Increased risks between Interleukin-10 gene polymorphisms and haplotype and head and neck cancer: a meta-analysis

Molecular epidemiological research suggests that interleukin-10 (IL-10) polymorphisms may be associated with an increased risk of head and neck cancer (HNC), but results remain controversial. To derive a more precise evaluation, we performed a meta-analysis focused on genetic polymorphisms of IL-10. PubMed, Embase, CNKI and Wanfang databases were searched for studies that examined the relationship between IL-10 polymorphisms or haplotypes and HNC risk. The odds ratio (OR) and 95% confidence interval (CI) were applied to assess the relationship strength. Publication bias, sensitivity and cumulative analyses were conducted to measure the robustness of our findings. Overall, nine related studies involving 2,258 patients and 2,887 control samples were analyzed. Significant associations between the IL-10-1082A > G polymorphism and HNC risk were observed (G vs. A: OR = 1.56, 95% CI = 1.27–1.92, P < 0.01, I2 = 69.4%; AG vs. AA: OR = 1.64, 95% CI = 1.32–2.05, P < 0.01, I2 = 55.6%; GG vs. AA: OR = 2.24, 95% CI = 1.69–2.97, P < 0.01, I2 = 38.5%; AG + GG vs. AA: OR = 1.70, 95% CI = 1.36−2.14, P = 0.02, I2 = 61.8%; GG vs. AA + AG: OR = 1.89, 95% CI = 1.23−2.90, P = 0.01, I2 = 46.3%) in the total population, as well as in subgroup analysis. Moreover, increased HNC risks were also associated with the IL-10 −819T > C polymorphism and the GCC haplotype. In conclusion, our meta-analyses suggest that IL-10 polymorphisms, specifically the −1082A > G polymorphism, may be associated with increased risk of HNC development.

in 1992-1996 to 66% in 2002-2006 4 . The development of prophylactic strategies remains critical for managing HNC. For example, many studies have demonstrated that HNC is associated with complex environmental factors, such as tobacco use, alcohol consumption, and intake of vitamins and minerals. In addition, infection with human papillomavirus (HPV) and dental trauma are also thought to be potential HNC risk factors [5][6][7][8][9][10] .
Interestingly, these factors do not explain the variation in susceptibility observed within different populations. Over the past decade, many studies have suggested that leukocytes and their relevant cytokines may play a central role in inflammatory infiltration and malignant transformation [11][12][13] . Interleukin-10 (IL-10) is a cytokine produced by monocytes, macrophages, lymphocytes and other human Thl cells 14 , that plays a functional role in inflammatory and immunoregulatory activities 15 . During carcinogenesis, IL-10 functions in both immune suppression (cancer promotion) and anti-angiogenesis (cancer inhibition) 16 .
IL-10 is an important inflammatory cytokine with anti-inflammatory properties. The IL-10 gene is located on chromosome 1 at q31-32, contains five exons and four introns that span a length of 4.8 kb and encode a 178-amino-acid protein [17][18][19] . Mutations in IL-10 have been detected at several loci, and the association between IL-10 SNPs and disease risk has been heavily studied. The three most common IL-10 SNPs are located in the promoter region (− 1082A > G (rs1800870), − 819T > C (rs1800871) and − 592A > C (rs1800872)) and have been reported to regulate IL-10 transcription and expression [20][21][22] . These mutations influence IL-10 gene transcription and translation, resulting in abnormal cell proliferation and cancer development 23,24 . Numerous molecular epidemiological studies have investigated the association betweenIL-10 gene polymorphisms and cancer risk, such as breast cancers 25 , lung cancer 26 , cervical cancer 27 , and digestive cancer 28 . In 2006, Pratesi et al. 29 published the first study describing the negative association between three IL-10 promoter polymorphisms and the risk of nasopharyngeal cancer in Italian populations. However, the results were not consistent with subsequent studies. To date, no published genome-wide association studies (GWAS) have been performed to explore the precise association between IL-10 polymorphisms and HNC risk. Given the important role of IL-10 in the development of HNC cancer, we conducted a meta-analysis to assess the relationship between IL-10 polymorphisms (− 1082A > G, − 819T > C and − 592A > C) and HNC risk.

Methods
This meta-analysis was designed according to the guidelines described in the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA Compliant) statement 30 . Search strategy. The Pubmed, Embase CNKI and Wanfang databases were searched to identify studies that examined the association between HNC and IL-10 polymorphisms, using the following search terms: "head and neck cancer", "oral cancer", "nasopharyngeal cancer", "pharynx cancer", "larynx cancer", "Interleukin-10", "IL-10", "polymorphism", and "variant". Eligible criteria. All selected studies met the following criteria: 1) the research design was a case-control study; 2) the study focused on the association between the IL-10 polymorphisms and HNC susceptibility; 3) the study included sufficient genotype distribution data to calculate odds ratios (ORs) and 95% confidence intervals (CIs); 4) the study was published in either Chinese or English. The largest sample size or most recent publication was preferentially selected in cases of overlapping data or duplicate publications.
Data extraction. The following information was extracted from all qualified studies by two independent researchers (Niu and Cai): first author, publication year, country, racial descent (categorized as either Asian or Caucasian), source of controls, number of cases and controls with different genotypes, genotyping method, Hardy-Weinberg equilibrium (HWE) for controls, cancer location and study quality assessment.
Quality assessment. Two independent authors (Niu and Du) assessed the quality of the included studies using quality scoring criteria modified from previous meta-analyses (Supplemental Table S1 online, which demonstrated the scale for quality assessment.) 31,32 . Modified criteria were based on traditional quality scoring used for observational studies in genetic epidemiological issues and ranged from 0 points (worst) to 9 points (best). Studies with a score of 6 or higher were classified as high quality, whereas studies with a score of 6 or less were classified as low quality.
Statistical analysis. ORs with 95% CIs were used to assess the strength of the association between the IL-10-1082A > G, − 819T > C and − 592A > C polymorphisms and HNC risk. For the IL-10-1082A > G polymorphism, pooled ORs were obtained for allele contrast (G vs. A), co-dominant model (AG vs. AA; GG vs. AA), dominant model (GA + GG vs. AA), and recessive model (GG vs. AA + GA). These similar genetic models were also used to assess the IL-10 − 819T > C and − 592A > C polymorphisms. Subgroup analyses were performed based on cancer location, HWE status of controls, ethnicity, and study design. ORs were calculated using the random-effects model (DerSimonian and Laird method) when the P-value was less than 0.10 or I 2 was greater than 40% 33 . Otherwise, a fixed-effect model (the Mantel-Haenszel method) was adopted 34 . Cumulative meta-analyses and sensitivity analysis were conducted to evaluate the overall robustness of the study's results. Publication bias was analyzed using Egger's linear regression and Begg's funnel plots 35 . In addition, heterogeneity was assessed using Cochran's Q statistic and the I 2 method, and meta-regression was conducted to analyze heterogeneity 36 . Statistical analysis was performed using STATA version 11.0 (Stata Corporation, College Station, TX, USA) with a two-sided P-value, P < 0.05 was considered significant.

Study characteristics.
A systematic review of the literature identified 111 relevant studies. Figure 1 and PRISMA flow diagram (Supplemental Table S2 and S3 online) show a flow chart of the studies selection procedure. One hundred and two studies were excluded. Ultimately, nine studies 29,37-44 satisfied the outlined inclusion criteria and characteristics (Table 1 and Supplemental Table S4 online), including the quality assessment score that was generated based on the Newcastle-Ottawa Scale of studies. Each of the nine studies focused on the association of the − 1082A > G polymorphism with HNC risk 29,[37][38][39][40][41][42][43][44] , and six studies investigated the association of the − 819T > C and − 592A > C variants with HNC risk 29,37,39,[42][43][44] . Three studies focused on Caucasian populations 29,38,40 , and six studies focused on Asian populations 37,39,[41][42][43][44] . Within the distribution of genotypes in the control groups, only three studies exhibited HWE in the − 1082A > G polymorphism 29,41,44 , and only one study deviated from HWE in the − 819T > C and − 592A > Cvariants 42 .
Sensitivity analysis revealed that no single study qualitatively changed the pooled ORs, indicating that the results of this meta-analysis were stable (Fig. 3 for AG + GG vs. AA model). Cumulative analysis by publication date demonstrated that cancer risk increased gradually and became positive following the study conducted by Farhat et al. in 2008 (Fig. 4 for AG + GG vs. AA model).
Funnel plots were conducted to assess the publication bias, and no evidence of asymmetry was observed ( Fig. 5 for AG + GG vs. AA model). This result was further supported by the analysis using Egger's test (G vs. A: P = 0.09; AG vs. AA: P = 0.17; GG vs. AA: P = 0.69; AG + GG vs. AA: P = 0.12; GG vs. AA + AG: P = 0.57).
Association between the IL-10 − 819T > C polymorphism and HNC risk Six studies consisting of 1,676 cases and 2,230 controls were included in the analysis to determine whether the IL-10 − 819T > C polymorphism was associated with HNC risk. A significant increase in HNC risk was observed in the overall population (C vs. T, OR = 1.15, 95% CI = 1.04-1.21, P = 0.01, I 2 = 20.1%; CC vs. TT, OR = 1.28, 95% CI = 1.03-1.59, P = 0.03, I 2 = 0%), as well as among Asian populations (C vs. T, OR = 1. 16 Table 1. Characteristics of case-control studies on IL-10 −1082A > G and −819T > C and −592A > C polymorphisms and cancer risk included in the meta-analysis. MAF: Minor allele frequency in control group. HN: All cancer locates in head and neck region, no specific description in original article. a HWE in control. Table 2). Furthermore, analysis restricted to HWE studies revealed elevated cancer risk in homozygous comparison (OR = 1.24, 95% CI = 1.07-1.45, P = 0.01, I 2 = 33.3%) and recessive model (OR = 1.53, 95% CI = 1.08-2.15, P = 0.02, I 2 = 0%). Moreover, these results were consistent with subgroup analysis of the population control group (Table 2) Association between the IL-10 − 592A > C polymorphism and HNC risk Six studies consisting of 1,676 cases and 2,230 controls were included in the analysis to determine whether the IL-10-592A > C polymorphism was associated with HNC risk. Overall, no significant association was observed in all five models ( Table 2). Only two genetic models (for CC vs. AA, OR = 1.24, 95% CI = 1.07-1.45, P = 0.01, I 2 = 33.3%; for CC vs. AA + AC, OR = 1.53, 95% CI = 1.08-2.15, P = 0.02, I 2 = 0%) revealed increased risk of HNC in the population control group. Further subgroup analysis of ethnicity and cancer type was conducted, and no significant association was identified. The pooled ORs did not exhibit any change with sensitivity and cumulative analysis, and no publication bias was observed (C vs. A: P = 0.26; AC vs. AA: P = 0.45; CC vs. AA: P = 0.43; AC + CC vs. AA: P = 0.85; CC vs. AA + AC: P = 0.52).

A/A A/G G/G A/A A/G G/G
Association between the IL-10 haplotype and HNC risk Of the selected studies, only four described the association between the IL-10 − 1082A > G, − 819T > C and − 592A > C haplotype and HNC risk (Table 3). Quantitative synthesis indicated that only the GCC haplotype was associated with a significant increase in HNC risk in the overall population (GCC vs. ATA, OR = 1.44, 95% CI = 1.04-2.00, P = 0.03, I 2 = 52.6%; Table 4).

Discussion
Genetic factors have been shown to influence the susceptibility of patients to various diseases and have attracted increasing attention 45,46 . Inflammation and immune cytokines play an important role during the malignant progression of normal epithelium to cancer by driving angiogenesis, cell metastasis and immune-suppression [47][48][49] . Genetic mutations influence the transcription and translation of genes, resulting in abnormal expression of corresponding mRNAs and proteins and deregulation of various cellular processes, such as apoptosis and proliferation.
To date, numerous molecular epidemiological studies have been conducted to evaluate the association between polymorphisms of IL-10 and the risk of HNC development, but results have remained conflicting. Regarding the IL-10 − 1082A > G polymorphism, Wei et al. reported that the AG and GG genotypes were associated with a significant increase in HNC risk compared with the AA genotype in a Chinese population (95% CI = 1.37-3.48 and 1.33-10.84) 37 . A similar increase in HNC risk was also observed in  72 1.37-2.15 <0.01 60.7 1.59 1.22-2.07 <0.01 58.7 3.15 2.21-4 47 1.10-1.96 0.01 70.5 1.62 1.15-2.28 0.01 62.2 1.72 1.18-2.49 <0.01 37.1 1.65 1.17-2 other studies that focused on different ethnicities 39,40,42,43 , whereas other studies identified no significant association between the IL-10 − 1082A > G polymorphism and HNC risk 29,38,41 .   In 2014, a meta-analysis that included four studies focused on nasopharyngeal cancer was published and reported an increased risk with the IL-10 − 1082A > G polymorphism 50 . To our knowledge, this is the first and most comprehensive meta-analysis to date that has explored the association between the IL-10 polymorphisms and HNC risk, more relative studies of HNC and more polymorphisms of IL-10 are collected to make a precise conclusion. Our meta-analysis includes nine studies, consisting of 2,258 patients and 2,887controls, and revealed that the IL-10-1082A > G polymorphism is associated with a significant increased risk of HNC. In subgroup analyses by ethnicity, we found that individuals with the G allele and mutated genotypes had a significant HNC risk compared with healthy individuals in Asian populations, suggesting that the increased HNC risk may be ethno-specific. Six case-control studies of IL-10 − 819T > C and− 592A > C polymorphisms were examined, including 1,676 cases and 2,230 controls. Positive correlations were observed in the − 819T > C polymorphism analyses but not for the − 592 A > C polymorphism.
In the current analysis, we found that the frequency of GCC haplotype was higher in the HNC patients than in controls and was associated with a significant increased HNC risk. The GCC haplotype of the IL-10 promoter may result in increased transcription and IL-10 expression. These effects down-regulate the expression of Th1 cytokines, thus allowing tumor cells to escape immune surveillance 37 . However, due to the small sample size and the limited number of studies examined, it remains unclear whether the GCC haplotype is indeed a marker of HNC patients. Further research is warranted to investigate this relationship.
Stratified analysis was successfully used to relieve moderate heterogeneity bias in the IL-10-1082A > G polymorphism analysis within the Asian population and the oral cancer group, suggesting that ethnicity and cancer location may influence heterogeneity. Moreover, we did not observe any publication bias in the three polymorphisms, demonstrating that the results of this meta-analysis are stable.
It is important to note the limitations of our meta-analysis. First, all of our results may be influenced by casualness due to the small number of studies included and the limited sample size of each study. Larger sample sizes are necessary to accurately clarify the association between the IL-10 polymorphisms and HNC risk. Second, only articles published in English or Chinese were selected, potentially causing a language bias. Third, HNC is a multi-factorial malignant tumor that likely arises from complex interactions between genetic mutations, environmental changes, lifestyle, diet, age and gender. Meta-analysis  Pratesi  2006  89  130  46  79  53  0  66  110  84  0  Nasopharyngeal   Wei  2007  198  210  235  84  69  8  278  51  86  5  Nasopharyngeal   Yao  2008  280  300  319  106  104  31  377  73  125  25  Oral   Hsu  2015  145  112  167  16  107  0  157  16  51  0  Oral   Table 3. Characteristics of case-control studies on IL-10 −1082A > G and −819T > C and −592A > C haplotype and head and neck cancer risk included in the meta-analysis.  Table 4. Meta-analysis of the IL-10 −1082A > G, −819T > C and −592A > C haplotype and head and neck cancer risk. NA: not available.
is a retrospective approach 51 , and the fundamental underlying mechanisms cannot be explained clearly due to unadjusted databases.

Conclusion
Despite these limitations, our results suggest that the IL-10 − 1082A > G polymorphism is a risk factor for HNC, especially in Asian populations. Our findings also indicate that the IL-10 − 819T > C polymorphism also plays an important role in HNC development. No significant association was detected between the IL-10 − 592A > C polymorphism and HNC risk. Moreover, the GCC haplotype was associated with an increased risk of HNC. In the future, additional studies with larger sample sizes are needed to identify the precise pathogenesis of IL-10 polymorphisms in HNC.