this. Third, not all of the authors of the included studies agreed to provide their data and exact genotype data were reported in a minority of the studies. The analysis was therefore only conducted with an additive model (per-allele risk analysis). Fourth, in this meta-analysis, the power of several subgroup results was < 80%, indicating that additional high-level studies are still needed.
In conclusion, this meta-analysis suggests that the TERT genetic polymorphism rs2853676 is associated with an increased risk of glioma, lung adenocarcinoma and ovarian cancer among Caucasians, suggesting that the association may be cancer-type and ethnically specific. To validate this association and investigate our findings further, functional studies are warranted.