CASP-8 −652 6N ins/del polymorphism and cancer risk: A literature-based systematic HuGE review and meta-analysis

Caspase-8 (encoded by the CASP-8 gene) is crucial in generating cell death signals and eliminating potentially malignant cells. Genetic variation in CASP8 may affect susceptibility to cancer. The CASP-8 −652 6N ins/del (rs3834129) polymorphism has been previously reported to influence the progression to several cancers. However, the overall reported studies have shown inconsistent conclusions. In this human genome epidemiology (HuGE) review and meta-analysis, the aim was to identify the association between CASP-8 −652 6N ins/del polymorphism and cancer risk. According to the inclusion criteria, 19 case-control studies with a total of 23,172 cancer cases and 26,532 healthy controls were retrieved. Meta-analysis results showed that the del allele, del allele carrier and ins/del genotype of −652 6N ins/del in the CASP-8 gene were negatively associated with cancer risk (OR=0.91, 95% CI=0.84–0.98, P=0.01; OR=0.88, 95% CI=0.80–0.96, P=0.005; OR=0.91, 95% CI=0.85–0.98, P<0.001; respectively, while no significant correlation was observed between the del/del genotype of −652 6N ins/del and cancer risk (OR=0.89, 95% CI=0.79–1.01, P=0.08). In the subgroup analysis by ethnicity, the meta-analysis indicated that Caucasian populations harboring the del allele, del allele carriers and ins/del genotype had a lower cancer risk (OR=0.96, 95% CI=0.93–1.00, P=0.05; OR=0.86, 95% CI=0.75–1.00, P=0.05; OR=0.91, 95% CI=0.84–0.98, P=0.01; respectively). In addition, a negative association was found between the del allele of −652 6N ins/del in the CASP-8 gene and cancer risk in the Asian population (OR=0.89, 95% CI=0.83–0.97, P=0.005). In conclusion, this meta-analysis suggests that the del allele, del allele carrier and ins/del geno-type of the −652 6N ins/del polymorphism in the CASP-8 gene may be protective factors for cancer risk.


Introduction
Cancer is a leading cause of death worldwide, with millions of individuals succumbing to various types of cancer annually (1). Therefore, it is of utmost importance to identify anticancer prevention and treatment strategies. According to epidemiology, cell apoptosis plays a role in the incidence of cancers. Apoptosis, also known as programmed cell death, is a fundamentally important biological process triggered by a variety of stimuli, including deprivation of growth/survival factors, exposure to cytotoxic drugs or DNA damaging agents, activation of death receptors and activity of cytotoxic cells, that is involved in controlling cell number and eliminating harmful or virus-infected cells to maintain cell homeostasis (2)(3)(4). The inappropriate process of apoptosis potentially results in various pathological disorders (5). The caspase family (cysteine and aspartic proteases) is mainly involved in the regulation of cell apoptosis (6), and has two major functions: caspase-1, -4, -5 and -11, as initiator caspases, are primarily involved in the processing and activation of pro-inflammatory cytokines, while caspase-2, -3, -6, -7, -8 and -9, as executor caspases, play a role in the execution phase of apoptosis (6,7). CASP activation has two dinstinct albeit converging pathways: the extrinsic or receptor-mediated pathway, and the intrinsic or mitochondrial pathway. These two pathways possess an independent group of initiator caspases despite using the same group of effector caspases (8)(9)(10). Caspase-8 (CASP-8) is essential for the extrinsic cell death pathways initiated by the TNF family members with the formation of the death-inducing signaling complex (11).
Single-nucleotide polymorphisms (SNPs) are the most common form of human genetic variation, leading to susceptibility to cancer. Findings of previous studies showed that some variants in CASP-8 gene are associated with susceptibility to various human cancers (12,13). A case-control study in a Chinese population found that CASP-8 -652 6N del/del genotypes showed a multiplicative joint effect with FasL and Fas in attenuating susceptibility to pancreatic cancer (14). However, relevant studies on -652 6N del in CASP-8 are inconclusive and inconsistent. Therefore, a human genome epidemiology (HuGE) review and meta-analysis were conducted, including the most recent and relevant articles in order to identify statistical evidence of the association between the CASP-8 -652 6N ins/del polymorphism and cancer risk that have been investigated. Inclusion and exclusion criteria. The included studies had to meet the following criteria: i) case-control study focused on the associations between CASP-8 -652 6N ins/del polymorphism and cancer risk; ii) all patients diagnosed with a malignant tumor confirmed by pathological examination of the surgical specimen; iii) the frequencies of alleles or genotypes in case and control groups could be extracted; iv) the publication was in English or Chinese. Studies were excluded when they were: i) not case-control studies about CASP-8 -652 6N ins/del polymorphism and cancer risk; ii) based on incomplete data; iii) useless or overlapping data were reported; iv) meta-analyses, letters, reviews or editorial articles.

Materials and methods
Data extraction. Using a standardized form, data from published studies were extracted independently by two reviewers to populate the necessary information. The information extracted from each of the articles included: first author, year of publication, country, language, ethnicity, study design, source of cases and controls, number of cases and controls, mean age, sample, cancer type, genotype method, allele and genotype frequency, and evidence of Hardy-Weinberg equilibrium (HWE) in controls. In case of conflicting evaluations, an agreement was reached following a discussion with a third reviewer.
Quality assessment of included studies. Two reviewers independently assessed the quality of papers according to modified STROBE quality score systems (15,16). Forty assessment items associated with the quality appraisal were used in this meta-analysis, scores ranging from 0 to 40. Scores of 0-20, 20-30 and 30-40 were defined as low, moderate and high quality, respectively. Disagreement was resolved by discussion.
Statistical analysis. The odds ratio (OR) and 95% confidence interval (95% CI) were calculated using Review Manager Version 5.1.6 (provided by the Cochrane Collaboration, available at: http://ims.cochrane.org/revman/download) and STATA Version 12.0 (Stata Corp., College Station, TX, USA) software. Between-study variations and heterogeneities were estimated using Cochran's Q-statistic (17,18) (P≤0.05 was considered to be a manifestation of statistically significant heterogeneity). The effect of heterogeneity, ranging from 0 to 100% and representing the proportion of inter-study variability that can be contributed to heterogeneity rather than to chance, was quantified using the I 2 test. When a significant Q-test (P≤0.05) or I 2 >50% indicated that heterogeneity among studies existed, the random-effects model was employed for the meta-analysis. Otherwise, the fixed-effects model was used. To establish the effect of heterogeneity on conclusions of the meta-analyses, a subgroup analysis was carried out. We also tested whether genotype frequencies of controls were in HWE using the χ 2 test. Funnel plots are often used to detect publication bias. However, due to its limitations caused by varied sample sizes and subjective reviews, Egger's linear regression test, which measures the funnel plot's asymmetry using a natural logarithmic scale of OR, was used to evaluate the publication bias (19). When the P-value is <0.1, publication bias is considered significant. All the P-values were two-sided. To ensure the reliability and accuracy of the results, two reviewers populated the data in the statistical software programs independently and obtained identical results.

Results
Characteristics of included studies. Subsequent to the initial screening a total of 105 relevant publications were identified. Nineteen studies (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) appeared to have met the inclusion criteria and were subjected to further examin ation. The flow chart of study selection is shown in Fig. 1. In the pooled analysis, a total of 23,172 cancer cases and 26,532 healthy controls from 19 studies were included and addressed. The   Twelve of these studies were conducted in Asian popul ations, 6 in Caucasian populations and 1 in African populations. The HWE test was performed on the genotype distribution of the controls in all the included studies, 2 of these studies were out of HWE (34,37) and the remaining studies showed to be in HWE (P>0.05). Quality scores of included studies were >20 (moderate-high quality). The characteristics and methodological quality of the included studies are shown in Table I. The genotype distribution of the CASP-8 -652 6N ins/del polymorphism in the case and control groups is shown in Table II.
Main results and subgroup analysis. A summary of the metaanalysis findings of the association between CASP-8 -652 6N ins/del polymorphism and cancer risk is provided in      test. Egger's linear regression test was used to measure the asymmetry of the funnel plot. The graphical funnel plots of included studies appeared to be symmetrical (Fig. 5). Egger's test also showed that there was no statistical significance for all evaluations of publication bias (all P>0.05). Findings of Egger's publication bias test are shown in Table IV.

Discussion
CASP-8, located on chromosome 2q33-q34, encoded by the CASP-8 gene, is a caspase protein that plays a key role in the execution-phase of cell apoptosis (28). When induced by Fas and various apoptotic stimuli, this protein is involved in apoptosis (29). Caspase-8 is known to activate during death receptor-initiated apoptosis, inducing apoptosis and maintaining immune homeostasis and immune surveillance, while the single genetic variants in CASP-8 and their function in human cancer susceptibility remain to be elucidated (21). Second, the control subjects of the present study might not be representative of the general population, necessitating welldesigned population-based studies with large sample sizes and detailed exposure information to validate our findings. Third, there was significant between-study heterogeneity from studies of the -652 6N ins/del polymorphism, while the genotype distribution also showed deviation from HWE in some studies. Fourth, our meta-analysis was based on unadjusted OR estimates as not all published studies presented adjusted ORs, or when they did, the ORs were not adjusted by the same potential confounders, such as age, gender, ethnicity and exposures. In addition, our analysis did not consider the possibility of gene-gene or SNP-SNP interactions or the possibility of linkage disequilibrium between polymorphisms. Therefore, our conclusions should be interpreted with caution.
In conclusion, findings of this study have shown a common insertion-deletion variation in the promoter region of the CASP-8 gene as a low penetrance susceptibility locus for certain common types of human cancers. The del allele, del allele carrier and ins/del genotype of the -652 6N ins/del polymorphism in CASP-8 gene may serve as protective factors for cancer risk. However, these findings should be validated by large-scale, prospective studies investigating more diverse ethnic groups and more detailed environmental exposure data.